The invention concerns a feed additive of antimicrobial properties, a concentrate, use of the feed additive in the process of manufacturing the concentrate and the production method of the feed additive. More specifically, the discussed solution refers to a feed additive of antimicrobial properties capable of limiting the endogenous microflora activity of the gastrointestinal tract, in particular: of Listeria, Salmonella and Clostridium. According to the invention, the action of the applied supplements involves the improvement of poultry rearing results (body weight gains, feed utilisation) by limiting the activity of endogenous microflora, in particular Listeria, Salmonella and Clostridium.

Poultry meat is one of the most frequently consumed meat types. Broiler chickens, which constitute the source of this meat, are also a rich store of many pathogenic microorganisms such as, for example: Salmonella sp., Campylobacter jejuni, Clostridium perfringesns or Listeria sp. The above microorganisms pose a very serious hazard both to humans as well as to poultry infected by them. They may cause very many quite serious diseases, among others, of the digestive system, frequently of high mortality. Until recently, the main factor employed to protect poultry against bacterial infections having their source in the alimentary canal was antibiotics. The ban on antibiotics in poultry nutrition (inclusion of antibiotics in feeds for poultry) introduced in 2006 made it necessary to look for effective replacers of these compounds. One of a good and, at the same time, safe - both to man and the environment - alternatives for antibiotics can include protein metabolites of many bacteria, also known as bacteriocins which can be synthesised by numerous strains of lactic fermentation bacteria applied commercially and which are generally recognised as safe (GRAS). Bacteriocins can have either bactericidal or bacteriostatic properties. Many of them exhibit antagonistic properties in relation to both human and animal pathogenic microorganisms. Among advantages of bacteriocins is their high specificity of action as majority of them act against only some groups of microorganisms. In addition, bacteriocins active against pathogens are, most frequently, inactive in relation to microorganisms essential for the proper functioning of human and animal organisms (Abee et al., 1995; Cleveland et al., 2001; Montville et al., 1995, Galvez et al., 2007), whereas antibiotics are not so selective in their action. Moreover, bacteriocin application, in contrast to the use of antibiotics, fails to trigger off the development of resistant forms of microorganisms and does not interfere with the natural equilibrium of the intestinal ecosystem and, consequently, bacteriocins appear to be a good substitute for antibiotics. Some researchers claim that there are possibilities of bacteriocin application for medicinal purposes, i.e. to treat intestinal infections, especially those caused by antibiotic-resistant microorganisms. However, bacteriocins are employed most frequently to protect food articles against the development of undesirable microorganisms. These compounds, due to their high selectivity of action as well as very high antimicrobial activity, also arouse interest of manufacturers of poultry feeds.

Investigations which have been carried out so far on possibilities of bacteriocin use as feed additives corroborate the usefulness of such solution. Numerous researchers showed that they can act as a good tool reducing the development of various pathogenic microorganisms. For example, Wooley et al. (1999) reported reduced colonisation of poultry alimentary tract by a strain of Salmonella typhimurium dangerous both for birds and humans following the inclusion in the feed of microcin 24, a bacteriocin produced by Escherichia coli, whereas Audisio et al. (2000) isolated an endogenous Enterococcus faecium J96 strain from the crop of chickens and using a bacteriocin manufactured by it, they controlled the expansion of another intestinal pathogen, namely Salmonella pullorum. On the other hand, Gaenzle et al. (1999) reported reduced counts of Listeria innocua and inactivation of Escherichia coli in poultry gastrointestinal tract following the application of curvacin manufactured by Lactobacillus curvatus. Recent reports by Stern et al. (2005) and Cole et al. (2006) indicating a possibility of influencing the development of Campylobacter jejuni and Campylobacter col in the gastrointestinal tract of chickens and turkeys with the assistance of B602 (NRRL B-30509) and OR7 (NRRL33, B3514) bacteriocins appear to be particularly interesting. Reports of the above-mentioned researchers deserve particular attention because counts of Campylobacter strains occurring in poultry gastrointestinal tract are not affected by competitive exclusion (CE) processes, i.e. processes which comprise one of the major mechanisms of action of probiotics (Stern et al., 2001; Mead, 2002). In addition, in their experiments on turkeys, Cole et al. (2006) also found changes in the histological picture of the duodenum, namely crypt depth reduction which, according to the above authors, may have been directly connected with the reduction in Campylobacter sp. numbers.

The use of bacteriocins as factors eliminating or limiting the development of pathogenic microorganisms in broiler chickens is not easy because bacteriocins - as protein compounds - are digested (degraded) by proteolytic enzymes in the gastrointestinal tract of poultry which results in a complete or partial loss of the biological activity, i.e. ability to affect intestinal pathogens. Therefore, in order to increase the effectiveness of action of bacteriocins, it is essential to elaborate a method which will protect these compounds against the destructive impact of proteolytic enzymes. There is no information about investigations carried out in this area in the available literature. So far, majority of bacteriocins used in poultry nutrition was introduced into feeds without additional protective agents. Carriers elaborated by us (on the basis of lipids) can be employed for encapsulating bacteriocins irrespective of their form (liquid or dried). They are digested only in the small intestine and, therefore, such carriers protect well bacteriocins against the action of proteolytic enzymes (trypsin and chymotrypsin) and, thanks to that, transport them to their destination, i.e. to the lower part of the gastrointestinal tract (small intestine and caecum) of poultry in active form. The carriers, according to the invention, make it possible to obtain the most active form of bacteriocins which, in contrast to non-encapsulated ones, operates best in the lower part of the gastrointestinal tract, i.e. in the place colonised in great numbers by pathogenic microorganisms. Among measurable effects of the reduction of pathogenic microorganisms following the application of encapsulated bacteriocins is the improvement of health and welfare of broiler chickens.

On the other hand, non-encapsulated bacteriocins can be utilised to regulate microflora composition of the crop and the creation - already in this section of poultry digestive tract - of a specific bactericidal barrier. This kind of approach is quite desirable because the crop is also strongly colonised by several dangerous pathogens and is considered to be a major gateway for infection. Therefore, any reduction in the occurrence of pathogenic microorganisms in the initial sections of the gastrointestinal tract decreases risks of poultry intestinal infections and, consequently, also infections in humans. This is confirmed by investigations carried out by Rosenquist et al. (2003) who found that even a twofold decrease in numbers of Campylobacter jejuni in broiler chickens reduced the risk of human infections by 30 times.

In the context of possibilities of the utilisation of bacteriocins as feed supplements in poultry (feed supplements in broiler chicken feeding), a bacteriocin manufactured by bacteria Carnobacterium divergens known as divercin deserves special attention. Divercin is a thermoresistant protein (it can withstand 30-minute heating at the temperature of 100°C as well as a 10-minute treatment at the temperature of 121°C) of 4227 Da molecular mass. This compound remains stable in the environment of pH ranging from 2 to 8 as well as in SDS, Tween 80 and urea solutions. It has a bactericidal effect on a number of bacteria not related with its producers and exhibits a particularly high activity against Listeria monocytogenes, Listeria innocua, Enterococcus fecalis as well as some strains of Clostridium bacteria. Divercin is not active in relation to lactic fermentation bacteria applied as starter cultures in many branches of food industry as well as dietary supplements (probiotics) for broiler chickens. Divercin activity against Listeria and Clostridium deserves special attention, especially bearing in mind alarming reports informing about numerous food poisonings with these bacteria as well as the fact that they occur in poultry digestive tract. Despite its bactericidal effect on many pathogenic bacteria occurring in poultry and the fact that it is resistant to a number of physico-chemical factors (temperature of 80°C to 85°C and pressure of 2-3 bars) to which feed is exposed during the manufacturing process, divercin has not yet been utilised as a feed additive in the feeding of this group of farm animals.

There is also no information in the available literature on the subject concerning the impact of divercin on the endogenous microflora of poultry gastromtestinal tract. On the other hand, investigations carried out by the authors revealed that this compound limits numbers of several strains of zoonotic bacteria causing dangerous, frequently fatal diseases for humans. Therefore, the application of divercin as a feed additive contributes directly to the improvement in chicken health condition and, consequently, also to the improvement of poultry meat quality. This fact also exerts an important impact on human health since feed supplementation with divercin indirectly reduces the infection risk of people with bacteria transferred in poultry products.

The second bacteriocin which can find application in chicken nutrition and contribute to the improvement, among others, of their health and, consequently, also to the improvement of the microbiological safety of products obtained from them is nisin. This bacteriocin is manufactured by Lactococcus lactis ssp. lactis bacteria. It exhibits activity in relation to many LABs as well as some bacteria from Staphylococcus, Micrococcus, Corynebacterium, Mycobacterium, Listeria, Clostridium and Bacillus genera. It exerts a bactericidal effect of vegetative forms of bacteria and acts on their spores. In addition, nisin markedly increases sensitivity of many sporulating forms of bacteria to the action of high temperature (it reduces D by 50-60%). It strongly restricts thermal resistance to thermal treatment of bacterial spores of: Clostridium botulinum, Clostridium sporogenes, Bacillus coagulans and Bacillus stearothermophilus bacteria. On the other hand, this bacteriocin is not active in relation to gram-negative bacteria because the external layer of their cell walls is impermeable for it. However, gram- negative bacteria with damaged lipopolysaccharide layer of the cell wall are sensitive to its action. Among causes of such damages are: thermal treatment, osmotic chock, pascalisation, action of metal chelating factors, e.g. EDTA, compounds of pimaricin lactoperoxidase or lysozyme. Salmonella typhimurium mutants deprived of the oligosaccharide core in the external layer of the cell wall are also sensitive to this bacteriocin. Toxicological experiments carried out on experimental animals showed that this substance does not affect their physiological functions. In Great Britain in 1959, nisin was allowed to be used in food articles. In 1969 this bacteriocin was granted WHO FAO acceptance and was registered as a safe food additive (WHO, 1969). At the same time, Food Additives Committees operating within the framework of the WHO/FAO Food Code Commission determined that the maximum nisin acceptable daily intake (ADI) per kilogram body weight of a consumer must not exceed 33 000 IU. On the basis of long-term investigations carried out in the USA, in 1988 the Food and Drug Agency (FDA) found nisin as a compound safe for human health and accepted the possibility of its introduction into food articles (ADA, 1988). At present, nisin is used in over 50 countries to conserve various food articles. In Poland, in accordance with the regulation issued by the Ministry of Health and Social Care, the use of nisin preparations (E234) is acceptable to conserve ripening and melted cheeses in the amount of 100 mg/kg of product. There is no data in the literature on the subject concerning the utilisation of nisin in poultry nutrition. Commercial nisin preparations known as Nisaplin ^® are employed to destroy undesirable microorganisms in finished poultry products. On the other hand, our investigations revealed that nisin administration to broiler chickens reduced risks of their infection with pathogenic bacteria (it modifies the composition of intestinal microflora) and, consequently, allows obtaining poultry products of higher microbiological quality, hence safer for man.

The patent application P-369985 (published 20-03-2006) describes the method of obtaining bacteriocins manufactured by bacteria from the Camobacterium genus. In the method described in the invention, bacteriocins are synthesised by bacteria from the Camobacterium divergens and/or Camobacterium piscicola genera during their growth in a production bioreactor employing well known culturing methods and maintaining anaerobic conditions and the medium for bacteriocin production is thermally sterilised at the temperature higher than 100°C for the period of at least 30 minutes and the culturing is conducted mamtaining the following parameters: the initial density of bacterial populations in the medium amounts to at least 105 cfu/ml, temperature ranging from 20-37°C, pH ranging from 5.5-7.0, most favourably at constant level. When the periodic or periodic-top up method is employed, culturing is conducted throughout the period covering the logarithmic cell growth phase. When the continuous method is used, the velocity of medium dilution (D) is maintained at the range from 0.02 h-1 to 0.08 h-1. When the continuous method with cell recirculation is applied, the velocity of medium circulation in the membrane filter is kept at more than 0.1 m/s and the medium is diluted with the velocity (D) ranging from 0.08 h-1 to 0.25 h-1. The biomass is removed with the velocity of over 10% in relation to the velocity of medium dilution. At the termination of culturing, bacteriocins are separated from bacterial cells by subjecting them to a shock pH reduction and/or reduction of water activity. Next bacteriocins are thickened and fixed using one of the widely known methods.

The patent application P-369986 (published 20-03-2006) describes the method of obtaining listeriocidal bacterial culture from the Camobacterium genus. The invention concerns the method of obtaining listeriocidal culture of bacteria from the Camobacterium genus according to which inoculum is produced on medium from pure cultures of C. diver gens and C. piscicola bacteria until cell density at the level of 106- 108 cfu/ml is achieved and then the proper culture is inoculated in it which is conducted using either a periodic method, a continuous method or a continuous method with cell recirculation mamtaining strictly defined conditions of the process including temperature ranging from 20-37°C, depending on the employed method, pH ranging from 5.5-7.0 at the initial density of bacterial population in the medium not less than 10 ufc/ml. The patent application P-369984 (published 20-03-2006) describes a new strain of Carnobacterium diver gens Sl-KKP 2012 p bacteria as well as biopreparations for the control of bacteria, especially from the Listeria genus in food articles and feeds employing this strain as well as a disinfecting agent utilising metabolites of this strain. In 1 g of dry matter, the preparation contains more than 105 viable bacterial cells of the new C. diver gens SI strain, preferably 2-5x108 and/or at least 106 live cells of lactic bacteria commonly used as starters in pharmaceutical industry and/or from 1-99% filler and/or from 0.1 to 10% starter substances, preferably l%.The disinfecting agent for bacterial control, especially from the Listeria genus, contains metabolites from the new bacterial strain C. diver gens SI, preferably divercin SI in the amount exceeding 4x105 JA/ml, possibly other disinfecting agents and/or washing agents. The above-mentioned new strain of bacteria was deposited at the Collection of Industrial Microorganism Cultures at the Institute of Agro-Food Industry Biotechnology and was assigned the number: KKP 2012p.

The patent application W08912399 (published 28-12-1989) describes nisin compositions used as bacteriocins. These compositions are made up of lanthionine containing bacteriocins as well as other constituents which are not bacteriocins. Once such bacteriocins are mixed with an appropriate carrier and at the correct doses, the composition is effective against gram-negative and gram-positive bacteria.

The patent application EP 1686185 (published 2006-08-02) describes lactic fermentation bacteria containing bacteriocins resistant to the action of proteases. In addition, the application also describes their addition to food articles and the impact on their shelf-life.

Despite the above-described existing state of art, there is a need to employ in poultry nutrition natural protein metabolites of lactic fermentation bacteria characterised by bactericidal activities such as bacteriocins and that is why the application of divercin and nisin fits very well into the strategy of improvement of animal health and welfare as one of the links in the trophic chain. The above-mentioned bacteriocins as well as probably other bacteriocins of specific scopes of activities clearly limit poultry infections, especially in large commercial farms and make it possible to obtain from them food free of pathogenic bacteria. By destroying pathogenic bacteria, bacteriocins proposed as feed supplements also improve markedly the health of broiler chickens and, in addition, their application brings some other advantages of economic nature, namely, it improves rearing results (increases body weight gains and improves feed utilisation. The subject of this invention is to provide means which will help eliminate from the gastrointestinal tract of chickens of a number of pathogenic bacteria and this elimination will be a direct result of the action of nisin and divercin by increasing the absorption surface of the poultry intestine which additionally will also improve digestibility and absorption of dietary nutrients. Furthermore, the implementation of the invention involving the supplementation of feeds with the above-mentioned bacteriocins will also exert a positive impact on waste management (reduces poultry mortality and, consequently decreases the number of animal intended for utilisation) and following decreased need for the application of chemical disinfection agents, it will also contribute to the protection of natural environment.

Unexpectedly, it turned out that the inclusion in poultry diets of the above bacteriocins may also make it possible to reduce or even eliminate completely the necessity to apply therapeutic antibiotics whose residues, unfortunately, are still detected in poultry meat and eggs. Therefore, the introduction into the marketplace of safe and very effective antibiotic replacers such as divercin and nisin preparations (as well as other obtained bacteriocins) should contribute to increased competitiveness of Polish manufacturers of poultry products on the EU market.

The subject matter of the invention is a feed additive of antimicrobial properties limiting the activity of the endogenous activity of intestinal microflora, in particular: Listeria, Salmonella and Clostridium characterised by that it contains in its composition 10-90% by weight of unpurified bacteriocin preparations, in particular divercin and/or purified bacteriocin preparations.

Preferably, when the additive to concentrates as component A, contains bacteriocins of C. divergens AS7 bacteria as well as their bactericidal metabolites, in particular divercin.

Preferably, when the additive is in a non-fixed form, i.e. in the form of postfermentation fluids, alternatively, their ultrafiltrates and is added directly to concentrates or water, or in a fixed form either as freeze-dried or spray-dried preparations, or in a fixed form with a carrier, or in the form of encapsulated non-fixed and/or fixed preparations of bacteriocins.

Preferably, when the additive is intended for gallinaceous and water fowl as well as ornamental birds.

The next subject of the invention is a supplementary concentrate prepared on the basis of typical feed constituents containing a feed additive of antimicrobial properties which restricts the activity of endogenous microflora of the gastrointestinal tract, in particular of Listeria, Salmonella and Clostridium and which is characterised in that it contains the additive described above in the amount of 0.1-2% by weight and/or antibacterial metabolites in the amount of 0.1-2% by weight as well as 99.1-98% by weight of plant-derived components containing 20-30% of wheat or maize bran, 10- 40% maize gluten; 2-20% fodder chalk; 5-15% wheat; 5-15% maize; 1-4% plant oil. Preferably, when the plant-derived constituents include grain like barley, maize, wheat; bran like wheat, oat in the amount from 0-60%, wheat meal in the amount of 0-30%, oil seed flour in the amount of 0-30%, molasses in the amount of 0-10%, mineral components in the amount of 0-10% and plant oil in the amount of 0-5%.

Preferably, the concentrate as a component contains post-extractive and post-distillation raw materials as well as screenings from herb production.

Preferably, the concentrate is intended for gallinaceous and water fowl as well as ornamental birds.

Preferably, the concentrate restricts the endogenous microflora activity, in particular of Listeria, Salmonella and Clostridium.

The next subject of the invention is the use of a feed additive of antimicrobial properties limiting the activity of endogenous microflora of the gastrointestinal tract, in particular, of Listeria, Salmonella and Clostridium as described above to manufacture a concentrate for gallinaceous and water fowl as well as ornamental birds.

Preferably, the additive is in a non-fixed form, i.e. in the form of postfermentation fluids, alternatively, their ultrafiltrates and is added directly to concentrates or water, or in a fixed form either as freeze-dried or spray-dried preparations, or in a fixed form with a carrier, or in the form of encapsulated non-fixed and/or fixed preparations of bacteriocins.

The next subject of the invention is the production method of a feed additive of antimicrobial properties limiting the activity of endogenous microflora of the gastrointestinal tract and characterised in that the above mentioned additive is manufactured in a non-fixed form, i.e. in the form of postfermentation fluids, alternatively, their ultrafiltrates and is added directly to concentrates or water, or in a fixed form either as freeze-dried or spray-dried preparations, or in a fixed form with a carrier, or in the form of encapsulated non-fixed and/or fixed preparations of bacteriocins.

EXAMPLES Example I

Scope of divercin activity - activity of divercin preparations in in vitro conditions

MATERIAL AND METHODS

Microorganisms

The following bacteria were used as indicator microorganisms to determine divercin activity: Listeria monocytogenes Scott A, 537, 540, 845, 4A i 50034, Listeria innocua F, Carnobacterium piscicola VI, Lactobacillus curvatus, Lactobacillus sake, Lactobacillus plantarum, Leuconostoc mesenteroides, Lactooccus lactis subsp. cremoris, Pediococcus acidilactici, Pediococcus pentosaceus, Enterococcus faecalis, Streprococcus thermophilus, Bacillus cereus, Clostridium tyrobutyricum, Clostridium perfringens, Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Pseudomonas fluorescens. They were obtained from the Culture Collection of the Department of Biotechnology and Microbiology in Poznan. The above-mentioned bacteria were cultured in enriched broth (BTL, Lodz) at temperatures optimal for their growth.

Divercin

Divercin was obtained according to the technology developed by Sip and Grajek (patent application P-369984 from 9.09.2004) with C divergens AS7 (Deposit in the Collection of Cultures of Industrial Microorganisms, IBPRS Warsaw, 07.09. 2004; deposit No. KKP 2012p). In the performed experiments, divercin water solutions of 819 200 AU/ml activity determined in relation to C piscicola bacteria were applied. These solutions were stored at the temperature of -20°C and were gently defrosted at room temperature.

Determination of divercin activity

The activity of divercin preparations in relation to individual bacteria was established using the point diffusion method. The result was assumed as positive when brighter areas of diameters greater than 10 mm were observed.

The sensitivity of different Listeria strains to divercin activity was determined employing the method of critical dilutions [Pilet et al., 1995].

Results

Sensitivity of different Listeria sp. to divercin activity

The performed experiments, the results of which are shown in Tab. 1, demonstrated different sensitivity levels of various strains of the Listeria genus to the action of divercin. Divercin was most active in relation to the most strongly pathogenic Listeria monocytogenes Scott A bacteria (1 638 400 AU/ml). The activity of the examined compound was two times weaker in relation to Listeria monocytogenes 537, Listeria monocytogenes 540, Listeria monocytogenes 845, Listeria monocytogenes 4A as well as to Listeria innocua F bacteria, while its weakest action was recorded against Listeria monocytogenes 50034 bacteria indicating that these bacteria were most resistant to the action of divercin.

Scope of divercin activity

It was determined that in in vitro conditions, divercin exerts a bactericidal effect on the following bacteria: Listeria monocytogenes, Listeria innocua, Enterococcus faecalis as well as on some strains of Carnobacterium piscicola, Carnobacterium divergens, Clostridium tyrobutiricum and Clostridium perfringens bacteria. On the other hand, it was not active against lactic fermentation bacteria from Lactobacillus, Lactococcus, Leuconostoc and Pediocccus genera as well as against gram-negative bacteria (Tab. 2).

Table 1. Activity of the standard divercin solution in relation to bacteria from Listeria genus

Table 2. Scope of divercin activity

Indicator strains Activity

Carnobacterium divergens V41

Carnobacterium piscicola VI +

Lactobacillus curvatus

Lactobacillus sake

Lactobacillus plantarum

Leuconostoc mesenteroides

Lactooccus lactis subsp. cremoris Pediococcus acidilactici -

Pediococcus pentosaceus -

Enterococcus faecalis +

Lactoococcus thermophilus -

Listeria innocua F +

Listeria monocytogenes 537 +

Listeria monocytogenes 540 +

Listeria monocytogenes 545 +

Listeria monocytogenes 50034 +

Listeria monocytogenes 4A +

Bacillus cereus -

Clostridium tyrobutyricum ATCC 25755 +

Clostridium perfringens +

Staphylococcus aureus -

Pseudomonas fluorescens -

Salmonella typhimurium -

Escherichia coli -

„+" sensitive strains,„-" strains resistant to divercin action

Example Π

Impact of divercm on rearing results, counts and activity of endogenous microflora of broiler chicken gastrointestinal tract

Experiment 1

The objective of the performed investigations was to determine the influence of divercin addition on rearing results of broiler chickens, selected populations of endogenous intestinal microflora as well as its activity expressed as the concentration of short-chain fatty acids in the chyme of chickens.

MATERIAL AND METHODS

In order to investigate divercin impact, a growth trial was conducted in pens on 300 one-day old ROSS 308 cockerels in 15 replications of 10 birds/group. The birds were randomly divided into the control group receiving the ionophore coccidiostatic drug - salinomycin in the amount of 60 ppm (S); control C - without addition of divercin and the experimental group D - with divercin addition. The aim of the application of the additional control group (S) was to document bacteriostatic action of salinomycin (Bjerrum et al., 2006; Engberg et al., 2000; Johansen et al., 2007). Throughout the rearing period, experimental birds were fed "provocative" diets, i.e. diets containing high proportions of feed constituents stimulating the development of undesirable intestinal microflora, e.g. Clostridiacae (Tabela 3).

Chyme samples (n=21) were diluted in a cellobiose-glucose broth. Next, cultures were performed on a selective medium of MacConey (MCC) type agar in order to determine potentially pathogenic populations {Enterobacteriacae); MRS agar was used for lactic fermentation bacteria (Jozefiak et al., 2008; Jozefiak et al., 2007). Concentrations of short-chain fatty acids were determined with the assistance of the gas chromatograph (Jozefiak et al., 2007). Differences between diets were established using Duncan test at the significance level of P<0.05.

Table 3. Composition and nutritive value of provocative diets used in trials on broiler chickens

Component Proportion [%]

Wheat 32.68

Barley 25.00

Soybean meal 46.8% 21.54

Beef tallow - animal fat 3.00

Lard - animal fat 5.37

Rapeseed meal 6.00

Fish meal 70% 3.00

Monocalcium phosphate 1.10

bro starter 1% px 1.00

Fodder chalk 0.42

L-lysine HC198 0.28

Salt (NaCl) 0.26

DL-methionine 99 0.21

Sodium carbonate (Na ₂ C0 ₃ ) 0.10

L-threonine 0.03

Formulation nutritive value

Crude protein % 22.000

Crude fat % 9.996

Crude fibre % 3.453

Dry matter % 88.631

Crude ash % 5.160

Salt - NaCl % 0.448

Sodium - Na % 0.1700

Calcium - Ca % 0.850 Total phosphorus % 0.703

Lysine % 1.300

Methionine % 0.550

Cystine % 0.381

Threonine % 0.815

Tryptophan % 0.273

Arginine % 1.312

Phosphorus re g kg 4.2000

AMEn Kcal/kg 3105.7634

DEB (MEq/kg) 205.9223

RESULTS AND DISCUSSION

Divercin addition caused improvement of chicken rearing results comparable with the group in which the ionophore coccidiostatic drug - salinomycin was applied (Tab. 3). Throughout the fattening period (days 1-35), chicken body weight gains were statistically significantly higher in comparison with the control group (C). In comparison with the control group, diet supplementation with divercin reduced pH value in the crop (P=0.001) and caeca (P=0.043) chyme. Irrespective of the type of the applied selective media as well as the segment of the gastrointestinal tract, tendencies for reduced populations of endogenous microflora were observed (Tab. 5). However, these differences in the case of lactic fermentation bacteria were confirmed statistically only in the crop and caeca. In comparison with the control group, in the experimental group reductions in acetic, lactic and succinic acid concentrations were observed in the crop, small intestine and caeca chyme. These differences were corifirmed statistically in the case of lactic and succinic acids in the crop and small intestine (Tab. 6). In addition, also concentrations of butyric, isobutiric, pentanoic and isopentanoic acids in the caeca of the experimental group were found reduced when compared with the control group. Concentrations of short-chain fatty acids were found lower in all the examined segments of the digestive tract; however, the differences were not confirmed statistically (Tab. 6). Table 4. Results of rearing of broiler chickens fed diets containing ionophore coccidiostatics (salinomycin); without feed additives (control ) and with divercin

Table 5. Populations of endogenous intestinal microflora and pH values segments of the gastrointestinal tract of broiler chickens

Table 6. Concentrations of short-chain fatty acids in various segments of the gastrointestinal tract of broiler chickens

ND— not found Experiment 2

The objective of the performed trials was to determine the impact of divercin supplementation on the intestinal digestibility of crude protein and crude fat. In addition, intestinal and total value of apparent metabolisable energy (AMEn), nitrogen retention and total fat digestibility were estimated.

MATERIAL AND METHODS

In order to investigate divercin impact, a growth trial was conducted in pens on 300 one-day old ROSS 308 cockerels in 15 replications of 10 birds/group. The birds were randomly divided into the control group receiving the ionophore coccidiostatic drug - salinomycin in the amount of 60 ppm (S); control C - without addition of divercin and the experimental group D— with divercin addition.

For this purpose, an indicator - 0.3% titanium dioxide (Ti0 ₂ ) - was added to all experimental diets. On the 28 ^th day of birds' life, about 50 g of excreta were collected from each replication and immediately frozen with the aim to carry out further laboratory analyses. Samples were freeze-dried (Christ 1825 Medizinische apparatebau §326 Osterode/Harz). Next, the collected excreta as well as experimental diets were ground in a laboratory mill with mesh size 1 mm. The experimental material prepared in this way was subjected to analysis on the content of titanium dioxide in accordance with appropriate methodology (Short et al., 1996) during which 100 mg samples were burnt at the temperature of 580°C for 13 hours and then boiled in sulphuric acid solution. After filtration of solutions and addition of H ₂ 0 ₂ , the intensity of the obtained colour was determined with the assistance of absorption spectrophotometry using 419 nm wave length in relation to a blank test. Coefficients of total apparent digestibility, nitrogen retention as well as AME and AMEn of the applied diets were calculated on the basis of the content of Ti0 ₂ , nutrients and gross energy in the excreta and diets.

RESULTS AND DISCUSSION

In comparison with the control group, the addition of divercin increased the value of the apparent metabolisable energy in the small intestine of broiler chickens (2779 vs. 2396 kcal). Despite the fact that this difference was statistically nonsignificant, a significant impact on broiler rearing results can be expected, especially on the feed conversion ratio (FCR). In addition, the AMEn value in industrial concentrates for poultry is one of the main factors affecting their raw material costs. It can be stated, on the basis of the obtained results, that the application of divercin exerts a significant influence on the reduction of production costs of feeds for broilers. Moreover, a tendency was observed in the discussed balance trial for improvement in the intestinal total protein digestibility (Tab. 7) which should also be considered as a positive aspect of divercin application.

Table 7. Results of the balance trial carried out on broiler chickens

CONCLUSIONS

1. Object of bacteriocin action - pathogenic microflora of poultry gastrointestinal tract. Supplementation of feeds with divercin either in free and/or encapsulated form reduces counts of pathogenic microorganisms in poultry digestive system.

2. Impact of bacteriocin action, i.e. of introduction into feeds for poultry of divercin preparations. Results of the regulation of poultry intestinal tract microflora composition and exclusion from or reduction in the system of proportions of specific groups of pathogenic microorganisms, in particular bacteria from Salmonella, Campylobacter, Listeria and Clostridium genera, include:

• Improvement of broiler health and welfare (reduction in mortality and decrease in the incidence of digestive tract diseases;

• Improvement in broiler chicken production results (rearing results) - improvement in nutrient retention (higher body gain increments) and concentrate utilisation (reduced feed consumption);

• Lower risk of the occurrence of pathogenic microorganisms in poultry products - improvement in microbiological quality of poultry articles; • Reduced risk of human contamination with pathogenic bacteria occurring in poultry gastrointestinal tract;

• Improved microbiological safety of poultry products for their consumers;

• Reduced use or total elimination of antibiotics;

• Reduction in contaminations introduced into the environment as a result of poultry production;

• Since the applied preparations were not purified, they also contained trace quantities of other metabolites which show synergistic activity.

LITERATURE

Bjerrum, L., R. M. Engberg, T. D. Leser, B. B. Jensen, K. Finster, and K. Pedersen. 2006. Microbial community composition of the ileum and cecum of broiler chickens as revealed by molecular and culture-based techniques. Poult. Sci. 85:1151-1164.

Engberg, R. M., M. S. Hedemann, T. D. Leser, and B. B. Jensen. 2000. Effect of zinc bacitracin and salinomycin on intestinal microflora and performance of broilers. Poult. Sci. 79:1311-1319.

Johansen, C. H., L. Bjerrum, and K. Pedersen. 2007. Impact of salinomycin on the intestinal microflora of broiler chickens. Acta Vet. Scand. 49:30.

Jozefiak, D., S. Kaczmarek, and A. Rutkowski. 2008. The effects of benzoic acid supplementation on the performance of broiler chickens. J Anim Physiol Anim Nutr (Berl).