IE20160180A1 - A composition and a method for controlling bacterial infection - Google Patents

Abstract

The invention relates to new and useful improvements in meat production and processing to reduce and/or eliminate bacterial contamination. The present invention relates more particularly to a composition for controlling bacterial infection in the gastro-intestinal tract of an animal comprising an oxidoreductase enzyme and its corresponding oxidisable substrate which are capable of producing and releasing hydrogen peroxide in the gastro-intestinal tract of the animal.

Description

“A composition and a method for controlling bacterial infection” Introduction The present invention relates to the field of meat production and meat processing. More particularly the present invention relates to reducing or eliminating bacterial contamination during meat production and processing.

Zoonoses are infectious diseases of animals that can naturally be transmitted to humans. Zoonoses can be caused by a range of disease pathogens such as viruses, bacteria, fungi and parasites. The most significant zoonotic pathogens causing foodborne diseases are Escherichia coli O157:H7, Campylobacter, Caliciviridae, Cryptosporidium and Salmonella.

Campylobacteriosis is the most frequently reported gastrointestinal bacterial illness in humans in Ireland and across the EU. The European Food Safety Authority (EFSA) is of the view that there is considerable under-ascertainment and under-reporting of campylobacteriosis within the EU, and that the true incidence is likely to be 10-100 times higher than the reported number. In 2004, campylobacteriosis became a notifiable disease in humans in Ireland under the Infectious Diseases Regulations (S.l. No. 707 of 2003). The annual incidence rate reported in Ireland in 2008 was 41.4 per 100,000 of the population; the average EU incidence that year was 40,7 per 100,000. In Ireland, the highest incidence has been observed in children aged 1-4 years, followed by adults aged 20-24 years.

A number of risk factors have been associated with human campylobacteriosis. These include the consumption and/or handling of raw or undercooked poultry or other meats, cross-contamination of ready-to-eat foods during food preparation as well as direct contact with animals. Poultry is regarded as one of the most important reservoirs for Campylobacter and constitutes a very significant vehicle for the transmission of Campylobacter to humans. An EFSA opinion estimated that handling, preparation and consumption of chicken meat may account for 20% to 30% of cases, while 50% to 80% may be attributed to the chicken reservoir as a whole. An EFSA baseline survey on the prevalence of Campylobacter species in broiler batches and of -2Campylobacter spp. and Salmonella spp. on broiler carcasses in the EU during 2008 found the community prevalence of campylobacter-colonised broiler batches was 71.2% and the prevalence of campylobacter-contaminated broiler carcasses was 75.8%. In Ireland, the prevalence in broiler batches was 83.1% and the prevalence of contaminated carcasses was 98.3%. The results of the Campylobacter enumeration on broiler carcasses revealed that 65.2% of Irish broiler carcasses had counts between 100 cfu/g and 10,000 cfu/g and 8.9% greater than 10,000 cfu/g. In addition to this it has been reported that Campylobacteriosis is the most common recognized bacterial zoonosis in the European Union and United States (Jaap A. Wagenaar, Nigel P. French, and Arie H. Havelaar, 2013, FOOD SAFETY, 57(11), 1600-1606).

Campylobacteriosis in humans is mainly caused by Campylobacter jejuni (C. jejuni) and, to a lesser extent, by Campylobacter coli (C. coli). Other Campylobacter species (eg, C. fari, C. upsaliensis, C. fetus) are also reported to cause disease in humans, but the reported number of these non-jejuni/coli infections worldwide is a small fraction of all Campylobacter infections. In the European Union, 198 252 cases were reported in 2009, but the true incidence was estimated at 9.2 million cases. In the United States the estimated annual incidence was 1.3 million cases; and in the United Kingdom an incidence of 570 000 cases. High seroconversion rates indicate that asymptomatic Campylobacter infection is a frequent event, occurring approximately once every year in any adult person in The Netherlands. Hence, only a small fraction of infections lead to symptomatic illness. Approximately 1 of 4 symptomatic cases in the Dutch population visit a general practitioner, and 1% are hospitalized.

Depending on severity of the infection, campylobacteriosis in the acute phase is characterized by diarrhea with abdominal cramps, nausea, fever, and bloody stools. The disease is usually self-limiting, and antimicrobial treatment is only indicated in severe cases. In rare cases, C. jejuni/coli can cause a bloodstream infection. Campylobacter infection is aiso associated with long-term sequelae, inciuding Guillain- Barre syndrome, reactive arthritis, postinfectious irritable bowel syndrome, and possibly inflammatory bowel disease. In the United States, campylobacteriosis was estimated to cause a burden of 13 300 quality-adjusted life-years (QALYs), second only to Salmonella enterica, and cost of illness of $1.7 billion annually. Campylobacter and poultry was the highest ranking food-pathogen combination with 608 231 illnesses, 6091 hospitalizations, 55 deaths, a burden of 9541 QALYs, and -3cost of illness of $1.3 billion in the United States. Therefore control and reduction of Campylobacter contamination is a public health issue.

The intestines of warm-blooded animals (mammals and birds) are the amplification vessel for Campylobacter. Manure from animals may contaminate surface water through runoff from pasture, presenting a risk for humans when consumed as (untreated) drinking water. Furthermore, humans can be exposed to surface water through direct contact (swimming) or indirect contact (consumption of raw products irrigated with surface water). Campylobacter can be isolated from the faeces of healthy food-producing animals (e.g. poultry, pigs, cattle, sheep), wild animals (e.g. birds), and companion animals (e.g. dogs, cats). Prevalence estimates vary from 71% C. jejuni/C. coli in broilers, 45% C. jejuni in dogs, 36% C. jejuni in beef cattle, and 42% C. coli in pigs. Presence of Campylobacter in these animals is usually asymptomatic, although in cattle and sheep C. jejuni has been reported to cause sporadic abortions. In the United States, a highly pathogenic C. jejuni clone is emerging in ruminants, causing abortion in sheep with evidence for transmission to humans. Except for this specific clone, humans are considered to be the only host species that becomes ill after oral ingestion with Campylobacter. The pathogenesis of C. jejuni disease in humans and the absence of clinical manifestations in most species is still unexplained. The lack of Campylobacter-associated disease or mortality in poultry flocks means there is no economic incentive for farmers to invest in prevention of flock contamination.

Human exposure from animal reservoirs is possible via multiple pathways including food (in particular, poultry meat), the environment, and direct animal contact. Based on molecular typing in several countries, it is estimated that the majority (50%-80%) of strains infecting humans originate from the chicken reservoir, 20%-30% from cattle, and the remainder from other reservoirs including sheep, pigs, and wildliving animals. The chicken reservoir includes both broiler chickens and laying hens, and pathways are not limited to consumption and preparation of meat but also include environmental transmission and direct animal contact. A meta-analysis of case30 control studies of sporadic cases suggested that traveling abroad, eating undercooked chicken, environmental sources, eating in a restaurant (particularly chicken), and direct contact with farm animals were significant risk factors for human campylobacteriosis. -4Broilers, turkeys, ducks, and all other types of poultry can become colonized with Campylobacter. Once introduced into a flock, Campylobacter spreads rapidly. Virtually all animals become colonized, shedding up to 108 Campylobacter/g of cecal contents. These counts remain at a similar level till slaughter (42 days in conventional production systems). Chickens serve as natural reservoir hosts for Salmonella Enteritidis and C. jejuni, and they colonize the intestinal tract of chickens, with the cecum being the primary colonization site (Van Immerseel et al., 2004, intermittent long-term shedding and induction of carrier birds after infection of chickens early posthatch with a low or high dose of Salmonella Enteritidis. Poult. Sci. 83:19111916). Once colonized, the pathogens are excreted to the environment via droppings, thereby potentially contaminating the environment, especially the grow-out houses infecting healthy flock (horizontal transmission). On the other hand, colonization of broiler chickens by C. jejuni is widespread and is difficult to prevent. In chickens, C. jejuni primarily colonizes the mucus overlying the epithelial cells in the ceca and small intestine. I-Fucose, the major carbohydrate component present in the mucin of chicken cecal mucus, can be used by C. jejuni as a sole substrate for growth. Thus, the cecal environment in chickens is favorable for the survival and proliferation of C. jejuni (Beery J. T. et al., 1988. Colonization of gastrointestinal tracts of chicks by Campylobacter jejuni. Appl. Environ. Microbiol. 54:2365-2370) and selects its colonization in the birds.

As broiler meat is the largest identified source of human exposure to Campylobacter, food safety authorities and producers are seeking cost-effective ways to intervene in the poultry production chain. However, once flocks are colonized, there are no methods commercially available to reduce the number of Campylobacter in the cecal contents.

In the poultry industry, antibiotics are used worldwide to prevent poultry pathogens and disease so as to improve meat and egg production. However, the use of dietary antibiotics results in common problems such as development of drug resistant bacteria, drug residues in the body of the birds and imbalance of normal microflora.

Despite considerable scientific investments, no vaccine is available that prevents or reduces Campylobacter colonization in poultry. Furthermore, the use of competitive exclusion, establishing a stabilized gut flora in young animals, effective in the control -5of Salmonella, has not been effective against Campylobacter.

The use of plant-derived antimicrobials for improving the safety of poultry products has also been proposed as feed supplements for reducing cecal populations of Salmonella Enteritidis and C. jejuni in chickens. The plant-derived antimicrobials include for example caprylic acid, trans-cinnamaidehyde, eugenol, carvacrol, and thymol (K. Venkitanarayanan , et al., 2013, Use of plant-derived antimicrobials for improving the safety of poultry products. Poultry Science 92 : 493-501). However the efficacy of such plant-derived antimicrobials has not been confirmed under field conditions. Furthermore these molecules might alter the sensory attributes of meat and eggs from treated birds.

Hydrogen peroxide (H2O2) is a well-known antimicrobial substance with many advantages. Indeed, there are no known microbial evasion mechanisms by which microbes can escape its effects and it has a short lifetime. Therefore hydrogen peroxide does not accumulate to dangerous levels. There are, however, problems associated with the use of hydrogen peroxide. Hydrogen peroxide solution is very unstable and is readily oxidised to water and oxygen. Furthermore, hydrogen peroxide at high concentration can be damaging to normal tissue and to cells. Thus, it is very difficult or even impossible to use hydrogen peroxide as an antimicrobial for animals or humans: its instability would make for a product with an impossibly short shelf-life, and dosing at the point of application would still not provide a sustained delivery over a usefully prolonged period. When it is used in wound treatment (as described in the British Pharmacopoeia, for example) very high concentrations (typically 3%) are needed to achieve a powerful antimicrobial effect over a very short time interval. Even this type of short burst can be effective, because of the great effectiveness of hydrogen peroxide, but there is the further disadvantage that such high concentrations can be relatively damaging to host cells and can impede the normal cellular process. For this reason, use of hydrogen peroxide tends to be restricted to initial clean-up and sterilisation of wounds.

In a commercial poultry slaughter line, up to 13 000 animals per hour are processed.

The process is completely automated, providing a challenge for hygienic slaughter and carcass preparation. Due to the high concentration of Campylobacter in the intestines, chicken carcasses can become contaminated at the surface during -6processing (e.g. after defeathering or rupture of the gut during evisceration). Technical improvement of the slaughtering process to prevent contamination of meat is expected to have an effect in the reduction of Campylobacter contamination, but evidence-based interventions are not yet available. Unlike many other bacteria, Campylobacter is unable to multiply outside the intestines of warm-blooded animals. This implies that when carcasses leave the slaughterhouse, bacteria will die and/or be removed from products and only a small fraction of bacteria initially present on the meat will ultimately reach the consumer. Hence, a zero-toierance approach for Campylobacter on fresh chicken meat is not necessary to achieve a high degree of consumer protection. Several risk assessment studies have demonstrated that consumer risks are mainly associated with highly contaminated products and that preventing these from reaching the consumer is both effective and efficient.

Currently, proposed antimicrobial treatments during poultry slaughter and dressing consist in the immersion of the carcasses in Lactic acid or Acetic acid or Trisodium phosphate, Chlorine/Acidified chlorine, Electrolysed water, Ozone (03). However all these chemical decontaminant treatments are not currently permitted under EU law.

Alternative options are the chemical and physical decontamination of meat, but their effectiveness is typically limited to a reduction of 1-2 log units which is not sufficient as a satisfying reduction must be of at least 4 log units. Moreover, in the European Union, chemical decontamination is allowed only for specific, approved compounds whereas in the United States, several decontaminating agents including organic acids, quaternary ammonium compounds, acidified sodium chlorite, and trisodium phosphate are being applied in practice. Physical decontamination (e.g. ultraviolet light, irradiation) is allowed in the European Union, but effectiveness is expected to be limited, particularly when implemented in high-volume slaughter lines. Consumer preferences (e.g. for fresh, untreated poultry products), acceptance (e.g. consumers do not like irradiated products), and economic arguments (higher number of animals per hour reduces costs but puts more stress on care for the individual carcass) prevent implementation of potentially effective interventions.

Currently, it is very difficult to keep broiler flocks Campylobacter free till the slaughter age, and there are no effective and technically implementable tools to reduce the colonization levels under field conditions. Generic interventions in the slaughterhouse - 7are not yet available, and site-specific improvement of slaughter hygiene is a high priority.

Therefore there is a need to provide a solution to control bacterial infection during the production and processing of meat.

In view of the above, it is an aim of the present invention to provide an effective and safe composition that can control bacterial infection of animals before slaughtering. Yet another aim is to provide a method of controlling bacterial infection of meat, Statements of Invention The present invention relates to a composition for controlling bacterial infection in the 10 gastro-intestinal tract of an animal comprising an oxidoreductase enzyme and its corresponding oxidisable substrate which are capable of producing and releasing hydrogen peroxide in the gastro-intestinal tract of the animai.

In an embodiment, the oxidoreductase enzyme is selected from one or more of the following glucose oxidase, hexose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, choline oxidase, pyruvate oxidase, glycollate oxidase and aminoacid oxidase. It will be understood that each oxidoreductase enzyme acts on a specific substrate. The corresponding substrates for these oxidoreductase enzymes are D-glucose, hexose, cholesterol, D-galactose, pyranose, choline, pyruvate, glycollate and aminoacid respectively. It will be understood that a mixture of one or more oxidoreductase enzymes and one or more substrates for the oxidoreductase enzymes may be used.

Preferably, the oxidoreductase enzyme is glucose oxidase, hexose oxidase, galactose oxidase and/or pyranose oxidase and the respective substrate for the oxidoreductase enzyme is D-glucose, hexose, D-galactose and/or pyranose.

According to a preferred embodiment of this aspect of the invention, the oxidoreductase enzyme is glucose oxidase and the substrate is D-glucose.

Preferably, the oxidoreductase enzyme is present in an effective amount at an activity of between 5 to 300 U/mg. The quantity of enzyme may be between 0.001 to 10% w/w. -8Ideally, the corresponding oxidisable substrate is present from 1 % to approximately 90% by weight based on the weight of the total system.

In an embodiment the composition further comprises an oxygen generator. Conveniently the oxygen generator is provided by a peroxide of an organic acid. The peroxide of an organic acid may be one or more of benzoyl peroxide, Methyl ethyl ketone peroxide, acetone peroxide, diethyl ether peroxide, Acetyl acetone peroxide, Acetyl benzoyl peroxide, tert-Butyl hydroperoxide, Diacetyl peroxide, Ethyl hydroperoxide, Methyl isobutyl ketone peroxide.

In a preferred embodiment the peroxide of an organic acid is benzoyl peroxide 10 (BPO). Preferably, the quantity of benzoyl peroxide may be between 0,01 to 5% w/w.

In an embodiment the composition further comprises L-cysteine. Preferably, the quantity of L-Cysteine may be between 0.001 to 0.5% w/w.

In an embodiment, the composition is in the form of a granule. Preferably, the composition is in the form of powdered granules formed with the inclusion of a binding agent.

In a preferred embodiment, the composition is in the form of a granule coated with an enteric coating. The enteric coating will allow passage through the stomach and permit dissolution in the intestinal cavity.

Various additional ingredients such as disintegrants, binders and bulking agents may 20 be added to the composition prior to forming the granules.

The granules may be compressed into a solid dosage form such as a tablet or lozenge. in another aspect the invention provides a method for controlling bacterial infection of meat comprising administering a composition according to the invention to an animal for a period of time immediately prior to slaughtering the animal and processing its meat.

In an embodiment the bacterial infection is caused by Campylobacter spp., Salmonella spp., Cryptosporidium spp., Coccidiosis spp., Clostridium difficile, -9Enteropathogenic and enterohemmoragic E coli, Bacillus cereus, Listeria monocytogenes, Shigella spp., Staphylococcus aureus, Staphylococcal enteritis, Streptococcus, Vibrio cholerae, including 01 and non-O1, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica and Yersinia pseudotubercuiosis, Brucella spp., Corynebacterium ulcerans, Coxiella burnetii or Q fever, Plesiomonas shigelloides, avian adenovirus, avian reovirus, endovirus-like viruses, runting stunting syndrome, turkey enteric corona virus, marble spleen disease virus, hemorrhagic enteritis virus.

In an embodiment the composition is administered in animal feed for a period between 24 to 48 hours before the animal is slaughtered.

In an embodiment the composition is administered to domestic fowl, pigs, sheep or cattle.

In another aspect it is envisaged that it may be possible to use the composition of the invention in medicaments for controlling or eliminating harmful gastric bacterial infections in animals, including humans.

Brief description of the drawings The invention will be more clearly understood from the following description of some embodiments thereof, given by way of example only, with reference to the accompanying figures, in which: Fig. 1 shows the CFU microbial count in chicken faeces in aerobic conditions after treatment with a composition according to the invention without BPO, or a composition according to the invention with BPO obtained according to Example 1, compared to a control without treatment; Fig. 2 shows the CFU microbial count in chicken faeces in anaerobic conditions after treatment with a composition according to the invention without BPO, or a composition according to the invention with BPO obtained according to Example 1, compared to a control without treatment. - 10Detailed Description of Preferred Embodiments of the Invention In this specification, it will be understood that the term “antimicrobial” or “antibacterial3’ are used interchangeably herein and cover biocidal or biostatic activity against various types of micro-organisms including but not limited to bacteria, fungi, viruses, protozoans, yeasts, parasitic or pathogenic micro-organisms and/or moulds colonizing the gastro-intestinal tract of domestic animals reared for their meat, milk or eggs such as domestic fowls or cattle and pigs. More particularly, the composition of the invention is active against bacteria that can be pathogenic for humans if contracted during meat production and processing or after ingestion. In a preferred embodiment the composition of the invention is active against Campylobacter spp., Salmonella spp., Cryptosporidium spp., Coccidiosis spp., Clostridium difficile, Enteropathogenic and enterohemmoragic E coli, Bacillus cereus, Listeria monocytogenes, Shigella spp., Staphylococcus aureus, Staphylococcal enteritis, Streptococcus, Vibrio choierae, including 01 and non-O1, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica and Yersinia pseudotubercuiosis, Brucella spp., Corynebacterium utcerans, Coxiella burnetii or Q fever, Plesiomonas shigelloides, avian adenovirus, avian reovirus, endovirus-like viruses, runting stunting syndrome, turkey enteric corona virus, marble spleen disease virus, and hemorrhagic enteritis virus.

By “controlling bacterial infection” is meant significantly reducing or maintaining the bacterial concentration at a threshold considered safe for the persons handling the meat or carcasses during meat production and processing and for the persons eating the processed meat.

For example, in Ireland, according to the Recommendations for a Practical Control Programme for Campylobacter in the Poultry Production and Slaughter Chain (Food Safety Authority of Ireland, 2011) on-farm and in the slaughterhouse microbiological criteria are recommended for reducing the risk of Campylobacteriosis in humans. Onfarm a microbiological level of <7 Iog10 cfu/g of Campylobacter spp. in 10 pooled cecal contents is recommended. Sampling should be carried out on-farm seven days (or less if feasible) before slaughter. In the slaughter house, a target level of £4 Iog10 cfu/g of Campylobacter spp. on neck skin samples taken post-chill is recommended. -11 In this specification the term “by weight”, “percentage by weight” or “w/w %” refers to the weight of the final composition or system. These w/w values are interchangeable with w/v.

The present invention relates to a composition for controlling bacterial infection in the gastro-intestinal tract of an animal comprising an oxidoreductase enzyme and its corresponding oxidisable substrate which are capable of producing and releasing hydrogen peroxide in the gastro-intestinal tract of the animai.

Advantageously, the composition according to the invention is a storage stable, single component system. The composition according to the invention overcomes the problems of incompatibility of hydrogen peroxide with other ingredients and the problem of hydrogen peroxide reacting in the upper gastro-intestinal tract and being lost as hydrogen peroxide is only produced on-site.

One of the main advantages of the composition according to the invention is that it provides hydrogen peroxide specifically at the required dosage at the precise site of bacterial colonization. This produces specifically in the gastro-intestinal tract producing immediately available hydrogen peroxide and an immediate antimicrobial effect.

Advantageously, hydrogen peroxide is an effective antimicrobial agent lethal to most microorganisms. Additionally, hydrogen peroxide is a natural and safe antimicrobial which degrades quickly and does not produce residues or bacterial resistance. Therefore there are no toxicity problems. This is one of the significant advantages of the present invention over known antibiotic treatments.

Furthermore, the composition according to the invention provides an antimicrobial agent that can be applied through feed which advantageously represents the most practical and economically viable method for adoption on farms. Advantageously, the composition according to the invention enables reducing the prevalence of bacterial colonization in living animals on farms which decreases the introduction of high concentrations of bacteria such as Campylobacter into the slaughterhouse. This advantageously results in a low concentration or absence of Campylobacter on the final food product. The composition according to the invention provides a means to control bacterial contamination on the farm and thus impacts reducing the -12transmission of pathogens not only via meat but also via other (environmental) pathways. Therefore the composition according to the invention provides a solution having a higher public health impact than other types of known interventions later in the chain, such as washing the carcasses with chemical agents.

The oxidoreductase enzyme may be selected from one or more of the following: glucose oxidase, hexose oxidase, cholesterol oxidase, galactose oxidase, pyranose oxidase, choline oxidase, pyruvate oxidase, glycollate oxidase and aminoacid oxidase. It will be understood that each oxidoreductase enzyme acts on a specific substrate. The corresponding substrates for these oxidoreductase enzymes are D10 glucose, hexose, cholesterol, D-galactose, pyranose, choline, pyruvate, glycollate and aminoacid respectively. It will be understood that a mixture of one or more oxidoreductase enzymes and one or more substrates for the oxidoreductase enzymes may be used. Glucose Oxidase (GOX), for example, catalyses the conversion of glucose into gluconic acid and hydrogen peroxide (H2O2). Xanthine oxidase, present in milk, is also capable of generating hydrogen peroxide.

Ideally, the oxidoreductase enzyme is present in the system at an activity of at least 1U per mg of the system. Generally speaking, one unit (II) is that amount of enzyme causing the oxidation of one micromole of substrate per minute at 25°C and pH 7,0. It will be understood that there must be sufficient oxidoreductase enzyme present to catalyze the substrate and form hydrogen peroxide as needed. Preferably, the oxidoreductase enzyme is present in the system at an activity of at least 1U, 14U or even 300U per mg of the system. The quantity of enzyme may be between 0.001 to 10% w/w.

Ideally, the corresponding oxidisable substrate is present from 1% to approximately 90% by weight based on the weight of the total system.

Preferably, the oxidoreductase enzyme is selected from one or more of glucose oxidase, hexose oxidase, galactose oxidase and pyranose oxidase and the respective associated substrate for the oxidoreductase enzyme is selected from Dglucose, hexose, D-galactose and pyranose.

According to a preferred embodiment of this aspect of the invention, the oxidoreductase enzyme is glucose oxidase and the substrate is D-glucose. -13In an embodiment the composition further comprises a peroxide of an organic acid.

Surprisingly, the inventors have found that the combination of an oxidase and its substrate with a peroxide of an organic acid produces a synergistic antimicrobial effect in anaerobic conditions. Advantageously, the inventors found that peroxides act as O2 generators and provide a source of O2 for enzyme reaction to proceed with the generation of hydrogen peroxide thus significantly increasing the antimicrobial effect in anaerobic conditions.

The peroxide of an organic acid may be one or more of benzoyl peroxide, Methyl ethyl ketone peroxide, acetone peroxide, diethyl ether peroxide, Acetyl acetone peroxide, Acetyl benzoyl peroxide, tert-Butyl hydroperoxide, Diacetyl peroxide, Ethyl hydroperoxide, Methyl isobutyl ketone peroxide In a preferred embodiment the peroxide of an organic acid is benzoyl peroxide. Preferably, the quantity of benzoyl peroxide may be between 0.01 to 5% w/w.

Advantageously, benzoyl peroxide (BPO) is a peroxide of an organic acid and a food 15 composition with a Generally Regarded As Safe (GRAS) rating under sections 201 (s) and 409 of the Federal Food, Drug, and Cosmetic Act and the 59th meeting Joint Expert Committee on Food Compositions (JECFA) concluded that benzoyl peroxide was of no safety concern.

In an embodiment the composition further comprises L-cysteine. Preferably, the 20 quantity of L-Cysteine may be between 0.001 to 0.5% w/w.

Advantageously L-Cysteine acts as an enhancer to the effectiveness of hydrogen peroxide under anaerobic conditions. The antimicrobial activity of hydrogen peroxide is advantageously enhanced by L-cysteine, particularly under anaerobic conditions whereby the exposure of microorganisms to hydrogen peroxide in the presence of L25 cysteine results in a 100-fold increase in hydrogen peroxide sensitivity to the microorganisms.

The composition of the present invention may be in the form of a solid or semi-solid preparation. Solid or semi-solid preparations include but are not limited to granules, capsules, pellets, gel caps, hydrogels, pills, pillules, tablets, lozenges and/or globules. -14Conveniently the composition may be provided in the form of powdered granules formed with the inclusion of a binding agent.

The granules may be added to the animal feed or to water immediately prior to feeding the animals, Any suitable binding agent could be used, and conveniently the binding agent may comprise one or more of polyvinylpyrrolidone (PVP), carboxymethy! cellulose (CMC), microcrystaliine cellulose (MCC), and polyethylene oxide (POE).

Optionally, the composition may comprise a bulking agent. The bulking agent may be one or more of sucrose, maltose, malto dextrin, lactose. The bulking agent may be present from 10% to 99% by weight based on the weight of the total system.

Advantageously sucrose acts as a binder and allows the granules to have a longer disintegration time.

Thus, according to a preferred embodiment of the present invention, the substrate for the oxidoreductase enzyme, preferably glucose, is present from 1 to 90 w/w% and sucrose, is present from 10 to 99 w/w %.

In a preferred embodiment, the composition is in the form of a granule coated with an enteric coating. The enteric coating advantageously protects the enzyme and its substrate from being degraded after ingestion before entering the gastro-intestinal tract of the animal. Additionally, the enteric coating enables the controlled delivery and sustained-release of hydrogen peroxide produced by the oxidoreductase enzyme and its substrate in the gastro-intestinal tract. Advantageously, this sustained release aspect is particularly important for the treatment of Campylobacter or Salmonella infections in domestic fowl and/or in pigs.

In a preferred embodiment, the composition comprises Glucose between 1 to 90% w/w, Sucrose between 10 -99% w/w, GOX between 0.001 to 10% w/w, BPO between 0.01% to 5.0% w/w, and L-Cysteine between 0.001- 0.5% w/w.

Advantageously, the composition according to the invention allows the production and release of between 1 to 200 mmols of hydrogen peroxide, for 48 hours following ingestion. - 15A second aspect of the invention concerns a method for controlling bacterial infection of meat comprising administering a composition according to the invention to an animal for a period of time immediately prior to slaughtering the animal, In one embodiment, the composition comprises an oxidoreductase enzyme and its 5 corresponding oxidisabie substrate and further comprises Benzoyl peroxide and/or LCysteine.

The bacterial infection may be Campylobacter spp., Salmonella spp., Cryptosporidium spp., Coccidiosis spp., Clostridium difficile, Enteropathogenic and enterohemmoragic E coli, Bacillus cereus, Listeria monocytogenes, Shigella spp., Staphylococcus aureus, Staphylococcal enteritis, Streptococcus, Vibrio cholerae, including 01 and non-O1, Vibrio parahaemolyticus, Vibrio vulnificus, Yersinia enterocolitica and Yersinia pseudotuberculosis, Brucella spp., Corynebacterium ulcerans, Coxiella burnetii or Q fever, Plesiomonas shigelloides, avian adenovirus, avian reovirus, endovirus-like viruses, runting stunting syndrome, turkey enteric corona virus, marble spleen disease virus, and hemorrhagic enteritis virus.

In another embodiment the composition is a powdered granulate coated with an enteric coating.

Typically the composition is administered in the animal feed for a period between 24 to 48 hours before the animal is slaughtered.

EXAMPLES Example 1: Process for manufacturing granules of a composition according to the invention In the following example L-Cysteine and Benzoyl peroxide are optionally added.

Furthermore the order of the mixing steps of each ingredient is not essential, however it is preferable to add the enzyme at as late a stage as possible to the mixing. 200 grams of D-Glucose are mixed with 200 grams of Sucrose.

Then 20 grams of the binding agent PVP K29-32 is dissolved into 130 grams of -16isopropyl alcohol (IPA). The dissolved binding agent is then sprayed onto the powders of D-Glucose and Sucrose and mixed. The mixture is then placed in an incubator at 37°C for 18 hours.

The resulting hard slab granulate is then pushed through a screen to produce a first 5 powder granulate having 2 mm fines. grams of Glucose Oxidase (GOX) are dissolved in 50g IPA, and 3 grams of LCysteine are dissolved into 20 grams deionized water.

Then the dissolved Glucose oxidase and L-Cysteine are added to 315 grams of the first powder granulate to obtain a mixture. To 150 grams of this mixture are added 3 grams of benzoyl peroxide (BPO) powder finely milled to obtain a second powdered granules mixture.

The second powdered granules mixture is dried at 25°C overnight then placed in an incubator at 37°C for 12 hours approximatively.

Separately an enteric coating mixture is prepared: 50 grams of IPA are mixed with 50 15 grams of Acetone. Then 12.5 grams of CAP (Cellulose acetate phthalate also known as cellacefate) and 2.5 grams of Propylene glycol (plasticizer) are added to the mixture and then stirred for 30 minutes until dissolved.

The obtained enteric coating mixture is then sprayed onto the granules of the second powdered granules mixture until the granules are fully covered with enteric coating.

The final powdered granules coated with enteric coating are then sieved through a 4 mm sieve and dried for 2 hours at 37 °C.

The obtained final powdered granules comprise: Glucose between 1 to 90% w/w, Sucrose between 10 -99% w/w, GOX between 0.001 to 10% w/w, and optionally: BPO between 0.01% to 5.0% w/w and L-Cysteine between 0.001- 0.5% w/w.

These granules allow the on-site production and release of between 1 to 200 mmols of hydrogen peroxide, for 48 hours following ingestion. - 17Exatrtple 2: Antimicrobial activity of the composition according to the invention in aerobic conditions Objective: The aim of this study is to assess the antimicrobial effect of granules incorporating the composition of the invention in aerobic conditions on chicken faeces.

Method: 10% of chicken faeces was added to nutrient broth, then 5% w/w of the granules was added to the mixture. The containers were then sealed and incubated at 37°C on a shaking table to simulate movement. Samples were removed after 48 hrs and cfu count was determined by plate counting and a kill curve established.

Results: as shown by Figure 1, in aerobic conditions, the treatment with granules according to the invention comprising glucose oxidase and D-glucose, and the treatment with granules according to the invention comprising glucose oxidase, Dglucose and BPO obtained according to Example 1 showed after 24 hours a 9 log reduction in bacterial CFU/mL.

Conclusion: The granules according to the invention have a significant antimicrobial effect in an aerobic environment.

Example 3: Antimicrobial activity of the composition according to the invention in anaerobic conditions Objective: The aim of this study is to assess the antimicrobial effect of granules incorporating the composition of the invention in anaerobic conditions that are similar to the gastro-intestinal tract of animals, for example poultry.

Method: 10% of chicken faeces was added to nutrient broth, then 5% w/w of the granules was added to the mixture. The containers were then sealed in an anerobic jar with and anaerobic generator and incubated at 37°C on a shaking table to simulate movement. Samples were removed after 48 hrs and cfu count was determined by plate counting and a kill curve established.

Results: as shown by Figure 2, in anaerobic conditions, the treatment with granules according to the invention comprising glucose oxidase and D-glucose showed after 48 hours a 5 log reduction in bacterial CFU/mL. The treatment with granules -18according to the invention comprising glucose oxidase, D-glucose and BPO prepared according to Example 1 showed a 7 log reduction in bacterial CFU/mL, which is an overall improvement of approximately 50% after 48 hours compared to the composition comprising glucose oxidase and D-glucose only.

Conclusion: The granules according to the invention have a significant antimicrobial effect in an anaerobic environment. Furthermore the combination of glucose oxidase and D-glucose with BPO produces a synergistic antimicrobial effect that is a significant reduction in the bacterial infection.

The invention provides a novel formulation which releases a therapeutically active 10 concentration of hydrogen peroxide into the gut of chickens for the reduction of colonisation by a range of pathogenic organisms including Salmonella Enteritidis and Campylobacter Jejuni which are the two major foodborne pathogens transmitted through poultry products.

Advantageously the invention provides a composition and method for eliminating or 15 reducing the intensity of harmful bacteria in animals prior to slaughter, thus reducing the level of contamination which might get onto the meat during processing.

In this specification the terms ''include and comprise” and any grammatical variations thereof are used interchangeably and should be accorded the widest possible interpretation.

The invention is not limited to the embodiments hereinbefore described with reference to the Examples and accompanying drawings but may be varied in construction and detail within the scope of the appended claims.

Claims (10)

1. A composition for controlling bacterial infection in the gastro-intestinal tract of an animal comprising an oxidoreductase enzyme and its corresponding oxidisable substrate which are capable of producing and releasing hydrogen peroxide in the 5 gastro-intestinal tract of the animal.

2. The composition according to claim 1 further comprising a peroxide of an organic acid.

3. The composition according to claim 2 wherein the peroxide of an organic acid is benzoyl peroxide. 10

4. The composition according to any one of claims 1 to 3 further comprising LCysteine.

5. The composition according to any one of claims 1 to 4 wherein the oxidoreductase enzyme is glucose oxidase and the substrate is D-glucose.

6. The composition according to any one of claims 1 to 5 in the form of granules 15 coated with an enteric coating.

7. A method for controlling bacterial infection of meat comprising administering a composition according to any one of claims 1 to 6 to an animal for a period of time immediately prior to slaughtering the animal and processing its meat.

8. The method according to claim 7 wherein the bacterial infection is caused by 20 Campylobacter spp. or Salmonella spp. or Cryptosporidium spp.

9. The method according to claim 7 or claim 8 wherein the composition is administered to the animal for a period between 24 to 48 hours immediately before the animal is slaughtered.

10. The method according to any one of claims 7 to 9 wherein the composition is 25 administered to poultry.