Patent Grant
9061046
This application is the U.S. national stage of International Application No. PCT/EP2008/063044, filed Sep. 29, 2008, which claims the benefit of UK Patent Application No. GB0718974.9, filed Sep. 28, 2007.
The present invention relates to the use of oligosaccharides derived from specific arabinoxylans in the treatment and/or prevention of gastrointestinal infections in animals and human beings. More particularly the invention relates to a method for preventing or reducing the gastrointestinal infection of an animal or a human being with bacteria associated with gastroenteritis by supplementing their diets with the said oligosaccharides.
Gastroenteritis is a significant problem in animal husbandry as well as in human medicine. Gastroenteritis is primarily caused by food-borne pathogens, such as bacterial species belonging to the Salmonella, Campylobacter, Escherichia, Listeria, Clostridium, or Shigella geni, which typically ingress through the lower parts of the gastrointestinal tract. In animal husbandry, gastrointestinal infections can be relatively well controlled by inclusion of sub-therapeutic doses of antibiotics in the feed. However, the preventative use of antibiotics is either banned or strongly discouraged in many countries due to the growing emergence of antibiotic-resistant pathogens. Likewise, antibiotics in human medicine are nowadays subject to more strict use policies. Alternative treatments include the use of dietary supplements such as short or medium chain organic acids, manno-oligosaccharides which interfere with mucosal attachment of pathogens, or fructan-based oligosaccharides which stimulate beneficial gut microbiota. Inoculation of animals with competitive-exclusion microbiota is another form of treatment.
There is a need to extend the range of preventive and therapeutic treatment methods against gastrointestinal infections, especially since some of the proposed control agents do not yield consistent results. For instance inulin and oligofructose have been reported to decrease the incidence of systemic infection by Salmonella enterica serovar Typhimurium and Listeria monocytogenes in mice, whereas in rats it increased the levels of S. enterica ser. Enteritidis in the gut and promoted systemic translocation. Likewise, inclusion of a oligofructose from Jerusalem artichokes in chicken diet promoted colonisation of chicken caeca by Salmonella enterica ser. Typhimurium, whereas oligofructose from chicory roots had the opposite effect.
Arabinoxylans are polysaccharides that occur in the cell wall of most monocotyledonous plant species, and are therefore abundantly present in cereal-based diets. They comprise a main chain of β-1,4-linked D-xylopyranosyl units to which O-2 and/or O-3 □-L-arabino-furanosyl units are linked, together with minor substituents such a alpha-(methyl)glucuronyl, acetyl, feruoyl, and/or p-coumaryl groups. Arabino-oligosaccharides (hereinafter referred as AXOS), i.e. oligosaccharides derived by partial hydrolysis of arabinoxylan, have been demonstrated to stimulate the growth and improve the feed intake when added to the diet of animals (WO03/015533), whereas uncleaved arabinoxylan is well known to hamper efficient food utilisation. AXOS has also been shown to stimulate beneficial microflora in the caeco-colonic compartment of the gastrointestinal tract (WO06/002495), while arabinoxylan promotes fermentation in the ileum. The effect of dietary supplement with arabinoxylan on the attachment S. enterica ser. Enteritidis to the ileum of broiler chicks has been investigated. It was found that dietary addition of 0.5% arabinoxylan from corn hull (average molecular weight 500,000) slightly promoted Salmonella attachment when the chicks were raised at 23° C., whereas a slight decrease in attachment was observed in chicks raised at the suboptimal temperature of 29° C., although none of the effects were significant.
In a first object the present invention provides a method for preventing or reducing the infection of an animal or a human being with bacteria associated with the occurrence of gastroenteritis, comprising enterally administering arabinoxylo-oligosaccharides having an average degree of polymerization between 3 and 50 and an average degree of substitution between 0.15 and 0.35. The present invention further provides the use of such arabinoxylo-oligosaccharides in the production of a human or veterinary medicine for preventing or reducing the infection of a subject with bacteria associated with the occurrence of gastroenteritis.
The pathogenic sequence of a bacterial gastrointestinal infection starts with the survival and growth of a pathogen in the lumen of the gastrointestinal tract. Therefore, the strengthening of a subject's colonization resistance against pathogens is an important strategy in the prevention of gastrointestinal infections. Moreover, an improved colonization resistance not only decreases the occurrence of infections but also reduces the severity of an incurred infection. The present invention shows that the oral administration of an appropriate amount of certain arabinxylo-oligosaccharides significantly improves the gastrointestinal colonization resistance of a subject against pathogenic bacteria. Indeed, supplementing such arabinxylo-oligosaccharides to the feed of a model animal orally infected with a gastrointestinal pathogen resulted in a clearly lower fecal excretion of that model pathogen in the faeces in the period following the infection. Moreover, the oral administration of such arabinoxylo-oligosaccharides reduced the translocation of the pathogen towards internal organs. Therefore, in a first object the present invention provides a method to reduce or prevent the infection of a human being or an animal with gastro-intestinal pathogenic bacteria. Said method comprises enterally administering a therapeutically effective amount of specific arbinoxylo-oligosaccharides to said human being or animal prior to or during the occurrence of an infection. Amongst others, the method of the present invention can be used to reduce or prevent the infection by gastro-intestinal pathogenic bacteria belonging to the Salmonella, Campylobacter, Escherichia, Listeria, Clostridium, cholera, Vibrio, Yersinia, Aeromonas, Pleisomonas or Shigella geni.
As used herein “arabinoxylo-oligosaccharides” refers to oligosaccharides derived from arabinoxylans comprising a main chain of β-1,4-linked D-xylopyranosyl units to which O-2 and/or O-3 α-L-arabino-furanosyl units are preferably linked. For the purpose of the present invention it is preferred that the average degree of substitution of the arabinoxylo-oligosaccharides varies between 0.15 and 0.35. Preferably, the average degree of polymerisation of the arabinoxylo-oligosaccharides used in the present invention is at least 3, more preferably at least 4, for instance at least 5. Preferably, the average degree of polymerisation of the arabinoxylo-oligosaccharides used in the present invention is at most 50, more preferably at most 20, most preferably at most 10. The average degree of substitution and the average degree of polymerisation can be determined by any method known in the art, e.g. as previously described by K. Swennen et al. in J. Sci. Food Agric. (2006) 86:722-731. In short, the carbohydrates in an arabinoxylo-oligosaccharides-containing preparation were hydrolysed using trifluoric acid and the resulting monosaccharides were reduced and converted to alditol acetates, which were then analyzed using gas chromatography. The total arabinoxylan-content was calculated as being 0.88 times the sum of the monosaccharides xylose and arabinose, while the average degree of substitution was determined as the arabinose-to-xylose ratio. The average degree of polymerization was calculated as the sum of the total xylose and arabinose divided by the amount of reducing end xylose.
Arabinoxylo-oligosaccharides suitable for use in the method according to the present invention can be obtained by partial hydrolysis of arabinoxylans extracted from cereals or cereal derived material. More preferably, these arabinoxylo-oligosaccharides may be obtained by hydrolysis of arabinoxylans extracted from bran, for instance wheat or rye bran.
When using the method according to the present invention for the prevention or treatment of the infection of an animal, e.g. a mammal, with gastro-intestinal pathogenic bacteria it is preferred to administer a therapeutically effective amount of the specific arabinoxylo-oligosaccharides through (together with) the feed. Preferably the animal feed is supplemented with at least 1 g, for instance at least 2 g, of arabinoxylo-oligosaccharides per kg feed. Preferably the animal feed is supplemented with at most 50 g, more preferably at most 10 g, most preferably at most 5 g, for instance at most 4 g of arabinoxylo-oligosaccharides per kg feed. The method of the present invention is useful as an element in the prophylaxis of livestock animals, poultry and pets. It more particularly contributes to the prophylactic management of the breeding of calves, chickens, turkeys, pigs and horses amongst others.
When using the method of the present invention in pet animals, such as cats and dogs, it may be advantageous to incorporate the daily effective amount of arabinoxylo-oligosaccharides in a daily serving of a pet food. Preferably, such pet food comprises at least 0.25 g, more preferably at least 0.5 g of arabinoxylo-oligosaccharides per daily serving of said pet food. Preferably, such pet food comprises at most 10 g, more preferably at most 5 g, most preferably at most 2.5 g of arabinoxylo-oligosaccharides per daily serving of said pet food. In a particular embodiment, it is preferred to include between 0.25 and 2 g, more preferably between 0.25 and 1 g of arabinoxylo-oligosaccharides per daily serving of a domestic cat food. In another particular embodiment, between 0.25 and 3 g, more preferably between 0.5 and 2 g of arabinoxylo-oligosaccharides are included per daily serving of food for dogs with a body weight below 10 kg, while between 0.25 and 5 g, more preferably between 0.5 and 3 g of arabinoxylo-oligosaccharides are included per daily serving of food for dogs with a body weight above 10 kg.
When using the method of the present invention in pet animals, such as cats and dogs, the daily effective amount of arabinoxylo-oligosaccharides can alternatively be included in a treat food for pets, such as biscuits or cookies. Typically, such pet treat food comprises between 0.25 and 5 g, more preferably between 0.25 and 2.5 g, for instance between 0.25 and 1 g of arabinoxylo-oligosaccharides per serving of said treat food.
When using the method according to the present invention for the prevention or treatment of the infection of a human with gastro-intestinal pathogenic bacteria, the arabinoxylo-oligosaccharides can be administered in admixture with a drink (e.g. water, milk, soft drink or beer), or in the form of a tablet, dietary supplement, candy, chewing gum or a food product. For the treatment of infants the arabinoxylo-oligosaccharides can be incorporated into an infant formula. The present invention can also be accomplished by incorporating the specific arabinoxylo-oligosaccharides in yoghurts, dairy products, fruit juice and cereal based products, such as bread, breakfast cereals, cookies and crackers amongst others. One aspect of the present invention relates to the finding that the consumption of between 0.25 and 10 g, preferably between 1 and 5 g, for instance between 1 and 3 g of arabinoxylo-oligosaccharides per day per human significantly reduces the presence in feces of several bacteria species associated with gastroenteritis. The reduced presence in the feces reflects a decrease of the presence of these bacteria in the intestinal flora, reducing the occurrence and severity of gastro-intestinal infections.
In a second object, the present invention relates to the use of said arabinoxylo-oligosaccharides in the production of a veterinary medicine for preventing or reducing the infection of an animal, e.g. a mammal, by gastro-intestinal pathogenic bacteria. Preferably said veterinary medicine can be incorporated into the feed of an animal in need thereof at a dose corresponding to between 1 and 50 g, more preferably between 1 and 10 g, most preferably between 1 and 5 g, for instance between 2 and 4 g of said arabinoxylo-oligosaccharides per kg feed. Preferably such veterinary medicine comprises at least 8% (w/w), more preferably at least 20% (w/w), most preferably at least 40% (w/w), for instance more than 60% by weight or more than 80% (w/w) of said arabinoxylo-oligosaccharides. Alternatively, said veterinary medicine may be added to the drinking water of the animal, preferably the veterinary medicine is added to water in order to obtain a weight concentration of said arabinoxylo-oligosaccharides between 0.25 and 20% (w/w), more preferably between 0.25 and 10% (w/w), most preferably between 0.25 and 5%. Furthermore, the said veterinary medicine may also be included into pet foods or in treat food for pets in order to supplement such pet foods with said arabinoxylo-oligosaccharides as indicated above.
In a third object the present invention relates to the use of said arabinoxylo-oligosaccharides in the production of a pharmaceutical composition for preventing or reducing the infection of a human being with gastro-intestinal pathogenic bacteria. The pharmaceutical composition of said medicament allows for the administration of a daily dose of between 0.25 and 10 g, preferably between 1 and 5 g, for instance between 1 and 3 g of arabinoxylo-oligosaccharides. Preferably said medicament comprises at least 8% (w/w), more preferably at least 20% (w/w), most preferably at least 40% (w/w), for instance more than 60% by weight or more than 80% (w/w) of said arabinoxylo-oligosaccharides.
Pharmaceutical compositions for the oral administration of the said arabinxylo-oligosaccharides can take the form of solid formulations such as, for example, tablets or capsules prepared by conventional means with one or more pharmaceutically acceptable excipients such as, but not limited to, binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g. lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g. magnesium stearate, talc or silica); disintegrants (e.g. potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). The tablets can be coated by methods well known in the art. Liquid formulations for oral administration can take the form of, for example, solutions, syrups or suspensions, or they can be presented as a dry product, such as powders for dispersion or dissolution into water and/or other suitable liquid vehicles before use. Such liquid formulations can be prepared by conventional means with one or more pharmaceutically acceptable additives such as, but not limited to, suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g. lecithin or acacia); aqueous or non-aqueous vehicles (e.g. almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g. methyl- or propyl-p-hydroxybenzoates or sorbic acid). These formulations can also contain buffer salts, flavouring agents, colouring agents and/or sweetening agents, as appropriate.
In a particular embodiment of this invention, said medicament may be formulated as a powder that can be admixed with a food or drink product, wherein said powder comprises between 5 and 80% (w/w), more preferably between 10 and 60% (w/w), for instance between 30 and 50% (w/w) of said arabinoxylo-oligosaccharides. Said powder preferably further comprises a flowing agent, such as silica and optionally a filler. In another particular embodiment said medicament may be formulated as a syrup, preferably comprising between 8 and 50% (w/w), more preferably between 10 and 50% (w/w), for instance between 20 and 40% (w/w) of said arabinoxylo-oligosaccharides. Said syrup may further comprise an aqueous or non-aqueous vehicle, a suspending agent, an emulsifying agent, a preservative, a buffer salt, a flavouring agent, a colouring agent and/or a sweetening agent.
The pharmaceutical formulation of this invention may also take the form of, for example, a chewing gum, a confectionery, a gel, and the like. Next to arabinoxylo-oligosaccharides, a chewing gum composition of the present invention may include a gum base and one or more of the other typical chewing gum composition components such as sweeteners, softeners, flavouring agents and the like. The chewing gum compositions of the present invention, may be coated or uncoated and may be in the form or slabs, sticks, pellets, balls amongst other forms. In a further aspect of the present invention, the chewing gum compositions may include an insoluble gum base generally comprising one or more elastomers, plasticizers, waxes, fats, oils, emulsifiers, fillers and/or texturizers. Other ingredients used in chewing gum compositions may further include both natural and artificial, sugar-based and sugarless sweeteners. Sugarless sweeteners include, but are not limited to, sugar alcohols such as sorbitol, mannitol, xylitol, hydrogenated starch hydrolysates, maltitol and the like. High intensity sweeteners such as sucralose, aspartame, neotame, salts of acesulfame and the like may also be present. Flavouring agents for use in chewing gum compositions are well known in the art and include citrus oils, peppermint oil, spearmint oil, oil of wintergreen, menthol and the like. Softeners may be present to modify the texture of the chewing gum composition. Other materials which may be present in the gum composition of the present invention include antioxidants (e.g. butylated hydroxyanisole, butylated hydroxytoluene, β-carotenes, tocopherols, colorants, flavorants and the like.
The term “confectionery” as used herein includes, but is not limited to, nougats, candies, panning goods, gel confections, fondants, lozenges, hard boiled candies, mints, troches, pastilles, microcapsules, and other solid forms including freeze dried forms (cakes, wafers, and tablets) and fast dissolving solid forms including compressed tablets and water soluble thin films. Lozenges may include discoid shaped solids comprising said arabinxylo-oligosaccharides in a flavoured base. The base may be a hard sugar candy, glycerinated gelatin, or a combination of sugar with sufficient mucilage to provide a stable form at room temperature. Lozenge compositions (compressed tablet type) typically include one or more fillers (compressible sugar), flavouring agents and/or lubricants. Details regarding the preparation of confectionery compositions can be found in Skuse's Complete Confectioner (13th Edition) (1957) the content of which is incorporated herein by reference.
It is preferred that the said daily effective dose of the arabinoxylo-oligosaccharides is between 1 to 10, more preferably 1 to 7, most preferably 1 to 5, for instance 3 or 4, tablets, capsules, chewing gums, or confectionery used as a pharmaceutical composition for the administration of said arabinoxylo-oligosaccharides.
A preparation comprising said arabinoxylo-oligosaccharides, which is in particular suitable for use in animal feed can be produced starting from the ‘thin stillage’ derived from the industrial ethanol production using cereals such as wheat, corn, barley or rye. When ethanol-containing fermented mash from cereals, produced by yeast-mediated fermentation after mashing and enzymatic liquefaction and saccharification of the starch, is distilled to obtain ethanol, a slurry comprising an aqueous phase and solids is obtained, which is typically referred to as ‘whole stillage’. The whole stillage can be separated through physical separation, such as filtration, centrifugation or decantation, in a liquid aqueous fraction called ‘thin stillage’ and a solid-enriched fraction called ‘wet distilled grains’. On dry matter basis, the thin stillage from cereal fermentation contains between 20% and 40% (w/w) of said arabinoxylo-oligosaccharides. This product can be dried by a sequence of water evaporation and drying steps, either with or without mixing with a carrier product suitable for nutritional us, such as cereal bran, to facilitate the drying process. The ratio of the thin stillage dry matter to the dry matter of the carrier product can vary between 1:0 and 1:4 (w/w on dry matter basis). The arabinoxylo-oligosaccharides content of the final dry product depends on the ratio of thin stillage over the drying carrier, and preferably contains between 8 and 40% (w/w), more preferably between 15 and 40% (w/w), most preferably between 20 and 40% (w/w), for instance between 25 and 35% of said arabinoxylo-oligosaccharides. Preferably, such arabinoxylo-oligosaccharides-containing preparations are added to the feed in amounts allowing to supplement the feed with between 1 and 50 g, more preferably between 1 and 10 g, most preferably between 1 and 5 g of said arabinoxylo-oligosaccharides. For instance, when a feed is supplemented with such thin stillage derived arabinoxylo-oligosaccharides-containing preparation comprising 20% of arabinoxylo-oligosaccharides said preparation is preferably added to the feed in an amount varying between 5 and 250 g, more preferably between 5 and 50 g, most preferably between 5 and 25 g per kg of feed. Therefore, in a fourth object the present invention provides a method for the production of a preparation between 8 and 40% of arabinoxylo-oligosaccharide. This method comprises the separation of the aqueous phase (thin stillage) from a mash of yeast fermented cereals after distillation of the ethanol from said mash (whole stillage) and subsequently drying said isolated thin stillage.
Materials and Methods
AXOS Preparations.
AXOS-9-0.25 was prepared from wheat bran by extraction with a glycosyl hydrolase family (GHF) 10 endoxylanase essentially as previously described (Swennen et al. cited supra), except that the step of protease treatment of the bran was omitted. This preparation has a content in polymeric xylose and arabinose of 61% on a dry matter basis, the average degree of polymerisation of AXOS is 9, and the arabinose to xylose ratio is 0.25. AXOS-3-0.25 was prepared by enzymatic extraction from wheat bran as previously described (Swennen et al. cited supra), except that that the xylanase treatment was performed for 10 hours at 50° C. with a GHF 11 xylanase (Grindamyl H640, commercially available from Danisco, Denmark) at 1.2 units per g destarched and deproteinised wheat bran, and for another 10 hours at 50° C. after addition of a GHF 10 xylanase (Shearzyme 500L, commercially available from Novozymes, Denmark) at 21 units per g destarched and deproteinised wheat bran. This preparation has a content in polymeric xylose and arabinose of 81% on a dry matter basis, the average degree of polymerisation of AXOS is 3, and the arabinose-to-xylose ratio is 0.25.
Feed Composition.
The chicks were fed a single wheat-corn-soybean diet throughout the experiment. Four different diets were prepared. The control ration contained no added AXOS and had the composition shown in Table 1. The 0.2% AXOS-3-0.25 diet consisted of the control ration with addition of 0.25% (w/w) AXOS-3-0.25, which corresponds to 0.2% AXOS content after correction for purity. The 0.2% AXOS-9-0.25 diet consisted of the control ration with 0.33% (w/w) of added AXOS-9-0.25, corresponding to 0.2% AXOS content after correction for purity. The 0.4% AXOS-9-0.25 diet consisted of the control ration with 0.66% (w/w) of added AXOS-9-0.25, corresponding to 0.4% AXOS content after correction for purity.
Salmonella Strain.
Salmonella enterica ser. Enteritidis strain SE147, a well-characterized streptomycin-resistant strain isolated from a poultry farm was used for challenge of the chickens. The strain was grown for 6 hours in Luria-Bertoni medium (LB). The number of colony-forming units (cfu) per milliliter was determined by plating 10-fold dilutions of the bacterial suspension on brilliant green agar (BGA, commercially available from Oxoid, Basingstoke, United Kingdom) containing streptomycin (100 μg/ml), whereafter the plates were incubated aerobically for 20 hours at 37° C. The initial bacterial suspensions were kept at 4° C. during plate counting, and they were diluted in phosphate buffered saline (PBS) to the appropriate density prior to use in the in vivo trial.
In Vivo Trial.
One-day-old chicks of mixed sex (224 in total, Ross breed) were housed in a bio-contained poultry house. The birds were randomly divided in 4 groups of 56 each, and each group was reared in a 1 m2 surface pen with a steel rod floor. Optimal house temperature was provided by a central water heating unit and infrared bulbs and a continuous light regime was used throughout the experiment. All birds received feed and water (1 drinker per pen) ad libitum. They were orally vaccinated against coccidiosis (Paracox-5, commercially available from Schering-Plough Animal Health, Brussels, Belgium) at 3 days post-hatch. The birds were orally inoculated with 2.5×109 cfu Salmonella per animal at 14 days post-hatch. At the day of Salmonella inoculation, cloacal swabs were taken from all animals. At days 1, 3, 7, 11, 15, 19, and 23 after inoculation, cloacal swabs were taken from all animals remaining in the pens. At days 1, 3, 7, 11, 15, 19, and 23 post inoculation, a total of 8 birds per treatment group were euthanized by intravenous T61 injection and samples of caecum and spleen were taken for bacteriological analysis. The experiment was performed under supervision of the ethical committee of the Faculty of Veterinary Medicine, Ghent University.
Bacteriological Analysis.
Cloacal swabs were directly inoculated on BGA plates containing 100 μg/ml streptomycin, which were incubated overnight at 37° C. Swab cultures were scored 0 for no colonies, 1 for 1 to 10 colonies, 2 for 11 to 100 colonies, 3 for 101 to 1000 colonies, or 4 for more than 1001 colonies of S. enterica. After plating, the swabs were enriched by immersing in buffered peptone water and incubating overnight at 37° C. For those samples that scored negative by direct plating of the swabs, the overnight buffered peptone water cultures were further enriched by addition of 1 mL of this suspension to 9 mL brilliant green tetrathionate broth. After overnight incubation at 37° C., a drop of this suspension was plated on BGA containing 100 μg/ml streptomycin. Samples of caeca and spleen were homogenized in 10 volumes of buffered peptone water, and 10-fold dilutions were made in buffered peptone water. For each dilution 6×20 μL drops were inoculated on BGA containing 100 μg/ml streptomycin and after overnight incubation (37° C.) the number of colony-forming units per gram of tissue was determined after counting the bacterial colonies. For samples that were negative after titration, pre-enrichment was performed as described above for the cloacal swabs. Samples that were negative for Salmonella presence after titration but positive after Salmonella enrichment were presumed to contain 0.5×101 cfu/g. Samples that were negative after enrichment were presumed to have 0 cfu/g. The SPSS 9.0 software was used for statistical analysis. Datasets on Salmonella score or Salmonella counts were analysed by multivariate ANOVA with days after inoculation and treatment group as independent variables, using the Scheffe error protection for inter-variable comparisons. The nonparametric Kruskal-Wallis test was used to check for inter-treatment effects on Salmonella score or Salmonella counts at each time point, and in case inter-treatment effects were detected (P<0.05), the nonparametric Mann-Whitney test was used to determine significant differences between the control and each of the treatment groups. Datasets on percentages of Salmonella-positive swabs were analysed by logistic regression with days after inoculation and treatment group as independent variables. The Cochran-Q test was used to check for inter-treatment effects on percentages of Salmonella-positive swabs at each time point, and in case inter-treatment effects were detected (P<0.05), the McNemar change test was used to determine significant differences between the control and each of the treatment groups.
To assess the effect of AXOS on intestinal colonisation by S. enterica ser. Enteritidis, as a representative of a enteric pathogen, an in vivo infection trial was performed on chickens. Four different treatment groups were compared; the control group was fed a regular wheat-corn-soybean based diet, the second group was fed the control diet base with addition of the short chain AXOS-3-0.25 at 0.2%, and the third and fourth groups received the control diet base supplemented with the medium chain AXOS-9-0.25 at 0.2% and 0.4%, respectively. Inoculation with Salmonella was performed when the chicks were 14 days of age, and colonisation of the intestines by Salmonella was monitored by taking cloacal swabs from all living animals and by enumerating Salmonella in the caeca from a sample of euthanized animals. Cloacal swabs were taken from all animals at the day of inoculation. None of these swabs revealed the presence of Salmonella, indicating that there was no infection with Salmonella prior to the artificial inoculation.
During the period from day 1 to 11 days post infection (dpi), more than 80% of the cloacal swabs in the control group tested positive for Salmonella presence, while the percentage of positive swabs steadily declined in the 15-23 dpi period (
Measurement of Salmonella colonisation of the caecum yielded similar results as provided by the cloacal swab scores (
The extent of Salmonella translocation to internal organs was assessed by measuring the Salmonella levels in spleen at different time points after infection (
In conclusion, dietary addition of AXOS helps to protect animals from intestinal colonisation of an orally applied bacterial pathogen, as well as systemic translocation of the pathogen to organs. The protection is dose-dependent, since the level of protection was, for all examined tissues, higher in the animals receiving 0.4% dose than those receiving 0.2% of AXOS-9-0.25.
AXOS Preparation.
AXOS-5-0.20 was prepared from wheat bran by extraction with a glycosyl hydrolase family (GHF) 10 endoxylanase as previously described (Swennen et al. cited supra), except that the step of protease treatment of the bran was omitted. This preparation had an average degree of polymerisation of 5 and an arabinose to xylose ratio of 0.20.
Study Design.
6-week-old male rats (Wistar) are purchased from Elevage Janvier (Le Genest-St-Isle, France) and randomly assigned to 3 groups of 8 rats each. The rats are housed in stainless steel wire-bottom cages in an environmentally controlled room (22° C.) with a 14-10 hours light-dark cycle. Rats are given free access to water and to pellets (10 mm) of the ‘control’ diet (Table 4) during 6 days. After 6 days of adaptation on the control diet, the rats are randomly assigned to one of 3 different treatment groups, and the groups are each given free access during 10 days to pellets (10 mm) of one of the 3 diets described in Table 4, respectively, a control diet, a 2.5% AXOS diet and a 5% AXOS diet.
Animals were weighed and feed intake is measured 3 times per week. After said 10 days, all animals are orally infected with a clinical isolate of Salmonella enteridis, as described by Bovee-Oudenhoven et al. in Gastroenterology (1997) 113:550-7. The animals are infected in the morning by gastric gavage with 0.5 ml of saline containing 3% (wt/vol) sodium bicarbonate with 5×108 viable S. Enteridis. The day before infection and 1, 3, 6 and 9 days after Salmonella infection, fresh faecal samples are collected. The number of Salmonella in the faeces are quantified by plating on Modified Brilliant Green Agar as previously described (Giaffer et al., 1991). Throughout the infection experiment the rats of each experimental group are fed their respective experimental diet.
In a second infection experiment with rats the effect of administration of AXOS in the feed on translocation to the spleen at day 2 after infection is investigated. The experimental set-up is similar to that described above. At the section day the animals are sacrificed and the spleen is aseptically removed, weighed and homogenized in sterile saline (10-fold dilution) and plated on Brilliant Green Agar for counting as described in (Oudenhove et al., 1994). Viable Salmonella counts are expressed as the total log 10 colony-forming units in the spleen.
The study shows a reduction of the fecal Salmonella excretion in the groups fed an arabinoxylo-oligosaccharide-containing diet, illustrating a stimulating effect of the arabinoxylo-oligosaccharides on the colonization resistance against Salmonella. The study further provides an indication that the oral administration of arabinoxylo-oligosaccharides to rats reduces the translocation of Salmonella to internal organs and more particularly to the spleen.
AXOS Preparation.
AXOS-5-0.20 was prepared from wheat bran by extraction with a glycosyl hydrolase family (GHF) 10 endoxylanase as previously described (Swennen et al. 2006), except that the step of protease treatment of the bran was omitted. This preparation had an average degree of polymerisation of 5 and an arabinose to xylose ratio of 0.20.
Subjects
Sixty healthy volunteers (men and women; age ranging between 18 and 80 years) are included in the study. None of the subjects had severe gastrointestinal complaints in the 3 months prior to the start of the study and had ever had abdominal surgery. They have been free of antibiotics or any other medical treatment influencing gut transit or the intestinal flora for at least 3 months before the start of the study. During the study the volunteers are urged to have a regular eating pattern (3 meals per day) and the intake of fermented milk products containing living bacteria or the intake of compounds known to modulate the composition of the intestinal flora is forbidden.
Study Design.
The duration of the entire clinical trial is 19 weeks. The clinical trial starts with a run-in period of 1 week, followed by 4 administration periods of 3 weeks each, with a wash out period of 2 weeks between the administration periods. In the course of the study all volunteers receive three dosages of AXOS-5-0.20 (2.5 g/day, 5.0 g/day and 10 g/day) and a placebo. The volunteers are divided into four groups in a random manner, with the same number of persons in each group, following a double blind protocol. The various treatments are assigned to the groups according to a 4×4 ‘Latin Square design’, allowing the order of the treatments to be different for every group. The study design is schematically presented in
During the administration periods, the volunteers are asked to consume 20 ml of a specific type of syrup at breakfast and in some administration periods an additional 20 ml at diner, this according to the intake scheme specified in Table 2. The compositions of the syrups used in the study is given in Table 3.
During the one-week run-in period and the last week of each intake period (AXOS or placebo), the volunteers are asked to fill in a questionnaire on a daily basis. The questions herein are designed to evaluate the occurrence and gravity of symptoms reflecting gastrointestinal complaints. The questions interrogate the volunteers on the occurrence of mild gastrointestinal discomfort, such as bloating or increased flatulence, as well as on more serious complaints, such as abdominal cramps, diarrhea and vomiting.
The outcome of the study indicates that the oral administration of arabinoxylo-oligosaccharides improves to overall gastrointestinal well being and lowers the occurrence of gastrointestinal discomfort and attenuates the gravity thereof. This suggests a better protection against gastrointestinal infection as a result of the intake of the arabinoxylo-oligosaccharides.
| Number | Date | Country | Kind |
|---|---|---|---|
| 0718974.9 | Sep 2007 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2008/063044 | 9/29/2008 | WO | 00 | 3/26/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2009/040445 | 4/2/2009 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 4494530 | Jansma et al. | Jan 1985 | A |
| 5362502 | Slade et al. | Nov 1994 | A |
| 5429828 | Fodge et al. | Jul 1995 | A |
| 5614501 | Richards | Mar 1997 | A |
| 5622738 | Takeuchi et al. | Apr 1997 | A |
| 6033712 | Greenshields et al. | Mar 2000 | A |
| 6132716 | Morgan et al. | Oct 2000 | A |
| 6147206 | Doner et al. | Nov 2000 | A |
| 6162473 | Fodge et al. | Dec 2000 | A |
| 6241983 | Paul et al. | Jun 2001 | B1 |
| 6558930 | Hwang et al. | May 2003 | B2 |
| 6720014 | Short et al. | Apr 2004 | B1 |
| 7033633 | Poulsen et al. | Apr 2006 | B2 |
| 20010020091 | Buchanan et al. | Sep 2001 | A1 |
| 20020037331 | Hwang et al. | Mar 2002 | A1 |
| 20020195213 | Izumi et al. | Dec 2002 | A1 |
| 20050118299 | Vickers et al. | Jun 2005 | A1 |
| 20070207132 | Speelmans et al. | Sep 2007 | A1 |
| 20080102162 | Delcour et al. | May 2008 | A1 |
| 20100035302 | Broekaert et al. | Feb 2010 | A1 |
| 20110020498 | Broekaert et al. | Jan 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| 0 856 259 | Aug 1998 | EP |
| 0 904 784 | Mar 1999 | EP |
| 1 175 905 | Jan 2002 | EP |
| 0 463 706 | Mar 2003 | EP |
| 1 137 424 | Apr 2004 | EP |
| 1 418 819 | Nov 2005 | EP |
| 2 811 997 | Jan 2002 | FR |
| 02-001701 | Jan 1990 | JP |
| 2303459 | Dec 1990 | JP |
| 4309501 | Nov 1992 | JP |
| 5112455 | May 1993 | JP |
| 06-217761 | Aug 1994 | JP |
| 10120704 | May 1998 | JP |
| 10237107 | Sep 1998 | JP |
| 2000-501935 | Feb 2000 | JP |
| 2005-500807 | Jan 2005 | JP |
| WO 9104673 | Apr 1991 | WO |
| WO 9523514 | Sep 1995 | WO |
| WO 9606935 | Mar 1996 | WO |
| WO 9709423 | Mar 1997 | WO |
| WO 9720920 | Jun 1997 | WO |
| WO 9727293 | Jul 1997 | WO |
| WO 9854305 | Dec 1998 | WO |
| WO 9930570 | Jun 1999 | WO |
| WO 0133973 | May 2001 | WO |
| WO 0142433 | Jun 2001 | WO |
| WO 0167891 | Sep 2001 | WO |
| WO 0189317 | Nov 2001 | WO |
| WO 0224926 | Mar 2002 | WO |
| WO 02051264 | Jul 2002 | WO |
| WO 02067698 | Sep 2002 | WO |
| WO 2006002495 | Jan 2006 | WO |
| WO 2006027529 | Mar 2006 | WO |
| WO 2008000050 | Jan 2008 | WO |
| WO 2008098320 | Aug 2008 | WO |
| WO 2009117790 | Oct 2009 | WO |
| Entry |
|---|
| Gove et al., definition of prevent, Webster's Third New International Dictionary, 1963, p. 1798. |
| “Overview of Gastroenteritis”, Merck Manual Online Edition, [retrieved on Apr. 3, 2012] from the Internet http://www.merckmanuals.com/home/print/. Revision Sep. 2007. |
| Karmali et al., CMA Journal, 1979, 23, 1525-1532. |
| Bedford, “Factors Influencing the Use of Enzymes in Cereal-Based Diets,” In Recent Advances in Enzymes in Grain Processing, 19:1-9 (2003). |
| International Search Report WO 2008/000050 A3, Jan. 29, 2008 for PCT/BE2007/000072. |
| Carr et al., “Viability of Bifidobacteria in Yogurt Products,” Abstracts of the General Meeting of the American Society for Microbiology, 102nd General Meeting of the American Society for Microbiology, Salt Lake City, UT, May 19-23, 2002, Session 102:Abstract O-7, May 20, 2002. |
| Gibson et al., “Regulatory Effects of Bifidobacteria on the Growth of Other Colonic Bacteria,” J. Appl. Bacteriol. 77:412-420, 1994. |
| Grootaert et al., “Microbial Metabolism and Prebiotic Potency of Arabinoxylan Oligosaccharides in the Human Intestine,” Trends in Food Science and Technology 18:64-71, 2007. |
| International Search Report for PCT/EP2008/063044, mailed May 18, 2009. |
| Written Opinion for PCT/EP2008/063044, mailed May 18, 2009. |
| International Preliminary Report on Patentability for PCT/EP2008/063044, mailed Apr. 8, 2010. |
| Communication pursuant to Article 94(3) EPC for European Patent Application No. 08804894.7, dated Aug. 23, 2013 (5 pages). |
| Gibson, “Functional foods: probiotics and prebiotics,” Culture 28:1-3 (2007). |
| Gibson et al., “Prebiotics and resistance to gastrointestinal infections,” Br J Nutr. 93 Suppl. 1:S31-S34 (2005). |
| Niewold et al., “Dietary inclusion of arabinoxylan oligosaccarides (AXOS) down regulates mucosal responses to a bacterial challenge in a piglet model,” Journal of Functional Foods 4:626-635 (2012). |
| Abe et al., “Effect of Administration of Bifidobacteria and Lactic Acid Bacteria to Newborn Calves and Piglets,” J. Dairy Sci. 78:2838-2846 (1995). |
| Advisory Action for U.S. Appl. No. 10/487,184, dated Nov. 15, 2007. |
| Advisory Action for U.S. Appl. No. 10/487,184, dated Apr. 20, 2010. |
| Annison et al., “Analysis of Wheat Arabinoxylans from a Large-Scale Isolation,” Carbohydrate Polymers 19: 151-159 (1992). |
| Aust et al., “Estimation of Available Energy of Dietary Fibres by Indirect Calorimetry in Rats,” Eur. J. Nutr. 40:23-29 (2001). |
| Bach Knudsen and Hansen, “Gastrointestinal Implications in Pigs of Wheat and Oat Fractions, 1. Digestibility and Bulking Properties of Polysaccharides and other Major Constituents” Br. J. Nutr. 65(2):217-232 (1991). |
| Bedford, “Factors Influencing the Use of Enzymes in Cereal-Based Diets,” In Recent Advances in Enzymes in Grain Processing, 19:1-9 (2002). |
| Bedford and Classen, “Reduction of Intestinal Viscosity through Manipulation of Dietary Rye and Pentosanase Concentration Is Effected Through Changes in the Carbohydrate Composition of the Intestinal Aqueous Phase and Results in Improved Growth Rate and Food Conversion Efficiency of Broiler Chicks,” J. Nutr. 122:560-569 (1992). |
| Bird, “Performance Improvements Following Enzyme Supplementation of Wheat- and Barley-Based Poultry Diets,” in Enzymes in Poultry Swine Nutr. Proceedings of the First Chinese Symposium on Feed Enzymes, Nanjing Agricultural University, Nanjing, P.R. China, May 6-8, 1996. pp. 73-87. Published Jun. 1997. |
| Bonneau and Laarveld, “Biotechnology in Animal Nutrition, Physiology and Health,” Livest. Prod. Sci. 59.223-241 (1999). |
| Boros and Marquardt, “Improvement of Nutritional Value of Rye by Enzyme Application”, Hodowla Roslin Aklimatyzacja I Nasiennictwo, 38:95-110 (1994). |
| Brennan and Singh, Economic Assessment of Improving Nutritional Characteristics of Feed Grains, Report prepared for Grains Research and Development Corporation, Economic Research Report No. 3, Wagga Wagga (2000). |
| Brief filed on behalf of DF3 SAS and Cosucra Groupe Warcoing in opposition of EP 1 758 470, dated Feb. 17, 2011, including Annex 1 and Annex 2. |
| Burton-Freeman, “Dietary Fiber and Energy Regulation,” J. Nutr. 130(2S Suppl.): 272S-275S (2000). |
| Caprita et al., “Biochemical Aspects of Non-Starch Polysaccharides,” Scientific Papers: Animal Science and Biotechnologies 43:368-375 (2010). |
| Carvalheiro et al., “Optimization of Brewery's Spent Grain Dilute-Acid Hydrolysis for the Production of Pentose-Rich Culture Media,” Appl. Biochem. Biotechnol. 113-116:1059-1072 (2004). (Abstract only). |
| “Cereal Grains and Their By-Products,” www.asasea.com abstract, printed Mar. 20, 2003. |
| Choct, “Feed Non-Starch Polysaccharides: Chemical Structures and Significance”, ASA Southeast Asia, p. 1-9.; www.asasea.wm/an08.97.html, date omitted on IDS but reference of record has date of Feb. 18, 2004. |
| Choct and Annison, “Anti-Nutritive Effect of Wheat Pentosans in Broiler Chickens: Roles of Viscosity and Gut Microflora,” Br. Poult. Sci. 33:821-834 (1992). |
| Choct and Kocher, “Non-Starch Carbohydrates: Digestion and its Secondary Effects in Monogastrics,” Proc. Nutr. Soc. Aust. 24:31-38 (2000). |
| Cloetens, “In Vivo Evaluation of the Gastrointestinal Effects of Arabinoxylan-Oligosaccharides in Healthy Subjects,” Doctoral Thesis in Medical Sciences, Katholieke Universiteit Leuven (Apr. 2009). |
| Close, “Producing Pigs Without Antibiotic Growth Promoters,” Advances in Pork Production 11:47-56 (2000). |
| Communication from Examiner for GB Application No. 0612976.1, dated Nov. 1, 2006. |
| Cosucra, “Quantités d'oligosaccharides prébiotiques vendus par la société,” dated Jul. 3, 2009; cited in opposition of European Patent Application No. EP 02766984.5 on Aug. 17, 2009. |
| Courtin et al., “Determination of Reducing End Sugar Residues in Oligo- and Polysaccharides by Gas-Liquid Chromatography”, Journal of Chromatography A, 866:97-104 (2000). |
| Courtin et al., “Dietary Inclusion of Wheat Bran Arabinoxylooligosaccharides Induces Beneficial Nutritional Effects of Chickens,” Cereal Chem. 85(5):607-613 (2008). |
| Courtin and Delcour, “Physicochemical and Bread-Making Properties of Low Molecular Weight Wheat-Derived Arabinoxylans,” J. Agric. Food Chem. 46:4066-4073 (1998). |
| Crittenden et al., “In Vitro Fermentation of Cereal Dietary Fibre Carbohydrates by Probiotic and Intestinal Bacteria,” J. Sci. Food Agric. 82:781-789 (2002). |
| Data Sheet of Cellulase <Onozuka> RS from Trichoderma viride, Copyright 2007/SERVA GmbH. |
| Data Sheet of Pentopan® Mono BG, Novozymes A/S Denmark (2008). |
| De Preter et al., “The In Vivo Use of the Stable Isotope-Labelled Biomarkers Lactose-[15N]ureide and [2H4]Tyrosine to Assess the Effects of Pro- and Prebiotics on the Intestinal Flora of Healthy Human Volunteers,” British Journal of Nutrition 92:439-446 (2004). |
| Egi et al., “Arabinoxylans and Their Behavior During Malting and Brewing,” MBAA TQ 41(3):248-267 (2004). |
| E-mail Communication between Joël Bazin of Novozymes France S.A. and Joseph Fockedey of Cosucra Groupe Warcoing and its English translation, dated Sep. 1, 2010 and Sep. 6, 2010. |
| English Language Translation of the Office Action issued in connection with Chinese Patent Application No. 200580022193, dated Feb. 12, 2010. |
| Fastinger, “Development of a Novel Value-Added Distillers Dried Grains with Solubles: Effects on Amino Acid and Energy Digestibility in Pigs,” Dissertation, Ohio State University (2005). |
| Faulds et al., “Specificity of Feruloyl Esterases for Water-Extractable and Water-Unextractable Feruloylated Polysaccharides: Influence of Xylanase,” Journal of Cereal Science 38:281-288 (2003). |
| Glitsøet al., “Degradation of Rye Arabinoxylans in the Large Intestines of Pigs,” J. Sci. Food Agric. 79:961-969 (1999). |
| Grootaert et al., “Microbial Metabolism and Prebiotic Potency of Arabinoxylan Oligosaccharides in the Human Intestine,” Trends in Food Science & Technology 18:64-71 (2007). |
| Groot Wassink et al., “Fractionation of Crude Pentosanase (Arabinoxylanase) for Improvement of the Nutritional Value of Rye Diets for Broiler Chickens,” J. Sci. Food Agric. 46(3): 289-300 (1989). |
| Hoffmann et al., “1H-N.M.R. Study of Enzymically Generated Wheat-Endosperm Arabinoxylan Oligosaccharides: Structures of Hepta-to Tetradeca-Saccharides Containing Two or Three branched Xylose Residues,” Carbohydr. Res. 223: 19-44 (1992). |
| Hoffmann et al., “Structural Characteristics of the Warm-Water-Soluble Arabinoxylans from the Tailings of the Soft Wheat Variety Kadet,” Carbohydrate Polymers 16:275-289 (1991). |
| Instruction Manual for Cellulase “Onozuka” R-10 from Trichoderma viride cited in brief filed on behalf of DF3 SAS and Cosucra Groupe Warcoing in opposition of EP 1 758 470, dated Feb. 17, 2011. |
| International Preliminary Report on Patentability for PCT/BE2007/000072, issued Jan. 6, 2009, mailed Jan. 15, 2009. Includes written opinion, mailed Jan. 29, 2008. |
| Interview Summary for U.S. Appl. No. 10/487,184, dated Dec. 10, 2008. |
| Kitta (Shigeki Kitta, Executive General Manager, Consumer Health Products Division, Suntory Limited Dojimahama 2-1-40, Kita-ku, Osaka, Osaka 530-8203, Japan). “Beginning of Sales,” [Letter]. Letter to: Joseph Fockedey (Cosucra, Rue de la Sucrerie, 1, 7740 Warcoing, Belgium). Sep. 5, 2007. |
| Kontula et al., “Oat Bran β-gluco- and Xylo-Oligosaccharides as Fermentative Substrates for Lactic Acid Bacteria,” Int. J. Food Microbiol. 45: 163-169 (1998). |
| Kurmann and Rasic, Chapter 6, “The Health Potential of Products Containing Bifidobacteria,” in Therapeutic Properties of Fermented Milks, Robinson ed., pp. 117-157 (1991). |
| Livesey, “The Energy Values of Dietary Fibre and Sugar Alcohols for Man,” Nutr. Res. Rev. 5(1):61-84 (1992). |
| Maes et al., “Relative Activity of Two Endoxylanases Towards Water-Unextractable Arabinoxylans in Wheat Bran,” Journal of Cereal Science 39:181-186 (2004). |
| Maes, “Structural Features of Wheat (Triticum aestivum) Bran Arabinoxylan Fractions Extracted with Endoxylanases or Alkaline Media,” Doctoral Dissertation in Agriculture, Katholieke Universiteit Leuven, Belgium (Aug. 2004). |
| Majerowicz, (Bredema, 38 Avenue de l'Opéra 75002 Paris, France, mm@bredema.com); Joseph Fockedey (Cosucra, Rue de la Sucrerie, 1, 7740 Warcoing, Belgium, Jfockedey@cosucra.com ). “Opposition au Brevet EP 1 418 819 Bl,” [electronic mail on the internet]. Message to: Stephanie Patarin (Bredema, 38 Avenue de l'Opéra 75002 Paris, France; stp@bredema.com) and Eric Enderlin (Bredema, 38 Avenue de l'Opéra 75002 Paris, France; een@bredema.com), Marc Majerowicz, (Bredema, 38 Avenue de l'Opéra 75002 Paris, France; mm@bredema.com), Yann Dugenet (yann.dugenet@wanadoo.com), Mingan Choct (Professor (seconded to Poultry CRC), Faculty of Arts and Sciences, School of Environmental and Rural Sciences, University of New England, Armidale NSW 2351 Australia, mchoct@une.edu.au), Joseph Fockedey (Cosucra, Rue de la Sucrerie, 1, 7740 Warcoing, Belgium; Jfockedey@cosucra.com ). Sep. 10, 2007, 9:45 AM. French and English languages. The product is a food additive containing about 70 of arabinoxylans, having a molecular mass between 5000 and 758000, and is obtained in the starch industry by wet milling. |
| Modler, “Bifidogenic Factors—Sources, Metabolism and Applications,” Int. Dairy J. pp. 383-389 and 391-407 (1994). |
| Moers et al., “Endoxylanase Substrate Selectivity Determines Degradation of Wheat Water-Extractable and Water-Unextractable Arabinoxylan,” Carbohydrate Research 340:1319-1327 (2005). |
| Mroz et al., “Effects of Dietary Carbohydrates and Buffering Capacity on Nutrient Digestibility and Manure Characteristics in Finishing Pigs,” J. Anim. Sci. 78(12):3096-3106 (2000). |
| Mul and Perry, “The Role of Fructo-Oligosaccharides in Animal Nutrition,” In Recent Advances in Animal Nutrition, Proceedings of the 28th University of Nottingham Feed Conference, Cole ed., Nottingham University Press, pp. 57-79 (1994). |
| Notice of Allowance and Notice of Allowability for U.S. Appl. No. 12/306,943, dated Jul. 14, 2011. |
| Office Action for Australian Patent Application No. 2005259856, dated Mar. 11, 2010. |
| Office Action for Canadian Application No. 2,456,304, dated Dec. 18, 2009. |
| Office Action for Indian Application No. 417/DELNP/2004, dated Feb. 4, 2010. |
| Office Action for European Patent Application No. EP 07 784 889.3, dated Jun. 24, 2009. |
| Official Communication from European Patent Office for European Patent Application 07 784 889.3, dated Dec. 9, 2010. |
| Official Communication from European Patent Office for European Patent Application 08 804 894.7-2123, dated Feb. 9, 2011. |
| Office Action for U.S. Appl. No. 10/487,184, dated Jan. 4, 2007. |
| Office Action for U.S. Appl. No. 10/487,184, dated Jun. 14, 2007. |
| Office Action for U.S. Appl. No. 10/487,184, dated Sep. 17, 2008. |
| Office Action for U.S. Appl. No. 10/487,184, dated Jun. 17, 2009. |
| Office Action for U.S. Appl. No. 10/487,184, dated Jan. 5, 2010. |
| Office Action for U.S. Appl. No. 10/487,184, dated Mar. 10, 2011. |
| Office Action for U.S. Appl. No. 10/487,184, dated Aug. 18, 2011. |
| Office Action for Chinese Patent Application No. 200980116562.X, dated Oct. 20, 2011 (with Translation). |
| Pawlik et al., “Improvement of the Nutritional Value of Rye by the Partial Hydrolysis of the Viscous Water-Soluble Pentosans Following Water-Soaking or Fungal Enzyme Treatment,” Br. Poult. Sci. 31(3):525-538 (1990). |
| Prosky and Hoebregs, “Methods to Determine Food Inulin and Oligofructose,” J. Nutr. 129(Suppl):1418S-1423S (1999). |
| Reply to the Official Communication Under A96(2) for EP 02766984.5, submitted Jan. 10, 2005. |
| Rijnen et al., “Effects of Dietary Fermentable Carbohydrates on Energy Metabolism in Group-Housed Sows,” J. Anim. Sci. 79(1):148-154 (2001). |
| Roberts et al., “The Influence of Dietary Composition on Energy Intake and Body Weight,” J. Am. Coll. Nutr. 21(2): 140S-145S (2002). |
| Rokey, G.J., “Petfood and Fishfood Extrusion,” The Technology of Extrusion Cooking 144-145 (1995). |
| Ross Tech 99/37, “Role of Antibiotic as Growth Promoter,” printout from world.rossbreeders.com (1999); cited in opposition of European Patent Application No. EP 02766984.5 on Aug. 17, 2009. |
| Schooneveld-Bergmans, “Physicochemical Characteristics of Wheat Bran Glucuronoarabinoxylans,” J. Cereal Sci. 29(1):49-61 (1999). |
| Search Report for GB Application No. 0612976.1, mailed Oct. 30, 2006, date of search Oct. 27, 2006. |
| Shiiba et al., “Purification and Characterization of Two Arabinoxylans from Wheat Bran,” Cereal Chem. 70(2):209-214 (1993). |
| Smith et al., “Energy Balance and Thermogenesis in Rats Consuming Nonstarch Polysaccharides of Various Fermentabilities,” Am. J. Clin. Nutr. 68(4):802-819 (1998). |
| Sørensen et al., “Efficiencies of Designed Enzyme Combinations in Releasing Arabinose and Xylose from Wheat Arabinoxylan in an Industrial Ethanol Fermentation Residue,” Enzyme and Microbial Technology 36:773-784 (2005). |
| Sørensen et al., “Optimization of Reaction Conditions for Enzymatic Viscosity Reduction and Hydrolysis of Wheat Arabinoxylan in an Industrial Ethanol Fermentation Residue,” Biotechnol. Prog. 22:505-513 (2006). |
| Supplemental Search Report for British Application No. GB0612976.1, dated Nov. 1, 2006. |
| Swennen et al., “Ultrafiltration and Ethanol Precipitation for Isolation of Arabinoxylooligosaccharides with Different Structures,” Carbohydrate Polymers 62:283-292 (2005). |
| Van Craeyveld et al., “Structurally Different Wheat-Derived Arabinoxylooligosaccharides Have Different Prebiotic and Fermentation Properties in Rats,” Journal of Nutrition 138:2348-2355 (2008). |
| Van Laere et al., “Fermentation of Plant Cell Wall Derived Polyaccharides and Their Corresponding Oligosaccharides by Intestinal Bacteria,” J. Agric. Food Chem. 48(5):1644-1652 (2000). |
| Van Loo et al., “Functional Food Properties of Non-Digestible Oligosaccharides: A Consensus Report from the ENDO Project (DGXII AIRII-CT94-1095),” Br. J. Nutr. 81(2):121-132 (1999). |
| Voragen, “The Cell Wall and its Components in Cereals and Pulse Grains, Consequences on Nutritional Value,” Cah. Options Mediterr. 26:119-123 (1997). |
| Yamada et al., “Preparation of a New Arabinoxylooligosaccharide from Wheat Bran Hemicellulose and Its Structure,” Biosci. Biotech. Biochem. 58(2):288-292 (1994). |
| Cloetens et al., “The Bifidogenic Potential of Arabinoxylo-Oligosaccharides in Healthy Volunteers Depends on the Degree of Polymerisation,” Abstract W1382, Gastro. 134(4):A-692 (2008). |
| Cloetens et al.,“The Effect of Two Different Doses of Arabinoxylo-Oligosaccharides and Oligofructose Enriched Inulin on the Colonic Ammonia Metabolism in Healthy Volunteers,” Abstract 1383, Gastro. 134(4): A-693 (2008). |
| International Preliminary Report on Patentability for International Application No. PCT/BE2009/000020 dated Mar. 18, 2010 (date of completion and mailing of report). |
| International Search Report WO 2008/000050 A3, Jan. 29, 2008. |
| International Search Report for PCT International Application No. PCT/BE2009/000020 dated Sep. 23, 2009 (date of completion of search) and Oct. 13, 2009 (date of mailing of report). |
| Swennen et al., “Large-Scale Production and Characterisation of Wheat Bran Arabinoxylooligosaccharides,” J. Science and Food Agric. 86(11):1722-1731 (2006). |
| Written Opinion of the International Searching Authority of PCT International Application No. PCT/BE2009/000020 dated Sep. 23, 2009 (date of completion of this opinion) and Oct. 13, 2009 (date of mailing of this opinion). |
| Yamada et al., “Structure and Properties of Oligosaccharides from Wheat Bran,” Cereal Foods World 38(4):490-492 (1993). |
| Reply to Written Opinion for International Patent Application No. PCT/BE2009/000020 dated Jan. 29, 2010. |
| Association des Amidonneries de Céréales de l'Union Européenne, printout from http://www.authorstream.com/Presentation/miloung-16703-7-Labelling-wheat-starch-derivatives-AAC-members-Why-new-EU-rules-specific-forwheat-seedcount-presentation-002-News-Reports-ppt-powerpoint/, “Changes in the EU Labeling Legislation,” (Nov. 19, 2003). |
| Beaugrand et al., “Impact and Efficiency of GH10 and GH11 Thermostable Endoxylanases on Wheat Bran and Alkali-Extactable Arabinoxylans,” Carbohydrate Research 339:2529-2540 (2004). |
| Clayton, “The Extraction and Ion-Exchange Chromatography of Buffer-Soluble and Gluten Proteins of Wheat Flour,” Cereal Chemistry 43:495-516 (1966). |
| Fang et al., “Influence of Temperature on the Retention Behaviour of Proteins in Cation-Exchange Chromatography,” Journal of Chromatography A 729(1-2):49-66 (1996), Abstract only. |
| Katapodis amd Christakopoulos,“Enzymic Production of Feruloyl Xylo-Oligosaccharides from Corn Cobs by a Family 10 Xylanase from Thermoascus aurantiacus,” LWT 41:1239-1243 (2008), e-published Sep. 1, 2007. |
| Official Communication from the European Patent Office for European Patent Application No. 09725460.1, dated Feb. 15, 2011. |
| Official Communication from the European Patent Office for European Patent Application No. 09725460.1, dated Feb. 21, 2011. |
| Official Communication from the European Patent Office for European Patent Application No. 09725460.1, dated Feb. 28, 2011. |
| Purolite Company, “Corn Sweetener Refining with Ion Exchange Resins,” (2007). |
| Roberfroid and Slavin , “Nondigestible Oligosaccharides,” Critical Reviews in Food Science and Nutrition 40(6):461-480 (2000). |
| Swennen, “Production, Characterisation, and Functionality of Arabinoxyooligosaccharides with Different Structures,” Dissertationes de Agricultura, Katholieke Universiteit Leuven (Feb. 2007). |
| Yuan et al., “Antioxidant Activity of Feruloylated Oligosaccharides from Wheat Bran,” Food Chemistry 90:759-764 (2005). |
| Number | Date | Country | |
|---|---|---|---|
| 20120015902 A1 | Jan 2012 | US |
