WO2022214741A1

WO2022214741A1 - Feed ingredient composition, use thereof and feed comprising the same

Info

Publication number: WO2022214741A1
Application number: PCT/FI2022/050223
Authority: WO
Inventors: Jyrki LEPPÄLÄ
Original assignee: Nordic Caraway Oy
Priority date: 2021-04-07
Filing date: 2022-04-06
Publication date: 2022-10-13
Also published as: FI20215415A1

Abstract

The present invention relates to a feed ingredient composition for animals, comprising Carvone and at least one surfactant. The present invention also relates to a use of said composition and feed comprising said feed ingredient composition for animals.

Description

FEED INGREDIENT OOMP09TION, USE THEREOF AND FEED OOMPRI9NGTHE SAME

FI ELD OF THE INVENTION

The present invention relates to an animal feed ingredient composi tion for improving the quality and balance of the microflora in the animal gastro intestinal tract.

The present invention further relates to a use of theanimal feed ingre dient composition as an intestinal microbiota modulator.

The present invention further also relates to an animal feed compris- ingtheanimal feed ingredient composition.

The present invention further also relates to adrinking water or pellet feed for animals, as well asamethod for making awheat-soy-based feed pellet.

BACKGROUND OF THE I NVENTI ON

Companies producing feeds for production animals are continuously seeking new feed ingredients that would improve the health and productivity of the animals. Antimicrobial growth promoters have been routinely used in the in dustry for decades the rationale being that the overall bacterial load and the pres ence of pathogens hamper the productivity of the animals. Snce the ban of the antibiotic growth promoters in EU, feed companies are even more desperately looking for alternative intestinal microbiota modulators.

BRI EF DESCRI PTI ON OF TH E I NVENTI ON

An object of the present invention is to provide an animal feed ingre dient composition so as to overcome the above problems. The objects of the in vention areachieved by an animal feed ingredient composition, use of said animal feed ingredient and an animal feed comprising said animal feed ingredient, drink ing water or pellet feed or method of making a wheat-soy-based feed pellet ac cording to what is stated in the independent claims. The preferred embodiments of theinvention aredisclosed in thedependent claims.

The feed ingredient composition for animals according to the inven tion comprises Carvone, and at least one surfactant.

In some embodiments of the invention, the term “Carvone” may be un derstood as comprising R-(-)-Carvoneor S(+) -Carvone, or a combi nation thereof. In some embodiments of the invention, Carvone is present in the ani mal feed ingredient composition as a mixture thereof with at least one lipophilic ingredient such as e.g. limonene and/or essential oils. For example, Carvone may be present in the animal feed ingredient composition in the form of an essential oil having a Carvone content or component.

According to a preferred embodiment of the animal feed ingredient composition the at least one surfactant is selected from at least one of a Cl 2-C15 alcohol ethoxylate; lecithins, such as hydrolysed lecithin (lysol ecithi n ; lysophos- phatidyl chol i ne) and rapeseed lecithin; PEG glyceryl ricinoleate; ethoxylated cas tor oil ; glycerol monooleate; and sorbitan monooleate.

In a first preferred embodiment of the animal feed ingredient compo sition according to the invention Carvone is S-(+)-Garvone i.e. D-carvone. Prefera bly, the carvone is provided in combination with limonene.

In a certain embodiment the animal feed ingredient composition ac cording to the invention is a mixture of asurfactant and caraway oil.

In a further preferred embodiment of the feed ingredient composition according to the invention the R-(-)-Carvone or S-(+)-Carvone, or a combination thereof, is/ are included individually or in combination as such, i.e. each of them as essentially pure isolated compounds. R-(-)-Carvone or S-(+)-Garvone may be e.g. obtained by synthesis from suitable starting material. Preferably, D-carvone i.e. (S)-(+)-carvoneis used.

In astill further preferred embodiment the feed ingredient composi tion of the invention consists of R-(-) -Carvone or S(+)-Garvone, or a combination thereof, and at least one surfactant. In one of the possible different embodiments only one surfactant is used.

In another preferred embodiment of the animal feed ingredient com position the at least one surfactant has a hydrophilic-lipophilic balance (HLB) value of 3-25, preferably 7-20, such as 8-12, most preferably 12-20.

In yet another preferred embodiment of the animal feed ingredient composition the Carvone, such as the R-(-)-Carvone or S-(+)-Carvone, or a combi nation thereof, is included in the composition as such or in the form of an essen tial oil having a Carvone content, e.g. caraway oil, at a dose of up to 10 g/ L, prefer ably in the range of 0.07-5 g/ L, more preferably 1 .25-2.5 g/ L in the emulsion, and the surfactant is included at a dose of up to 10 g/ L, preferably in the range of 0.07-5 g/ L, more preferably 0.25-2.5 g/ L, most preferably 0.25-1.0 g/ L in the emulsion. The essential oil having a Carvone content may be used as such or fur ther purified or refined by rectifying (by distillation) it to desired content with respect of Carvone.

According to another aspect, the invention provides a use of the ani mal feed ingredient composition according to the invention as a microbial growth inhibitor in animal feed or animal drinking water.

In a preferred embodiment of said use, it is provided for microbial growth inhibition of at least one of Salmonella enterica, Escherida coli, Campylo bacter jejuni, Qostridi urn perfringens, and Enter ococcusfaedum.

According to afurther aspect, the invention provides a use of the feed ingredient composition according to the invention as an intestinal microbiota modulator in animal feed or animal drinking water.

In a preferred embodiment of said use, it is provided for microbial growth inhibition of at least one of Salmonella enterica, Escherida coli, Campylo bacter jejuni, Qostridi urn perfringens, and Enterococcus faed urn, without essential growth inhibition of intestinal commensal microorganisms such as Bifidobacte rium pseudolonaum.

According to afurther aspect, the invention also provides afeed com prising afeed ingredient composition according to the invention.

The feed according to the invention can be in the form of a liquid, paste, powder or pellets . In a particular preferred embodiment said animal feed is for animals, such as chickens and pigs, and domestic animals, such as cats and dogs.

The animal feed according to the invention in any one of its embodi ments may bewheat-soy-based.

According to ayet further aspect, the invention also provides drinking water or pellet feed for animals comprising afeed ingredient composition accord- ingto theinvention.

Yet further, the invention provides a method of making a wheat-soy- based feed pellet, comprising grinding wheat grains and mixing the resulting wheat particles with the animal feed ingredient composition according to the in vention and soybean oil, and pelleting the obtained mixture. In a particular em bodiment theanimal feed ingredient composition used in this method isan essen tial oil having a Carvone content, such ase.g. caraway oil. The invention is based on the realization of a positive interaction, a synergistic effect, between asurfactant and caraway oil which is both a surprising and significant finding.

It was found in teststhat an emulsion of Caraway oil and aC12-C15 al cohol ethoxy I ate surf act ant (acommercial product under the tradename “SUrfac® LM70/90” was used in the tests) emulsion was the most superior in inhibiting the growth of most tested pathogens, while it had no inhibition effect on commensal Lactobacillus strains.

Caraway oil (carvone content 60%) without added surfactant was sig nificantly less efficient growth inhibitor for most of the pathogenic strains. The basic theory behind the findings could be to increase surface area between hy drophilic (water= cells) and lipophilic components (carvone-based or carvone- containing products) Both of the tested antibiotics showed dose-dependent inhi bition of commensal bacteria, while many of the tested pathogens were not inhib ited.

Most of the tested bacteriawerenot inhibited by caraway powder (the exception was slight inhibition of Campylobacter jejuni at higher product doses).

An advantage of afeed ingredient, a use thereof and afeed comprising said feed ingredient of the invention is that no antibiotics are needed for inhibit ing the growth of pathogens.

As already indicated above, Cl 2-C15 alcohol ethoxylate has been test ed as surfactant with positive results in the form of the commercial product “Sur face LM70/ 90” (study 1 ). The combination of caraway oil and Rapeseed lecithin improved slightly the G jejuni inhibition as compared to caraway oil alone (study 4).

Further, also the following commercial surfactants were tested: “SABO-Nutreem R495” and “EL 33 Castor oil” (study 4).

A suitable surfactant has a hydrophilic-lipophilic balance (HLB) value selected depending on the HLB value required in the feed. Surfactants having HLB values of 3.5-6 are suitable for water-based emulsions (referred to as o/w- emulsions (oil-in-water)). Surfactants having HLB values in the range of 8-20, such as 8-12 are suitable for oil-based emulsions (referred to as w/ o-emulsions (water-in-oil)). SUrfac® LM70/90 has an HLB-valueof approximately 12. Throughout this application the terms “D-carvone”, “(+) -carvone” and “(SH+)- carvone” are used interchangeably. Likewise, throughout this application, “L- carvone”, “(-)-carvone” and “(R)-(-)-carvone” are used interchangeably. Throughout this application terms “hydrolysed lecithin”, “lysol ecithi n” and “lysophosphatidylcholine” are used interchangeably.

Throughout this application terms Έ 484”, “ethoxylated castor oil”, and “glyceryl polyethylene glycol ricinoleate” (castor oil 33 EO) are used as syno nyms.

In Study 6 was used a commercial surfactant called “Skbopal EL 200”, which may be described as Polyoxyl 200 castor oil or as Ethoxylated castor oil (200 ethoxy groups) Polyethylene glycol castor oil . It could also be described as a mixture of triricinoleate esters of ethoxylated glycerol with small amounts of pol yethyleneglycol (macrogol) ricinoleate and the corresponding free glycols.

BRI EF DESCRI PTI ON OF TH E DRAWI NGS

In the following the invention will be described in greater detail by means of preferred embodiments with reference to the attached drawings, in which

Figure 1 A shows the growth of Salmonella enterica at 4-hour time point;

Figure 1 B shows the growth of Escherichia col i 156/ 97 F4 at 4-hour time point;

Figure 1 C shows the growth of Escherichia col i KBAK1601/ 15 at 4- hour time point;

Figure 2 shows the growth of Qostridium perfringens at the 24-hour time point;

Figure 3 shows the growth of Campylobacter jejuni at the 24-hour time point;

Figure 4 shows the growth of Campylobacter jejuni at the 24-hour time point - caraway powder treatments;

Figure 5 shows the growth of Bifidobacterium pseudolongum at 7- hour time point;

Figure 6A shows the growth of Lactobacillus sal ivari us at 6-hour time point;

Figure 6B shows the growth of Lactobacillus reuteri at 6-hour time point;

Figure 7 shows the growth of Enterococcus faecium at 4-hour time point;

Figure 8 shows the growth of Escherichia col i KBAK1601/ 15 strain at 4-hour time point;

Figure 9 shows the growth of Bifidobacterium pseudolongum at 6- hour time point;

Figures 10A-10C show the effect of the test products on the body weight of broiler chickens at day 14, 21 and 35, respectively;

Figures 11 A shows the effect of test products on the body weight gain of broiler chickensduring daysO-14;

Figures 11 B shows the effect of test products on the body weight gain of broiler chickensduring days 14-21 ;

Figures 11 Cshows the effect of test products on the body weight gain of broiler chickensduring 21 -35;

Figures 11 D show the effect of test products on the body weight gain of broiler chickensduring days 0-35;

Figure 12A shows the effect of the test products on feed consumption of broiler chickensduring days0-14;

Figure 12B shows the effect of the test products on feed consumption of broiler chickensduring days 14-21 ;

Figure 12C shows the effect of the test products on feed consumption of broiler chickensduring days 21 -35;

Figure 12D shows the effect of the test products on feed consumption of broiler chickensduring days 0-35;

Figure 13 shows the effect of test products on mortality of broiler chickens;

Figure 14A shows the effect of test products on mortality-corrected feed conversion efficiency of broiler chickensfor days0-14;

Figure 14B shows the effect of test products on mortality-corrected feed conversion efficiency of broiler chickensfor days 14-21 ;

Figure 14C shows the effect of test products on mortality-corrected feed conversion efficiency of broiler chickensfor days21-35;

Figure 14D shows the effect of test products on mortality-corrected feed conversion efficiency of broiler chickensfor daysO-35;

Figure 15 shows the quantity of limonene remaining in feed at differ ent timepoints presented as a percent from timepoint 0 (before pelleting);

Figure 16 shows the quantity of carvone remaining in feed at different timepoints presented as a percent from timepoint 0 (before pelleting);

Figure 17 shows the quantity of limonene remaining in feed at differ - ent timepoints presented as a percent from timepoint 0 (before pelleting);

Figure 18 shows the quantity of carvone remaining in feed at different timepoints presented as a percent from timepoint 0 (before pelleting);

Figures 19A-19B shows the growth of Escherichia col i KBAK1601/ 15 at a4-hour timepoint;

Figures 20A-20B show the growth of Bifidobacterium pseudolongum at a6-hour timepoint;

Figures 21 A-21 B show the growth of Enterococcus faeci urn strain at 3- hour timepoint; Figures 22A-22B show the growth of Campylobacter jejuni strain at

24-hour timepoint;

Figure 23 shows the growth of Campylobacter jejuni strain at 24-hour time point with caraway oil when tested at different concentrations;

Figure 24 shows the growth of broiler chicken pathogenic E. col i KBAK1601/ 15 at a4-hour timepoint;

Figure 25 shows the growth of commencial and beneficial B. pseudo longum DSM 20099 at a6-hour timepoint;

Figure 26 shows the growth of Enterococcus faed urn at a 3-hour time point; and Figure 27 shows the growth of C&mpylobacter jejuni strain DSM 4688 at a24-hour timepoint.

DETAI LED DESCRI PTI ON OF TH E I N VENTI ON Sx studies are presented in the detailed description. The following commercial surfactants have been used in the studies:

I . SUrfac® LM70/ 90 from SUrfachem (studies 1 -5)

2. SABGNUTREEM R495 from S^BO (study 5)

3. EL 33 CASTOR GL from Mosselman (study 5) 4. RAPESEED LECITHIN from BICservices (study 5)

Study 1 : study of antimicrobial properties of caraway-based products

In this study the antimicrobial properties of caraway-based products from their production line and fractions thereof were studied. The test products and fractions were evaluated for their potential effect on the growth of relevant intestinal microbes in an in vitro bacterial culturing study. In addition to pathogenic microorganisms, selected commensal bacte ria were tested in this study. O e of the pivotal questions was whether the mini mum inhibitory concentration (MIC) of the products would be lower for the path ogens than for commensal intestinal bacteria. In general, the rationale of this piece of work was to shed light on the susceptibility of the common enteric path ogens as well as selected commensal intestinal bacteria to different caraway preparations and concentrations as well as to provide valuable information on the mode-of-action of the products tested.

Materialsand methods

Microbial cultures and treatments

The microbial growth inhibition was tested with pure microbial cul tures in synthetic liquid growth media at 37°G The microbial species, strains and the growth media as well as conditions utilised in the study are listed in Table 1. Anaerobic and microaerophilic bacteria; Lactobacillus, Campylobacter jejuni and Qostridium perfringens were cultured in anaerobic media and under anaerobic or microaerophilicconditions.

The first five strains in the Table 1 are obvious pathogens for the pro duction animals and/or the end-consumer. L. salivarius and L. reuteri are the most common commensal lactobaci 11 i in the chicken small-intestine and E. faeci- um (an opportunistic pathogen) and B. pseudolongum also belong to the normal intestinal microbiota of warm-blooded animals. Growth kinetics of each bacterial strain was initially analysed in a pre-test to find out the late exponential/ early stationary growth phase (data not shown), si nee this time point provides a widest dynamic range for thegrowth monitoring.

A total of four different caraway products/ fractions were included in the study, each of which was tested at eight doses (2 c dilution series). In addi tion, two commonly used antimicrobial growth promotors with different spec trum (bacitracin methylene di salicylate (BMD) and monensin) were included as positive controls. Moreover, SUrfac® LM70/90 surfactant was used as an emulsi fier in the Treatment 7 (combination with caraway oil). Moreover, SUrfac® LM70/90 alone was induded as an additional control to monitor its possible ef fect on bacterial growth.

Table 1 . Char act i sties of microbial strainsused in thisstudy.

List of test products/ treatments and the starting concentrations (strongest final doses) :

1 . Negative CTRL (no amendments) 2. Bacitracin (BMD) ( Positi ve CTRL), 40g/ L- strongest concentration (undiluted)

3. Monensin (Positive CTRL), 20mg/ L-strongest concentration (undiluted)

4. Caraway powder, 10g/ L-strongest concentration (undiluted)

5. Water extract of caraway powder, 10g/ L (assuming 100% solubility) - strong est concentration (undiluted) 6. Caraway oil, 10g/ L- strongest concentration (undiluted)

7. Caraway oil & SUrfac® LM70/ 90 emulsifier, 10g/ L- strongest concentration (undiluted)

8. SUrfac® LM70/ 90 emulsifier, 10g/ L — strongest concentration (undiluted) It should be noted that all OD measurements of two treatments- ‘Car away powder’ and ‘Water extract of caraway powder’ were significantly affected by the accumulation of the insoluble powder particles on the bottom of the cultur ing wells; therefore, optical density result interpretation was challenging. Bacterial growth inhibition test

Initially, different doses of test products were added in the bacterial growth medium containing 10% overnight cultured microbial inoculum. Moreo ver, for each microbial species tested, a culture without added test product was used as a negative control. All testswererun in three replicate cultures. The level of growth inhibition was generally assessed by measuring the optical density (OD) of the culture at wavelength of 600 nm, where the in crease in OD is an indicator of the proliferation of bacterial cells. For Campylobac ter jejuni, which is a very small-sized bacterium and does not elidt an increase in OD 600 nm, a method utilising DNA binding dye SYT3R G een was used to assess the changes in bacterial density during the incubation period. The microbial growth was monitored for 4 to 24 hours depending on the microbial strain (the optimal culturing times were defined in the pre-test).

Calculations The growth (in percentages to Control without any additions i.e. Neg.

CTRL) was calculated with thefollowingformula:

Growth (percentage, %) = 100 c - - -

VOD 600 nm negative control average/

Statistical analyses consisted of two-tailed Student’s t-tests where the inhibition percentage values from treatments were compared against those achieved from the negative controls.

Sgnificance according to Student’s t-test is shown in the figures as follows:

- p-value< 0.05 ^* - p-value< 0.01 ^**

- p-value< 0.001 ^*** Major findings

Sal monel I a enterica & Escherichiacoli (Figures 1A-1 C)

Both S enterica and E col i belong to Enterobacteriaceae, which is a large family consisting of only C am-negative bacteria. In addition to E col i and Salmonella, Enterobacteriaceae includes also many other well-known pathogenic genera such as Yersinia, Klebsiella and Shigella. In this study, we evaluated the inhibitory potential of test compounds against S enterica serovar Typhimurium aswell as two pathogenic E. col i strains.

- The growth of Salmonella or E. coli was not considerably inhibited by the tested antibiotics (BMD & monensin). - Pure caraway oil showed a moderate dose-dependent inhibition of both Salmo nella and E. coli. The maximum inhibition was obtained with the highest 10g/ L dose (Sal monel I a -30% from the CTRL; E. coli -37% or -24% from CTRL).

- Caraway oil/ SUrfac® LM70/90 emulsion inhibited significantly both Salmonella and E. coli growth dose-dependently. The maximum inhibition was obtained with the highest 10g/ L dose (Salmonella -64% from CTRL; E. coli -59% or -62% from CTRL).

- The results showed that SUrfac® LM70/90 emulsifier alone did not show con sider ableinhibition of Saimonellaor E. coli.

- Hence, it is evident that inclusion of SUrfac® LM70/90 facilitates the inhibitory potential of caraway oil most likely by improving itswater-solubility.

Figure 1 A shows the growth of Salmonella enterica, Figure 1 B Esche richiacoli 156/ 97 F4+ and Rgure CEscherichiacoli KBAK1601/ 15 at 4-hour time point. Error bars indicate the standard error of 3 replicate well s and asterisks the statistical significance of the difference to negative CTRL with no test product ac- cording to the Student’s t-test. Hundred % growth (dashed line) is the growth of negative CTRL i n the absence of any test products.

Clostridium perfringens f Figure 2)

Qostridium perfringens is a bacterium that is nearly always present in the intestine of chickens at low numbers. However, partly dueto unknown factors itsnumberscan occasionally increase several ordersof magnitude and it can start producing toxins that cause significant tissue damage. When the outbreak is seri- ousenough it leads to necrotic enteritis and increased mortality of animals.

- The growth of Qostridium perfringenswas not considerably inhibited by any treatment.

- However, a slight inhibition (maximum of -13% from CTRL) was observed with BMD- and SUrfac-containing treatments.

Figure 2 shows the growth of Qostridium perfringens at the 24-hour time point. Error bars indicate the standard error of 3 replicate wells and aster isks the statistical significance of the difference to negative CTRL with no test product according to the Student’s t-test. Hundred % growth (dashed line) is the growth of negative CTRL in the absence of any test products.

Campylobacter jejuni (Figures 3 and 4)

Campylobacter jejuni generally colonise poultry or swine as commen sal organisms. Qi the contrary, in humans the infection is assodated with acute enteritis. It is generally assumed that G jejuni contaminate poultry or pig meat during processing from individual animals carrying the pathogen. Hence, it can survivethroughout thefood chain supply to constitute a risk to human health.

Due to its small size, a different method utilising DNA binding dye SYBR G"een was used to assess the densities of Campylobacter jejuni cultures. The method is based on epifluorescence rather than absorbance and therefore, measurements were less affected by ‘powder particle sedimentation’ effect as compared to OD measurements of other target microbes.

- Neither of the antibiotics tested showed growth inhibition of Campylobacter jejuni.

- Qi the other hand, caraway oil alone showed a dose-dependent inhibition with the three strongest inclusion levels (-20% to -64% from neg CTRL).

- Caraway oil/ SUrfac® LM70/90 emulsion was the most efficient treatment to inhibit G jejuni growth, as inhibition was observed already with the most diluted concentration. With the strongest concentration, the magnitude of inhibition was nearly -90% of the CTRL treatment.

- In addition, SUrfac® LM70/90 alone inhibited to some extent the G jejuni growth; however, the difference to caraway oil/ SUrfac® LM70/90 emulsion was considerable. Therefore, it is evident that most of the observed inhibitory effect in emulsion was in fact derived from caraway oil, not Surfac.

- The results also showed that caraway powder treatment inhibited G jejuni growth by 35% with the strongest inclusion level (Figure 4). However, we still cannot completely exclude the possible ‘powder particle’ effect which could ham per the inhibition result.

- Interestingly, the caraway powder water extract did not have a considerable inhibitory effect on G jejuni growth. Hence, it is likely that the effective compo nents of the powder are not water soluble, possibly oil.

Figure 3 shows the growth of Campylobacter jejuni at the 24-hour time point. Error bars indicate the standard error of 3 replicate wells and aster isks the statistical significance of the difference to negative CTRL with no test product according to the Student’s t-test. Hundred % growth (dashed line) is the growth of negative CTRL in the absence of any test products.

Figure 4 shows the growth of Campylobacter jejuni at the 24-hour time point - caraway powder treatments. Error bars indicate the standard error of 3 replicate wells and asterisks the statistical significance of the difference to negative CTRL with no test product according to the Student’s t-test. Hundred % growth

(dashed line) isthe growth of negative CTRL in the absence of any test products.

Bifidobacterium pseudolongum (Rgure5)

Bifidobacteriaarethought to exert a protective role against pathogenic microorganisms such as Campylobacter and salmonella via production of antimi crobial agents as well as preventing pathogen colonisation and enhancing the host immune response in warm-blooded animals.

- The growth of Bifidobacterium pseudolongum was inhibited by both tested an- tibiotics already at the lower doses tested. The maximum inhibition was approx imately -50% from CTRL.

- Qi the other hand, caraway oil alone did not have an inhibitory effect at the doses tested.

- However, when caraway oil was combined with SUrfac® LM70/90 inhibition was observed and its magnitude was rather similar than with SUrfac® LM70/90 alone.

- Therefore, it is evident that the B. pseudolongum inhibition of the caraway oil/ SUrfac® LM70/90 emulsion was derived completely from SUrfac.

Figure 5 shows the growth of Bifidobacterium pseudolongum at 7- hour time point. Error bars indicate the standard error of 3 replicate wells and asterisks the statistical significance of the difference to negative CTRL with no test product according to the Student’s t-test. Hundred % growth (dashed line) isthe growth of negative CTRL in the absence of any test products.

Lactobacillus reuteri & Lactobacillussalivarius (Figures6Aand 6B)

Lactobacillus is the dominating bacterial genus in the small-intestine of warm-blooded animals, showing either homo- or heterofermentative lactic acid metabolism. In general, high lactobaci 11 i levels in small-intestine may increase physical barrier effect against pathogenic bacteria. In addition, lacticadd secreted by lacticadd bacteria tends to lower the pH of the intestinal tract.

Low pH in the small-intestine is known to be beneficial for the animal performance and health in several ways.

- In general, both Lactobacillus strains showed rather similar inhibition patterns with the tested products

- The lactobaci IN growth was inhibited by both antibiotics with all dosestested (maximum inhibition of -65 to -80% from the CTRL).

- Interestingly, no inhibition was observed with caraway oil alone, caraway oil/ SUrfac® LM70/ 90 emulsion or SUrfac® LM70/ 90 at any of the doses tested.

- This lack of inhibition with the caraway products is indeed promising, since Lactobaci 11 us is an important commensal bacterial genus in thesmall-intestineof warm-blooded animals.

Figure 6A shows the growth of Lactobacillus salivarius and Figure 6B the growth of Lactobacillus reuteri at 6-hour time point. Error bars indicate the standard error of 3 replicate wells and asterisks the statistical significance of the difference to negative CTRL with no test product according to the Student’s t-test. Hundred % growth (dashed line) is the growth of negative CTRL in the absence of any test products.

Enterococcus faed urn (Figure 7)

The metabolism of enterococci is like that of the homofermentative lactobacilli. A characteristic feature of enterococci isthat they tend to possess and transfer antibiotic resistance genes and are thus considered potentially risky group of bacteriaif therapeutic or prophylactic antibiotics are being or have been used.

Enterococcus faecium is a G'am-positive bacterium in the genus En terococcus. It can be commensal in the gastrointestinal tract of humans and ani- mals, but it may also be an opportunistic pathogen. Therefore, enter ococcosis of ten occurs secondary to another disease.

- Thegrowth of E. faecium wasdose-dependently inhibited by both tested antibi otics (maximum inhibition of -48% to -66% from the CTRL)

- Also, caraway oil/ SUrfac® LM70/ 90 emulsion showed a dose-dependent inhibi tion of E. faecium (maximum inhibition of -70% from the CTRL)

- Qi the contrary, caraway oil or SUrfac® LM70/90 alone had no considerable effect on thegrowth of Enterococcus faecium.

- Therefore, it isevident that indusion of SUrfac® LM70/90 increases significant ly the inhibitory potential of caraway oil most likely by improving its water- solubility.

Figure 7 shows the growth of Enterococcus faecium at 4-hour time point. Error bars indicate the standard error of 3 replicate well s and asterisks the statistical significance of the difference to negative CTRL with no test product ac cording to the Student’s t-test. Hundred % growth (dashed line) is the growth of negative CTRL i n the absence of any test products.

Study 2: Determination of optimal dose of Surfac® LM70/90 emulsifier in caraway oil/ Surfac® LM70/90 emulsion and evaluation of inhibitory effect of specific caraway oil components car vone and limonene

Major findings:

• The caraway oil with Surfac® LM70/90 increased the inhibition of pathogenic E. col i compared to caraway oil alone.

• The highest rate of E. col i inhibition was obtained with the SUrfac® LM70/90 doses of 0.13 to 0.5 g/ L.

• Both isomers of car vone inhibited significantly the growth of pathogenic E. col i, while limonene had no effect on E. col i proliferation.

• Inhibition of B. pseudolongum with thecaraway oil/ Surfac® LM70/ 90 emulsion was derived completely from Surfac.

• Surfac® LM70/90 doses ranging from 0.03 to 1 .0 g/ L SUrfac® LM70/90 inhib ited B. pseudolongum growth 50 to 70% from the Neg CTRL, while the lowest Surfac® LM70/ 90 dose (0.01 g/ L) did not haveaconsiderableinhibitory effect.

• Isomers of carvone and limonene had no effect of B. pseudolongum growth when tested alone.

• In conclusion, it is challenging to determine the Surfac® LM70/90 dose with caraway oil that would be optimal for both bacterial species tested. However, B. pseudolongum is a bacterium residing in the lower intestinal tract and it is possi ble that caraway oil/ SUrfac® LM70/ 90 product would be absorbed from the gas trointestinal tract before entering the lower intestine. Background and rationale of the study

In study 1 , we showed that the caraway oil/ SUrfac® LM70/90 emul sion was superior in inhibiting the growth of most tested pathogens, while it had a smaller effect on commensal bacteria. Hence, it was concluded that the addition of a surfactant was essential for boosting the inhibitory potential of caraway oil most likely by increasing the solubility of the effective inhibitory components present in theoil.

The rationale of study 2 was to find out the SUrfac® LM70/90 emulsi fier concentration in the caraway oil/ SUrfac® LM70/90 emulsion, which will be optimal for inhibiting of the pathogenic bacterial growth, while not affecting con- siderably the growth of commensal intestinal bacteria. An additional target of this work was to find out how the addition of two main components of caraway oil - isomers of carvone and I imonene - affect the growth of selected microbes in the presence and absence of SUrfac® LM70/ 90.

Materialsand methods

Microbial cultures and treatments

The microbial growth inhibition was tested with pure microbial cul tures in synthetic growth liquid media at 37°G Two microbial species, pathogenic chicken isolate Escherichia col i KBAK 1601/ 15 and commensal Bifidobacterium pseudolongum DSM 20099, were cultured in suitable media under appropriate conditions. Growth kinetics of each bacterial strain was analysed during the pre vious study 1 .

List of treatments and starting concentrations (strongest final doses):

1 . Negative CTRL (no amendments)

2. Caraway oil (2.5 g/ L)

3. Caraway oi I ( 1 .25 g/ L)

4. Caraway oil (2.5 g/ L) & SUrfac® LM70/90 at 8 doses (from 1 .0 g/ L)

5. Caraway oil (1 .25 g/ L) & SUrfac® LM70/ 90 at 8 doses (from 1 .0 g/ L)

6. SUrfac® LM70/ 90 at 8 doses (from 1 .0 g/ L)

7. R-(-)-Garvone (1 25g/ L)

8. R-(-)-Carvone (1 25g/ L) & SUrfac® LM70/ 90 (1 .0 g/ L)

9. R-(-)-Garvone (1 25g/ L) & SUrfac® LM70/ 90 (0.25 g/ L)

10. S-(+)-Carvone (1 25g/ L)

11. S-(+)-Carvone (1.25g/ L) & SUrfac® LM70/90 (1.0 g/ L)

12. S-(+)-Garvone (1.25g/ L) & SUrfac® LM70/90 (0.25 g/ L)

13. (R)-(+)-Limonene (1.25g/ L)

14. (R)-(+)-Limonene (1.25g/ L) & SUrfac® LM70/ 90 (1 .0 g/ L)

15. (R)-(+)-Limonene (1.25g/ L) & SUrfac® LM70/ 90 (0.25 g/ L)

16. (S)-(-)-Limonene (1.25g/ L)

17. (S) -(-)-□ monene (1.25g/ L) & SUrfac® LM70/ 90 (1.0 g/ L)

18. (S)-(-)-Limonene (1.25g/ L) & SUrfac® LM70/ 90 (0.25 g/ L)

Bacterial growth inhibition test

Microbial culture media were supplemented with caraway oil alone or with an emulsion of two doses of the caraway oil with final concentrations of 2.5 g/ L and 1 .25 g/ L and SUrfac® LM70/90 at eight doses (strongest dose 1 .0 g/ L) and inoculated with 10% (v/v) of actively growing microbial cultures. For each microbial species, a culture without added test product was used as a negative control. Also, the same eight doses of the SUrfac® LM70/90 emulsifier were test ed alone as a control in order to reveal the inhibitory effect of the pure SUrfac. In ‘Carvoneand Limonene’ test, both (+)- and (-)-isomersof each com pound were tested at a concentration of 1 .25 g/ Lwith and without theaddition of SUrfac® LM70/ 90, which was applied at concentrations of 1 .0 and 0.25 g/ L.

All tests were run in at least with three replicates in 96-wells plates. The level of growth inhibition was generally assessed by measuring the optical density (OD) of the culture at a wavelength of 600 nm, where the increase in OD is an indicator of the proliferation of bacterial cells.

Calculations

The growth (in percentages to control with no product amendments i.e. Neg. CTRL) was calculated with the foil owing formula: f . __{/ L L} ( OD 600 nm treatment X \

Growth (percentage, %) = 100 c -

\OD 600 nm negative control average /

Statistical analyses consisted of two-tailed Student’s t-tests, where the inhibition percentage values from treatments were compared against those achieved from negative controls.

Sgnificance according to Student’s t-test is shown in the figures as follows:

- p-value< 0.05 ^*

- p-value< 0.01 ^**

- p-value< 0.001 ^***

Major findings

Escherichiacoli (Figure8)

The results of E. col i KBAK1601/ 15 strain at 4-hour time point is shown in Figure8.

- Pure caraway oil showed a moderate dose-dependent inhibition of E. coli. The higher 2.5 g/ L dose inhibited growth 34% and smaller 1 .25 g/ L dose only 11 % from the CTRL.

- SUrfac® LM70/90 alone showed no inhibition of E. col i growth. In fact, the in crease of SUrfac® LM70/ 90 concentration appeared stimulate dose-dependently the E. col i proliferation. - Emulsion of the lower caraway oil concentration (1 .25 g/ L) with different SUr- fac® LM70/90 concentrations showed generally smaller rate of E. col i inhibition than the higher dose. The highest rate of inhibition (-45% from Neg CTRL) was obtained with the SUrfac® LM70/90 doses of 0.13 to 0.5 g/ L, while the smaller SUrfac® LM70/ 90 doses showed only moderate rate of inhibition.

- Emulsion of the higher caraway oil dose (2.5 g/ L) with different SUrfac® LM70/90 concentrations inhibited the growth of E. col i dose-dependent I y. Again, the highest inhibition (-63% from Neg CTRL) was obtained with the SUrfac® LM70/90 doses of 0.13 to 0.5 g/ L, while the lowest SUrfac® LM70/90 dose (0.01 g/ L) showed theinhibition rateof 32%.

- Neither of the limonene isomers showed considerable inhibition of E. col i either in a presence or absence of SUrfac.

- Qi the other hand, both (+) and (-) isomers of carvone showed dose-dependent and rather comparable inhibition pattern with around 40% without SUrfac® LM70/90 and 52% (+)/45% (-) with the higher (1.0 g/ L) dose of SUrfac® LM70/90 from Neg CTRL. The highest rate of inhibition (60% from Neg CTRL) was observed when (+)-Carvone was combined with lower (0.25 g/ L) dose of SUrfac.

Figure 8 shows the growth of Escherichia col i KBAK1601/ 15 strain at 4-hour time point. Error bars indicate the standard error of 3 replicate wells and asterisks the statistical significance of the difference to Negative CTRL with no test product according to the Student’s t-test. Hundred % growth (dashed line) is the growth of Negative CTRL in the absence of any test products.

Bifidobacterium pseudolongum (Rgure9)

The growth of B. pseudolongum DSM 20099 strain at 6-hour time point is shown in Figure 9.

- Results of this study confirmed the previous test results i.e. the inhibition of B. pseudolongum with the caraway oil/ Surfac® LM70/90 emulsion was derived completely from Surfac.

- As expected, the growth of B. pseudolongum was not inhibited by thetwo doses of caraway oil alone. - The rate of inhibition was nearly identical with caraway oil/ SUrfac® LM70/90 emulsion and SUrfac® LM70/ 90 alone: 50 to 70% from the Neg CTRLwith 0.03 to 1 .0 g/ L SUrfac® LM70/90 concentration. Qi the other hand, the lowest SUrfac® LM70/90 dose (0.01 g/ L) did not have a considerable effect on the B. pseudo- longum growth.

- The results showed that the isomers of carvoneand limonene had no effect of B. pseudolongum growth when tested alone.

- As expected, the rate of B. pseudolongum inhibition was very significant when the isomers of carvone and limonene were included in conjunction with SUrfac. Interestingly, the lower SUrfac® LM70/90 dose showed slightly higher rate of inhibition when combined with carvone isomers, while both SUrfac® LM70/90 doses showed equal inhibition when combined with limonene isomers.

Figure 9 shows the growth of Bifidobacterium pseudolongum at 6- hour time point. Error bars indicate the standard error of 3 replicate wells and asterisks the statistical significance of the difference to Negative CTRL with no test product according to the Student’s t-test. Hundred % growth (dashed line) is the growth of Negative CTRL in the absence of any test products.

Concluding remarks

The emulsion of caraway oil and SUrfac® LM70/90 was confirmed to increase the growth inhibition of pathogenic E. col i , while the inhibition of com mensal B. pseudolongum was same for corresponding doses of SUrfac® LM70/90 alone and caraway oi I/ SUrfac® LM70/90 emulsion. This supports the ear Her find ings that inhibitory effect of B. pseudolongum was derived completely from SUr fac® LM70/90 emulsifier.

The highest rate of E. coli inhibition was observed at the dose range of 0.13 to 0.50 g/ L of the SUrfac® LM70/ 90 in the caraway oi I/ SUrfac® LM70/90 emulsion. However, these SUrfac® LM70/90 concentrations appeared to inhibit considerably the growth of commensal B. pseudolongum. Therefore, it is challeng ing to find and determine the SUrfac® LM70/90 dose with caraway oil that would be optimal for both bacterial species tested. It is noteworthy that B. pseudo longum is a bacterium residing in the lower intestinal tract, while E. coli is found in high quantitiesin small -intestine of warm-blooded animals. It is possible that caraway oil/ SUrfac® LM70/ 90 product exerts its inhibitory effect in small-intestine and would be absorbed from the gastrointesti nal tract before entering the lower intestine. In this case, the beneficial bifidobac- teriawould not be negatively affected by thefeed additive, and SUrfac® LM70/90 concentration showing a significant boosting effect on E col i inhibition (e.g. 0.1 g/ L) could be considered to be applied in combination with caraway oil in the broiler chicken feeding trial.

The results showed that both isomers of carvone inhibited significant ly the growth of pathogenic E col i, while limonene had no effect on E col i prolif eration. This clearly indicates that carvone is the active inhibitory substance against gram-negativeenterobacteriaof thetwo dominant components present in caraway oil.

Study 3: Broiler chicken feeding trial with caraway oil preparations

Major conclusions:

- Both caraway oil and D-carvone improved body weight gain when added to gether with an emulsifier.

- Duringthe first threeweekstheemulsifier used appeared to increase feed consumption.

- Feed conversion efficiency tended to be improved by caraway oil whether or not added together with the emulsifier; D-carvone tended to improve feed conversion only when added together with the emulsifier.

- In the future studies the role of an emulsifier should be investigated in more detail and thefeed grade emulsifiersin the marketplace should be surveyed.

Background

The target of this study was to see whether caraway oil and its main component D-carvone have an effect on broiler performance. Furthermore, the importance of an emulsifier on the efficiency of the oily products was studied.

Trial outline

Dietary treatments The pelleted diet was wheat-soy based feed for broiler chickens. The starter formulation was used for the first 2 weeks and the grower diet for the fol lowing 3 weeks of the trial. The feeds were formulated and manufactured by Ali- metrics Ltd. The trial was conducted with 11 dietary treatments and 8 replicate pens (Table 2). The test diets were sampled (pooling of 3 x 300g /diet) for the proximate analysis and for the own pur poses of the client.

Proximate analyses were performed. Nutrient composition and ana lysed values of diets areshown in Table3.

Table2.Treatmentsand sampling control

Figures 10A-1 OQ 11 A-11 D, 12A-12D, 13, 14A-14D.

Table 3. Calculated and analysed values feeds

Starter % Grower %

Wheat 59.52 65.98

Soybean meal (high pro) 32.00 26.00

Soya oil 4.40 4.40

Monocalciumphosphate 1.70 1.50

Limestone 1.05 0.92 Nag 0.38 0.39

Mineral premix^* 0.20 0.20

Vitamin premix^** 0.23 0.20

DL-Methionine 0.27 0.21

L-Lysine HQ 0.18 0.20

Threonine_ 0.07 0.00

Total_ 100 100

Metabolisable energy (MJ kg) 12.43 12.69

Crude protein 226.73 204.53

Lysine 12.40 11.08

Methionine 5.69 4.86

Threonine 8.77 7.16

Methionine + cysteine 9.31 8.25

Calcium 9.50 8.39

Non-phytate phosphorus 4.68 4.22

Analysed values (g/ kg) Starters (0-14 days)

Treatment Tr Tr Tr Tr Tr Tr Tr 7 Tr 8 Tr Tr Tr

1 2 3 4 5 6 9 10 11

87.8 87.5 87.3 87.2 87.2 87.4 87.8 87.7 87.5 87.5 88.1

Crude pro 223 235 237 229 233 233 234 236 229 224 233 tein

Crude fat 64 66 65 58 59 59 60 61 60 60 61

Crude 30 32 37 40 43 41 32 39 31 34 36 fiber

Ash 55 56 54 54 55 57 53 54 57 59 59 Nitrogen 507 487 481 490 482 485 500 486 498 499 492 free extrac tives

Growers ( 14-35days)

Treatment Tr Tr Tr Tr Tr Tr Tr 7 Tr 8 Tr Tr Tr 1 2 3 4 5 6 9 10 11

Crude fat 65 64 66 68 65 65 66 68 66 68 68

Crude 37 38 37 36 33 46 47 42 36 36 37 fiber

Ash 56 54 56 59 53 54 55 55 57 57 54

Nitrogen 533 521 516 523 536 512 526 524 515 517 521 free extrac tives

Table3 continued from previous page.

^*) Contents of the mineral premix: calcium 296.9 g/ kg, zinc 32.5 g/ kg, manganese 25.0 g/ kg, iron 12.5 g/ kg, copper 4.0 g/ kg, iodine 225 mg/ kg, selenium 100 mg/ kg. *^*) Contents of the vitamin premix: calcium 331 .3 g/ kg, all-rac-a-tocopheryl ace tate 30.0 g/ kg, niacin 20.1 g/ kg, panthotenic acid 7.51 g/ kg, riboflavin 3.0 g/ kg, pyridoxine 2.01 g/ kg, retinol 1.8 g/ kg, menadione 1505 mg/ kg, thiamine 1257 mg/ kg, folic acid 504 mg/ kg, biotin 75.0 mg/ kg, cholecalciferol, 56.3 mg/ kg, co- balamin 12.5 mg/ kg.

Animals and housing

The 35-day trial was conducted in the broiler house of Alimetrics in Southern Finland, on August -Sept ember 2019, in accordance with EU Directive 2010/ 63/ EU and Alimetrics standard operating procedures for the running of animal studies. The temperature of the hall was raised to 32 °Ctwo days before the chicks arrived. Luminosity was adjusted to 20 lux and air humidity was fixed to 60 %. Brooder lamps were adjusted to provide extra heating to the chicks dur ing the first week. The temperature was gradually decreased to 22 “C over the rearing period. Temperature, ventilation and humidity were monitored and rec orded throughout thetrial on adaily basis. The dark hours were increased by one hour daily, until light-dark cyde was 18 hours light and 6 hoursdark.

Newly hatched, male Ross 308 broiler chicks were randomly allocated into feeding treatments. Birds were housed in 88 pens (1.125 m2 each) with wood shavings litter. In the beginning there were 15 birds in each pen and the total number of birds was 1320. The maximum allowed stocking density (33 kg/ m2) was not exceeded. The chicks had free access to feed and drinking water throughout the trial. A veterinarian checked the health of the chicks in the begin ning of the trial. The birds were observed twice a day throughout the trial and birdswith compromised health were euthanized.

Performance parameters

The birds and feeds were weighed on days 0, 14, 21 and 35. Feed in- takeper pen and thefeed conversion ratio (FCR) were measured for thefollowing periods:

- Days 0-14, starter diet period

- Days 14-21 early grower diet period

- Days 21 -35 late grower diet period

FCR was calculated both corrected and uncorrected for mortality.

Sampling

Qi days 14 and 35 two randomly selected birds from all pens were euthanised by cervical dislocation, abdominal cavity opened and digesta samples were collected from ileum and caecum. All samples were stored and preserved in accordancewith the requirements for possible later analysis. Altogether 352 digesta samples were taken. The samples will be stored for 3 months after completion of the trial and then discarded unless no analyses have been agreed on.

Statistical analysis

All data were analysed by Tukey’s test using SPSS statist! cal software package (IBM version 22). Obtained p-values smaller than 0.1 are shown as sym bols in the graphs.

Main findings on performance

Effect of test products on mean body weight

- Qi day 14, none of the tested products gave statistically significant benefit on mean body weight. Caraway oil alone tended to impair body weight dose de pendency. This was also the case with D-carvone whether or not the emulsifier was used. When combined with SUrfac® LM70/ 90 caraway oil had a positive nu merical effect (~ 14 grams) at low and high doses. (Figure 10A)

- Again, on day 21 , no statistically significant effects were seen between the treatments on body weight. However, at this time point, Caraway oil at high dose with emulsifier improved body weight by 46 grams and the effect was statistically significant when analysed by t-test (p = 0.022). In this present trial we have no corresponding dose of D-carvone, but D-carvone at low dose with SUrfac® LM70/90 improved body weight by 33 grams (p = 0.092 by t-test). (Figure 10B)

- The same trend continued also on day 35. With emulsifier, caraway oil at high dose improved body weigh by 128 grams (p = 0.008 by t-test) and D-carvone at low dose by 100 grams (p = 0.056 by t-test). (Figure 10C)

Figure 10 shows the effect of the test products on the body weight of broiler chickens. Panels A, B and Cshow the body weight on days 14, 21 and 35, respectively. Error bars show SEM.

Effect of test products on mean body weight gain

- There were not significant differences in mean body weight gain during the days 0-14 between the tested products. Caraway oil if used with emulsifier improved body weight gain numerically at low and high doses compared to control. Cara way oil used alone, tended to have dose dependent negative effect on growth of broiler chickens. This was the case also with D-carvone whether or not SUrfac® LM70/ 90 was used with the product. (Figure 11 A) - During the early grower diet period (days 14-21), caraway oil with emulsifier differed statistically significantly at medium dose from high dose. The magnitude of improvement at high dosewas35 gramswhen compared to control. The dose dependent negative effect of D-carvoneno longer existed. (Figure 11 B)

- Compared to control treatment, all test products improved numerically mean body weight gain during the final two weeks of the trial (days 21 -35) by 35 to 80 grams. The most positive effect was seen when products were combined with SUrfac. The effects were not statistically significant by Tukey’s test but the treat ments improving body weights in section 3.1 improved also body weight gain when analysed by thet-test. (Figure 11 C)

- Thenumerical improvement was seen also during thewhole35-day trial with all products, especially when emulsifier was present; 127 grams at high dose of car away oil and 99 grams with low dose of D-carvone. These effects were statistically significant when analysed by t-test (p-values of 0.008 and 0.058, respectively). (Figure 11 D)

Figure 11 shows the effect of test products on the body weight gain of broiler chickens. Panels A, B, Cand D show the body weight gain during the days 0-14, 14-21 , 21 -35 and 0-35, respectively. Error bars show SEM and letters above columns the difference between the treatments by Tukey HSDtest.

Effect of test products on feed consumption

Test products did not differ from each other during any feeding period statistically significantly when analysed by Tukey FISD test. Flowever, some nu meric effect s w er e seen .

- Feed consumption during the starter diet period (0-14 days) increased parallel to body weight gain, but thereafter the phenomenon gradually subsided. /erall, during the whole trial, D-Carvone alone increased feed consumption per pen by about 1 .4 kg compared to pens of the control treatment. (Figures 12A-12D)

Figure 12 shows the effect of the test products on feed consumption of broiler chickens. Panels A, B, Cand D show the feed consumption during the peri ods 0-14, 14-21 , 21-35 and 0-35 days, respectively. Error bars show SEM.

Effect of test products on mortality

- There were no significant differences between the test products on mortality, during any growth period. (Figure 13) Figure 13 shows the effect of test products on mortality of broiler chickens. Figure shows the total mortality during the whole 35-day trial. Error bars show SEM.

Effect of test products on mortality-corrected feed conversion efficiency

Oily the results on mortality-corrected feed conversion ratio are shown due to almost identical results without mortality correction.

- During the starter diet period (0-14 days), none of the treatments differed sig nificantly from the control. The numeric improvement with caraway oil without emulsifier was4 points. (Figure 14A)

- Again, during the early grower diet period (days 14-21), caraway oil alone at medium dose improved statistically significantly mortality corrected FCR by 9 points compared to use at low dose. The positive effect can be explained by in creased body weight gain. Almost equal improvement was seen also with caraway oil + SUrfac® LM70/90 at high dose and D-carvone + surface at low dose. With all these the effect surpassed control diet by ~4 points. (Figure 14B)

- The effects and the magnitude of improvement of the test products remained similar during the days 21 -35 as seen during the previous period, though without statistical significance dueto high variation within the treatments. (Figure 14C)

- As seen earlier, during the whole 35-day trial several test products improved mortality-corrected FCR numerically, by 6 points. The positive effect was seen with caraway oil regardless of whether emulsifier was used or not. With D-carvonethe use of surfac® LM70/90 is more justified to improve FCR. (Figure 14D)

- Overall, it seemed that SUrfac® LM70/90 was efficient in increasing feed intake, and, consequently body weight gain. The benefit of Surfac® LM70/ 90 for FCRwas not as obvious.

Figures 14A-14D show the effect of test products on mortality- corrected feed conversion efficiency of broiler chickens. Figures 14A-14D show the feed conversion efficiency with mortality correction during the growth peri ods 0-14, 14-21 , 21-35 and 0-35 days, respectively. Error bars show SEM and let ters above columns the difference between the treatments by Tukey FISDtest. Study 4: Monitoring of possible caraway oil evaporation in pelleted feed during processing and storage

Major findings:

• Results showed that carvone- the effective component of the caraway oil in mi crobial inhibition - remained stable and did not evaporate from feed during pel leting and storage.

• Qi the other hand, approximately 70% of I imonene was evaporated from pellet ed feed during 4-week storage. It is noteworthy, however, that limonene was shown to have no inhibitory effect on the growth of pathogenic bacteria in the previous study 2.

• Therefore, the fact that carvone was not evaporated from feed during pro cessing and storage is promising and critical for proceeding with the planned an imal feeding trials.

• There were no considerable differences between the rate of evaporation in feed with caraway oil only or feed with caraway oil/ SUrfac® LM70/90 combination.

• As part of this study, an accurate and sensitive method for quantification of in dividual caraway oil components in feed matrix was developed.

• The analysis of caraway oil in feed showed that the carvone/ 1 imonene ratio in the product was 65:35 at Timepoint 0 (before pelleting).

Rationale of the study

The aim of this work was to find out whether caraway oil is evapo rated from pelleted feeds during processing and storage when applied alone or in combination with SUrfac® LM70/90 emulsifier. This information is essential in order to deter mi net he most suitable procedure to use caraway oil as an additive.

Materialsand methods

Caraway oil inclusion and feed manufacturing

Two 200 kg batches (with caraway oil only or caraway oil/ SUrfac® LM70/90 combination) of pelleted wheat-based broiler diet were manufactured for this study. Initially, wheat grains were ground to fine particles using a ham- mermill and transferred to a batch mixer. Subsequently, the rest of the feed in gredients induding caraway oil (concentration of 2 kg/ton) and SUrfac® LM70/90 (concentration of 0.2 kg/ ton) pre-mixed with soybean oil was intro duced gradually and mixed thoroughly with mashed feed. Both mixed batches of wheat-based feed were pelleted using a pelleting machine with a 4 mm matrix and the temperature was monitored throughout the process. Pelleted feeds were rapidly cooled and placed into three 25 kg paper bags per batch. The bags were stored at room temperature prior to thesampling.

Feed sampling and monitoring of caraway oil evaporation

A total of 4 replicate representative feed subsamples were collected from the bags at each timepoint for the analysis of both caraway oil compounds (I i monene & carvone) . The ti me poi nts of sampl i ng were as fol I ows:

1 . Timepoint 0 = Mash feed sampling prior to the pelleting process (feed with car away oil & caraway oil/ SUrfac® LM70/ 90 combination)

2. Timepoint 1 = Pelleted feed sampling immediately after processing and before packing (feed with caraway oil & caraway oil/ SUrfac® LM70/ 90 combination)

3. Timepoint 2 = Pelleted feed sampling after one week of storage in 25 kg paper bags (feed with caraway oil only)

4. Timepoint 3 = Pelleted feed sampling after two weeks of storage in 25 kg paper bags (feed with caraway oil only)

5. Timepoint 4 = Pelleted feed sampling after four weeks of storage in 25 kg paper bags (feed with caraway oil & caraway oil/ SUrfac® LM70/ 90 combination)

Method development for caraway oil component analyslsin feed

In order to accurately measure the concentration of caraway oil in feed and monitor its possible evaporation, an analytical method for caraway oil com ponents (I i monene & carvone) was developed and validated for Agilent gas chro matography (7890A) mass spectrometer (5975D). During the developmental work, caraway oil composition was characterised and possible analyte losses dur ing sample pre-treatment were determined. The relevant characteristics of the method were validated according to the general guidelines for animal feed analy sis laboratories (Quality assurance for animal feed analysis laboratories. FAO An imal Production and Health Manual No. 14. Rome).

In brief: a representative feed sample (5g) was mixed with 10 mL of water, 1 mL of methanol and 1 mL of heptane containing the internal standard (1- tert-Butyl-1 -cyclohexene, 2.5 mg/ mL). Resulting mixture was extracted by strong agitation with 24 mL of heptane for 40 min. After centrifugation (3 500 rpm for 5 min), supernatant was collected for QC- MS analysis. To match the condition with samples, procedural standards were used in calibration. The data (single ion monitoring) were collected for limonene and carvone, which are the most rele vant components in caraway oil. Calculations

The quantity of the remaining caraway oil (GO.) components (limo- nene & carvone) (in percentages to timepoint 0) was calculated with the follow- ingformula:

Remaining GO. component (percentage, %) =

100

Results

Limoneneand carvone stability in feed with a mixture of caraway oil and SUrfac® LM70/90

- Approximately 40% of I imonene evaporated from the feed with a mixture of caraway oil and SUrfac® LM70/90 during pelleting and before packing (Figure 15).

- At the end of storage time at 4-week timepoint, about 70% of the original limo- nene present in feed had evaporated (Figure 15).

Figure 15 shows the quantity of limonene remaining in feed at differ ent timepoints presented as a percent from timepoint 0 (before pelleting). Hun- dred % (orangeline) isthequantity of the limoneneat timepoint 0.

Error bars indicate standard error of the mean of four replicates.

- Qi the contrary, carvone showed only minimal evaporation from the mixture of caraway oil and SUrfac® LM70/90 during the feed pelleting and storage of four weeks (reduction of 5 % from the unpelleted control ; Timepoint 0) (Figure 16).

Figure 16 shows the quantity of carvone remaining in feed at different timepoints presented as a percent from timepoint 0 (before pelleting). Flundred % (orange line) is the quantity of the carvone at timepoint 0. Error bars indicate standard error of the mean of four replicates.

Limoneneand carvone stability in feed with caraway oil only

- Similarly to thefeed with caraway oil/ SUrfac® LM70/90 mixture, approximate ly 40% of limonene evaporated from the feed with caraway oil during pelleting and before packing (Figure 17).

- The limonene evaporation continued gradually and about 70% of original limo nene was evaporated at theend of the 4-week storage period (Rgure 17).

- Qi the other hand, carvone concentration remained very stable in feed with caraway oil only (Figure 18). Flence, it is evident that carvone was not affected by pelleting and subsequent storage of 4 week. Figure 17 shows the quantity of limonene remaining in feed at differ ent timepoints presented as a percent from timepoint 0 (before pelleting). Hun dred % (orange line) is the quantity of limonene at timepoint 0. Error bars indi cate standard error of the mean of four replicates.

Figure 18 shows the quantity of carvone remaining in feed at different timepoints presented as a percent from timepoint 0 (before pelleting). Hundred % (orange line) is the quantity of carvone at timepoint 0. Error bars indicate standard error of the mean of four replicates.

Conclusions

Of the two major components of caraway oil, considerable part (about 70%) of limonene evaporated from pelleted feed during processing and storage, while concentration carvone remained very stable. The results also showed that no considerable differences in the rate of evaporation was observed between the two tested feeds - feed with caraway oil only or feed with caraway oil/ SUrfac® LM70/90 combination.

According to results of the previous bacterial inhibition tests, carvone (not limonene) was determined as an effective inhibitor of the growth of patho genic microorganisms. Therefore, the fact that carvone was not evaporated from feed during processing and storage is promising and critical for proceeding with the planned animal feeding trials.

Study 5: Comparison of different surfactant emulsifiers in enhancing the bacterial inhibitory potential of caraway oil

Major findings:

- Unlike SUrfac® LM70/90 that was used in the previous experiments, the new surfactant candidates alone did not inhibit the growth of B. pseudolongum and E. faecium.

- Generally, the three new surfactants alone did not show significant growth inhi bition of E. col i and G jejuni ; however, slight trend-like inhibition growth was ob served at the highest 1 g/ L eon cent rat ion of Rapeseed lecithin.

- The addition of SABO-Nutreem and Castor oil surfactants did not significantly change the bacterial inhibition profile when tested with caraway oil as compared to caraway oil alone. - The combination of caraway oil and Rapeseed lecithin improved slightly the G jejuni inhibition as compared to caraway oil alone.

- Of the new surfactant candidates tested, the most promising results were ob tained with Rapeseed lecithin.

1 . Background and rationale of the study

In the previous studies, it was shown that the caraway oil/ SUrfac® M70/90 emulsion was effective in inhibiting the growth of tested pathogens, while it had a smaller effect on commensal bacteria. Hence, it was concluded that the addition of a surfactant was essential for boosting the inhibitory potential of caraway oil most likely by increasing the solubility of the effective inhibitory components present in theoil.

The rationale of the proposed work is to test SUrfac® M70/ 90 and 3 other surfactants and find out the surfactant emulsifier concentrations in the car away oil/ surfactant emulsions, which will be optimal for inhibiting the pathogen ic bacterial growth while not affecting considerably the commensal intestinal bac teria.

2. Materialsand methods

2.1 Microbial cultures and treatments

The microbial growth inhibition was tested with pure microbial cul tures in synthetic growth liquid mediaat 37°G Four microbial species, pathogenic chicken isolate Escherichia coli KBAK 1601/ 15, opportunistic pathogen Entero coccus faecium, pathogen Campylobacter jejuni DSM 4688, and commensal Bifidobacterium pseudolongum DSM 20099, were cultured in suitable media un der appropriate conditions. The growth kinetics of each bacterial strain was evaluat ed during the previous projects.

List of treatments and starting concentrations (strongest final doses) :

1 . Negative CTRL (no amendments)

2. Caraway oil at 0.312-0.75 g/ L

3. Caraway oil & SUrfac® LM70/ 90 at 8 doses from 1 g/ L

4. Caraway oil & surfactant - SABO-NUTREEM R495 at 8 doses from 1 g/ L

5. Caraway oil & surfactant - EL 33 CASTOR GL at 8 doses from 1 g/ L

6. Caraway oil & surfactant - RAPESEED LECITHIN at 8 doses from 1 g/ L 7. SUrfac® LM70/ 90 at 8 doses from 1 g/ L

8. Surfactant - SABGNUTREEM R495 at 8 doses from 1 g/ L

9. Surfactant - EL 33 CASTOR GL at 8 doses from 1 g/ L

10. Surfactant - RAPESEED LEO THIN at 8 doses from 1 g/ L

2.2. Bacterial growth inhibition test

Microbial culture media were supplemented with caraway oil alone or with an emulsion of selected doses of the caraway oil with final concentrations from 0.312 g/ L to 0.75 g/ L depending on bacterial species and 4 surfactants at eight doses (strongest dose 1 .0 g/ L) and inoculated with 5-10% (v/ v) of actively growing microbial cultures. Also, the same eight doses of the surfactant emulsifi ers were tested alone as a control to find out the inhibitory effect of the surfac tants themselves. For each microbial species, aculturewithout added test productswas used as a negative control.

All tests (except Campylobacter jejuni) were run in at least with three replicates in 96-wells plates. The level of growth inhibition was generally as sessed by measuring the optical density (OD) of the culture at a wavelength of 600 nm for Escherichia col i , Bifidobacterium pseudolongum, and Enterococcus faecium, where the increase in OD is an indicator of the proliferation of bacterial cells. For Campylobacter jejuni, which is a small-sized bacterium and does not elicit asignificant increase in OD 600 nm, a method utilising DNA binding dyeSYBRGeen was used to assess the changes in bacterial density during the incubation period.

An important modification of the protocol from the previous experi ments was that in this study caraway oil and surfactants were mixed and vor- texed with growth medium in tubes to the final desired concentration before loading to 96-well plate, allowing the improved blending of oil, surfactants, and media. In the previous trials, growth medium was added to the 96-wells plate with aloaded 10X-concentrated solution of oil and surfactant.

2.3. Calculations

Growth (percentage, %) = 100 c -

\OD 600 nm negative control average / Statistical analyses consisted of two-tailed Student’s t-tests, where the inhibition percentage values from treatments were compared against those achieved from negative controls.

Sgnificance according to Student’st-test isshown in the figures as follows:

- p-value< 0.05 ^*

- p-value< 0.01 ^**

- p-value< 0.001 ^***

3. Major findings

3.1 . Escherichia col i (Figures 19Aand 19B)

The growth of broiler chicken pathogenic E. col i KBAK1601/ 15 at a 4- hour time point isshown in Figures 19Aand 19B.

- The growth of E. col i was not significantly inhibited by any of the tested surfac tants alone, except minor inhibition at the highest 1 g/ L concentration of rape- seed lecithin.

- Pure caraway oil at 0.75 g/ L dose showed 50% inhibition from negative CTRL.

- The addition of any of the surfactants did not significantly change the bacterial inhibition profile when tested with 0.75 g/ L caraway oil as compared to caraway oil alone.

Figures 19A and 19B show the growth of Escherichia col i KBAK1601/ 15 at a 4-hour time point. Error bars indicate the standard error of 3 replicate wells and asterisks the statistical significance of the difference to nega tive CTRL with no test product according to the Student’s t-test. Hundred % growth (dashed line) is the growth of negative CTRL in the absence of any test products.

3.2. Bifidobacterium pseudolongum ( Figures 20A and 20B)

The growth of commencial and beneficial B. pseudolongum DSM 20099 at a6-hour time point isshown in Figures20Aand 20B.

- The growth of B. pseudolongum was not inhibited by the three new surfactant candidates alone. In contrast, the previously tested SUrfac® LM70/90 inhibited bifidobacterial growth significantly, by 60% already at 0.06 g/ L dose and by 85% at 1 g/ L dose.

- Pure caraway oil at 0.63 g/ L dose showed a moderate 30% growth inhibition of B. pseudolongum from negative CTRL. - The addition of three new surfactants did not significantly change the bacterial inhibition profile when tested with 0.63 g/ L caraway oil as compared to caraway oil alone, while the addition of SUrfac® LM70/90 inhibited growth at the similar rateasSurfac® LM70/ 90 alone.

Figures 20A and 20B show the growth of Bifidobacterium pseudo- longum at a 6-hour time point. Error bars indicate the standard error of 3 repli cate wells and asterisks the statistical significance of the difference to negative CTRL with no test product according to the Student’s t-test. Hundred % growth (dashed line) isthe growth of negative CTRL in the absence of any test products.

3.3. Enterococcus faed urn (Figures 21 A and 21 B)

The growth of Enterococcus faeci urn at a3-hour time point isshown in Figures 21 A and 21 B.

- Similar to B. pseudolongum, the growth of Enterococcus faeci urn was not inhib ited by thethreenew surfactant candidates alone, in contrast to previously tested SUrfac® LM70/90 which dose-dependent I y inhibited growth by 30% at 0.02 g/ L dose and by 45-55% at 0.03-1 g/ L dose.

- Pure caraway oil at 0.75 g/ L dose showed 35% growth inhibition from negative CTRL.

- The addition of SABO-Nutreem did not significantly change the bacterial inhibi tion profile when tested with 0.75 g/ L caraway oil as compared to caraway oil alone, while the addition of Castor oil and Rapeseed lecithin inhibited growth maximum by 60% at 0.03-0.25 g/ Lof surfactant.

- As expected, the addition of SUrfac® LM70/ 90 to the oil inhibited the growth of Enterococcus faed urn significantly, by 60% already at 0.01 g/ L of surfactant.

Figures 21Aand 21 B show the growth of Enterococcus faeci urn strain at 3-hour time point. Error bars indicate the standard error of 3 replicate wells and asterisks the statistical significance of the difference to negative CTRL with no test product according to the Student’s t-test. Hundred % growth (dashed line) is the growth of negative CTRL in the absence of any test products.

3.3. Campylobacter jejuni (Figures 22A-22B and 23)

The growth of Campylobacter jejuni strain DSM 4688 at a 24-hour time point isshown in Figures 22A, 22Band 23.

- The growth of Campylobacter jejuni was slightly inhibited by SABO-Nutreem and Castor oil surfactants alone at the highest 1 g/ L concentration. Small inhibi- tion was observed at the highest 0.5-1 g/ L concentration of Rapeseed lecithin and average (up to 35% from negative CTRL) inhibition at 0.13-1 g/ L by previously tested SUrfac® LM70/ 90.

- The addition of SABO-Nutreem and Castor oil did not affect the bacterial inhibi tion profile when tested with 0.31 g/ L caraway oil as compared to caraway oil alone, while the addition of SUrfac® LM70/90 and Rapeseed lecithin inhibited growth by an additional 10-20% at maximum surfactant dose.

- Pure caraway oil at 0.31 g/ L dose showed asignificant 50% inhibition from neg ative CTRL and nearly complete inhibition of Campylobacter jejuni growth at 0.63 g/ L dose (Figure 5).

Figures 22A and 22B show the growth of Campylobacter jejuni strain at 24-hour time point. Error bars indicate the standard error of 3 replicate wells and asterisks the statistical significance of the difference to negative CTRL with no test product according to the Student’s t-test. Flundred % growth (dashed line) is the growth of negative CTRL in the absence of any test products.

Figure 23 shows the growth of Campylobacter jejuni strain at 24-hour time point with caraway oil when tested at different concentrations. Error bars indicate the standard error of 3 replicate well sand asterisks the statistical signifi cance of the difference to negative CTRL with no test product according to the Student’s t-test. Flundred % growth (dashed line) is the growth of negative CTRL in the absence of any test products.

It will be obvious to a person skilled in the art that, as the technology advances, the inventive concept can be implemented in various ways. The inven tion and its embodiments are not limited to the examples described above but may vary within the scope of theclaims.

Study 6: Comparison of various surfactant emulsifiers in enhancing the bacterial inhibitory potential of caraway oil

Major findings:

- 90/ 10 oil/ surfactant treatments showed comparableor better pathogen growth inhibition than caraway oil alone.

- The most promising results were obtained when caraway oil was mixed with Sabopal EL200. This mixture showed better growth inhibition of all tested patho gens than pure caraway oil. The second best, particularly for inhibition of the Campylobacter jejuni, was Oil/ Gycerol monooleate mixture, especially at higher doses tested.

1 . Background and rationale of the study

In the previous studies, it was shown that the caraway oil and surfactant emul sion was superior in inhibiting the growth of tested pathogens, while it had a smaller effect on commensal bacteria. Hence, it was concluded that theaddition of asurfactant was relevant for boosting the inhibitory potential of caraway oil most likely by increasing the solubility of the effective inhibitory components present in the oil.

The reasoning of the work reported is to test the effect of several potential emul sifiers in combination with caraway oil on the chosen commensal and pathogenic intestinal bacteria.

2. Materialsand methods

2.1 Microbial cultures and treatments Four microbial species, pathogenic chicken isolate Escherichia col i KBAK 1601/ 15, opportunistic pathogen Enterococcus faed urn, pathogen C&mpylobacter jejuni DSM 4688, and commensal Bifidobacterium pseudolongum DSM 20099, were cultured in correspondent media under appropriate conditions. The growth kinetics of each bacterial strain was evaluated during the previous projects. All treatments were tested at 7 doses.

Treatments 3, 4, 5, and 7 were performed with 90 to 10 caraway oil to surfactant ratioBacitracin (BMD) and monensin wereapplied as positive controls.

List of treatments:

1 . Negative CTRL (no amendments) 2. Caraway oil alone (new batch provided by theclient)

3. Caraway oil & Gycerol monooleate (Feed grade; HLB 3) 4. Caraway oil & Sbrbitan monooleate20 EO(Feed grade; HLB 15)

5. Caraway oil & Sabopal EL 200 (HLB 19)

6. Caraway oil & Raps lecithin (HLB 7-10)

7. Antibiotic 1 ; Bacitracin (BMD) (Positive CTRL)

5 8. Antibiotic2; Monensin (Positive CTRL)

2.2. Bacterial growth inhibition test

10

Microbial culture mediawere supplemented with the combination of caraway oil and emulsifiers at seven different doses and inoculated with 5-10% (v/v) of ac tively growing microbial cultures. For each microbial species, a culture without added test products was used as a negative control.

15

All tests (except C&mpylobacter jejuni) were run in three replicates in 96-wells plates. The level of growth inhibition was generally assessed by measuring the optical density (OD) of the culture at awavelength of 600 nm for Escherichia col i, Bifidobacterium pseudolongum, and Enterococcus faedum, where the increase in 20 OD is an indicator of the proliferation of bacterial cells. For C&mpylobacter jejuni, which is a small-sized bacterium and does not elicit a significant increase in OD 600 nm, a method employing DNA binding dye SYT3R Or een was used to assess the changes in bacterial density during the incubation period.

25 2.3. Calculations

The growth (in percentages to control with no product amendments i.e. Neg. CTRL) was calculated with thefollowingformula:

„„ _f . _ _/ . _{L L} OD 600 nm treatment X \

30 Growth (percentage, %) = 100 c -

\OD 600 nm negative control average /

Statistical analyses consisted of two-tailed Student’s t-tests, where the inhibition percentage values from treatments were compared against those achieved from negative controls. Sgnificance according to Student’s t-test is shown in the fig- 35 ures as follows: - p-value< 0.05 ^*

- p-value< 0.01 ^**

- p-value< 0.001 ^***

3. Results

3.1 . Escherichia coli (Figure 24)

The growth of broiler chicken pathogenic E coli KBAK1601/ 15 at a 4-hour time point is shown in Figure 24.

- Pure caraway oil at 1 .5g/ L dose showed 90% and at 0.75 g/ L about 35% inhibi tion of the E coli growth from negative CTRL.

- The addition of Sabopal EL200 to caraway oil increased inhibition of E coli on 10% (from 89% to 99%) at a 1 .5 g/ L oil dose. Three other surfactants from the first treatment group showed similar growth inhibition profiles to the caraway oil alone.

- Controls BMD and Monensin only slightly (10-15%) inhibit the growth at high est doses.

Figure 24 shows the growth of Escherichia coli KBAK1601/ 15 at a 4-hour time point. Error bars indicate the standard error of 3 replicate well s and asterisks the statistical significance of the difference to negative CTRL with no test product ac cording to the Student’s t-test. Flundred % growth (dashed red line) is the growth of negative CTRL in the absence of any test products.

3.2. Bifidobacterium pseudolongum (Figure 25)

The growth of commencial and beneficial B. pseudolongum DSM 20099 at a 6- hour time point is shown in Figure 25.

- Pure caraway oil showed moderate, dose-dependent growth inhibition of B. pseudolongum from 75% and 68% (correspondent oil doses 6 and 3 g/ L) from negative CTRL.

- All other treatments (except oil/rapeseed lechitin mixture) inhibited bifidobac- terial growth at asimilar rate as pure oil.

- Oil/ rapeseed lechitin mixture inhibited the growth of B. pseudolongum by 10%- 20% more than pure caraway oil at the highest 1 .5-6 g/ Loil doses.

- Controls Monensin and especially BMD inhibited the growth very effectively even at smallest doses. Figure 25 shows the growth of Bifidobacterium pseudolongum at a 6-hour time point. Error bars indicate the standard error of 3 replicate well s and asterisks the statistical significance of the difference to negative CTRL with no test product ac cording to the Student’s t-test. Hundred % growth (dashed red line) is the growth of negative CTRL in the absence of any test products.

3.3. Enter ococcusfaedum (Figure 26)

The growth of Ent ococcusfaedum at a3-hour time point isshown in Figure26.

- Pure caraway oil showed modest dose-dependent growth inhibition of Entero coccus faedum by 65% and 45% (correspondent oil doses 3 and 1.5 g/ L) from negative CTRL.

- All four treatments from the first group at the oil doses 1 .5-3 g/ L showed 15- 30% better than pure oil inhibition rate - the best of them was Sabopal EL200, showing 20% better inhibition also at 0.75 g/ Ldose.

- Positive controls, Monensin and BMD, inhibited the growth effectively even at the smallest doses.

Figure 26 shows the growth of Enterococcus faedum strain at a 3-hour time point. Error bars indicate the standard error of 3 replicate well s and asterisks the statistical significance of the difference to negative CTRL with no test product ac cording to the Student’s t-test. Hundred % growth (dashed red line) is the growth of negative CTRL in the absence of any test products.

3.4. Campylobacter jejuni (Figure 27)

The growth of C&mpylobacter jejuni strain DSM 4688 at a 24-hour time point is shown in Figure 27.

- Pure caraway oil showed significant dose-dependent growth inhibition of C jejuni by 94% and 77% (correspondent oil doses 1 .5 and 0.75 g/ L) from negative CTRL.

- Inhibition profile of the G jejuni was almost identical to the Ent ococcusfaed um inhibition profile -better than pure oil inhibition for the first treatment group with Oil/ ¾bopal EL200 as the best from all treatment showing 20% inhibition at smallest tested dose.

- Control BMD inhibited the growth dose-dependent I y and effectively while monensin gave no growth inhibition even at the highest dose.

Figure 27 shows the growth of C&mpylobacter jejuni strain at 24-hour time point. Error bars indicate the standard error of 3 replicate well s and asterisks the statis- tical significance of the difference to negative CTRL with no test product accord ing to the Student’s t-test. Hundred % growth (dashed red line) is the growth of negative CTRL i n the absence of any test products.

Claims

1 . An animal feed ingredient composition, comprising Carvone and at least one surfactant.

2. An animal feed ingredient composition according to daim 1 , where in the Carvone is S-(+)-Carvonei.e. D-carvone.

3. An animal feed ingredient composition according to daim 1 or 2, wherein carvone is provided in combination with limonene.

4. An animal feed ingredient composition according to any one of claims 1 -3, wherein the Carvone is provided in the form of an essential oil with a content of carvone

5. An animal feed ingredient composition according to any one of the preceding claims 1 -4, wherein the composition is a mixture of a surfactant and carvone.

6. An animal feed ingredient composition according to daim 5, where in the at least one surfactant has a hydrophilic-lipophilic balance (HLB) value of 3-25, preferably 8-20.

7. An animal feed ingredient composition according to daim 5 or 6, wherein the D-Carvone is included in the composition as such or in the form of caraway oil or in combination with limonene at adoseof up to 10 g/ L, preferably in the range of 0.07-5 g/ L, more preferably 1 .25-2.5 g/ L in the emulsion, and the surfactant is included at adose of up to 10 g/ L, preferably in the range of 0.07-5 g/ L, more preferably 0.25-2.5 g/ L, most preferably 0.25-1 .0 g/ L in the emulsion.

8. An animal feed ingredient composition according to any one of the preceding claims, wherein the at least one surfactant is selected from a C12-C15 alcohol ethoxylate; at least one of lecithins, such as hydrolysed lecithin and rape- seed lecithin; PEG glyceryl ricinoleate; ethoxylated castor oil ; glycerol monoole- ate; and sorbitan monooleate.

9. An animal feed ingredient composition according to any one of the preceding claims, wherein the Caraway oil, comprises:

- 1-99 % carvone, preferably 50-98% carvone , more preferably 50- 65% carvone

- 1 -99% limonene, preferably 50-98% limonene, more preferably 50- 65% limonene

- 1-10% other lipophilic components.

10. Use of an animal feed ingredient composition according to any one of the claims 1 -9 as a microbial growth inhibitor in animal feed or animal drinking water.

11. Use according to daim 10, for microbial growth inhibition of at least one of Salmonella enterica, Eschericia coli, C&mpylobacter jejuni, Clostridium perfringens, Enterococcus faeci urn.

12. Use of an animal feed ingredient composition according to any one of the claims 1 -9 as an intestinal microbiota modulator in animal feed or animal drinking water.

13. Use according to claim 12 for growth inhibition of intestinal path ogenic microorganisms such as at least one of Salmonella enterica, Eschericia coli, C&mpylobacter jejuni, Clostridium perfringens, and Enterococcus faed urn, without essential growth inhibition of intestinal commensal microorganisms such as Bifidobacterium pseudolongum.

14. An animal feed comprising an animal feed ingredient composition accordi ng to any one of the clai ms 1 -9.

15. An animal feed according to daim 14, wherein the animal feed is in pellet form.

16. An animal feed according to daim 14 or 15, wherein the animal feed is for chicken.

17. An animal feed according to any one of the claims 14-16, wherein the animal feed is wheat-soy-based.

18. Drinking water or pellet feed for animals comprising an animal feed ingredient composition according to any one of the cl aims 1 -9.

19. Method of making a wheat-soy-based animal feed pellet, compris ing grinding wheat grains and mixing the resulting wheat particles with the ani mal feed ingredient composition according to any one of claims 1-9 and soybean oil, and pelletingtheobtained mixture.

20. Method according to daim 19, wherein the Carvone in the animal feed ingredient composition according to any one of claims 1 -9 is Carvone as such, preferably S-(+)-Carvone i.e. D-carvone, a combination of Carvone and Lim- onene, or caraway oil.