FI20215415A1 - Feed ingredient composition, use thereof and feed comprising the same - Google Patents

Abstract

Esillä oleva keksintö koskee eläinten rehun ainesosakoostumusta, joka käsittää R-(-)-karvonia tai S-(+)-karvonia, tai näiden yhdistelmää, ja ainakin yhtä pinta-aktiivista ainetta. Esillä oleva keksintö koskee lisäksi rehua, joka käsittää mainittua eläinten rehun ainesosakoostumusta.

Description

FEED INGREDIENT COMPOSITION, USE THEREOF AND FEED COMPRISING THE

SAME

FIELD OF THE INVENTION

The present invention relates to a feed ingredient composition for ani- mals.

The present invention further relates to a use of a feed ingredient com- position as a microbial growth inhibitor in animal feed.

The present invention further also relates to a feed comprising a feed ingredient composition.

The present invention further also relates to a drinking water or pellet feed for animals, as well as a method for making a wheat-soy-based feed pellet.

BACKGROUND OF THE INVENTION

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. Since the ban of the an- tibiotic growth promoters in EU, feed companies are even more desperately look- ing for alternative intestinal microbiota modulators.

BRIEF DESCRIPTION OF THE INVENTION

An object of the present invention is to provide a feed ingredient com- position for animals so as to overcome the above problems. The objects of the in- = vention are achieved by a feed ingredient composition, use of said feed ingredient

N 25 anda feed comprising said feed ingredient, drinking water or pellet feed or method x of making a wheat-soy-based feed pellet according to what is stated in the inde-

S pendent claims. The preferred embodiments of the invention are disclosed in the

I dependent claims. + The feed ingredient composition for animals according to the invention = 30 comprises R-(-)-Carvone or S-(+)-Carvone, or a combination thereof, and at least 2 one surfactant.

S According to a preferred embodiment of the feed ingredient composi- tion the at least one surfactant is selected from at least one of a C12-C15 alcohol ethoxylate, hydrolysed lecithin (lysolecithin; lysophosphatidylcholine), PEG glyceryl ricinoleate, and castor oil, preferably a C12-C15 alcohol ethoxylate.

In a first preferred embodiment of the feed ingredient composition ac- cording to the invention the R-(-)-Carvone or S-(+)-Carvone, or a combination thereof, is included in the composition in the form of a Caraway -based raw mate- rial. Preferably, the Caraway -based raw material is Caraway oil. Alternatively, the

Caraway -based raw material is Caraway powder. According to another alternative the Caraway -based raw material is a water-extract of Caraway powder.

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-(+)-Carvone may be e.g. obtained by synthesis from suitable starting material. Preferably, D-carvone i.e. (S)-(+)-carvone is used.

In a further preferred embodiment, the feed ingredient composition of the invention further comprises water.

In yet a further preferred embodiment the feed ingredient composition of the invention is an emulsion.

In a still further preferred embodiment the feed ingredient composition of the invention consists of R-(-)-Carvone or S-(+)-Carvone, or a combination thereof, and at least one surfactant, preferably only one surfactant.

In another preferred embodiment of the feed ingredient composition the at least one surfactant has a hydrophilic-lipophilic balance (HLB) value of 8-12.

In yet another preferred embodiment of the feed ingredient composi- tion the R-(-)-Carvone or S-(+)-Carvone, or a combination thereof, is/are included in the composition as such or in the form of caraway oil at a dose of 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,

S 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 emul- ? sion.

S 30 In still another preferred embodiment of the feed ingredient composi-

E tion wherein the at least one surfactant is selected from at least one ofa C12-C15

LO alcohol ethoxylate, hydrolysed lecithin, PEG glyceryl ricinoleate, and castor oil, s preferably a C12-C15 alcohol ethoxylate.

N According to another aspect, the invention provides a use of the feed

N 35 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, Eschericia coli, Campylobac- ter jejuni, Clostridium perfringens, Bifidobacterium pseudolongum, and Enterococcus faecium.

According to a further aspect, the invention also provides a feed com- prising a feed ingredient composition according to the invention.

The feed according to the invention is preferably in pellet form. In a par- ticular preferred embodiment said feed is for animals, in particular production an- imals, such as chickens and pigs, and domestic animals, such as cats and dogs.

The feed according to the invention in any one of its embodiments may be wheat-soy-based.

According to a yet further aspect, the invention also provides drinking water or pellet feed for animals comprising a feed ingredient composition accord- ing to the invention.

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 feed ingredient composition according to the invention and soy- bean oil, and pelleting the obtained mixture. The feed ingredient composition used in this method is preferably caraway oil.

The invention is based on the realization of a positive interaction be- tween a surfactant and caraway oil which is both a surprising and significant find- ing.

It was found in tests that an emulsion of Caraway oil and a C12-C15 al- cohol ethoxylate surfactant (a commercial product under the tradename “Surfac®

LM70/90” was used in the tests) emulsion was superior in inhibiting the growth of most tested pathogens, while it had no inhibition effect on commensal Lactobacil-

S lus strains. 4 Caraway oil without added surfactant was significantly less efficient

P growth inhibitor for most of the pathogenic strains. Therefore, it can be concluded

S 30 thatthe addition of a surfactant is essential for boosting the inhibitory potential of

E caraway oil. Without being bound to theory, this is most likely by increasing the

LO solubility of the effective inhibitory oil components. Both of the tested antibiotics s showed dose-dependent inhibition of commensal bacteria, while many of the

N tested pathogens were not inhibited.

N 35 Most of the tested bacteria were not inhibited by caraway powder (the exception was slight inhibition of Campylobacter jejuni at higher product doses).

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

As already indicated above, C12-C15 alcohol ethoxylate has been tested as surfactant with positive results in the form of the commercial product “Surfac®

LM70/90” (study 1). The combination of caraway oil and Rapeseed lecithin im- proved slightly the C. 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-emul- sions (oil-in-water)). Surfactants having HLB values of 8-12 are suitable for oil- based emulsions (referred to as w/o-emulsions (water-in-oil)). Surfac® LM70/90 has an HLB-value of approximately 12.

Throughout this application the terms “D-carvone”, “(+)-carvone” and “(S)-(+)-car- vone” are used interchangeably. Likewise, throughout this application, “L-car- vone”, “(-)-carvone” and “(R)-(-)-carvone” are used interchangeably.

Throughout this application terms “hydrolysed lecithin”, “lysolecithin” and “lysophosphatidylcholine” are used interchangeably.

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

BRIEF DESCRIPTION OF THE DRAWINGS

In the following the invention will be described in greater detail by

N means of preferred embodiments with reference to the attached drawings, in

N which

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

S Figure 1B shows the growth of Escherichia coli 156/97 F4 at 4-hour

E 30 time point;

LO Figure 1C shows the growth of Escherichia coli KBAK1601/15 at 4-hour s time point;

N Figure 2 shows the growth of Clostridium perfringens at the 24-hour

NN 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 5 time point;

Figure 6A shows the growth of Lactobacillus salivarius 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 coli 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 11A shows the effect of test products on the body weight gain of broiler chickens during days 0-14;

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

Figures 11C shows the effect of test products on the body weight gain of broiler chickens during 21-35;

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

Figure 12A shows the effect of the test products on feed consumption of

S broiler chickens during days 0-14; + Figure 12B shows the effect of the test products on feed consumption of ? broiler chickens during days 14-21;

S 30 Figure 12C shows the effect of the test products on feed consumption of

E broiler chickens during days 21-35;

LO Figure 12D shows the effect of the test products on feed consumption of s broiler chickens during days 0-35;

N Figure 13 shows the effect of test products on mortality of broiler chick-

N 35 ens;

Figure 14A shows the effect of test products on mortality-corrected feed conversion efficiency of broiler chickens for days 0-14;

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

Figure 14C shows the effect of test products on mortality-corrected feed conversion efficiency of broiler chickens for days 21-35;

Figure 14D shows the effect of test products on mortality-corrected feed conversion efficiency of broiler chickens for days 0-35;

Figure 15 shows the quantity of limonene remaining in feed at different 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 different 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 O (before pelleting);

Figures 19A-19B shows the growth of Escherichia coli KBAK1601/15 at a 4-hour time point;

Figures 20A-20B show the growth of Bifidobacterium pseudolongum at a 6-hour time point;

Figures 21A-21B show the growth of Enterococcus faecium strain at 3- hour time point;

Figures 22A-22B show the growth of Campylobacter jejuni strain at 24- hour time point; and

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

S x DETAILED DESCRIPTION OF THE INVENTION

S Five studies are presented in the detailed description. The following

I 30 commercial surfactants have been used in the studies: & 1. Surfac& LM70/90 from Surfachem (studies 1-5) 2 2. SABO-NUTREEM R495 from SABO (study 5) 5 3. EL 33 CASTOR OIL from Mosselman (study 5)

O 4. RAPESEED LECITHIN from BIC services (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 bacteria were tested in this study. One of the pivotal questions was whether the minimum inhibitory concentration (MIC) of the products would be lower for the pathogens 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 pathogens as — well as selected commensal intestinal bacteria to different caraway preparations and concentrations as well as to provide valuable information on the mode-of-ac- tion of the products tested.

Materials and methods

Microbial cultures and treatments

The microbial growth inhibition was tested with pure microbial cul- tures in synthetic liquid growth media at 37°C. 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

Clostridium perfringens were cultured in anaerobic media and under anaerobic or microaerophilic conditions.

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 lactobacilli in the chicken small-intestine and E. faecium (an opportunistic pathogen) and B. pseudolongum also belong to the normal intestinal microbiota of warm-blooded animals. Growth kinetics of each bacterial strain was

N initially analysed in a pre-test to find out the late exponential/early stationary 5 growth phase (data not shown), since this time point provides a widest dynamic 7 range for the growth monitoring. © 30 A total of four different caraway products /fractions were included in

E the study, each of which was tested at eight doses (2 x dilution series). In addition,

LO two commonly used antimicrobial growth promotors with different spectrum s (bacitracin methylene disalicylate (BMD) and monensin) were included as positive

N controls. Moreover, Surfac& LM70/90 surfactant was used as an emulsifier in the

N 35 Treatment 7 (combination with caraway oil). Moreover, Surfac® LM70/90 alone was included as an additional control to monitor its possible effect on bacterial growth.

Table 1. Characteristics of microbial strains used in this study. conditions

Salmonella Gram-negative patho- | IR 715 LB; aerobic enterica gen; serovar

Typhimurium perfringens

Campylobac- | Gram-negative pathogen | DSM 4688 BHI; microaero- ter philic jejuni

Escherichia Gram-negative patho- | 156/97 F4+ LB; aerobic coli gen; post- weaning diarrhea strain of piglets

Escherichia Gram-negative patho- | KBAK LB; aerobic coli gen; chicken 1601/15 isolate salivarius sal philic

O reuteri sal philic

K faecium istic pathogen 7 Bifidobacte- Gram-positive commen- | DSM 20099 DSMZ medium N58;

E rium sal anaerobic

O pseudo- longum 3 5

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

1. Negative CTRL (no amendments) 2. Bacitracin (BMD) (Positive 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) — strongest 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 - ‘Cara- way powder’ and ‘Water extract of caraway powder’ were significantly affected by the accumulation of the insoluble powder particles on the bottom of the culturing 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. Moreover, for each microbial species tested, a culture without added test product was used as a negative control. All tests were run 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 increase in

OD is an indicator of the proliferation of bacterial cells. For Campylobacter jejuni, which is a very small-sized bacterium and does not elicitan increase in OD 600 nm, a method utilising DNA binding dye SYBR Green was used to assess the changes in bacterial density during the incubation period. The microbial growth was moni-

N tored for 4 to 24 hours depending on the microbial strain (the optimal culturing 5 times were defined in the pre-test).

O

S 30 Calculations

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

LO CTRL) was calculated s with the following formula:

N

N 35 — Growth (percentage, %) = 100 x 0 KI

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.

Significance according to Student's t-test is shown in the figures as fol- lows: — p-value < 0.05 * - p-value < 0.01 ** — p-value < 0.001 ***

Major findings

Salmonella enterica & Escherichia coli (Figures 1A-1C)

Both S. enterica and E. coli belong to Enterobacteriaceae, which is a large family consisting of only Gram-negative bacteria. In addition to E. coli and

Salmonella, Enterobacteriaceae includes also many other well-known pathogenic genera such as Yersinia, Klebsiella and Shigella. In this study, we evaluated the in- hibitory potential of test compounds against S. enterica serovar Typhimurium as well as two pathogenic E. coli 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 (Salmonella -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

S CTRL). + — The results showed that Surfac& LM70/90 emulsifier alone did not show consid- 7 erable inhibition of Salmonella or E. coli. © 30 —- Hence, itis evident that inclusion of Surfac® LM70/90 facilitates the inhibitory

E potential of caraway oil most likely by improving its water-solubility.

LO Figure 1A shows the growth of Salmonella enterica, Figure 1B Esche- s richia coli 156/97 F4+ and Figure C Escherichia coli KBAK1601/15 at 4-hour time

N point. Error bars indicate the standard error of 3 replicate wells and asterisks the

N 35 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 in the absence of any test products.

Clostridium perfringens (Figure 2)

Clostridium perfringens is a bacterium that is nearly always present in the intestine of chickens at low numbers. However, partly due to unknown factors its numbers can occasionally increase several orders of magnitude and it can start producing toxins that cause significant tissue damage. When the outbreak is seri- ous enough it leads to necrotic enteritis and increased mortality of animals. — The growth of Clostridium perfringens was 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 Clostridium perfringens at the 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. 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 commensal organisms. On the contrary, in humans the infection is associated with acute enter- itis. It is generally assumed that C. jejuni contaminate poultry or pig meat during processing from individual animals carrying the pathogen. Hence, it can survive throughout the food chain supply to constitute a risk to human health.

Due to its small size, a different method utilising DNA binding dye SYBR

N Green was used to assess the densities of Campylobacter jejuni cultures. The

N method is based on epifluorescence rather than absorbance and therefore, meas- 3 urements were less affected by ‘powder particle sedimentation’ effect as compared 3 30 — to OD measurements of other target microbes.

I — Neither of the antibiotics tested showed growth inhibition of Campylobacter je- a K juni. = - On the other hand, caraway oil alone showed a dose-dependent inhibition with = the three strongest inclusion levels (-20% to -64% from neg CTRL).

S 35 —- Caraway oil/Surfac® LM70/90 emulsion was the most efficient treatment to in- hibit C. 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 C. jejuni growth; however, the difference to caraway oil/Surfac® LM70/90 emulsion was consider- able. Therefore, itis 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 C. jejuni growth by 35% with the strongest inclusion level (Figure 4). However, we still can- not completely exclude the possible ‘powder particle’ effect which could hamper the inhibition result. — — Interestingly, the caraway powder water extract did not have a considerable in- hibitory effect on C. jejuni growth. Hence, it is likely that the effective components 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 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 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) is the growth of negative CTRL in the absence of any test products.

Bifidobacterium pseudolongum (Figure 5)

Bifidobacteria are thought to exert a protective role against pathogenic microorganisms such as campylobacter and salmonella via production of antimi-

S crobial agents as well as preventing pathogen colonisation and enhancing the host + immune response in warm-blooded animals. 7 - The growth of Bifidobacterium pseudolongum was inhibited by both tested anti- © 30 — biotics already at the lower doses tested. The maximum inhibition was approxi-

E mately -50% from CTRL.

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

N - However, when caraway oil was combined with Surfac& LM70/90 inhibition was

N 35 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) is the growth of negative CTRL in the absence of any test products.

Lactobacillus reuteri & Lactobacillus salivarius (Figures 6A and 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 lactobacilli levels in small-intestine may increase physical barrier effect against pathogenic bacteria. In addition, lactic acid secreted by lactic acid 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 lactobacilli growth was inhibited by both antibiotics with all doses tested (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

Lactobacillus is an important commensal bacterial genus in the small-intestine of 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

N standard error of 3 replicate wells and asterisks the statistical significance of the 5 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

S 30 any test products.

E

LO Enterococcus faecium (Figure 7) s The metabolism of enterococci is like that of the homofermentative lac-

N tobacilli. A characteristic feature of enterococci is that they tend to possess and

N 35 transfer antibiotic resistance genes and are thus considered potentially risky group of bacteria if therapeutic or prophylactic antibiotics are being or have been used.

Enterococcus faecium is a Gram-positive bacterium in the genus Enter- ococcus. It can be commensal in the gastrointestinal tract of humans and animals, but it may also be an opportunistic pathogen. Therefore, enterococcosis often oc- curs secondary to another disease. — The growth of E. faecium was dose-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) — On the contrary, caraway oil or Surfac® LM70/90 alone had no considerable ef- fect on the growth of Enterococcus faecium. — Therefore, it is evident that inclusion of Surfac® LM70/90 increases significantly 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 wells 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 in 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 carvone and limonene

Major findings: * The caraway oil with Surfac® LM70/90 increased the inhibition of pathogenic E. coli compared to caraway oil alone. —« The highest rate of E. coli inhibition was obtained with the Surfac® LM70/90 doses of 0.13 to 0.5 g/L.

S » Both isomers of carvone inhibited significantly the growth of pathogenic E. coli, + while limonene had no effect on E. coli proliferation. 7 e Inhibition of B. pseudolongum with the caraway oil/Surfac& LM70/90 emulsion © 30 — was derived completely from Surfac.

E * Surfac® LM70/90 doses ranging from 0.03 to 1.0 g/L Surfac& LM70/90 inhibited

LO B. pseudolongum growth 50 to 70% from the Neg CTRL, while the lowest Surfac® s LM70/90 dose (0.01 g/L) did not have a considerable inhibitory effect.

N e Isomers of carvone and limonene had no effect of B. pseudolongum growth when

N 35 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 possible that caraway oil /Surfac® LM70/90 product would be absorbed from the gastroin- testinal tract before entering the lower intestine.

Background and rationale of the study

In study 1, we showed that the caraway oil /Surfac® LM70/90 emulsion 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 the oil.

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 limonene - affect the growth of selected microbes in the presence and absence of Surfac® LM70/90.

N

O

N

+

I

N

O

I a a

LO

5

LO

N

O

N

Materials and methods

Microbial cultures and treatments

The microbial growth inhibition was tested with pure microbial cul- tures in synthetic growth liquid media at 37°C. Two microbial species, pathogenic chicken isolate Escherichia coli 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 previ- ous 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 oil (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-(-)-Carvone (1.25g/L) 8. R-(-)-Carvone (1.25g/L) & Surfac& LM70/90 (1.0 g/L) 9. R-(-)-Carvone (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-(+)-Carvone (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)-(-)-Limonene (1.25g/L) & Surfac& LM70/90 (1.0 g/L)

S 18. (S)-(-)-Limonene (1.25g/L) & Surfac& LM70/90 (0.25 g/L) <+ 7 Bacterial growth inhibition test © 30 Microbial culture media were supplemented with caraway oil alone or

E with an emulsion of two doses of the caraway oil with final concentrations of 2.5

LO g/L and 1.25 g/L and Surfac® LM70/90 at eight doses (strongest dose 1.0 g/L) and s inoculated with 10% (v/v) of actively growing microbial cultures. For each micro-

N bial species, a culture without added test product was used as a negative control.

N 35 Also, the same eight doses of the Surfac® LM70/90 emulsifier were tested alone as a control in order to reveal the inhibitory effect of the pure Surfac.

In ‘Carvone and Limonene’ test, both (+)- and (-)-isomers of each com- pound were tested at a concentration of 1.25 g/L with and without the addition of

Surfac® LM70/90, which was applied at concentrations of 1.0 and 0.25 g/L.

All tests were run in atleast 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 indi- cator 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 following formula:

Growth (percentage, %) = 100 X (5. =)

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.

Significance according to Student's t-test is shown in the figures as fol- lows: —-p-value<0.05 * - p-value < 0.01 ** - p-value < 0.001 ***

Major findings — Escherichia coli (Figure 8)

O Theresults of F. coli KBAK1601/15 strain at 4-hour time pointis shown x in Figure 8.

K - Pure caraway oil showed a moderate dose-dependent inhibition of E. coli. The 7 30 — higher 2.5 g/L dose inhibited growth 34% and smaller 1.25 g/L dose only 11% i from the CTRL. 0 - Surfac® LM70/90 alone showed no inhibition of E. coli growth. In fact, the in- 5 crease of Surfac® LM70/90 concentration appeared stimulate dose-dependently

O the E. coli 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. coli 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. coli dose-dependently. 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 the inhibition rate of 32%. — Neither of the limonene isomers showed considerable inhibition of E. coli either in a presence or absence of Surfac. — On 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 coli 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 (Figure 9)

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

N — Results of this study confirmed the previous test results i.e. the inhibition of B. 5 pseudolongum with the caraway oil/Surfac® LM70/90 emulsion was derived ? completely from Surfac.

S 30 - As expected, the growth of B. pseudolongum was not inhibited by the two doses

E of caraway oil alone.

LO - The rate of inhibition was nearly identical with caraway oil/Surfac& LM70/90 s emulsion and Surfac& LM70/90 alone: 50 to 70% from the Neg CTRL with 0.03 to

N 1.0 g/L Surfac& LM70/90 concentration. On the other hand, the lowest Surfac&

N 35 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 carvone and 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. In- terestingly, the lower Surfac® LM70/90 dose showed slightly higher rate of inhi- bition 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. coli, while the inhibition of com- mensal B. pseudolongum was same for corresponding doses of Surfac® LM70/90 alone and caraway oil /Surfac® LM70/90 emulsion. This supports the earlier 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 oil/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. pseudolongum

N is a bacterium residing in the lower intestinal tract, while E. coli is found in high 5 quantities in small-intestine of warm-blooded animals. 7 Itis possible that caraway oil /Surfac® LM70/90 product exerts its in- © 30 hibitory effect in small-intestine and would be absorbed from the gastrointestinal

E tract before entering the lower intestine. In this case, the beneficial bifidobacteria

LO would not be negatively affected by the feed additive, and Surfac® LM70/90 con- s centration showing a significant boosting effect on E. coli inhibition (e.g. 0.1 g/L)

N could be considered to be applied in combination with caraway oil in the broiler

N 35 chicken feeding trial.

The results showed that both isomers of carvone inhibited significantly the growth of pathogenic E. coli, while limonene had no effect on E. coli prolifera- tion. This clearly indicates that carvone is the active inhibitory substance against gram-negative enterobacteria of the two dominant components presentin 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 together — with an emulsifier. - During the first three weeks the emulsifier 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 de- tail and the feed grade emulsifiers in 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 im- portance 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

N starter formulation was used for the first 2 weeks and the grower diet for the fol- 5 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

S 30 pens (Table 2). The test diets were sampled (pooling of 3 x 300g /diet) for the prox-

E imate analysis and for the own purposes of the client.

LO Proximate analyses were performed. Nutrient composition and ana- s lysed values of diets are shown in Table 3.

S

Table 2. Treatments and sampling control

Dose of Sampling of

Caraway birds

Tr | Treatment | Designation in Figures* oil/ Dose of Pens/ Birds/ 2 birds/pen # descrip- D-Carvone | emulsifier treat- treat- On days 14 and tion kg/tn kg/tn ment ment 35 a | Control | er | < | - | s | 20 | 164162 | Carawayoil | — Caraway-dose1 | 02 | oc | 8 | 20 | 16416 3 | Carawayoil | — Caraway-dosez | 05 | oc | 8 | 20 | 16416 14 | Carawayoil | — Caraway-dose3 | 125 | oc | 8 | 120 | 16416 - 5 | Caraway oil | Caraway + Surtac-dose1 | 02 | 002 | 8 | 120 | 16416

S 6 | Caraway oil | Caraway + Surtac-dose2 | 05 | 005 | 8 | 120 | 16416 3 7 | Caraway oil | Caraway +Surfac-dose3 | 125 | 0425 | 8 | 120 | 16416 = 8 | D-Carvone | — D-Carvone-dose1 | 043 | oc | 8 | 120 | 16416 : [9 | D-Carvone | — D-Carvone-dose2 | 081 | oc | 8 | 120 | 16416

E 10 | D-Carvone | D-Carvone + Surfac-dose1| 013 | 0013 | 8 | 120 | 16416 2 11] D-Carvone | D-Carvone + Surfac-dose2| 081 | 0081 | 8 | 120 | 16416 ? | Total | es | 4320 | sz

S 5 * Figures 10A-10C, 11A-11D, 12A-12D, 13, 14A-14D.

Table 3. Calculated and analysed values feeds

Starter % Grower % (d 0-14) (d 21-42)

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

NaCl 0.38 0.39

Mineral premix* 0.20 0.20

Vitamin premix** 0.23 0.20

DL-Methionine 0.27 0.21

L-Lysine HCl 0.18 0.20

Threonine 0.07 0.00

Total 100 100

Metabolisable energy (M]/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

Calcium 9.50 8.39

Non-phytate phosphorus 4.68 4.22

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

N Treat- Tr Tr Tr Tr Tr Tr Tr 7 Tr 8 Tr Tr Tr ment 1 2 3 4 5 6 9 10 11 <Q Dry 87.8 875 873 87.2 87.2 874 878 87.7 87.5 87.5 881

S matter

E Crude 223 235 237 229 233 233 234 236 229 224 233

LO protein s Crude 64 66 65 58 59 59 60 61 60 60 61

N fat

N 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

Nitro- 507 487 481 490 482 485 500 486 498 499 492 gen free extrac- tives

Growers (14-35days)

Treat- Tr Tr Tr Tr Tr Tr Tr 7 Tr 8 Tr Tr Tr ment 1 2 3 4 5 6 9 10 11

Dry 89.2 89.2 89.2 89.2 89.2 89.3 897 89.5 89.0 89.2 89.1 matter

Crude 201 214 217 207 205 217 204 207 214 213 211 protein

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

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

Nitro- 533 521 516 523 536 512 526 524 515 517 521 gen free extrac- tives

Table 3 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-

S 5 tate 30.0 g/kg, niacin 20.1 g/kg, panthotenic acid 7.51 g/kg, riboflavin 3.0 g/kg, 4 pyridoxine 2.01 g/kg, retinol 1.8 g/kg, menadione 1505 mg/kg, thiamine 1257 <Q mg/kg, folic acid 504 mg/kg, biotin 75.0 mg/kg, cholecalciferol, 56.3 mg/kg, cobal-

S amin 12.5 mg/kg.

E

W 10

S

Animals and housing

The 35-day trial was conducted in the broiler house of Alimetrics in

Southern Finland, on August-September 2019, in accordance with EU Directive 2010/63/EU and Alimetrics standard operating procedures for the running of an- imal studies. The temperature of the hall was raised to 32 °C two 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 during the first week. The temperature was gradually decreased to 22 °C over the rearing pe- riod. Temperature, ventilation and humidity were monitored and recorded throughout the trial on a daily basis. The dark hours were increased by one hour daily, until light-dark cycle was 18 hours light and 6 hours dark.

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 num- ber ofbirds 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 beginning of the trial.

The birds were observed twice a day throughout the trial and birds with compro- mised health were euthanized.

Performance parameters

The birds and feeds were weighed on days 0, 14, 21 and 35. Feed intake per pen and the feed conversion ratio (FCR) were measured for the following peri- ods: - Days 0-14, starter diet period - Days 14-21 early grower diet period

S - Days 21-35 late grower diet period + FCR was calculated both corrected and uncorrected for mortality.

O

S 30 Sampling

E On days 14 and 35 two randomly selected birds from all pens were eu-

LO thanised by cervical dislocation, abdominal cavity opened and digesta samples s were collected from ileum and caecum. All samples were stored and preserved in

N accordance with the reguirements for possible later analysis.

N 35

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 statistical 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 - On 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- pendently. 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 numerical effect (~14 grams) at low and high doses. (Figure 104) - Again, on day 21, no statistically significant effects were seen between the treat- ments 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 sig- nificant when analysed by t-test (p = 0.022). In this present trial we have no corre- sponding dose of D-carvone, but D-carvone at low dose with Surfac& LM70/90 im- proved 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

N broiler chickens. Panels A, B and C show the body weight on days 14, 21 and 35, 5 respectively. Error bars show SEM.

O

S 30 Effect of test products on mean body weight gain

E - There were not significant differences in mean body weight gain during the days

LO 0-14 between the tested products. Caraway oil if used with emulsifier improved s body weight gain numerically at low and high doses compared to control. Caraway

N oil used alone, tended to have dose dependent negative effect on growth of broiler

N 35 chickens. This was the case also with D-carvone whether or not Surfac& LM70/90 was used with the product. (Figure 11A)

- During the early grower diet period (days 14-21), caraway oil with emulsifier dif- fered statistically significantly at medium dose from high dose. The magnitude of improvement at high dose was 35 grams when compared to control. The dose dependent negative effect of D-carvone no longer existed. (Figure 11B) - 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 Sur- fac. The effects were not statistically significant by Tukey's test but the treatments improving body weights in section 3.1 improved also body weight gain when ana- lysed by the t-test. (Figure 11C) - The numerical improvement was seen also during the whole 35-day trial with all products, especially when emulsifier was present; 127 grams at high dose of cara- way 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 11D)

Figure 11 shows the effect of test products on the body weight gain of broiler chickens. Panels A, B, C and 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 HSD test.

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 HSD test. However, some nu- meric effects were seen. — - Feed consumption during the starter diet period (0-14 days) increased parallel to body weight gain, but thereafter the phenomenon gradually subsided. Overall,

S 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) 7 Figure 12 shows the effect of the test products on feed consumption of © 30 broiler chickens. Panels A, B, C and D show the feed consumption during the peri-

E ods 0-14, 14-21, 21-35 and 0-35 days, respectively. Error bars show SEM.

LO s Effect of test products on mortality

N - There were no significant differences between the test products on mortality, dur-

N 35 ingany growth period. (Figure 13)

Figure 13 shows the effect of test products on mortality of broiler chick- ens. 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

Only 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 signif- icantly from the control. The numeric improvement with caraway oil without emul- — sifier was 4 points. (Figure 14A) - Again, during the early grower diet period (days 14-21), caraway oil alone at me- dium dose improved statistically significantly mortality corrected FCR by 9 points compared to use at low dose. The positive effect can be explained by increased body weight gain. Almost egual improvement was seen also with caraway oil + Sur- fac&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 sim- ilar during the days 21-35 as seen during the previous period, though without sta- tistical significance due to high variation within the treatments. (Figure 14C) -Asseen earlier, during the whole 35-day trial several test products improved mor- tality-corrected FCR numerically, by 6 points. The positive effect was seen with caraway oil regardless of whether emulsifier was used or not. With D-carvone the 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 FCR was not as obvious.

N Figures 14A-14D show the effect of test products on mortality-cor- 5 rected feed conversion efficiency of broiler chickens. Figures 14A-14D show the 7 feed conversion efficiency with mortality correction during the growth periods O- © 30 14, 14-21, 21-35 and 0-35 days, respectively. Error bars show SEM and letters

E above columns the difference between the treatments by Tukey HSD test.

O

5

S

Study 4: Monitoring of possible caraway oil evaporation in pelleted feed during processing and storage

Major findings: e Results showed that carvone - the effective component of the caraway oil in mi- crobial inhibition - remained stable and did not evaporate from feed during pellet- ing and storage.

On the other hand, approximately 70% of limonene was evaporated from pelleted 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 processing and storage is promising and critical for proceeding with the planned animal feed- ing 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 indi- vidual caraway oil components in feed matrix was developed. e The analysis of caraway oil in feed showed that the carvone/limonene ratio in the product was 65:35 at Timepoint O (before pelleting).

Rationale of the study

The aim of this work was to find out whether caraway oil is evaporated from pelleted feeds during processing and storage when applied alone or in com- bination with Surfac& LM70/90 emulsifier. This information is essential in order — to determine the most suitable procedure to use caraway oil as an additive. = Materials and methods

S Caraway oil inclusion and feed manufacturing x Two 200 kg batches (with caraway oil only or caraway oil/Surfac&

N 30 LM70/90 combination) of pelleted wheat-based broiler diet were manufactured 7 for this study. Initially, wheat grains were ground to fine particles using a ham-

E mermill and transferred to a batch mixer. Subsequently, the rest of the feed ingre-

O dients including caraway oil (concentration of 2 kg/ton) and Surfac& LM70/90 5 (concentration of 0.2 kg/ton) pre-mixed with soybean oil was introduced gradually

O 35 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 tem- perature prior to the sampling.

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 (limonene & carvone). The time points of sampling were as follows: 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 analysis in feed

In order to accurately measure the concentration of caraway oil in feed and monitor its possible evaporation, an analytical method for caraway oil compo- nents (limonene & carvone) was developed and validated for Agilent gas chroma- tography (7890A) mass spectrometer (5975D). During the developmental work, caraway oil composition was characterised and possible analyte losses during sam- ple pre-treatment were determined. The relevant characteristics of the method were validated according to the general guidelines for animal feed analysis labora-

N tories (Ouality assurance for animal feed analysis laboratories. FAO Animal Pro- 5 duction and Health Manual No. 14. Rome). 7 In brief: a representative feed sample (5g) was mixed with 10 mL of wa- © 30 ter, 1 mLofmethanoland 1 mL of heptane containing the internal standard (1-tert-

E Butyl-1-cyclohexene, 2.5 mg/mL). Resulting mixture was extracted by strong agi-

LO tation with 24 mL of heptane for 40 min. After centrifugation (3 500 rpm for 5 min), s supernatant was collected for GC-MS analysis. To match the condition with sam-

N ples, procedural standards were used in calibration. The data (single ion monitor-

N 35 ing) were collected for limonene and carvone, which are the most relevant compo- nents in caraway oil.

Calculations

The quantity of the remaining caraway oil (C.0.) components (limonene & carvone) (in percentages to timepoint 0) was calculated with the following for- mula:

Remaining C. 0. component (percentage, %) = 100 x (2 of C.0.component at timepoint X ) g of C.0.component at timepoint 0)

Results

Limonene and carvone stability in feed with a mixture of caraway oil and Surfac& 1M70/90 — Approximately 40% of limonene 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 guantity of limonene remaining in feed at different timepoints presented as a percent from timepoint O (before pelleting). Hundred % (orange line) is the quantity of the limonene at timepoint 0.

Error bars indicate standard error of the mean of four replicates. — On 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 O (before pelleting). Hundred % (orange line) is the quantity of the carvone at timepoint 0. Error bars indicate standard error of the mean of four replicates.

N

N Limonene and carvone stability in feed with caraway oil only

S — Similarly to the feed with caraway oil/Surfac® LM70/90 mixture, approximately

S 40% of limonene evaporated from the feed with caraway oil during pelleting and z 30 before packing (Figure 17). + - The limonene evaporation continued gradually and about 70% of original limo- = nene was evaporated at the end of the 4-week storage period (Figure 17). = - On the other hand, carvone concentration remained very stable in feed with car- away oil only (Figure 18). Hence, itis evident that carvone was not affected by pel- leting and subsequent storage of 4 week.

Figure 17 shows the quantity of limonene remaining in feed at different timepoints presented as a percent from timepoint 0 (before pelleting). Hundred % (orange line) is the quantity of limonene at timepoint 0. Error bars indicate stand- ard 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 O (before pelleting). Hundred % (orange line) is the quantity of carvone at timepoint O. 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 bac- terial inhibitory potential of caraway oil

Major findings:

N — Unlike Surfac& LM70/90 that was used in the previous experiments, the new 5 surfactant candidates alone did not inhibit the growth of B. pseudolongum and E. 7 faecium. © 30 — — Generally, the three new surfactants alone did not show significant growth inhi-

E bition of E. coli and C. jejuni; however, slight trend-like inhibition growth was ob-

LO served at the highest 1 g/L concentration of Rapeseed lecithin. s — The addition of SABO-Nutreem and Castor oil surfactants did not significantly

N change the bacterial inhibition profile when tested with caraway oil as compared

N 35 to caraway oil alone.

— The combination of caraway oil and Rapeseed lecithin improved slightly the C. 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 ad- dition 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 the oil.

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 cara- — way oil/surfactant emulsions, which will be optimal for inhibiting the pathogenic bacterial growth while not affecting considerably the commensal intestinal bacte- ria. 2. Materials and methods 2.1 Microbial cultures and treatments

The microbial growth inhibition was tested with pure microbial cul- tures in synthetic growth liquid media at 37°C. Four microbial species, pathogenic chicken isolate Escherichia coli KBAK 1601/15, opportunistic pathogen Enterococ- cus faecium, pathogen Campylobacter jejuni DSM 4688, and commensal Bifidobac- — terium pseudolongum DSM 20099, were cultured in suitable media under appropriate conditions. The growth kinetics of each bacterial strain was evaluated

S during the previous projects. < 7 List of treatments and starting concentrations (strongest final doses): © 30 — 1. Negative CTRL (no amendments)

E 2. Caraway oil at 0.312-0.75 g/L

LO 3. Caraway oil & Surfac& LM70/90 at 8 doses from 1 g/L s 4. Caraway oil & surfactant - SABO-NUTREEM R495 at 8 doses from 1 g/L

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

N 35 — 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 - SABO-NUTREEM R495 at 8 doses from 1 g/L 9. Surfactant - EL 33 CASTOR OIL at 8 doses from 1 g/L 10. Surfactant - RAPESEED LECITHIN 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 emulsifiers were tested alone as a control to find out the inhibitory effect of the surfactants themselves. For each microbial species, a culture without added test products was 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 assessed by measuring the optical density (OD) of the culture at a wavelength of 600 nm for

Escherichia coli, Bifidobacterium pseudolongum, and Enterococcus faecium, where the increase in OD is an indicator of the proliferation of bacterial cells. For Campyl- obacter jejuni, which is a small-sized bacterium and does not elicit a significant increase in OD 600 nm, a method utilising DNA binding dye SYBR Green 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 vortexed 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 a loaded 10X-

S concentrated solution of oil and surfactant. < ? 2.3. Calculations

S 30 The growth (in percentages to control with no product amendments i.e.

E Neg. CTRL) was calculated with the following formula:

LO

3 Growth (percentage, %) = 100 X (ror) 3

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.

Significance 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 *** 3. Major findings 3.1. Escherichia coli (Figures 19A and 19B)

The growth of broiler chicken pathogenic E. coli KBAK1601/15 at a 4- hour time point is shown in Figures 19A and 19B. - The growth of E. coli was not significantly inhibited by any of the tested surfac- tants alone, except minor inhibition at the highest 1 g/L. concentration of rapeseed 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 coli

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

N The growth of commencial and beneficial B. pseudolongum DSM 20099 5 at a 6-hour time point is shown in Figures 20A and 20B. ? - The growth of B. pseudolongum was not inhibited by the three new surfactant

S 30 candidates alone. In contrast, the previously tested Surfac& LM70/90 inhibited

E bifidobacterial growth significantly, by 60% already at 0.06 g/L dose and by 85%

LO at 1 g/L dose. s - Pure caraway oil at 0.63 g/L dose showed a moderate 30% growth inhibition of

N B. pseudolongum from negative CTRL.

N 35 — - 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 rate as Surfac& 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 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. Enterococcus faecium (Figures 21A and 21B)

The growth of Enterococcus faecium at a 3-hour time point is shown in

Figures 21A and 21B. - Similar to B. pseudolongum, the growth of Enterococcus faecium was not inhib- ited by the three new surfactant candidates alone, in contrast to previously tested

Surfac® LM70/90 which dose-dependently 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 max- imum by 60% at 0.03-0.25 g/L of surfactant. - As expected, the addition of Surfac® LM70/90 to the oil inhibited the growth of

Enterococcus faecium significantly, by 60% already at 0.01 g/L of surfactant.

Figures 21A and 21 B show the growth of Enterococcus faecium 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

N product according to the Student's t-test. Hundred % growth (dashed line) is the 5 growth of negative CTRL in the absence of any test products.

O

S 30 3.3.Campylobacter jejuni (Figures 22A-22B and 23)

E The growth of Campylobacter jejuni strain DSM 4688 at a 24-hour time

LO point is shown in Figures 22A, 22B and 23. s - The growth of Campylobacter jejuni was slightly inhibited by SABO-Nutreem and

N Castor oil surfactants alone at the highest 1 g/L concentration. Small inhibition was

N 35 observed at the highest 0.5-1 g/L concentration of Rapeseed lecithin and average (up to 35% from negative CTRL) inhibition at 0.13-1g/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 a significant 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. Hundred % 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 in- dicate the standard error of 3 replicate wells and asterisks the statistical signifi- cance of the difference to negative CTRL with no test product according to the Stu- dent's t-test. Hundred % 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 the claims.

N

O

N

+

I

N

© 30

I a a

LO

5

LO

N

O

N 35

Claims (18)

1. A feed ingredient composition for animals, comprising R-(-)-Carvone or S-(+)-Carvone, or a combination thereof, and at least one surfactant.

2. A feed ingredient composition according to claim 1, wherein the R-(- )-Carvone or S-(+)-Carvone, or a combination thereof, is included in the composi- tion in the form of a Caraway -based raw material.

3. A feed ingredient composition according to claim 2, wherein the Car- away -based raw material is Caraway oil.

4. A feed ingredient composition according to any one of the preceding — claims 1-3, wherein the composition further comprises water.

5. A feed ingredient composition according to any one of the preceding claims 1-4, wherein the composition is an emulsion.

6. A feed ingredient composition according to claim 5, wherein the at least one surfactant has a hydrophilic-lipophilic balance (HLB) value of 8-12.

7. A feed ingredient composition according to claim 5 or 6, wherein the R-(-)-Carvone or S-(+)-Carvone, or a combination thereof, is/are included in the composition as such or in the form of 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- 5g/L more preferably 0.25-2.5 g/L, most preferably 0.25-1.0 g/L in the emulsion.

8. A feed ingredient composition according to any one of the preceding claims 1-7, wherein the atleast one surfactant is selected from atleast one ofa C12- C15 alcohol ethoxylate, hydrolysed lecithin, PEG glyceryl ricinoleate, and castor oil, preferably a C12-C15 alcohol ethoxylate.

9. A feed ingredient composition according to claim 3, wherein the Car- - away -based raw material is Caraway powder or a water-extract thereof. O

10. Use of a feed ingredient composition according to any one of the + claims 1-9 as a microbial growth inhibitor in animal feed or animal drinking water. 7

11. Use according to claim 10, for microbial growth inhibition of atleast S 30 = one of Salmonella enterica, Eschericia coli, Campylobacter jejuni, Clostridium E perfringens, Bifidobacterium pseudolongum, Enterococcus faecium. 10

12. A feed comprising a feed ingredient composition according to any > one of the claims 1-9. N

13. A feed according to claim 12, wherein the feed is in pellet form. N 35

14. A feed according to claim 12 or 13, wherein the feed is for animals, such as chicken.

15. A feed according to any one of the claims 12-14, wherein the feed is wheat-soy-based.

16. Drinking water or pellet feed for animals comprising a feed ingredi- ent composition according to any one of the claims 1-9.

17. Method of making a wheat-soy-based feed pellet, comprising grind- ing wheat grains and mixing the resulting wheat particles with the feed ingredient composition according to any one of claims 1-9 and soybean oil, and pelleting the obtained mixture.

18. Method according to claim 17, wherein the feed ingredient compo- sition according to any one of claims 1-9 is caraway oil. N O N + I N O I a a LO 5 LO N O N