WO2015096904A1

WO2015096904A1 - Drug comprising oligo-(2-7)-galacturonate and therapeutic use thereof

Info

Publication number: WO2015096904A1
Application number: PCT/EP2014/003485
Authority: WO
Inventors: Markus PAULMICHL
Original assignee: Paulmichl Markus
Priority date: 2013-12-27
Filing date: 2014-12-23
Publication date: 2015-07-02
Also published as: DE102013022109A1

Abstract

The invention relates to a drug for oral administration, said drug comprising oligo-(2-7)-galacturonic acid, for use in a method for treating or preventing bacterial infections of the gastro-intenstinal tract. The invention further relates to a drug for oral administration, said drug comprising oligo-(2-7)-galacturonic acid, for use in a method for treating or preventing adiposity.

Description

OLIGO (2-7) GALACTURONATE COMPREHENSIVE MEDICAMENT AND

THE THERAPEUTIC USE

The invention relates to a medicament for oral administration for use in a method of treating or preventing bacterial infections of the gastrointestinal tract. The invention further relates to a medicament for oral administration for use in a method for treating or preventing obesity.

The intestinal microbiota contains over 10 ¹⁴ bacteria and plays a role in health and disease. The interaction of microbiota and host plays a key role in maintaining intestinal homeostasis. Changes in either gut microbiota or host genetics can lead to changes in normal gut flora, termed microbial dysbiosis. In an attempt to control the gut microbiota, the use of antibiotics has become a routine procedure for treating both humans and livestock. Studies have shown that antibiotic treatment of chickens, cattle and pigs reduces Salmonella and E. coli infection. Antibiotics multiply but demonstrably Proteobacteria, a strain that contains a large number of pathogenic bacteria. Furthermore, the long-term use of antibiotics has also been shown to contribute to the increased prevalence of antibiotic-resistant bacteria. An additional risk lies in the potential transfer of antibiotic resistance genes to the existing microflora, which contributes to the spread of antibiotic-resistant bacteria. Livestock serves in particular as a reservoir for bacteria that are resistant to antibiotics. These resistant bacteria tend to spread from manure, which is often used as fertilizer, to the water supply and to plants consumed by other animals or humans.

As a result of the adverse effects of continuous antibiotic use, studies have begun to explore alternative methods of controlling and maintaining the normal gut microbiota while preventing infection. In recent years, attention has been focused on dietary supplements. Soluble dietary oligosaccharides derived from various sources have been shown to promote the proliferation of certain beneficial bacterial groups and are therefore referred to as prebiotics. Examples of prebiotics include fructooligosaccharides, galactooligosaccharides, arabinose, galactose, inulin, raffinose, mannose, lactulose, stachyose, annanoligosaccharides, xylooligosaccharides, palatinose, lactosukrose, glycooligosaccharides, isomaltooligosaccharides and soybean oligosaccharides.

A nutritional supplement strategy has always been used for the prevention and treatment of diarrhea. Ernst Moro, an Austrian physician and pediatrician, proved in 1908 that a carrot soup drastically lowered the infant mortality rate from diarrhea in Germany (Kunz C: Historical aspects of human milk oligosaccharides, Adv Nutr 2012; 3: 430S-439S). It was later assumed that oligo (2-7) -galacturonic acid is responsible for the anti-diarrheal effect of carrot soup. There is already a drug that focuses on the effectiveness of substances in the treatment of diarrhea. It was said that the activity is based on the substance hindering the attachment of bacteria to the intestinal wall.

Notwithstanding this hypothesis, or at least complementary thereto, the inventors herein have found that by preventing the growth of certain microbiata in certain regions of the gastrointestinal tract, including inhibiting the growth of pathogens, the substance is actually used in the qualitative and quantitative alteration of the intestinal tract. Microbiota is effective. This was surprising since there was no evidence for such a mode of action. Of particular interest was the finding that the growth of such bacteria supporting the absorption of nutrients is reduced.

Understanding these effects suggests alternative medicinal uses for the substance oligo- (2-7) -galacturonic acid which is the subject of the present invention described below.

The invention relates to a medicament for oral administration comprising oligo- (2-7) -galacturonic acid for use in a method of treating or preventing bacterial infections of the gastrointestinal tract. The drug may also be in the form of a dietary supplement. It can be used in humans or animals. Particularly preferred is the use of the medicament of the invention in the prevention of bacterial infections of the gastrointestinal tract of animals, such as livestock (including cattle, pigs, poultry and fish). The prevention or treatment may be, for example, by administration at regular intervals, for example once a day. In the treatment of animals, the drug can be added to the food of the animals, eg once or twice daily. The drug may be used in place of antibiotics, which would be particularly preferred in the drug treatment of animals, or in addition to antibiotics. Use without antibiotics may be particularly attractive in the preventive administration of the medicament of the invention. The invention further relates to a medicament for oral administration comprising oligo- (2-2) -galacturonic acid for use in a method for treating or preventing obesity. Particularly preferred is the use of the medicament of the invention in the treatment of obesity in humans. The prevention or treatment may be, for example, by administration at regular intervals, for example once a day. In the treatment of animals, the drug can be added to the food of the animals, eg once or twice daily.

The acidic oligosaccharide oligo- (2-7) -galacturonic acid is also referred to below as Galursan HF 7K or GHF7K. Preferably, the substance is used in its isolated form (purity> 90% or> 95%) or synthesized form. In one embodiment, the medicament does not include any further ingredients isolated from carrot.

In one embodiment, the medicament comprises greater than 5% by weight of isolated Galursan HF 7K. Preferred medicaments comprise more than 10, 20 or 40% by weight of isolated Galursan HF 7K.

In one embodiment, the medicament further comprises pharmaceutically acceptable adjuvants such as, for example, cellulose, starch, lactose, hydroxymethyl starch sodium, magnesium stearate, talc or polyvinylpyrrolidone; and / or further includes pharmaceutically acceptable additives, such as diluents, adhesives, disintegrating agents, lubricants, dyes, antioxidants and / or flavoring agents.

In one embodiment, the medicament is Galursan HF 7K and optionally one or more of the additives described above.

In one embodiment, the medicament is in the form of tablets or powder. If the drug is in the form of powder, packages of predetermined doses may be provided for single administration. Further details of the invention will become apparent from the experiments and figures described below.

Method:

Mice.

All experiments used mice with a weakness towards Friend Virus B / NIH (FVB / N) WT. 6-8 weeks after weaning, the mice were either maintained on a control mouse diet (CT, 7922 NIH-07 Mouse Diet, Harlan Laboratories, Indianapolis, IN) or a GFH7K diet (Richter Pharma AG, Wels, Austria) consisting of 2% GFH7K which was added to the control diet and drinking water. The CT diet consisted of 22.5% crude protein, 5.2% fat, 3.7% crude fiber, 3.1 kcal / g energy density, 29% calories from protein, 15% calories from fat and 56% calories from carbohydrates. The diet was fed for 14 days and water was replenished daily. This experiment was repeated twice separately (1: CT n = 8, GHF7K n = 7, 2 .: CT n = 6, GHF7K n = 4). On day 15, the CT and GFH7K-fed mice received ~ 5 cm segments of duodenum, jejunum, terminal ileum (hereafter referred to as ileum), cecum and colon (proximal and distal). The individual intestinal segments were rinsed with phosphate buffered saline (PBS, pH 7.4) and mucosal swabs taken as described above. The intestinal segments were rinsed briefly with 500 μM PBS. The segments were then opened lengthwise, washed thoroughly with PBS and slides were used to scrape the epithelial and mucosal layers. Luminal rinses and mucosal swabs were processed to total DNA stored at -20 ° C until the samples were evaluated by quantitative real-time PCR (qPCR).

Bacterial strains and rearing conditions. Pure cultures of bacterial strains were used to generate standard curves to correlate the number of bacterial cells with qPCR cycle threshold (CT) values. Micrococcus luteus, Peptostreptococcus anaerobius, Staphylococcus aureus, Escherichia coli, Faecalibacterium prausnitzii, and Burkoholdena cepacia were a gift from dr. Daniel J. Hassett. Lactobacillus acidophilus and Rhibozium legaminosarum were purchased from Carolina Biological Supply Company (Carolina Biological Supply Company, Burlington, NC), bacteroidetes thetaiotaomicron ATCC 29741 and Prevotella melaninogenica ATCC 25845 purchased from Fisher Scientific (Thermo Fisher Scientific, Waltham, MA) and Ruminococcus productus ATCC 27340D-5 was purchased from ATCC (American Type Culture Collection, Manassas, VA). E. coli, S. aureus, M. luteus, B. cepacia, L. acidophilus and R. legaminosarum were grown in Luria-Burtani medium (LB; Thermo Fisher Scientific) at 37 ° C in a shaker incubator. R. productus and F. prausnitzii were grown in tryptone yeast excretion glucose medium (TYG; Thermo Fisher Scientific), P. melaninogenica and B. thetaiotaomicron were grown in Tryptone Soy Medium (TSB; Thermo Fisher Scientific) and Peptostreptococcus anaerobius was grown in Him Heart Infusion Medium (BHI) at 37 ° C in a dual port anaerobic chamber (Coy Systems, Coy Lab Products, Grass Lake, MI). The numbers of bacterial cells were determined by a Petroff-Hauser chamber (Hausser Scientific, Horsham, PA) and Colony Forming Units (CFU). qPCR amplification of bacterial 16S genomic DNA sequences.

To extract the total DNA, a QIAamp DNA Stool Kit (Qiagen, Valencia, CA, USA) was used according to the manufacturer's instructions and as described above with addition of lysozyme (10 mg / ml, 37 ° C, 30 min.) And 95 ° C lysis temperature used. qPCR was used to quantify the total bacterial and bacterial phyla, class, gene and species using a Step One Real Time PCR machine (Applied Biosystems, Carlsbad, California USA) with SYBR Green PCR Master Mix ( Applied Biosystems) and bacteria specific Primers to analyze 16S genomic DNA (Table 1) in a final volume of 20 μΙ. The standard curves were generated from pure bacterial cultures and used to calculate the bacterial count from a cycle of thresholds (CT). The bacterial phyla composition was determined using calculated CFU values for each bacterial phyla as a percentage of total bacteria. Total bacteria were calculated using the Universal Bacterial Primer and represent the total number of bacteria per tested intestinal segment. Ion concentration measurements.

Intestinal segments flushed with 100 μM double deionized water were used to analyze the ionic composition of the gut contents of the mice fed CT and GHF7K diets as described above. Irrigations were performed on gut segments of equal length used to remove bacterial load. The withdrawn mud samples were weighed and the intestinal volume was calculated from the total weight minus the 100 μΙ rinse water assuming a density of one. GHF7K diet did not alter intestinal fluid volume (data not shown). The rinses were then at 1400 g for 10 min. centrifuged at 4 ° C to granulate intestinal solids. Flame photometry was used to determine the supernatant Na + and K + concentrations (digital flame photometer, single channel Digital Flame Photometer Model 02655-10, Cole-Parmer Instrument Company Vernon Hills, IL). Chloridometry was used to determine Cl ion concentration (Model 4425100 digital chloridometer, Labconco Kansas City, MO). The calculated ion concentration was normalized to intestinal volumes and expressed as mM. An electronic pH meter (Orion Mdel 720A, Thermo Fisher Scientific Waltham, MA) was used to measure the pH electrochemically.

Statistics. The data are shown herein as mean ± SEM. Differences between groups determined by two factors (food and intestinal region) were determined using two-way analysis of variance (ANOVA). The Holme-Sidak post-hoc test was used with the help of SigmaPlot (Systat Software Inc, San Jose, Calif.) To establish significance between pairwise comparisons. P <0.05 was considered significant, and n is the number of experiments.

Results:

WT-FVB / N mice were fed either with a control diet (CT) or a diet supplemented with 2% GHF7K in water and diet for a period of 2 weeks. The mouse weight was tested at the beginning and at the end of the study. No significant weight changes were observed during the course of the study (Table 2). Furthermore, mice supplemented with GHF7K consumed the same amount of food and water as CT fed mice, indicating that the sugar itself did not stimulate increased food or water consumption or weight gain. To test whether the total number of bacteria in the GHF7K-supplemented mice was altered, DNA was extracted from luminal rinses and mucosal swabs and analyzed by qPC using a universal 16S RNA primer. In the total number of luminal bacteria (total bacterial load) changes were not detected in any intestinal segment (Figure 1A). As with the luminal bacterial population, no changes were detected in the total bacterial load associated with the mucosa (Figure 1B). This indicates that in the diet supplemented with GHF7K, there was no overall overgrowth or decrease of bacteria. This is advantageous because increased total bacteria are attributed to increased bacterial translocation, sepsis and inflammation. Thus, minimal changes in total luminal and mucosal-associated bacterial populations are likely to be beneficial as it minimizes potentially damaging epithelial immune response interactions. To determine if GHF7K could alter the gut microbiota in a manner similar to antibiotics, the bacterial composition was analyzed by qPCR using specific primers for bacterial phyla. Studies have shown that the gastrointestinal tract of mice and humans is dominated by Firmicutes and Bacteroidetes, while actinobacteria, proteobacteria, fusobacteria and Verrucomicrobia-phyla are present in lesser abundance. The major intestinal bacterial phyla of mice were compared as a percentage of the total bacteria as shown in FIG. In the duodenum or cecum (Figure 2A), there were no significant changes in the luminal bacterial population due to the GHF7K diet. There was, however, a decrease in Firmicutes (Δ23.1 ± 3.7% between CT and GHF7K) and an increase in Bacteroidetes (22.3 ± 4.1%) in the jejunum of GHF7K-fed mice. Similarly, the mice fed GHF7K showed a decrease of Firmicutes (19.8 ± 2.4%) and an increase of Bacteroidetes (11, 2 ± 2.3%), whereas in the proximal colon a decrease of Bacteroidetes ( 15.8 ± 1.2%) but no significant change in Firmicutes (0.9 ± 0.2%). In the distal colon, there was a decrease in Firmicutes (10.7 ± 2.2%) as well as Bacteroidetes (14.0 ± 3.0%) in the experimental group. Actinobacteria showed no change in any of the intestinal segments of the GHF7K-fed mice. Interestingly, α, β, y proteobacteria increased in the ileum (8.6 ± 1.8%), proximal (15.0 ± 2.8%) and distal colon (24.2 ± 4.7%) , The increase in proteobacteria is similar to that seen with the use of antibiotics. In the GHF7K-fed mice, the increase in proteobacteria was due to an increase in γ-proteobacteria (ileum: 8.46 ± 1, 9%, proximal colon: 14.0 ± 2.5%, distal colon: 2.4 ± 0 , 8%) and α-proteobacteria (distal colon: 21, 1 ± 3.8%). Proteobacteria, particularly γ-proteobacteria, comprise a number of pathogenic bacteria, such as Escherichia coli, Salmonella, Yersinia, Vibrio and Pseudomonas. The increased γ-proteobacteria in the GHF7K-fed mice can minimize the growth of non-resident pathogenic species, and hence competition, because members of the same phyla occupy the same niche. In the luminal bacterial population, on the other hand, no significant changes in the mucosal-associated bacterial population were found in the lower intestinal segments (Fig. 2B). However, changes in the upper intestinal segments, duodenum and jejunum, were noted. In the GHF7K-fed mice, there was a decrease in the oesophageal o, ß, γ-proteobacteria (3.0 ± 1.5%) and a moderate increase in firmicutes (2.1 ± 0.7%). In the jejunum, there was an increase in actinobacteria (4.2 ± 1, 1%) with a decrease in other unspecified bacterial species (5.9 ± 1.1%). This shows that GHF7K primarily affects the luminal bacterial population and that these effects are region specific.

Next, subgroups of the major phyla from the luminal bacterial population were examined by qPCR using subgroup specific primers and bacterial cell numbers calculated by comparison to standard curves generated from each species. Antibiotics have been shown to reduce most of the Clostridium species, including C. coccoides clusters XlVa and C. leptum clusters IV. Further, antibiotics have been shown to reduce Bacteroides, Prevotella, Lactobacillus, Bifidobacteria and Prevotella and murine intestinal bacteroidetes (MIB) in earlier experiments. These groups were tested in the luminal bacterial population of GHF7K-fed mice to determine if GHF7K reduced these groups similar to antibiotics. In the Firmicutes subgroup Lactobacillus or the actinobacteria subgroup Bifidobacterium, no changes were found in any of the intestinal segments of the GHF7K-fed mice (Figure 3. Lactobacillus and Bifidobacterium are often used as probiotics because of their therapeutic and prophylactic properties. that probiotic bacteria may be beneficial in treating diarrhea and injection with pathogens, so it is beneficial that the GHF7K diet does not reduce any of these valuable bacteria. Examination of the Firmicutes subgroup C. coccoides cluster XlVa and C. leptum cluster IV revealed changes in the GHF7K diet (FIG. 4). In the jejunum of mice fed with GHF7K, reduced C. coccoides were found, while in the cecum, the proximal and distal colon increased C. coccoides appeared. Significantly decreased C. leptum was found in both the duodenum and jejunum of GHF7K-fed mice. The Bacteroidetes subgroups Bacteroides, Prevotella and MIB were also examined (Figure 5). MIB was decreased in the proximal and distal colon, and Prevotella was significantly reduced in the duodenum of GHF7K-fed mice. No changes in the bacteriodes were detected in any intestinal segment. With the exception of the reduced MIB, the changes in the Firmicutes and Bacteriodetes subgroups did not directly reflect changes observed in antibiotics. Together, these data suggest that the GHF7K diet may alter the gut microbiota at the phyla and subgroup levels.

Certain bacterial groups have the ability to alter ion transport. To determine whether GHF7K influenced the luminal ion concentration either directly or indirectly, Na + and K + concentrations of the intestinal contents were determined by flame photometry, and the Cl concentration was determined by chloride ionometry, as shown in FIG. Compared to CT fed mice, mice fed with GHF7K had an increased luminal concentration of Na + in the cecum, with no changes in any other segment (Figure 6A). K + concentration was also affected: K + was reduced in the cecum and in the distal colon of the experimental group (Figure 6B). Cl concentration was increased only in the duodenum and jejunum of GHF7K-fed mice (Figure 6C). The differences in the Na + and K + concentrations in the cecum and Cl concentrations in the upper section of the small intestine found between CT and GH7FK-fed mice may represent changes in intestinal reabsorption. However, these changes were probably corrected by the colon, as there were no differences in the concentration of Na + and Cl in this segment. Concentrations of K + in the distal colon declined found what may indicate a decreased secretion of K +. It is not clear whether this effect is mediated by the host or by bacteria, but represents another level of interaction between bacteria and host. Changes in ion composition may affect the gut microbiota, and altered ionic composition may act as a mechanism the newly proliferating bacteria can keep a niche. These data show that GHF7K supplementation may alter the luminal ion composition along with the altered bacterial composition. In no intestinal segment of the GHF7K-fed mice, changes in intestinal pH were detected (Figure 6D), similar to those observed with galacturonic acid-fed dogs. Taken together, these studies demonstrate that diet supplemented with GHF7K can induce specific regional changes in intestinal microbiota at the level of phyla and subgroups and the ionic composition of the intestinal contents corresponding to changes in the intestinal milieu.

discussion

It is well established that oligosaccharides are degraded in the intestinal tract of mammals and can beneficially act as a prebiotic by modifying the host microbiota. The concept of modulating gut health through nutrition has always been used, but more recently, scientific advances have begun to provide a mechanistic insight into how nutritional supplementation may be beneficial to the host. The aim of the present work was the assessment of the prebiotic properties of the acidic oligosaccharide GHF7K. Our data shows that supplementation of GHF7K prolongs the proteobacteria of the phylum by increasing a- and γ-proteobacteria. The GHF7K dietary supplement also changes the Firmicutes and Bacteriodetes subgroup members in a region-specific manner. This substance is of particular interest to the livestock industry, which currently uses antibiotics for animal health purposes. When GHF7K can reproduce the beneficial effects noted with antibiotics while it holds constant levels of beneficial bacteria, then it could serve as an antibiotic alternative. This would be very important to reduce the spread of antibiotic resistance in the food chain. In this work, we demonstrate in a mouse model that GHF7K mimics the use of antibiotics by multiplying the proteobacteria, and that it could potentially be used as an antibiotic alternative.

Bacteroidetes and Firmicutes are the two dominant phyla in all vertebrates. Studies have shown that mice and humans have a similar composition at high taxonomic levels (phyla, class, order), but that they differ greatly at the lower taxonomic levels (genera, species, subspecies). Despite low taxonomic differences, there are broad trends at the phylum level. Furthermore, the gut microbiota of higher vertebrates (including humans, mice and farm animals) have been found to be similar in core functions, further demonstrating the value of using mouse models to extrapolate large trends in the gut microbiota. Interestingly, no changes were detected in the cecum of GHF7K-fed mice. The cecum represents a "bioreactor" and has proven to be a relatively stable environment. Cecal conditions may prevent the occurrence of drastic changes, thereby preserving the gut microbiota. The cecum has been shown to include a complex microbial community, and although changes in the phyla or major subgroups in this area have not been detected, it is possible that changes will occur at the species level. However, sequencing would be required to fully address these changes.

Prebiotics have been shown to selectively stimulate the growth and / or activity of specific bacteria. Preclinical studies show that prebiotics have the potential to be used in the treatment of diseases and in the prevention of intestinal infections. In this work presented data and data of the literature suggest that prebiotics are the priority whereas leukocyte-associated bacteria appear to be more affected by modifications at the level of the host epithelial cells. In addition to prebiotics and probiotics, antibiotics are also used to modify the intestinal gut microbiota. Many antibiotics have been shown to reduce the beneficial bacteria lactobacillus and bifidobacteria. It is advantageous that GHF7K does not reduce these two groups, as Lactobacillus and Bifidobacteria have been shown to reduce luminal pH, produce short-chain fatty acids, excrete antimicrobial compounds (bacteriocins), induce the production of antimicrobial compounds (defensins) by the host epithelium, the adhesion of pathogenic Prevent bacteria from epithelial cells and actively compete for nutrients that could be used by pathogenic bacteria. Previous studies have shown that supplementation with acidic oligosaccharides, such as galacturonic acids, in baby food does not affect the levels of bifidobacteria or lactobacillus. This is consistent with the present study, which shows no change in Bifidobacteria or Lactobacillus in mice fed with GHF7K.

GHF7K administration also led to region-specific changes in the bacteroidetes and firmicutes of the phyla and subgroup? C. coccoides, C. leptum, Prevotella and MIB. Whether these changes are beneficial, harmful or neutral is not clear. Dietary changes have been shown to produce rapid changes in intestinal metagenomics, which represent all microbial genes of the gut and intestine, as well as related functions. However, as long as the metagenome core functions are maintained in a host, the changes in the specific species are balanced and have no deleterious effect. At this point, we can speculate that GHF7K has no negative effect on the intestinal microbiota, as it did not alter the gross morphology of the gut and did not result in large scale changes, such as weight gain, in the mice. However, further experiments are needed to address this issue comprehensively. Vancomycin, metronidazole, amoxicillin-clavulanic acid, clindamycin, ancomycin, amipenem and neomycin all have been shown to increase phylum proteobacteria. Although proteobacteria are a smaller taxon in the intestinal microbiota, this group has been shown to play a significant, active role in gut metabolism and overall host interaction. This study has demonstrated that GHF7K alters the intestinal microbiota by raising the levels of α and γ proteobacteria, providing evidence of its use as a potential antibiotic alternative.

A potential disadvantage of using antibiotics is that antibiotics can lead to increased susceptibility to further pathogen infections. Whether GHF7K increases susceptibility or protects against colonization with pathogens is currently unknown. In the future, it would be interesting to confront the GHF7K-fed mice with a pathogen, such as Salmonella typhymurium, Escherichia coli, or Clostridium difficile, to see if it actually offers an advantage. Although GHF7K does indeed propagate proteobacteria, it does not fully reflect antibiotic use, indicating that there may be some potential for benefit. Furthermore, preliminary studies with oligo (2-7) -galacturonic acids in poultry farming and the conventional use of Moros carrot soup suggest that GHF7K may benefit the host and prevent and / or remedy infection by pathogens. Therefore, it is plausible that supplementation with GHF7K can prevent infection by either (1) inducing the proliferation of settled proteobacteria, thereby limiting further colonization by other pathogenic proteobacteria, or (2) adding GHF7K to a preferred binding substrate of Proteobacteria members and luminal GHF7K can prevent binding of pathogenic γ-proteobacteria to oligosaccharides on the mucosa of the host.

In addition to the potential of using GHF7K as an antibiotic alternative, GHF7K may be useful for other conditions related to an altered gut milieu or microbial dysbiosis. One An example of this is recognizable in humans for gastric bypass surgery as a treatment for obese patients. Gastric bypass surgery has been shown to promote increased growth of γ-proteobacteria with a corresponding decrease in firmicutes and methanogens responsible for increased energy consumption in obese individuals. We demonstrate herein that GHF7K acts in a manner similar to gastric bypass surgery: reducing firmicutes in the small intestine and increasing γ-proteobacteria. This suggests that GHF7K could also be used as a nutritional supplement in obese patients and similarly produce "beneficial" effects as observed after gastric bypass surgery. Although many oligosaccharides have been shown to alter the intestinal microbiota, it is important to note that each oligosaccharide has its own properties and does not all act on the same bacterial groups or species. The data from this study suggests that the Galursan HF 7K sugar could potentially be used to alter the intestinal microbiota advantageously with fixed changes in the populations of luminal bacteria.

In the following the figures are discussed:

Figure 1. GHF7K-fed mice show no changes in total bacteria compared to mice fed control diet. Total bacteria were quantitated by qPCR using specific universal primers for the bacterial 16S genomic DNA sequence. The number of bacterial cells was calculated from a standard curve and normalized to intestinal flush volume. (A) Bacteria contained in the luminal rinses of mice fed control (n = 14, black bars) and GHF7K (n = 11, white bars) diets. (B) Mucosa-associated (adherent) bacterial stock between control mice (n = 14, black bars) and the GHF7K diet (n = 11, white bars). As expected, the number of total bacteria in the colon is significantly greater than the total number in the small intestine (P <0.001). The GHF7 K diet did not significantly change the number of total bacteria per given segment (P = 0.665), suggesting that the GHF7K diet was the most effective Total bacterial load in the intestine does not change overall. Analyzed by two-way ANOVA with Holme-Sidak post hoc test.

Figure 2. GHF7K-fed mice show region-specific changes in the luminal and mucosa-associated gut microbiota. The relative abundance was calculated as a percentage of bacterial phyla compared to total bacteria for luminal (A) and mucosa-associated bacteria (B). The lettering is shown in Fig. (C). In all bacterial populations, differences were found at the segmental level (P <0.001), indicating different colonization patterns depending on the region. Drastic changes were found in the luminal bacterial population compared to the mucosa-associated bacterial population. In the luminal population, Firmicutes had significant changes in the jejunum (P = 0.029), ileum (P = 0.030) and distal colon (P = 0.048). Bacteroidetes had significant changes in the jejunum (P = 0.042), ileum (P = 0.029), proximal (P <0.001) and distal colon (P <0.001). In the case of actinobacteria, no significant changes were found between diet and segment (P = 0.263). α-, β-, γ-protease bacteria showed significant increases in the ileum (P = 0.031), in the proximal (P = 0.022) and distal colon (P <0.001). In the mucosa-associated bacterial population, changes in firmicutes (P = 0.033) and a-, ß-, γ-proteobacteria in the duodenum (P = 0.025) and actinobacteria (P = 0.014) and non-specific bacteria (P = 0.02) in the jejunum. n = 14 at control diet and n = 11 in GHF7K-fed mice. Analyzed by two-way ANOVA with Holme-Sidak post-hoc test.

Figure 3. The GHF7 K diet does not alter the beneficial bacterial groups Lactobacillus and Bifidobacterium. The bacterial count was examined from luminal rinses of CT and GHF7K-fed mice by qPCR. The values of the beneficial bacteria groups Lactobacillus (A) and Bifidobacterium (B) were none in the luminal population of mice fed control diet (n = 14, black bars) or GHF7 K diet (n = 11, white bars) Differences found. Analyzed by two-way ANOVA with Holme-Sidak post-hoc test.

Figure 4. The GHF7K diet changes the clostridial groups in the colon. The bacterial count was examined from luminal rinses of CT and GHF7K-fed mice by qPCR. Regional changes in the values of Firmicutes members C. coccoides (shown in Figure 4) and C. leptum were noted. C. coccoides decreased in the jejunum, but increased in the cecum and colon of the GHF7K-fed mice (n = 11, white bars). Compared with control mice (n = 14, black bars), C. leptum only decreased in the duodenum and jejunum of the GHF7K-fed mice (n = 11, white bars). * P <0.05 by two-way ANOVA with Holme-Sidak post hoc test.

Figure 5. The GHF7 K diet changes MIB and Prevotella, but does not change bacteroides. The bacterial count was examined from luminal rinses of CT and GHF7K-fed mice by qPCR. No changes were detected in the bacteroidetes member Bacteroides (B), but regional changes were noted in the values of (C) Prevotella and (A) MIB. GHF7K (n = 11, white bars), control (n = 14, black bars). ^* P <0.05 by two-way ANOVA with Holme-Sidak post hoc test.

Figure 6. The GHF7K diet alters the intestinal milieu with regional changes in the concentrations of Na +, K + and Cl, but does not change the pH. The ion concentrations in luminal fluid from intestinal segments of mice fed with control diet (black bars) and GHF7K diet (white bars). (A) The Na + concentration determined by flame photometry increased only in the cecum of the GHF7K-fed mice. (B) The K + concentration determined by flame photometry decreased significantly only in the cecum and distal colon of GHF7K-fed mice. C) The chloridometry-determined Cl concentration increased significantly in the duodenum and jejunum of the GHF7K-fed mice. D) Along the length of the intestinal tract no mice were fed between GHF7K and control diet fed mice Changes in pH detected. GHF7K (n = 11, white bars), control (n = 14, black bars). * P <0.05 by two-way ANOVA with Holme-Sidak post-hoc test.

In summary, it was found that supplementation with GHF7K did not alter mouse weight or daily food consumption. In addition, no changes in the total number of luminal or mucosa-associated bacterial populations were found in the GHF7K-fed mice. GHF7K supplementation significantly altered the composition of luminal and, to a lesser extent, mucosal-associated bacterial populations at the phyla level with region-specific differences. Similar to antibiotic use, the number of proteobacteria in the ileum and colon of GHF7K-fed mice was increased without changes in the number of useful Lactobacillus and Bifidobacterium genera of the phylum Firmicutes. According to the altered intestinal microbiota, changes in the ionic composition of the intestinal fluid were noted. An elevated Cl concentration in the duodenum and jejunum was found, while the Na + concentration in the cecum of GHF7K-fed mice was increased. Decreases were noted at the K + concentration in the cecum and distal colon. It is thus believed that supplementation with GHF7K may alter the intestinal microbiota, which correlates with changes in the intestinal milieu. These data suggest that GHF7K supplementation may be used purposefully to alter the gut microbiota and thus potentially constitute an alternative approach to prophylactic antibiotic use.

Claims

claims

A medicament for oral administration comprising oligo- (2-7) -galacturonic acid for use in a method of treating or preventing bacterial infections of the gastrointestinal tract.

A medicament according to claim 1 for use in the prevention of bacterial infections of the gastrointestinal tract in animals, especially livestock (including cattle, pigs, poultry and fish).

A medicament according to claim 1 for use in the place of antibiotics or in addition to antibiotics.

A medicament for oral administration comprising oligo- (2-7) -galacturonic acid for use in a method for treating or preventing obesity.

5. A medicament according to claim 4 for use in the treatment of obesity in humans.

A medicament according to any preceding claim for use as a nutritional supplement.

A medicament according to any preceding claim which comprises oligo- (2-7) -galacturonic acid in its isolated form, preferably at a purity greater than 90% or greater than 95%, or in its synthesized form.

A medicament according to any preceding claim comprising more than 5%, preferably more than 10 or 20 or 40% by weight of isolated oligo- (2-7) -galacturonic acid.

A medicament according to any preceding claim which consists of isolated oligo- (2-7) -galacturonic acid and optionally one or more additives.

A medicament according to any preceding claim, which is in the form of tablets or powder for oral administration, preferably being provided as packages of predetermined doses for single administration.