WO2009092787A1

WO2009092787A1 - Lactylates for the prevention and treatment of infections caused by gram-positive bacteria in animals

Info

Publication number: WO2009092787A1
Application number: PCT/EP2009/050770
Authority: WO
Inventors: Anne Cazemier
Original assignee: Purac Biochem Bv
Priority date: 2008-01-25
Filing date: 2009-01-23
Publication date: 2009-07-30
Also published as: EP2249824B1; BRPI0906503B8; AU2009207626A1; MX2010008010A; CN101917984A; CA2712448C; ES2505140T3; CA2712448A1; CN101917984B; EP2249824A1; JP2011510045A; AU2009207626B2; US20100311832A1; US20190046494A1; EP2082739A1; BRPI0906503A2; AU2009207626B9; CN103655536A; PL2249824T3; US11517550B2

Abstract

The present invention pertains to the use for preventing or treating intestinal infections caused by gram-positive bacteria in animals of an antibacterial compound selected from lactylate in accordance with Formula (1), Formula (1) R2 -COO- [ -CH(CH3) -C00 ] n-R1 or a Na, K, Ca, Mg, Fe(II), Zn, NH4, or Cu(II) salt thereof, a glycosylate of Formula (2), Formula (2): R2-C00- ( -CH2-C00) n-R1 or a Na, K, Ca, Mg, Fe(II), Zn, NH4, or C(II) salt thereof, a lactate ester of Formula (3), Formula (3): HO-CH ( CH3) -C00-R2 and/or a glycolic acid ester of Formula (4), Formula (4): HO-CH2-COO-R2 wherein R1 is selected from H, n stands for an integer with a value of 1-10, and R2 stands for a C1-C35 alkyl or alkenyl chain which may be branched or unbranched. The compound, which preferably is a lactylate or a Na, K, Ca, Mg, Fe(II), Zn, NH4 or Cu(II) salt thereof, is particularly useful in the treatment or prevention of clostridia. An animal nutrition composition and a method for preventing or treating infections are also claimed.

Description

LACTYLATES FOR THE PREVENTION AND TREATMENT OF INFECTIONS CAUSED BY

GRAM-POSITIVE BACTERIA IN ANIMALS

The present invention pertains to a method for preventing or treating intestinal infections caused by gram-positive bacteria in animals, to specified compositions for preventing or treating intestinal infections caused by gratn-positive bacte- 5 ria in animals, to the jse of specified compositions for preventing or treating intestinal infections caused by gram- positive bacteria in animals, and ro a nutrition composition for animals comprising a specific compound in an amount effective for preventing or treating intestinal infections 10 caused by gram-positive bacteria in animals.

Gram-positive bacteria are stained dark blue or violet by gram staining, mainly due to a high amount of peptidoglycan in their ceil wall. Among the gram positive bacteria are the 15 pathogenic bacteria Enterococcus, Clostridium, Listeria, Staphylococcus, various Bacillus species, and Streptococcus. While some of these organism are mainly of concern as food contaminants, others can cause diseases in animals,

20 For example, Clostr idia are responsible tor causing a number of widely varying diseases of the intestine in animals. As it is a nearly ubjquitous bacteria readily found m soil, dust, faeces, and feed, it is extremely difficult to keep animals free from Clostridia.

25 Clostridium-related intestinal diseases may be quite severe. For example, Clostr > dia aze involved in causing necrotic enteritis in chickens. In cattle, Clostridia-related enteritis can take the form of "sudden death syndrome", which, in practice can resuit in the in overnight deaths of a number of cattle. Also in other animals, Clostridia-related diseases may cause severe damage.

It is known to administer antibiotics Lo animals Lo protect 5 them from intestinal infection. It is also know- to include such antibiotics in animal nutrition. However,, there is an increasing resistance against the use of antibiotics in animal feed, and nowadays many countries have legislation that prohibits the use of antibiotⁱcs in arJ.mai feed. Moreover, 10 antibiotics have to be administered in very control Jed amounts .

Accordingly,, there is therefore a need for a non-antibiotic method and composition for animal feed that will help to

L5 treat or prevent intestinal infections caused by grair- posiLivo bacteria in animals, in particu_ar m mammals, including ruminants {e.g. cattle, sheep, goat, deer) ano monogastrics (e.g. swine, horses, rabbits}; in birds (e.g. poultry, turkey, pheasant", quail,); in fish, including marine

.0 fish (e.g. salmon halibut, tuna), fresh water fish (e.g. trout, carp, tilapia); molluscs (e.g. oyster, mussels, clam, snail} and crustacean (e.g. crab, lobster, shrimp). The present^" invention may also find application in humans, and m fur animals such as mink, ermine, sabre, and foxes.

^>b

Further, it may be desirabJe to suppress specific Gram- positive bacteria in the intestine with a view to increasing the growth of animals. It is believeα that this may be of interest for Lactobacillus spp. The present" invention is also

>0 of interest for this application.

The present invention provides a method and composition for animal feed for treating or preventing intestinal infections caused by gram-positive bacteria in animals. In accordance with the present invention, use is made of an antibacterial compound selected from lactylate in accordance with formula

1, 5.

or a Na, K, Ca, Mg, Fe(II), Zn, NH₄, or Cu(II) salt thereof,

10 a glycosylate of formula 2,

or a Na, K, Ca, Mg, Fe(II), Zn, NH₄, or Cu(II) salt thereof 15 a lactate ester of formula 3,

and/or a glycolic acid ester of formula 4,

wherein in the above formulas R1 is selected from H, n stanos for an integer with a value of 1-10, and R2 stands for a C1- C35 alkyl or alkenyl chair, which may be branched or un- branched

for the prevention or treatment of intestinal infections caused by gram-positive bacteria.

The present invention pertains to the prevention or treatment of intestinal infections by gram-positive bacteria in am- mals. The invention is particularly attractive for use against intestinal infections with anaerobic or facultative anaerobic bacteria, even irore in particular anaerobic bacteria. Within the groαp of anaerobic bacteria, it is S particularly desirable to have a method for the prevention or treatment of intestinal infections by spore~.form.ing bacteria, as these organism tend to be difficult to control. The invention is of particular interest in the prevention and treatment of intestinal infections by Clostridia. 0 Tn one embodiment, the present invention pertains to the prevention or treatment of intestinal infections causeα by Clostridium, in particular by Clostridium perfringens in poultry, in particular in chicken. In another embodiment the present invention pertains to the5 prevention or treatment of intestinal infections caused by Clostridium, in particular by one or more of Clostridium tet- anii, novyi (type B) sept i cum, chauvii, sordelii, hemolyticum, di CCici Ie, botulinum, m cattle, In a further embodiment the present invention pertains to theC reducrion of intestinal growth of Lactobacillus spp.

It is noted that WO 2004/10 ''877 describes an antimicrobial composition comprising a mixture ot lactic acid or a derivative thereof and an inorganic acid. The composition is5 described as antimicrobial in general. The use against Salmonella and Escherichia CoIi is specified. While lactylates are mentioned as possible lactic acid derivatives, their αse is not further elucidated. There is nothing in this reference that teaches or suggests the particular efficacy that the use of lactyJates has been found to show against gram-positive bacteria in animals. It in further noted that GB115480 describes the use of acy- lated alpha- hydroxy carboxylic acids against bacteria and fungi, for instance moulds, mildews, and yeasts. It is .indicated that, the compound can be used for consumption by or application to humans or other animals, bur this is never elucidated. There is nothing in this reference that reaches or suggests the particular efficacy that the use of lacty- lates has beer, found to show against, gram-positive bacteria.

In the present invention, use may be made of an an antibacterial compound selected from one or more of a lactylate in accordance with formula 1, or a Na, K, Ca, Mg, Fe(II), Zn, NH₄, or Cu(II) salt "hereof, a glycolylate of formula 2, or a Na, K, Ca, Mg, Fe(II), Zn, NH₄, or Cu(II) salt mereof, a lactate ester of rormuia 3, and/or a glycolic acid ester of formula 4.

The use of a lactylate of formula 1 or a salt thereof has been found to be preferred.

In a preferred embodiment of the present invention, R2 is an alkyl or alkenyl chain with 6-20 caroon atoms. More in particular, R2 is an alkyl or alkenyl chain with 6-18 carbon atoms. In this embodiment, suitable substituents include groups with 6 carbon atoms (capronic) , 8 carbon atoms fcaprylici 10 carbon atoms (capric acid), 12 carbon atoms

(lauryl), 14 carbon atoms (myristyl) , 16 carbon atoms (cetyl, palmityl), 18 carbon atoms (stearyl). Mixtures of two or more compounds may also be used. Where a salt is used, the use of a Na, K, Ca, or Mg salt may be particularly preferred.

The value for n is preferably m the range of 1-5. More in particular n has a value of 1, 2, or 3. The use of lauroyi lactylate, myristolyl lactylate, and their sodium salts is particularly preferred. In one embodiment; a mixture is used comprising 5-95 wt . % of lauroyl lactylate and 95-5 wt .% of myristoyl lactylate, or the sodium salc(s} of ? these compounds are used, more in particular, a mixture is ased comprising 25-75 wt . % , more in particular 40-60 wt . % of lauroyl lactylate, and 75-25 wt.%, more in particular 40--6C wt . o of myristoyl lactylate, or the sodium salt(s) of these compounds .

10

Tn one embodiment, of the present invention, the anti bacterid.! compound, in particular the lactylates or salts thereof, are used in combination with one or more cocciαostatic components. This is of particular interest in poultry during Line

15 lmrrLJiosuppre^s: on period, which is the period in a chick' 3 lifetime where the immune system which protects the anima, in the egg has deteriorated but the immune system of the animal itself has not been completely developed. E¹Or chickens this is between day 10 and ?0 of the aninais lifetime.

?0 This io of particular interest increase the resistance of ⁺ he chicken to intestinal Clostridium infections. More in particular, in chicken it iε oelieved that the necrotic enteritis caused by Clostridium is often procedeo by an infection with Eimcria. The Eimena is oelieved to damage the

.5 wall of the intestines, which makes it less resistant to an infection with Clostridium. The use of a combination of lac- LyIaLe with one or more coccidostatic components wi J i therefore provide an increased resistance of the chicken against necrotic enteritis.

50 Suitable coccidosta t i c component 3 are known in Lhe art, as are the amounts in which they should De provided. Suitable components include maduramycme, diclrzil, narasin, nicarba- zine, mononsir., robenidine, lasalocid, halofuqinon, narasm, salmoirycine, decoquinaue, and semduramycine .

7 he composition may be administered to animals as a component 5 of a conventional animal feed composition. In the context: of this invention tne ierra "animal nutrition" includes solid feed and liquid feed, such as drinking water. Thαs, the composition may be administered to an anima± as a solid or liquid component of a conventional animal feed composition or 10 in their drinking water.

The composition may also be adra.ini3i.erec. to the animal in a separate step, independent from the provision of a conventional animal feed coxr.pos.ition.

15 Tn one embodiment of the invention, the antioacterial compound, in particular the lactylate or salt thereof, is attached to a support. This provides a convenient way Lo obtain che antimicrobial composition in solid

form. Suitable supports are selected from vegetaole fiber material,

^■O vegetable carbohydrates such as cellulose, and mineral supports such as silica, starch, gyp^un, and lime. In another embodiment, the antimicrobial compound is added in a mixture with a vegetable oil, e.g., a corn oil, soybean oil, or olive oil.

^S5 The ant i -microbial compound may also be in the form of a tablet or other shaped body known for provision of pharmaceutical components to animals.

The amount of antinicroD: al compound, in particular iacty- .0 late, administered to the animal is suc.α that it is effective to treat or prevent intestinal infections caused by gram- positive bacteria in tr.e ammal Lo whicr. the compound is administered. Sucr. an amount is suitably in the range from 0.0001-5% Octoβα on the tola! weight of each feed fed to the anirtαl. Tn a preferred embodiment, the amount may be in the range of 0,001 to 2%, based on the total weight of each feed fed to the animal. It has been found that as compared to the 5 use of lactic acid as αoscr ibeJ in WO 2004/107877 it may bo possible Lo ase lower concentrations of the effective component. While in the Examples of WO 2004/10/87/ 1.2 wt . % of lactic acid is used, the use of, for example, lactylates in accordance with tne present invention allows the use of a re~

10 duccd amount of active component. According] y, in one eiT.bodin.ent of fr.e present inversion the amount may be ir, the range of 0.001 to 1 wt.%, more m particular 0,001 to O.b wt. %, based on the total weight of each feed fed to the animal. It is within the scope of the skilled person to

Ib determine the amount necessary.

Tf so αesired, the amount may be higher than required for the compound to be effective to treat or prevent infections caused by gram-positive bacteria Clostridia-rolated enteritis .0 in the animal. Ir.±s may be the case if the compound, also acts to promote growth, improve feed to gair. ratio, and/or improves digestibility or ammo acids administered in animal feeds .

Ib As mentioned aoovc, the antibacterial compound nay be administered to animals as d component of α conventiona^l animal feeα composition. A conventional animaJ feed composition may comprise wheat, starch, meat and bone meal, maize, sunflower meal, corn, cereals, barley, soybean meal, tapioca, citrus

SO pulp, legumes, beet pulp, etcetera, in accordance with the present invention the provision of antibacterial compounds to the animal to treat or prevent intestinal infections with Gram-positive bacteria will in general not be combined with the provision of antibiotics.

In. WO 2004/107877 lactic acid or a .lactic acid derivative i.s 5 used in combination with an inorganic acid selected fron nitrogen, sulphur, and phosphorus -containing acids. It is indicated that the inorganic acid is believed to lower the pH in the chymus during total passage in the animal , thereby increasing the presence of non-dissociated lactic aicid, which

10 disrupts the outer membrane of the pathogens,

In contrast, the present .invention does not rely on the presence of non-dissociated lactic acid. Therefore,, the present invention does not require the presence of an inorqanic acid to lower the pH in the chymus .

15 Accordingly, the present invention also pertains to the use of antibacterial compounds as described above, in particular lactylates according to tormula 1, in the prevention or treatment of intestinal infections caused by gram-positive bacteria, wherein such use is not accompanied by the use of

20 an inorganic acid selected from nitrogen, sulphur, and phosphorus-containing acids for increasing the presence of non- dissociated lactic acid .

The invention is further illustrated by the following exarn- 25 ples, which show the inventive merits of this invention, without the invention being limited thereto or thereby.

Example 1: Efficacy of a mixture of sodiun lauroyl lactylate and sodium myristoyl lactylate against necrotic enteritis in 30 chicken

The efficacy of a mixture of sodium lauroyl lactylate and sodium myristoyl lactylate against necrotic enteritis in chicken has been evaluated by Schotnorst Feed Research in an experimental C. perfrinqens infect, ion mode,, which they have developed in which a coccidiosis infection is used as a pre- trigger for C. pertπngens to colonise the small intestine and cause necrotic enteritis. A coccidiosis infection is initiated by a pathogenic Eimeria maxima and, on the peak of the coccidiosis infection, birds are inoculated with a C. per- fringens strain that proved to be pathogenic to broiler chickens. A coccidiosis infection caused by E. maxima (re~ suiting in lesions m the iriddle segment of the small intestine) followed by a Clostridium infection results in a r.ighly reproducible model and an easy and accurate way of scoring for necrotic enteritis lesions, because lesions of E. maxima and Clostridium are easy to distinct while lesions of both pathogens do not occjr in the same intestinal segment. The experiments are performed in cooperation with the Animal Health Service fGϋ) .

The experiment consisted of one treatment and twc control treatments, AIi treatments consisted of six replicate caqe≤ with 19 broilers per cage, The treatments are given in Table

,

2) 1 x 10^s cfu C. perfringens in 1 ml

5 Animals? management and procedures

One day-old male Ross 308 broiler chickens were supplied by Probroed & Sioot B, V., the Netherlands. At day 0, broilers arrived at the laboratory facilities of the Animal Health Service (Deventer, the Netherlands) and were housed in di¬

10 gestibility cages after individual weighing. Based on a weight-class system 19 birds were allotted to 30 Schothorst litter floor digestibility cages, resulting in a similar mean weight per cage. Broilers were housed in these cages until the end of the experiment at day 20. At day 9, if no mortal¬

L5 ity occurred, the number of chickens was standardised to l^~l and bird weight was measured again. First, birds with obvious visual aberrations were removed and second, birds were removed at random to decrease the number to 17. Lighting and temperature schedule throughout the experimental period was as follows, 22 hours of light followed by 2 hours darkness in the first period from day 0 to 9 followed by 18 hours of light and 6 hours darkness throughout the rest of the experi- merit. The ambient temperature was gradually decreased from 32°C at the start Lo 25°C at the end of the experiment.

Feed was supplied for ad libiiuxα intake from day 0 onwards 5 with exception of the 5 hours prior to inoculations (days 9, 14, 15 and 16) and sections (days 15, 16 and day 20) . Water was available for ad libitum intake throughout the experiment .

10 Feed composition

The broilers were supplied a wheat /soybean meal -based starter diet from day of arrival until day 9. From day 9 onwards, a wheaL/bariey-based grower diet was fed until the end of the experiment (day 20}. Grower feeds were fed as meals because

15 of the necessity of homogenouslymixing in the test, product.-; after feed production. Diets did not contain any coccidio- ≤tat≤ or antimicrobial feed additives other than the test product. The nutrient composition of the experimental diets was according tc Dutch standards to meet nutrient require-

20 raentε of broilers (CVB, 2006) .

Inoculum

At day 9, broilers were inoculated with either I mi saline or

E. mdxnna (10.000 sporulateα oocyεts/chickon in 1 ml} after a

25 5 hours feed withdrawal period, from day 14 onwards, broilers were either inoculated with 1 ml liver oroth (DIFCO) or C, perfringens once per day persisting three days after a b hours feed withdrawal. A detailed overview of the different treatments is presented in Table 1.

30

The pathoqonic C. perfringens strain was obtained from the Animal Health Service in Deventer, the Netherlands (approx. 10⁸ cfu in 1 ml ) . The strain was grcwn on an agar of sheep blood and the culture is typed by ClDC (Central Institute of Animal Disease Control in Lelystad) as C, perfringens produc- ing type α and β2 toxins. Each day a treshly prepared inoculum was used. 5

Lesion scoring

Clostridium perfringens : Gross and microscopic lesions generally occur in the small intestine, particular in the proximal site. The following scoring method was used:

10 0: no lesions

1: 1 to 5 small lesions (spots of less than 1 mm diameter) 2: more than 5 small lesions ( spots of less than 1 mm diameter) or 1 to 5 larger lesions (spots of 1 to 2 mm diameter )

15 3: more than 5 larger lesions (1 to 2 mm diameter) or erosive zones

4: dead birds with positive necrotic enteritis diagnoses post mortem All birds were scored "blind", i.e. the person scoring the

20 birds for lesions did not have knowledge of the birds treatment .

Measurcments

During the experiment the following parameters were measured: 25 * Individual body weight at day of arrival and means per cage at day 9 and day 20 of the experiment * Body weight of the bird? prior to necropsy * Feed intake per cage in the periods from day 0 to 9 and daily teed intake from 9-20 days of age

30 * Coccidiosis lesions and necrotic enteritis lesions in the small intestinal mucosa of 24 birds per treatment, at day 15, day 16 and day 20 of the experiment {total of 72 birds per treatment).

. Mortality per cage from 0 to 20 days of age. Daily records were kept of all routine study activities, 5 inealth disorders and of mortality (with its most probable cause) .

Statistical analyses

Raw data were analysed for outliers. Significant outliers

10 were excluded from the statistical analysis. The incidence of NE-iesions (% of affected birds] was analysed by Fisher Exact Test, whereas the severity of lesions and daily feed intake measurements were analysed by analysis of variance (ANOVA) using Genstat statistical software. Treatment means were com-

15 pared by the least significant difference (LSD), P<0,05 was considered to be statistically significant, whereas 0.05<P<0.10 was considered to be a near-significant trend.

20

Incidence and severity of lesions

Lesion scoring at day 15 (1 day post infection)

In Table 2, the percentage of positive scored birds (birds

25 with NE, lesions) is given as well as the mean .lesion score of all positive scored birds. Because the mean lesion score or all examined birds, affected as well as unaffected, gives a more representative picture for the population, statistical analyses have been performed over these results {see the fifth

30 column of Table 2} . The severity of lesions in both positive and negative scored birds is indicated on a scale of 0 to 4 (see section "lesion scoring"). Table 2: Birds observed with NE (%) and the mean severity of lesions scored at day 15 (1 day p,i.).

Values with no common superscript in a column differ significantly (P≤O.05) .

^{1^}' Lesions severitv of NE-positive scored birds

A significant treatment effect was observed on the NE incidence. As expected, the lowest incidence was observed in the LO uninfected control treatment but results were comparable to the results of the treatments supplemented with the test mixture and unsuppleinented infected control.

Based on the ANOVA it was concluded that there was a signifi- .5 cant treatment effect on the severity of necrotic lesions on day 15 (P< 0.001). On lesion severity it was evident that lesions were more severe in the infected treatments, unsupplemented as well as supplemented, than the uninfected control treatment for there were no positive score birds in !0 the latter. Among the infected treatments no statistical differences were observed.

Lesion scoring at day 16 (2. days post infection) In Table 3, the percentage, of positive-scored birds and the 5 mean lesion score of birds is given for day 16.

' Values with no common superscript in a column differ 5 significantly (P≤O.05).

Comparing the results of NE incidence and lesion severity on day 16 to the results of day 15, it is clear that the severity of infection was higher 2 days post infection. Although again

LO a significant treatment effect was observed on the NE incidence, it is evident that this is due to the difference between the uninfected control treatment and infected treatments, which is as expected, whereas among infected treatments there was no significant difference observed.

L5 A sharp distinction can be drawn on lesion severity 2 days post infection. The treatment supplemented with the test mixture resulted m a clear reduction in lesion severity compared to the infected unsupplemented control, although mean lesion scores were still higher than the uninfected control.

Lesion scoring at (day 20 (S days post infection) At day 20 no significant differences was observed between treatments. All treatments recovered from NE, at least based on macroscopical evaluation, with 0% incidence and obviously 0.0 for lesion severity. Mo r t ality

Mortality is one of the parameters to measure the severity of an infection with Clostridium in a flock. In this experiment the mortality was compared among treatments. Mortality was 5 14.6% in the infected control treatment (treatment 2) and 0% in the uninfected control. Supplementation of the test mixture resulted in a reduction in mortality (5.1%).

Production parameters

10 Besides lesions scoring, production parameters like body weight and daily feed intake were measured during tne trial period.

Body weight of one day- old broilers was in all treatments approx. 47 grams. Because treatments from day 0 to 9 were

15 si mi Jar, no differences in body weight gain and ieed intake were observed m this period,

In the infection period from day 9 to 20 both production parameters were significantly affected by the individual treatments. Body weight gain was highest in the uninfected

20 control, as expected, while broilers in the infected unsupple mented treatments showed the lowest body weight gain, This resulted in a 30% lower final weight at day 20 (523 g versus 749 g) . The infected supplemented treatment resulted in a significantly higher feed intake and boαy weight gain compared to

25 the .infected unsupplemented control. Reduction in production performance could be reduced with 10% showing a loss in final weight of approx. 20% when compared to the uninfected control group (approx 583 g versus 749 g) . It was concluded that the test mixture significantly increased production performance

30 during a subclir.ial Clostridium infection. Example 2: in vitro tests of lactylates against Clostridium

Liquid cultures of Clostridium perfringens ATCC 13124 were grown m screw-capped Lubes (100 x 16 mm) containing 10 ml brain heart infusion broth (Oxoid CM?25, Basingstoke, united Kingdom) for 24 hours at 30 °C. Brain heart infusion broth was prepared with varying amounts of lactylates. The pH of the media was adjusted to 6.0 with 9 M sulphuric acid usinq a Handylab pH 12 pK meter equipped w.i ⁺ h a Blueline 16 pH (mi- cro) probe (no, ?85i 29163). All media were sterilised by filtration using 0.45 μm cellulose acetate filters (Minisart syringefilter, sterile and non-pyrogenic, no. 16555, Sarto- rius, Gottingen, Germany) (9). 300 μl of each medium was transferred Lo a panel of a sterile Bioscreen honeycombe ICO well plate (Thermo electron Oy, Vantaa, Finland) . Completed well plates were stored at -30 ⁰C until further use.

Well plates were inoculated with 3 μl culture usinq a sterile Hamilton repealing dispenser (Hamilton, Bonaduz, Switser- land) . The growth rate of the test organisms was determined at 30 ⁰C with the Bioscreen C culture system (Oy Growth Curves AB Ltd, Helsinki, Finland) . In order to assure low oxygen conditions the Bioscreen was placed inside an anaerobⁱc cabinet equipped with a type M-12 oxygen sensor (In Vivo₂ 400 hypoxia workstation, Biotrace International PIc, Brid- qend, United Kingdom). The oxygen tension was regulated at C % oxygen using a Ruskinn gas mixer module (Biotrace International PIc). The Bioscreen C kinetically mcαόureo the development of turbidity by vertical photometry in up to ?00 wells simultaneously. The optical density of the cultures was automatically measured at fixed time intervals at 420 - 580 nm usinα a wide band filter. Table 4 shows the MIC values for the various iactylates tested for Clostridium perfringens ATCC 13124 in brain heart infusion broth. In the parentheses the number of repeats is given. MIC stands for the Minimal Inhibitory Concentration, which is the lowest concentration where the increase in ab- sorbance of a culture did not exceed the threshold value, which was defined as the average increase in absorbance value of the blanks plus three times the standard deviation.

1.0 Tt appears that even at very low concentration Iactylates are capable of suppressing the growth of Clostridium perfringens .

MIC values of different Iactylates

Lactylates MIC values (%)

^"cTTac^'tylate ClO 1actylate^"

C12 lactylate O JD 02 % ( 2x ) Ϊ4 ϊactvlate _ θ^"_._2δT_T_^~T_2xf__ lb lactylate

C18:l lactylate 0.02

Mixture^~™of^"~TTΪ^'vv""ci0/Ci2 0.002 (3x) lactylate)

Mixture of 1:1 C12/C14 0,001 % (3x) lactvlate

example 3: Dose-response studies and prevention studies of a mixture of sodium lauroyl lactylate and sodium myristoyl lactvlate aqainst necrotic enteritis in chicken

Analogous to Example 1, the influence of the dose of the compound was studied, Further, the use of the mixture on chicken which were not previously infected with Emeria and Clostridium was studied. The treatments performed are summarised in Table 5:

The test mixture was made up cf 50 wt . % of sodium lauroyl 5 .lactylate and 50 wt . % of myristoyl lactylate.

The results iray be summarised as follows.

In this experiment a subsequent infection with E. maxima and

C. perfringens resulted in an incidence of necrotic enteritis

' 0 of 56% and an average lesion score of 1.6 during the first two days post infection. Supplementing bird diets with test mixture reduced the number of infected birds and a dose response effect was observed, showinα the highest efficacy in the 0.6% and 0.3% treatments. Lesion severity was significantly reduced

15 due to test mixture supplementation and tne dose response effect was also strongly present on this parameter. Lesions were less severe in the treatments with the highest doses of test mixture. The test mixture supplementeα in. a pure form resulted m a somewhat better response than the test mixture supplied

'0 via a silica carrier. Supplementation of test mixture with 0,6% resulted in a significant reduction m mortality (4.6%) and was not significantly hiqhcr than the rot infected control treatment. Results with 0.3% test mixture supported the results observed in the lesion scoring.

With regard 1.0 production performance an effect was observed when comparing healthy birds with or without; test mixture supplementation with each other. The provision of test, mixture tended to increase body weight in the starter phase and αrowor phase. Supplementing infected birds with higher doses of test irixture resulted in the b:. rds having a similar final weight (day 37) as birds that were not infected at all.

It can be conciuoed that the tost mixture,, especially in a dose of 0.3 wt . % or higher is effective in preventing necrotic enteritis development ir. broiler by showing a lower incidence and lesions that were less severe.

Claims

1. use for preventing or treating intestinal infections caused by gram-positive bacteria in animals of an antibacte- rial compound selected from lactylate in accordance with formula 1,

Formula 1 R2-CQO- [-CH (CH₃) -COO] _n-Rl

or a Ka, K, Ca, Mg, Fe(TT), Zn, KH₄, or Cu(TI) salt thereof,

a glycolylate of formula 2,

Formula 2: R2-COO- [-CH2-COO] _fi~Rl

or a Na, K, Ca, Mg, Fe(II), Zn, NH₄, or Cu(II) salt thereof

a lactate ester of formula 3,

Formula 3: HO-CH (CH₃) -CGO-R2

and/or a glycolic acid ester of formula 4,

Formula 4: HO-CH2-COO-R2

wherein in the above formulae Rl is selected from H, n stands for an integer with a value of 1-10, and R2 stands for a Cl- C35 alkyl or alkenyl chain which may be branched or un- branched.

2. Use according to claim 1, wherein the antibacterial compound is a lactylate of formula 1 or a Na, K, Ca, Mg, Fe(Il), Zn, NH₄, or Cu(TT) salt thereof.

5 3. Use according to claim 1 or 2, wherein R2 is a C6-C18 alky] or alkenyl chain.

4, Use according to claim 1 or 2, wherein n is 1, 2, or 3.

'0 5, Antioαeteπal compound as defined :n any one of claims 1-4 for preventing or treating intestinal infections caused by gram-positive bacteria in animals.

6. Use of an antimicrobial compound as defined in any one 15 of claims 1-4 for the manufacture of a composition for pre^¬ venting or treating intestinal infections caused by oram- positive bacteria in animals,

7. Animal nutrition composition for preventing or treating 0 intestinal infections caused by gram-posifive bacteria in animals conprising an antimicrobial compound as defined m any one of claims 1-4.

8. Method for preventing or treating intestinal infections 5 caused by gram-positive bacteria in animals comprising feed^¬ ing the animal with an effective amount of an antimicrobial compound as defined in any one cf claims 1-4.

9. Use, compound, animal nutrition composition, or method 0 according to any one of the preceding claims, wherein the gram-positive bacterium is of zhe genus Clostridia .

10. Use, compound, animal nutrition composition, or method according to any one of the preceding claims wherein the ani^¬ mal is selected from cattle or poultry.