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EP2453758A1 - Use of natural substances as feed additives for aquatic animals - Google Patents

Use of natural substances as feed additives for aquatic animals

Info

Publication number
EP2453758A1
EP2453758A1 EP10734483A EP10734483A EP2453758A1 EP 2453758 A1 EP2453758 A1 EP 2453758A1 EP 10734483 A EP10734483 A EP 10734483A EP 10734483 A EP10734483 A EP 10734483A EP 2453758 A1 EP2453758 A1 EP 2453758A1
Authority
EP
European Patent Office
Prior art keywords
alpha
feed
pinene
cinnamaldehyde
cresol
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP10734483A
Other languages
German (de)
French (fr)
Inventor
Marco Frehner
Viviane Verlhac-Trichet
Philippe Narbel
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
DSM IP Assets BV
Original Assignee
DSM IP Assets BV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by DSM IP Assets BV filed Critical DSM IP Assets BV
Priority to EP10734483A priority Critical patent/EP2453758A1/en
Publication of EP2453758A1 publication Critical patent/EP2453758A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/105—Aliphatic or alicyclic compounds
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/158—Fatty acids; Fats; Products containing oils or fats
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K40/00—Shaping or working-up of animal feeding-stuffs
    • A23K40/30—Shaping or working-up of animal feeding-stuffs by encapsulating; by coating
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics

Definitions

  • the present invention relates to the use of natural active substances selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, di- hydroeugenol, eugenol, meta-cresol and terpinolene in the manufacture of feed for aquatic animals including fish and shrimp, especially for cold water fish as for example salmon, bream, bass and for warm water fish as for example carp, tilapia, catfish.
  • this invention relates to the use of at least two substances as defined above for the improvement of the feed conversion ratio and/or daily weight gain in fish, for reducing mortality by regulating the micro flora of the gut and/or by protecting the animal against infections caused by pathogenic microorganisms.
  • the present invention relates to a novel fish feed composition
  • a novel fish feed composition comprising as active ingredients at least two, preferably at least three or four active compound(s) selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene.
  • feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal.
  • Turnover rate is deter- mined by how fast the fish grow to a harvestable size. As an example, it takes from 12 to 18 months to raise Atlantic salmon from smolt (the physiological stage when the Atlantic salmon can first be transferred from fresh water to sea water) to harvestable size. A fast turnover has several positive results. First, it helps cash - -
  • patent WO2004/091307 describes the use of polyphenols, and other natural actives in feed to increase survival rate of Artemia after hatching.
  • WO2004/091307 is however silent with regard to the selection of compounds to be used.
  • the application of polyphenols to reduce mortality is in the disclosed case above only useful at time of hatching. It therefore remains a need in aquaculture to prevent the development of diseases, thereby reducing mortality by any prophylactic means including antimicrobial activity at the gut level.
  • the inventors of the present application surprisingly found that substances as de- fined above have a great potential for use in fish feed, e.g. for improving the feed conversion ratio (FCR) and/or weight gain and/or for the modulation of the gut flora. Further, the inventors surprisingly found that the substances, which are hereinafter also referred to as compounds, have also antimicrobial activity result- - -
  • the invention relates to methods for using at least two active compounds selected from the group consisting of alpha- pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in fish feed for improving the Feed Conversion Ratio (FCR) and/or weight gain and/or for reducing mortality by modulation of the gut micro- flora and/or by preventing diseases caused by pathogenic microorganisms.
  • FCR Feed Conversion Ratio
  • selected compounds of the invention eg.: cinnamaldehyde, dihydroeugenol, eugenol, and meta-cresol exhibit excellent effects inhibiting the growth of Yersinia ruckeri, a pathogenic microorganism which causes Enteric Redmouth (ERM), a disease found especially in salmonids and causing high mortality.
  • ERM Enteric Redmouth
  • alpha-pinene and/or alpha-terpineol and/or cinnamaldehyde and/or dihydroeugenol and/or eugenol and/or meta-cresol and/or terpinolene is/are used to improve animal feed digestibility and/or maintain animal health by supporting immune system function.
  • the invention relates to methods for using at least three active compounds selected from the group consisting of alpha-pinene, alpha- terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in fish feed for improving the Feed Conversion Ratio (FCR) and/or weight gain and/or for reducing mortality by modulation of the gut microflora and/or by preventing diseases caused by pathogenic microorganisms.
  • FCR Feed Conversion Ratio
  • alpha-pinene and cinnamaldehyde are used to im- prove animal feed digestibility and/or maintain animal health by supporting immune system function.
  • the FCR may be determined on the basis of a fish growth trial comprising a first treatment in which a mixture of at least two compounds according to the invention is added to the animal feed in a suitable concentration per kg feed, and a second treatment (control) with no addition of the compound(s) to the animal feed.
  • an improved FCR is lower than the control FCR.
  • the FCR is improved (i.e., reduced) as compared to the control by at least 1.0 %, preferably at least 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2.0 %, 2.1 %, 2.2 %, 2.3 %, 2.4 %, or at least 2.5 %.
  • gut designates the gastrointestinal or digestive tract (also referred to as the alimentary canal) and it refers to the system of organs within multi-cellular animals which takes in food, digests it to extract energy and nutrients, and expels the remaining waste.
  • gut “microflora” refers to the natural microbial cultures residing in the gut and maintaining health by aiding in proper digestion.
  • module as used herein in connection with the gut microflora generally means to change, manipulate, alter, or adjust the function or status thereof in a healthy and normally functioning animal, i.e. a non-therapeutic use.
  • supporting immune system function refers to the immune stimulation effect obtained by the compounds.
  • the term "mortality" as used herein refers to the ratio of life animals at the end of the growth phase versus the number of animals originally included into the pond. It may be determined on the basis of a fish challenge trial comprising two groups of fish challenged by a particular fish pathogen with the aim to provoke a mortality of 40 to 80 % of the animals in the untreated group. However, in the challenge group fed with a suitable concentration per Kg of feed of a mixture of at least two compounds according to the invention, the mortality is reduced compared to the - -
  • untreated group by at least 5 %, preferably at least, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, or at least 50 %.
  • the inventors of the present application surprisingly found that the compounds according to the invention and mixtures thereof are effective against a number pathogenic microorganisms of cold and warm water fish.
  • Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, meta-cresol and terpinolene were shown to exhibit inhibitory effect against Vibrio anguillarum, a shrimp pathogen causing vibriosis.
  • Alpha-pinene, cinnamaldehyde, dihydroeugenol, eugenol, limonene, and meta- cresol were shown to exhibit inhibitory effect against Aeromonas salmonicida which is the pathogen causing a disease known as furunculosis.
  • Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta- cresol and terpinolene were shown to exhibit inhibitory effect against Edwardsellia tarda causing systemic infection in fish.
  • Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, and terpinolene were shown to exhibit inhibitory effect against Lactococcus garvieae which is the etiological agent of Latococcosis, an emergent disease which affects many fish species and causes important economic losses both in marine and freshwater aquaculture when water temperature increases over 16 0 C in summer months.
  • Cinnamaldehyde, dihydroeugenol, eugenol, and meta-cresol exhibit excellent inhibitory effects on the growth of Yersinia ruckeri, a pathogenic microorganism which causes Enteric Redmouth (ERM), a disease found especially in salmonids. Cinnamaldehyde and meta-cresol were shown to exhibit inhibitory effect against:
  • Aeromonas hydrophila causing ulcers and hemorrhagic septicaemia. This pathogen is very resistant to conventional simple antimicrobials like chlorine.
  • Photobacte ⁇ um damselae formerly Pasteurella piscicida a pathogen causing high losses in the culture industry of economically important marine fishes such as seriola and red grouper in Japan and striped bass and white perch in the United States.
  • the present invention provides a fish feed composition
  • a fish feed composition comprising at least two active compound selected from the group consisting of alpha- pinene (CAS 99-86-5), alpha-terpineol (CAS 98-55-5), cinnamaldehyde (CAS 14371 -10-9 / 104-55-2), dihydroeugenol (CAS 2785-87-7), eugenol (CAS 97-53- 0), meta-cresol (CAS 108-39-4) and terpinolene (CAS 554-61 -0).
  • the compounds according to the invention are commercially available or can easily be prepared by a skilled person using processes and methods well-known in the prior art.
  • the compounds of the invention can be used alone or in mixtures thereof, in the form of natural available extracts or extract-mixtures or in the form of a natural substance. - -
  • extract as used herein includes compositions obtained by solvent extraction (which are also known as “extracted oils”), steam distillation (which are also known as “essential oils”) or other methods known to the skilled person.
  • Suitable extraction solvents include alcohols such as ethanol.
  • natural is in this context understood a substance which consists of compounds occurring in nature and obtained from natural products or through synthesis.
  • active compound(s) or natural substance or extract further ingredients may be added in minor amounts.
  • examples of such ingredients are: capsaicin, tannin, piperin, trimethylamine, 3,4,xylenol, furfuryl alcohol and mixtures thereof.
  • cinnamal- dehyde and/or meta-cresol are used as a major component of the mixture.
  • the mixture contains 10-90 % by weight of meta-cresol and/or cinnamalde- hyde, 1 -50 % by weight of alpha-pinene, 1 -50 % by weight of dihydro-eugenol, wherein the amounts being calculated on the total amount of said components.
  • the total amount of these active ingredients may vary within wide limits but is fi- nally used in the fish feed from 10 to 5000 ppm, preferably between 100 and 1000 ppm, calculated on the dry weight of the fish feed.
  • the most preferred mixture of at least two compounds as specified above comprises, cinnamaldehyde and alpha-pinene as a major component of the mixture.
  • the mixture contains 30-70 % by weight of alpha-pinene, 30-70 % by weight cinnamaldehyde, 1 -20 % by weight of metacresol, 1 -20 % by weight of dihydro-eugenol, wherein the amounts being calculated on the total amount of said components.
  • the total amount of these active ingredients may vary within wide limits but is finally used in the fish feed from 10 to 5000 ppm, preferably between 100 and 1000 ppm, calculated on the dry weight of the fish feed.
  • All compounds defined herein above may be used in combination with an emulsifying surfactant.
  • agent can be selected advantageously from those of a rather hydrophilic nature, for example among polyglycerol esters of fatty acids such as estehfied ricinoleic acid or propylene glycol esters of fatty acids, saccharo-esters or saccharo- glycerides, polyethylene glycol, lecithins etc.
  • polyglycerol esters of fatty acids such as estehfied ricinoleic acid or propylene glycol esters of fatty acids
  • saccharo-esters or saccharo- glycerides polyethylene glycol, lecithins etc.
  • a mixture of active compounds may contain 20 % by weight of cinnamaldehyde, 20 % by weight of meta-cresol, 20 % by weight of dihydro-eugenol, 20 % by weight of alpha-pinene, 3 % by weight of trimethylamine, 1.8 % by weight of piperin and 4 % by weight of furfuryl alcohol.
  • a mixture of active compounds contains 40 to 60 wt.-% alpha-pinene, 40 to 60 wt.-% cinnamaldehyde, and may further contain 1 to 5 wt.-% trimethylamine, 1 to 5 % piperin, and 3 to 8 wt.-% furfuryl alcohol.
  • the incorporation of the fish feed composition containing the active compound(s) into the fish feed may be performed as described in example 1.
  • the mixture of active compounds is then prepared directly as an oil which is then mixed with the oil sprayed onto the feed pellets as described in example 1.
  • the incorporation of the fish feed composition containing the active compound(s) into the fish feed may alternatively be carried out by preparing a premix of the active ingredients and other suitable additives.
  • a premix may comprise 2-10 % by weight of the active mixture or natural substance or extract, 0-40 % by weight of other conventional additives, such as flavorings, and 50-98 % by weight of any conventional absorbing support.
  • the support may contain, for example, 40-50 % by weight of wood fibers, 8-10 % by weight of stearin, 4-5 % by weight of curcuma powder, 4-5 % by weight of rosemary powder, 22-28 % by weight of limestone, 1 -3 % by weight of a gum, such as gum Arabic, 5-50 % by weight of sugar and/or starch and 5-15 % by weight of water.
  • a gum such as gum Arabic
  • This premix is then mixed with vitamins, as for example vitamin C, mineral salts and other feed additive ingredients, as for example yeast extracts containing nucleotides, glucan and other gut microflora modulators and then finally added to the feed in such quantities that the feed will contain 10-5000 ppm, preferably 100- 1000 ppm or 100-500 ppm of the active ingredients according to the invention.
  • the composition of the present invention will be preferably used together with yeast extract containing nucleotides, and glucan.
  • feed-additive ingredients are coloring agents, e.g. carotenoids such as beta-carotene, astaxanthin, and lutein; aroma compounds; stabilisers; antimicrobial peptides; polyunsaturated fatty acids; and/or at least one enzyme selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4.), phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4);
  • lysophospholipase (EC 3.1.1.5); phospholipase C (EC 3.1.4.3); phospholipase D
  • amylase such as, for example, alpha-amylase (EC 3.2.1.1 ); and/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).
  • polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.
  • the fish feed as described herein has a proximate composition of 20-60 wt.-% protein, and 1 -45 wt.-% moisture and lipid.
  • the fish feed comprises one or more of sources of:
  • - protein, carbohydrate and lipid for example, fish meal, fish oil, blood meal, feather meal, poultry meal, chicken meal and/or other types of meal produced from other slaughterhouse waste
  • - vegetable meal e.g. soya meal, lupin meal, pea meal, bean meal, rape meal and/or sunflower meal
  • soya meal e.g. soya meal, lupin meal, pea meal, bean meal, rape meal and/or sunflower meal
  • - gluten e.g. wheat gluten or corn gluten
  • fish feed as used herein includes a fish feed composition according to the invention and components as described above.
  • fish feed includes fish meal as a component.
  • fish feed is in the form of flakes or pellets, for example extruded pellets.
  • the invention in a third aspect, relates to a feed composition for aquatic animals and to the use of this composition for feeding fish.
  • the feed is particularly suitable for feeding salmonids, including Atlantic salmon (Salmo salar), other salmon species and trout, and non-salmonids such as cod, sea bass, sea bream and eel.
  • salmonids including Atlantic salmon (Salmo salar), other salmon species and trout, and non-salmonids such as cod, sea bass, sea bream and eel.
  • it can be fed to all types of fish, for example turbot, halibut, yellow tail and tuna.
  • the main raw materials are ground and mixed. Microingredients are then added to the mixer and the homogenous mix is conditioned by adding water and steam to the mass in a preconditioner. This starts a cooking process in the starch fraction (the binding component).
  • the mass is fed into a pellet mill. The mass is forced through the mill's die and the strings are broken into pellets on the outside of the die. The moisture content is low and drying of the feed is not necessary.
  • Additional oil including a fish feed composition according to the present invention is then sprayed onto the surface of pellets, but as the pellets are rather compact, the total lipid content rarely exceeds 24 %.
  • the added oil may be fish oil or vegetable oils, for example rape seed oil or soy oil, or a mixture of vegetable oils or a mixture of fish oil and vegetable oils.
  • the pellets are cooled in a cooler and bagged.
  • the final pressed fish feed contains 10 to 5000 ppm of the composition as described in the invention.
  • the main raw materials are ground and mixed.
  • Micro ingredients incl. a fish feed composition according to the invention are added to the mixer.
  • the homogenous mix is conditioned by adding water and steam to the mass in a preconditioner. Additional oil may also be added to the mass at this stage. This starts a cooking process in the starch fraction (the binding component).
  • the mass is fed into an extruder.
  • the extruder may be of the single screw or the twin-screw type. Due to the rotational movement of the mass in the extruder, the mass is further mixed. Additional oil, water and steam may be added to the mass in the extruder. At the end of the extruder, the mass has a temperature above 100 0 C and a pressure above ambient pressure.
  • the mass is forced through the openings in the extruder's die plate. Due to the relief in temperature and pressure, some of the moisture will evaporate immediately (flash off) and the extruded mass becomes porous.
  • the strings are cut into pellets by a rotating knife. The water content is rather high (18-28 %) and the pellets are therefore immediately dried to approximately 10 % water content in a dryer.
  • oil may be added to the feed by spraying oil onto the surface of the feed, or by dipping the feed in oil. It is advantageous to add the oil to the feed in a closed vessel where the air pressure is below ambient (vacuum coating) so that the porous feed pellets absorb more oil. Feed containing more than 40 % lipid may be produced this way. After the coater, the feed is cooled and bagged. Oil may be added at several places in the process as explained above, and may - -
  • fish oil or vegetable oils by example rape seed oil or soy oil, or a mixture of vegetable oils or a mixture of fish oil and vegetable oils.
  • the dominant protein source in dry feed for fish has been fish meal of different qualities.
  • Other animal protein sources are also used for dry fish feed.
  • chicken meal typically cheaper than fish meal and fish oil.
  • vegetable protein such as wheat gluten, maize (corn) gluten, soya protein, lupin meal, pea meal, bean meal, rape meal, sunflower meal and rice flour.
  • Example 3 Evaluation of the effect of the active compounds according to the invention on survival of Yersinia ruckeri, Vibrio anguillarum, Vibrio sal- monicida, Aeromonas salmonicida, Aeromonas hydrophila, Edwardsellia tarda, Photobacterium damselae, Lactococcus garvieae, Streptococcus iniae. - -
  • composition of the invention The antimicrobial activity of the composition of the invention towards Yersinia ruckeri, Vibrio anguillarum, Vibrio salmonicida, Aeromonas salmonicida, Aeromo- nas hydrophila, Edwardsellia tarda, Photobacterium damselae, Lactococcus gar- vieae, and Streptococcus iniae were determined in vitro.
  • the essential oil combination was the following one: alpha-pinene, cinnamalde- hyde, dihydroeugenol and meta-cresol at equivalent doses.
  • the experimental diets were fed for four weeks prior to challenge and after challenge until the end of the experiment. Fish weight was recorded at the start of experimental feeding, before challenge and at the end of the experiment on the remaining fish. - -
  • Challenge experiment The challenge dose was determined in a preliminary experiment, on the same fish population and under the same conditions. After a period of 4 weeks of experimental feeding, 25 fish were randomly distributed into four 38 I glass aquaria. Fish from each experimental tank were then anaesthetized and challenged by i.p. injection of Aeromonas salmonicida at a dose of 1.2 * 102 cfu/fish determined by spectrophotometry. The daily mortality was then recorded over a period of three weeks. First dead fish from each glass aquarium were subjected to a bacteriological investigation to confirm the bacteriological aetiology causing death. At the end of the experiment, all surviving fish were assessed for external signs of infection and in addition from 6 fish per diet group a necropsy and bacteriology were performed.
  • Example 6 Effect of an essential oil combination comprising alpha-pinene and cinnamaldehyde on the mortality of rainbow trout challenged with Aeromonas salmonicida.
  • a challenge experiment with Aeromonas salmonicida has been set up with juvenile rainbow trout in order to test the effect of alpha-pinene and cinnamaldehyde supplemented to the feed.
  • Challenge experiment The challenge dose was determined in a preliminary experiment, on the same fish population and under the same conditions. After a period of 4 weeks of experimental feeding, 25 fish were randomly distributed into four 38 I glass aquaria. Fish from each experimental tank were then anaesthetized and challenged by i.p. injection of Aeromonas salmonicida at a dose of 5 * 10 3 cfu/fish determined by spectrophotometry. The daily mortality was then recorded over a period of three weeks. First dead fish from each glass aquarium were sub- - - -

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Abstract

The present invention relates to the use of natural active substances selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in the manufacture of a feed composition for aquatic animals, especially for cold water fish as for example salmon, bream, bass and for warm water fish as for example carp, tilapia, catfish. More particular, this invention relates to the use of a substance as defined above for the improvement of the feed conversion ratio and/or daily weight gain in fish, for regulating the micro flora of the gut and for protecting the animal against infections caused by pathogenic microorganisms.

Description

USE OF NATURAL SUBSTANCES AS FEED ADDITIVES FOR AQUATIC
ANIMALS
The present invention relates to the use of natural active substances selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, di- hydroeugenol, eugenol, meta-cresol and terpinolene in the manufacture of feed for aquatic animals including fish and shrimp, especially for cold water fish as for example salmon, bream, bass and for warm water fish as for example carp, tilapia, catfish.
More particular, this invention relates to the use of at least two substances as defined above for the improvement of the feed conversion ratio and/or daily weight gain in fish, for reducing mortality by regulating the micro flora of the gut and/or by protecting the animal against infections caused by pathogenic microorganisms.
Furthermore, the present invention relates to a novel fish feed composition comprising as active ingredients at least two, preferably at least three or four active compound(s) selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene.
The term feed or feed composition means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal.
One important factor in aquaculture is the turnover rate. Turnover rate is deter- mined by how fast the fish grow to a harvestable size. As an example, it takes from 12 to 18 months to raise Atlantic salmon from smolt (the physiological stage when the Atlantic salmon can first be transferred from fresh water to sea water) to harvestable size. A fast turnover has several positive results. First, it helps cash - -
flow. Second, it improves risk management. Especially, a high mortality rate is a substantial risk for fish farmers.
It is generally known that mortality rate increases by an unbalanced microflora and/or by infections caused by pathogenic microbes. Fish diseases are common, and the likelihood of an outbreak is higher over a long growing period. There is also a risk that fish will escape due to accidents, e.g. when shifting nets, or due to bad weather causing wrecked fish pens. For other farm animals it is well known to use antibiotics and vaccines to prevent the development of diseases. In aquaculture, antibiotics are not so much used - at least in cold water aquaculture - due to the fact that disease spread very quickly, diseased fish do not eat much and also due to the negative impact on the environment of the wasted medicated feed. Vaccines are widely used when available but they are not developed for all diseases.
As an alternative to synthetic drugs, the use of plant extracts and essential oils in animal feed is described in the literature. For example, patent WO2004/091307 describes the use of polyphenols, and other natural actives in feed to increase survival rate of Artemia after hatching. WO2004/091307 is however silent with regard to the selection of compounds to be used. Moreover, the application of polyphenols to reduce mortality is in the disclosed case above only useful at time of hatching. It therefore remains a need in aquaculture to prevent the development of diseases, thereby reducing mortality by any prophylactic means including antimicrobial activity at the gut level.
The inventors of the present application surprisingly found that substances as de- fined above have a great potential for use in fish feed, e.g. for improving the feed conversion ratio (FCR) and/or weight gain and/or for the modulation of the gut flora. Further, the inventors surprisingly found that the substances, which are hereinafter also referred to as compounds, have also antimicrobial activity result- - -
ing in a reduced mortality. The unique selection of active compounds of the present invention allows for the first time controlling a number of fish diseases caused by a number of different pathogens. Therefore, in a first particular embodiment, the invention relates to methods for using at least two active compounds selected from the group consisting of alpha- pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in fish feed for improving the Feed Conversion Ratio (FCR) and/or weight gain and/or for reducing mortality by modulation of the gut micro- flora and/or by preventing diseases caused by pathogenic microorganisms. For example, it has been shown that selected compounds of the invention (eg.: cinnamaldehyde, dihydroeugenol, eugenol, and meta-cresol exhibit excellent effects inhibiting the growth of Yersinia ruckeri, a pathogenic microorganism which causes Enteric Redmouth (ERM), a disease found especially in salmonids and causing high mortality.
In alternative embodiments, alpha-pinene and/or alpha-terpineol and/or cinnamaldehyde and/or dihydroeugenol and/or eugenol and/or meta-cresol and/or terpinolene is/are used to improve animal feed digestibility and/or maintain animal health by supporting immune system function.
In another embodiment, the invention relates to methods for using at least three active compounds selected from the group consisting of alpha-pinene, alpha- terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in fish feed for improving the Feed Conversion Ratio (FCR) and/or weight gain and/or for reducing mortality by modulation of the gut microflora and/or by preventing diseases caused by pathogenic microorganisms.
In a preferred embodiment, alpha-pinene and cinnamaldehyde are used to im- prove animal feed digestibility and/or maintain animal health by supporting immune system function. - -
The FCR may be determined on the basis of a fish growth trial comprising a first treatment in which a mixture of at least two compounds according to the invention is added to the animal feed in a suitable concentration per kg feed, and a second treatment (control) with no addition of the compound(s) to the animal feed.
As it is generally known, an improved FCR is lower than the control FCR. In particular embodiments, the FCR is improved (i.e., reduced) as compared to the control by at least 1.0 %, preferably at least 1.5 %, 1.6 %, 1.7 %, 1.8 %, 1.9 %, 2.0 %, 2.1 %, 2.2 %, 2.3 %, 2.4 %, or at least 2.5 %.
The term "gut" as used herein designates the gastrointestinal or digestive tract (also referred to as the alimentary canal) and it refers to the system of organs within multi-cellular animals which takes in food, digests it to extract energy and nutrients, and expels the remaining waste.
The term gut "microflora" as used herein refers to the natural microbial cultures residing in the gut and maintaining health by aiding in proper digestion.
The term "modulate" as used herein in connection with the gut microflora generally means to change, manipulate, alter, or adjust the function or status thereof in a healthy and normally functioning animal, i.e. a non-therapeutic use.
The term "supporting immune system function" as used herein refers to the immune stimulation effect obtained by the compounds.
The term "mortality" as used herein refers to the ratio of life animals at the end of the growth phase versus the number of animals originally included into the pond. It may be determined on the basis of a fish challenge trial comprising two groups of fish challenged by a particular fish pathogen with the aim to provoke a mortality of 40 to 80 % of the animals in the untreated group. However, in the challenge group fed with a suitable concentration per Kg of feed of a mixture of at least two compounds according to the invention, the mortality is reduced compared to the - -
untreated group by at least 5 %, preferably at least, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, or at least 50 %.
In particular, the inventors of the present application surprisingly found that the compounds according to the invention and mixtures thereof are effective against a number pathogenic microorganisms of cold and warm water fish. Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, meta-cresol and terpinolene were shown to exhibit inhibitory effect against Vibrio anguillarum, a shrimp pathogen causing vibriosis.
Alpha-pinene, cinnamaldehyde, dihydroeugenol, eugenol, limonene, and meta- cresol were shown to exhibit inhibitory effect against Aeromonas salmonicida which is the pathogen causing a disease known as furunculosis. Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta- cresol and terpinolene were shown to exhibit inhibitory effect against Edwardsellia tarda causing systemic infection in fish.
Alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, and terpinolene were shown to exhibit inhibitory effect against Lactococcus garvieae which is the etiological agent of Latococcosis, an emergent disease which affects many fish species and causes important economic losses both in marine and freshwater aquaculture when water temperature increases over 16 0C in summer months.
Cinnamaldehyde, dihydroeugenol, eugenol, and meta-cresol exhibit excellent inhibitory effects on the growth of Yersinia ruckeri, a pathogenic microorganism which causes Enteric Redmouth (ERM), a disease found especially in salmonids. Cinnamaldehyde and meta-cresol were shown to exhibit inhibitory effect against:
(i) Vibrio salmonicida which is a psychrophilic bacterium that is the
causative agent of cold-water vibriosis in Atlantic salmon. - -
(ii) Aeromonas hydrophila causing ulcers and hemorrhagic septicaemia. This pathogen is very resistant to conventional simple antimicrobials like chlorine.
(iii) Photobacteήum damselae formerly Pasteurella piscicida: a pathogen causing high losses in the culture industry of economically important marine fishes such as seriola and red grouper in Japan and striped bass and white perch in the United States.
(iv) Streptococcus iniae which is highly pathogenic in marine fish and is highly lethal: outbreaks may be associated with 30-50 % mortality.
Other aquatic pathogens such as
(i) Pisciήckettsia salmonis the causative agent of piscirickettsiosis or salmonid rickettsial septicaemia (SRS),
(ii) Vibrio viscosus recently renamed Moritella viscosa etiologically re- sponsible for the disease referred to as "winter ulcer",
(iii) lch (parasite) one of the most prevalent protozoan parasites of fish, (iv) Vibrio harveyi, responsible for luminous vibriosis, a disease that affects commercially-farmed prawns
will also be inhibited by the compound mixture described in the present invention.
In a second aspect, the present invention provides a fish feed composition comprising at least two active compound selected from the group consisting of alpha- pinene (CAS 99-86-5), alpha-terpineol (CAS 98-55-5), cinnamaldehyde (CAS 14371 -10-9 / 104-55-2), dihydroeugenol (CAS 2785-87-7), eugenol (CAS 97-53- 0), meta-cresol (CAS 108-39-4) and terpinolene (CAS 554-61 -0). The compounds according to the invention are commercially available or can easily be prepared by a skilled person using processes and methods well-known in the prior art.
As fish feed composition, the compounds of the invention can be used alone or in mixtures thereof, in the form of natural available extracts or extract-mixtures or in the form of a natural substance. - -
The term "extract" as used herein includes compositions obtained by solvent extraction (which are also known as "extracted oils"), steam distillation (which are also known as "essential oils") or other methods known to the skilled person. Suitable extraction solvents include alcohols such as ethanol.
The term "natural" is in this context understood a substance which consists of compounds occurring in nature and obtained from natural products or through synthesis. To the active compound(s) or natural substance or extract further ingredients may be added in minor amounts. Examples of such ingredients are: capsaicin, tannin, piperin, trimethylamine, 3,4,xylenol, furfuryl alcohol and mixtures thereof.
If a mixture of at least two compounds as specified above is preferred, cinnamal- dehyde and/or meta-cresol are used as a major component of the mixture. Suitably the mixture contains 10-90 % by weight of meta-cresol and/or cinnamalde- hyde, 1 -50 % by weight of alpha-pinene, 1 -50 % by weight of dihydro-eugenol, wherein the amounts being calculated on the total amount of said components. The total amount of these active ingredients may vary within wide limits but is fi- nally used in the fish feed from 10 to 5000 ppm, preferably between 100 and 1000 ppm, calculated on the dry weight of the fish feed.
The most preferred mixture of at least two compounds as specified above comprises, cinnamaldehyde and alpha-pinene as a major component of the mixture. Suitably the mixture contains 30-70 % by weight of alpha-pinene, 30-70 % by weight cinnamaldehyde, 1 -20 % by weight of metacresol, 1 -20 % by weight of dihydro-eugenol, wherein the amounts being calculated on the total amount of said components. The total amount of these active ingredients may vary within wide limits but is finally used in the fish feed from 10 to 5000 ppm, preferably between 100 and 1000 ppm, calculated on the dry weight of the fish feed.
All compounds defined herein above (active compounds and additional ingredients) may be used in combination with an emulsifying surfactant. The emulsifying - -
agent can be selected advantageously from those of a rather hydrophilic nature, for example among polyglycerol esters of fatty acids such as estehfied ricinoleic acid or propylene glycol esters of fatty acids, saccharo-esters or saccharo- glycerides, polyethylene glycol, lecithins etc.
In a preferred embodiment of the invention, a mixture of active compounds may contain 20 % by weight of cinnamaldehyde, 20 % by weight of meta-cresol, 20 % by weight of dihydro-eugenol, 20 % by weight of alpha-pinene, 3 % by weight of trimethylamine, 1.8 % by weight of piperin and 4 % by weight of furfuryl alcohol. In another preferred embodiment of the invention, a mixture of active compounds contains 40 to 60 wt.-% alpha-pinene, 40 to 60 wt.-% cinnamaldehyde, and may further contain 1 to 5 wt.-% trimethylamine, 1 to 5 % piperin, and 3 to 8 wt.-% furfuryl alcohol. The incorporation of the fish feed composition containing the active compound(s) into the fish feed may be performed as described in example 1. The mixture of active compounds is then prepared directly as an oil which is then mixed with the oil sprayed onto the feed pellets as described in example 1. The incorporation of the fish feed composition containing the active compound(s) into the fish feed may alternatively be carried out by preparing a premix of the active ingredients and other suitable additives. Such a premix may comprise 2-10 % by weight of the active mixture or natural substance or extract, 0-40 % by weight of other conventional additives, such as flavorings, and 50-98 % by weight of any conventional absorbing support.
The support may contain, for example, 40-50 % by weight of wood fibers, 8-10 % by weight of stearin, 4-5 % by weight of curcuma powder, 4-5 % by weight of rosemary powder, 22-28 % by weight of limestone, 1 -3 % by weight of a gum, such as gum Arabic, 5-50 % by weight of sugar and/or starch and 5-15 % by weight of water. - -
This premix is then mixed with vitamins, as for example vitamin C, mineral salts and other feed additive ingredients, as for example yeast extracts containing nucleotides, glucan and other gut microflora modulators and then finally added to the feed in such quantities that the feed will contain 10-5000 ppm, preferably 100- 1000 ppm or 100-500 ppm of the active ingredients according to the invention. Moreover, the composition of the present invention will be preferably used together with yeast extract containing nucleotides, and glucan.
Further, optional, feed-additive ingredients are coloring agents, e.g. carotenoids such as beta-carotene, astaxanthin, and lutein; aroma compounds; stabilisers; antimicrobial peptides; polyunsaturated fatty acids; and/or at least one enzyme selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase (EC 3.2.1.22); protease (EC 3.4.), phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4);
lysophospholipase (EC 3.1.1.5); phospholipase C (EC 3.1.4.3); phospholipase D
(EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC 3.2.1.1 ); and/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).
Examples of polyunsaturated fatty acids are C18, C20 and C22 polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoic acid, eicosapentaenoic acid and gamma-linoleic acid.
The fish feed as described herein has a proximate composition of 20-60 wt.-% protein, and 1 -45 wt.-% moisture and lipid.
In some specific examples, the fish feed comprises one or more of sources of:
- protein, carbohydrate and lipid (for example, fish meal, fish oil, blood meal, feather meal, poultry meal, chicken meal and/or other types of meal produced from other slaughterhouse waste),
- animal fat (for example poultry oil),
- vegetable meal (e.g. soya meal, lupin meal, pea meal, bean meal, rape meal and/or sunflower meal),
- vegetable oil (e.g. rapeseed oil, soya oil), - -
- gluten (e.g. wheat gluten or corn gluten) and
- added amino acids (e.g. lysine)
The term "fish feed" as used herein includes a fish feed composition according to the invention and components as described above. Typically, fish feed includes fish meal as a component. Suitably, fish feed is in the form of flakes or pellets, for example extruded pellets.
In a third aspect, the invention relates to a feed composition for aquatic animals and to the use of this composition for feeding fish. The feed is particularly suitable for feeding salmonids, including Atlantic salmon (Salmo salar), other salmon species and trout, and non-salmonids such as cod, sea bass, sea bream and eel. However, it can be fed to all types of fish, for example turbot, halibut, yellow tail and tuna.
The invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed, since these embodiments are intended as illustrations of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.
Example 1 : Preparation of Pressed Fish Feed
The main raw materials are ground and mixed. Microingredients are then added to the mixer and the homogenous mix is conditioned by adding water and steam to the mass in a preconditioner. This starts a cooking process in the starch fraction (the binding component). The mass is fed into a pellet mill. The mass is forced through the mill's die and the strings are broken into pellets on the outside of the die. The moisture content is low and drying of the feed is not necessary. - -
Additional oil including a fish feed composition according to the present invention is then sprayed onto the surface of pellets, but as the pellets are rather compact, the total lipid content rarely exceeds 24 %. The added oil may be fish oil or vegetable oils, for example rape seed oil or soy oil, or a mixture of vegetable oils or a mixture of fish oil and vegetable oils. After oil coating, the pellets are cooled in a cooler and bagged. The final pressed fish feed contains 10 to 5000 ppm of the composition as described in the invention.
Example 2: Method for Preparation of Extruded Fish Feed
The main raw materials are ground and mixed. Micro ingredients incl. a fish feed composition according to the invention are added to the mixer. The homogenous mix is conditioned by adding water and steam to the mass in a preconditioner. Additional oil may also be added to the mass at this stage. This starts a cooking process in the starch fraction (the binding component). The mass is fed into an extruder. The extruder may be of the single screw or the twin-screw type. Due to the rotational movement of the mass in the extruder, the mass is further mixed. Additional oil, water and steam may be added to the mass in the extruder. At the end of the extruder, the mass has a temperature above 100 0C and a pressure above ambient pressure. The mass is forced through the openings in the extruder's die plate. Due to the relief in temperature and pressure, some of the moisture will evaporate immediately (flash off) and the extruded mass becomes porous. The strings are cut into pellets by a rotating knife. The water content is rather high (18-28 %) and the pellets are therefore immediately dried to approximately 10 % water content in a dryer.
After the dryer, more oil may be added to the feed by spraying oil onto the surface of the feed, or by dipping the feed in oil. It is advantageous to add the oil to the feed in a closed vessel where the air pressure is below ambient (vacuum coating) so that the porous feed pellets absorb more oil. Feed containing more than 40 % lipid may be produced this way. After the coater, the feed is cooled and bagged. Oil may be added at several places in the process as explained above, and may - -
be fish oil or vegetable oils, by example rape seed oil or soy oil, or a mixture of vegetable oils or a mixture of fish oil and vegetable oils.
Fish need protein, fat, minerals and vitamins in order to grow and to be in good health. The diet of carnivorous fish is particularly important. Originally in the farming of carnivorous fish, whole fish or ground fish were used to meet the nutritional requirements of the farmed fish. Ground fish mixed with dry raw materials of various kinds, such as fish meal and starch, was termed soft or semi- moist feed. As farming became industrialized, soft or semi-moist feed was replaced by pressed dry feed. This was itself gradually replaced by extruded dry feed.
Today, extruded feed is nearly universal in the farming of a number of fish species such as various types of salmonid, cod, sea bass and sea bream.
The dominant protein source in dry feed for fish has been fish meal of different qualities. Other animal protein sources are also used for dry fish feed. Thus, it is known to use blood meal, bone meal, feather meal and other types of meal produced from other slaughterhouse waste, for example chicken meal. These are typically cheaper than fish meal and fish oil. However, in some geographic regions, there has been a prohibition against using such raw materials in the production of feeds for food-producing animals and fish.
It is also known to use vegetable protein such as wheat gluten, maize (corn) gluten, soya protein, lupin meal, pea meal, bean meal, rape meal, sunflower meal and rice flour.
Example 3: Evaluation of the effect of the active compounds according to the invention on survival of Yersinia ruckeri, Vibrio anguillarum, Vibrio sal- monicida, Aeromonas salmonicida, Aeromonas hydrophila, Edwardsellia tarda, Photobacterium damselae, Lactococcus garvieae, Streptococcus iniae. - -
The antimicrobial activity of the composition of the invention towards Yersinia ruckeri, Vibrio anguillarum, Vibrio salmonicida, Aeromonas salmonicida, Aeromo- nas hydrophila, Edwardsellia tarda, Photobacterium damselae, Lactococcus gar- vieae, and Streptococcus iniae were determined in vitro.
In the tests the following organisms, growth media, culture conditions and evaluation method were used:
Bacteria: All tested pathogenic strains belong to the strain collection of the Cen- tre for fish and wildlife health, Institute of Animal Pathology, University of Bern (Switzerland).
Determination of suitable bacteria dilution: From a 24 hour old subculture of bacteria on blood sheep agar (Biomeheux, Geneva) a small amount was trans- ferred to sterile NaCI until a McFarland value of 0.5 was obtained. From this solution 3, 1.5 and 0.75 % dilutions in TSB were made on 96 well plates with round bottoms. Each well received a total volume of 100 μl. After 24 hours at 22 0C the growth of bacteria was assessed. The dilution resulting in a well demarcated spot covering half of the round bottom well was selected for the experiments.
Determination of solvent effect: To solve test substances in TSB agar, alcohol (ETOH) was used. To determine a possible effect of alcohol, the calculated final concentrations of alcohol in the test wells (0.1 , 0.05 and 0.025 %) were tested with different concentrations of bacteria, and did not show any effect on bacterial growth.
Concentration of test substances: The in vitro dose range was estimated considering a probable dietary concentration of at least 1000 ppm and a daily feeding rate of 2 %. The potential concentration in the gut was established at maximum 0.1 μl/100 μl. A serial 2 dilution was then tested leading to final concentrations of the substance of 0.85 μg/ml; 0.42 μg/ml and 0.21 μg/ml when adjusted to average essential oil density. - -
From each substance a stock solution consisting of 2 μl substance, 18 μl ETOH and 180 μl PBS was prepared.
Preparation of Plates: Triplicates of three concentrations (0.21 , 0.42 and 0.85 μg/ml) of each substance were tested. On each plate a positive control consisting of bacteria in TSB and a blank control (PBS) was included. Further Triplicates of three dilutions of ETOH were also included on each plate.
Reading of plates: After an incubation of 24 hours at 22 0C the plates were read using a score of 0 (no bacterial growth = no dot on the bottom of the well) to 3 (normal growth = size of dot comparable to dot of positive control).
The following results were obtained and are summarized in Table 1 :
Suitable concentration of bacteria for test: A concentration of 1.5 % of 0.5 McFarland value resulted in a clearly visible spot covering 2/3 of a round bottom well. This concentration was therefore considered as suitable for the tests.
Effect of ETOH on bacterial growth: No effect of any of the applied alcohol concentrations (1 %, 0.5 % and 0.05 %) could be found.
Effect of tested substances: The results are summarized in Table 1. No major differences within triplicates nor between triplicates on different plates could be seen. All positive controls showed clearly visible growth of bacteria within 48 hours. As expected from the preliminary trials no negative effect of alcohol could be determined. Broadest spectrum effects on the bacteria were found with cinna- maldehyde, and alpha-pinene followed by meta-cresol and dihydro-eugenol.
Table 1 : Effect of different substances on growth of Yersinia ruckeri, Vibrio anguil- larum, Vibrio salmonicida, Aeromonas salmonicida, Aeromonas hydrophila, Ed- wardsellia tarda, Photobacterium damselae, Lactococcus garvieae, and Streptococcus iniae after 48 hours incubation at 22 0C. Values represent means of 5 individual triplicates, nd: not determined, 3: no effect, 0: maximum growth inhibition. - -
Table 1 :
Example 4: Evaluation of the effect of the active compounds according to the invention on survival of Vibrio anguillarum, and Aeromonas salmonicida.
The effect of combinations of different substances was tested for growth inhibition of Aeromonas salmonicida and Vibrio anguillarum after 48 hours of incubation at 22 0C. The conditions of the tests were as described in example 3 and concentrations tested were (0.21 μg/ml, 0.42μg/ml and 0.85μg/ml).Values of Table 2 and 3 - -
represent the mean of 3 individual triplicates on three different plates for Aeromo- nas salmonicida and Vibrio anguillarum, respectively. 3: means no effect, 0:
means maximum growth inhibition. The substances tested either alone or in combinations are meta-cresol, cinnamal- dehyde, alpha-pinene, dihydroeugenol. Mixtures of the substances were prepared in proportions 1 :1 for two substances, 1 :1 :1 for three substances and 1 :1 :1 :1 for the four substances. Results: All compounds and combinations of compounds have shown some inhibitory effect against the two pathogens tested at the highest concentration, and a clear dose dependence of the effects against the two bacterium species was observed and is shown in Table 2 and Table 3.
- 7-
Table 2: Results of inhibition assay of Aeromonas salmonicida
- -
Table 3: Inhibition of Vibrio anguillarum
Example 5: Effect of a combination of essential oils comprising alpha- pinene, cinnamaldehyde, dihydroeugenol and meta-cresol on the mortality of rainbow trout challenged with Aeromonas salmonicida.
A challenge experiment with Aeromonas salmonicida has been set up with juvenile rainbow trout in order to test the effect of three doses of a combination of four essential oils supplemented to the feed.
Fish: 600 young of the year rainbow trout were obtained from a commercial fish farm in Switzerland. - -
Fish rearing conditions: For the acclimatization period and the experimental feeding period before infection, fish from the same dietary treatment are kept in 130 I glass aquaria equipped with a tap water flow through system and constant aeration. Water temperature is maintained at 17 ± 1.0 0C.
At the time of infection, fish from each treatment are transferred into four 38 I glass aquaria equipped with a flow through system with tap water and constant aeration. Water temperature is maintained at 17.5 ± 1.0 0C.
Diets and feeding: One basal diet for juvenile rainbow trout was produced in 4 mm diameter. From these pellets, crumbles adapted to the size of the fish were produced and ca 5-kg batch of feed were produced by coating the essential oil combination at the different doses mixed with the oil onto the crumbles. Coating was performed using a peristaltic pump and a concrete mixer to insure homogenous distribution of the products.
Fish are fed daily at a rate of 3.3 % of the body weight. The diet rate was regularly adjusted to the weight gain of fish.
The four experimental diets were produced:
- Diet A: a control diet not supplemented with essential oil combination
- Diet B: basal diet supplemented with 500 ppm of the essential oil combination
- Diet C basal diet supplemented with 2000 ppm of the essential oil combination - Diet D: basal diet supplemented with 4000 ppm of the essential oil combination
The essential oil combination was the following one: alpha-pinene, cinnamalde- hyde, dihydroeugenol and meta-cresol at equivalent doses. The experimental diets were fed for four weeks prior to challenge and after challenge until the end of the experiment. Fish weight was recorded at the start of experimental feeding, before challenge and at the end of the experiment on the remaining fish. - -
Challenge experiment: The challenge dose was determined in a preliminary experiment, on the same fish population and under the same conditions. After a period of 4 weeks of experimental feeding, 25 fish were randomly distributed into four 38 I glass aquaria. Fish from each experimental tank were then anaesthetized and challenged by i.p. injection of Aeromonas salmonicida at a dose of 1.2*102 cfu/fish determined by spectrophotometry. The daily mortality was then recorded over a period of three weeks. First dead fish from each glass aquarium were subjected to a bacteriological investigation to confirm the bacteriological aetiology causing death. At the end of the experiment, all surviving fish were assessed for external signs of infection and in addition from 6 fish per diet group a necropsy and bacteriology were performed.
Results of cumulative mortality are presented in Table 4: Table 4: Mean + SD of cumulative mortality in relation to essential oil combination dose
- -
Results: Feed intake was normal and fish grew from 4 to ca 14 g during the experiment. The results reveal a dose dependent effect of essential oils in rainbow trout challenged with A. salmonicida. Lowest mean values were found in the two groups receiving the higher doses, while in the group receiving the lowest dose a slightly higher cumulative mortality than in the control was found. These results suggest that the mixture of 4 essential oils in the diet have a beneficial effect on rainbow trout infected with A. salmonicida. Differences were found in cumulative mortality and in the presence of bacteria in surviving fish at the end of the experiment.
Example 6: Effect of an essential oil combination comprising alpha-pinene and cinnamaldehyde on the mortality of rainbow trout challenged with Aeromonas salmonicida.
Aim: A challenge experiment with Aeromonas salmonicida has been set up with juvenile rainbow trout in order to test the effect of alpha-pinene and cinnamaldehyde supplemented to the feed.
Fish: 600 young of the year rainbow trout were obtained from DSM Nutritional Products
Fish rearing conditions: For the acclimatization period and the experimental feeding period before infection, fish from the same dietary treatment are kept in 130 I glass aquaria equipped with a tap water flow through system and constant aeration. Water temperature is maintained at 14.5 ± 1.0 0C. - -
At the time of infection, fish from each treatment are transferred into four 38 I glass aquaria equipped with a flow through system with tap water and constant aeration. Water temperature is maintained at 13.5 ± 1.0 0C. Diets and feeding: One basal diet for juvenile rainbow trout was produced in 4 mm diameter. From these pellets, crumbles adapted to the size of the fish were produced and ca 5-kg batch of feed were produced by coating the essential oil combination at the different doses mixed with the oil onto the crumbles. Coating was performed using a peristaltic pump and a concrete mixer to insure homoge- nous distribution of the products.
Fish are fed daily at a rate of 3.3 % of the body weight. The diet rate was regularly adjusted to the weight gain of fish. Three experimental diets were produced:
- Diet A: a control diet not supplemented with essential oil combination
- Diet B: basal diet supplemented with 4000 ppm of alpha-pinene
- Diet C: basal diet supplemented with 4000 ppm of alpha-pinene + cinnamalde hyde at equal doses
The experimental diets were fed for four weeks prior to challenge and after challenge until the end of the experiment.
Fish weight was recorded at the start of experimental feeding, before challenge and at the end of the experiment on the remaining fish.
Challenge experiment: The challenge dose was determined in a preliminary experiment, on the same fish population and under the same conditions. After a period of 4 weeks of experimental feeding, 25 fish were randomly distributed into four 38 I glass aquaria. Fish from each experimental tank were then anaesthetized and challenged by i.p. injection of Aeromonas salmonicida at a dose of 5*103 cfu/fish determined by spectrophotometry. The daily mortality was then recorded over a period of three weeks. First dead fish from each glass aquarium were sub- - -
jected to a bacteriological investigation to confirm the bacteriological aetiology causing death. At the end of the experiment, all surviving fish were assessed for external signs of infection and in addition from 6 fish per diet group a necropsy and bacteriology were performed.
Results: Feed intake was normal and fish grew from 1 to ca 8 g during the experiment.
The results reveal no effect of alpha-pinene alone but there is a positive effect of the combination of alpha-pinene + cinnamaldehyde in reducing mortality by 15 % against the control treatment. Results of cumulative mortality are presented in Table 5:
Table 5: Mean + SD of cumulative mortality in relation to essential oil combi- nation

Claims

- - CLAIMS
1. Use of at least two natural active substances selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in a feed composition for improving feed conversion ratio and/or daily weight gain in aquatic animals.
2. Use of at least two natural active substances selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in a feed composition for regulating the micro flora of the gut in aquatic animals.
3. Use of at least two natural active substances selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene in a feed composition for reducing mortality in aquatic animals.
4. Use according to any of claims 1 to 3, wherein the aquatic animal is a cold water fish as for example salmon, trout, bream or bass.
5. Use according to any of claims 1 or 3, wherein the aquatic animal is a warm water fish as for example carp, tilapia or catfish.
6. Use according to any of claims 1 to 5, wherein the feed composition contains alpha-pinene, cinnamaldehyde, dihydro-eugenol and meta-cresol.
7. Use according to claims 6, wherein the feed composition contains alpha- pinene, cinnamaldehyde, dihydro-eugenol and meta-cresol each in a concentration between 10 mg and 5 g per Kg of feed.
8. Use according to claim 6, wherein the feed composition contains alpha- pinene, cinnamaldehyde, dihydro-eugenol and meta-cresol each in a concentration between 0.1 g and 1 g per Kg of feed. - -
9. Use of at least two natural active substances selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene for treatment and prevention of dis- eases caused by pathogenic microorganisms in aquatic animals.
10. A feed composition or a premix composition, or a feed additive for aquatic animals thereof, comprising at least two natural active substances selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene as main ingredients.
11. A feed additive according to claim 10, wherein the feed additive composition is a natural extract which comprises alpha-pinene, cinnamaldehyde, dihydroeugenol or meta-cresol, as main ingredient.
12. A method for improving the feed conversion ratio in aquatic animals, wherein at least two natural active substances selected from the group consisting of alpha-pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene are added to the animal feed.
13. A method for reducing mortality in aquatic animals, wherein at least two
natural active substances selected from the group consisting of alpha- pinene, alpha-terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta- cresol and terpinolene are added to the animal feed.
14. A method claim according to claim 12 or 13 wherein the feed composition contains alpha-pinene, cinnamaldehyde, dihydroeugenol and meta-cresol each in a concentration between 10 mg and 5 g per Kg of feed.
15. A method for improving feed conversion ration and/or daily weight gain in aquatic animals and/or for regulating the micro flora of the gut and/or for protecting the aquatic animals against infections caused by pathogenic microorganisms, which method comprises providing to the animal for - -
ingestion of the feed an effective amount of at least two natural active substances selected from the group consisting of alpha-pinene, alpha- terpineol, cinnamaldehyde, dihydroeugenol, eugenol, meta-cresol and terpinolene, which are present as ingredients of the feed ingested by the animal.
EP10734483A 2009-07-17 2010-07-16 Use of natural substances as feed additives for aquatic animals Withdrawn EP2453758A1 (en)

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CN103143553B (en) * 2013-03-29 2015-05-20 广东大众农业科技股份有限公司 Method for biological disposal of animal carcasses in river channel
CN103815207B (en) * 2014-03-14 2015-09-09 河南聚丰饲料科技有限公司 A kind of health the Yellow River compound feed for carps
KR101746273B1 (en) * 2015-02-25 2017-06-12 양정자 -carotene The feed additive for abalone including -carotene
US10499664B2 (en) 2015-03-31 2019-12-10 Universidad De Santiago De Chile Fish feed formulation of Hypericum perforatum, Rosamarinus officianalis or a mixture thereof
CN108289478A (en) * 2015-10-13 2018-07-17 帝斯曼知识产权资产管理有限公司 Include the feed addictive for aquatic animal of essential oil and lysozyme
CN108289469A (en) * 2015-12-02 2018-07-17 日清奥利友集团株式会社 Cladding fat or oil composition
CN105432972A (en) * 2015-12-04 2016-03-30 全椒县城西畜牧养殖专业合作社 Selenium-rich feed additive for weever
CN108424814A (en) * 2018-03-20 2018-08-21 甘肃烟草工业有限责任公司 A kind of preparation method of matrimony vine essence and the application in cigarette
CN109258984A (en) * 2018-10-08 2019-01-25 天津农学院 Application of the cinnamic acid with immune growth promoting function in Cynoglossus semilaevis feed
EP3747280A1 (en) * 2019-06-07 2020-12-09 Lucta, S.A. Antimicrobial combination for use in animal feeds
CA3197578A1 (en) * 2020-12-04 2022-06-09 Lucta, S.A. A feed additive for increasing omega-3/omega-6 polyunsaturated fatty acids ratio in aquatic animals
CN114009626A (en) * 2021-10-21 2022-02-08 广东信豚生物科技有限公司 Fish meal substitute containing yeast extract and aquatic animal premix composition

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CL2012000096A1 (en) 2012-07-06
CA2767873C (en) 2018-08-21
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JP2012533289A (en) 2012-12-27
AU2010272494B2 (en) 2015-04-09
CA2767873A1 (en) 2011-01-20
AU2010272494A1 (en) 2012-02-09
CN102469808B (en) 2014-01-15
JP5881175B2 (en) 2016-03-09

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