EP2170100A1 - Produit destiné à l'alimentation animale - Google Patents

Produit destiné à l'alimentation animale

Info

Publication number
EP2170100A1
EP2170100A1 EP08762394A EP08762394A EP2170100A1 EP 2170100 A1 EP2170100 A1 EP 2170100A1 EP 08762394 A EP08762394 A EP 08762394A EP 08762394 A EP08762394 A EP 08762394A EP 2170100 A1 EP2170100 A1 EP 2170100A1
Authority
EP
European Patent Office
Prior art keywords
animal feed
animal
feed additive
thymol
additive according
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Withdrawn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
EP08762394A
Other languages
German (de)
English (en)
Inventor
Hagen Klaus Schulze
Robert Barry Taylor
Hannele Kettunen
Arthur C. Ouwehand
Markku Saarinen
Nina Rautonen
Seppo Peuranen
Maria Helena Lino Bento
Kirsti Kaarina Tiihonen
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.)
DuPont Nutrition Biosciences ApS
Original Assignee
Danisco AS
Danisco US Inc
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 Danisco AS, Danisco US Inc filed Critical Danisco AS
Publication of EP2170100A1 publication Critical patent/EP2170100A1/fr
Withdrawn legal-status Critical Current

Links

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
    • A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
    • A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
    • 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
    • A23K20/111—Aromatic 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
    • A23K20/189—Enzymes
    • 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
    • A23K40/10—Shaping or working-up of animal feeding-stuffs by agglomeration; by granulation, e.g. making powders

Definitions

  • the present invention relates to an animal feed additive, an animal feed comprising this additive, methods using this animal feed additive and methods of preparation relating thereto.
  • Essential oils are concentrated volatile oils having the characteristic odour of the plant from which they are derived. Typically, essential oils are obtained by distillation of the plant and comprise a mixture of component compounds. These component compounds of essential oils include anethole, beta-ionone, capsaicin, carvacrol, cinnamaldehyde, citral, cresols, eugenol, guaiacol, limonene, thymol, tannin and vanillin. The use of component compounds of essential oils in animal feeds have recently been proposed.
  • VVO 96/13175 discloses the use of a mixture of cresols, guaiacol, thymol, anethole, eugenol, capsaicin and tannin with an emulsifying surfactant in a feed additive for poultry. This feed additive was used for the reduction of coccidiosis.
  • WO 99/59430 discloses the use of a mixture of guaiacol, thymol, eugenol, capsaicin, tannin and at least one cresol to alleviate, cure or prevent necrotic enteritis.
  • Fernandez, F., et al. (2001) Cellular and Molecular Life Sciences, Vol. 57, pages 1793-1801 disclosed that supplementing a diet with xylanase lowered the numbers of Campylobacter jejuni in the intestine of chicks.
  • the present invention alleviates the problems of the prior art.
  • the present invention provides an animal feed additive obtainable by:
  • the present invention provides an animal feed additive comprising: (i) cinnamaldehyde, and
  • the animal feed additive of this aspect may further comprise a physiologically acceptable carrier.
  • the present invention provides an animal feed comprising a feed substance and an animal feed additive according to the present invention.
  • the present invention provides a method of inhibiting the growth, or reducing the population, of pathogenic bacteria in an animal comprising a step of orally administering to the animal an animal feed additive according to the present invention, or an animal feed according to the present invention, in an amount effective to inhibit the growth, or reduce the population, of the bacteria in the animal.
  • the present invention provides a method of inhibiting the growth, or reducing the population, of an Eimeria or Histomonas parasite on an animal comprising a step of orally administering to the animal an animal feed additive according to the present invention, or an animal feed according to the present invention, in an amount effective to inhibit the growth, or reduce the population, of the parasite on the animal.
  • the present invention provides a method of inhibiting the growth, or reducing the population, of pathogenic bacteria in an animal, and of increasing the growth of, or increasing the population, of beneficial bacteria in an animal wherein the beneficial bacteria is selected from the group consisting of Lactobacillus and bifidobacteria; comprising a step of orally administering to the animal an animal feed additive according to the present invention, or an animal feed according to the present invention, in an amount sufficient to affect the growth, or the population, of the bacteria in the animal.
  • the present invention provides a method of accelerating the growth of an animal which comprises a step of orally administering to said animal a growth-accelerating effective amount of an animal feed additive according to the present invention, or an animal feed according to the present invention.
  • the present invention provides a method for increasing the immune maturation of an animal which comprises a step of orally administering to said animal an effective amount of an animal feed additive according to the present invention, or an animal feed according to the present invention.
  • the present invention provides a method for reducing biogenic amine production of an animal which comprises a step of orally administering to said animal an effective amount of an animal feed additive according to the present invention, or an animal feed according to the present invention.
  • the present invention provides a method for reducing animal mortality which comprises a step of orally administering to an animal an effective amount an animal feed additive according to the present invention, or an animal feed according to the present invention.
  • the present invention provides a method for increasing dietary nutrient and energy digestibility of an animal feed which comprises a step of orally administering to an animal: (i) an effective amount an animal feed additive according to the present invention and an animal feed; or
  • the present invention provides a method of preparing an animal feed additive comprising the steps of:
  • the present invention provides a method of preparing an animal feed additive comprising the steps of:
  • cinnamaldehyde (i) cinnamaldehyde, and (ii) an essential oil selected from the group consisting of carvacol, citral, eugenol, limonene, thymol, vanilen and combinations thereof;
  • the present invention provides an animal feed additive obtainable by:
  • the present invention provides a method of preparing an animal feed additive comprising the steps of:
  • cinnamaldehyde (i) cinnamaldehyde, and (ii) an essential oil selected from the group consisting of carvacol, citral, eugenol, limonene, thymol, vanilen and combinations thereof;
  • step (b) the components of the oil blend (for example, cinnamaldehyde and thymol) may be added separately to the gum solution, or the components of the oil blend may first be combined together and then added to the gum solution.
  • the components of the oil blend for example, cinnamaldehyde and thymol
  • the cinnamaldehyde may be trans-cinnamaldehyde.
  • the cinnamaldehyde may be synthetic, or isolated and/or purified cinnamaldehyde from natural sources.
  • the animal feed additive may comprise a mixture of cinnamaldehyde and thymol.
  • the compound (ii) includes thymol.
  • the ratio of thymol to cinnamaldehyde may be from 0.1 :1 to 10:1 , or from 1 :1 to 5:1; or from 2:1 to 4:1 , such as 3:1.
  • the compounds (ii) may be a combination of citral, eugenol, limonene and thymol.
  • the compounds (ii) may be a combination of citral, limonene and thymol
  • the compounds (ii) may be synthetic, or isolated and/or purified from natural sources.
  • the animal feed additive may comprise further components such as carvacol.
  • the animal feed additive may also comprise (iii) a feed enzyme.
  • Feed enzymes are enzymes that increase the digestibility of nutrients, leading to greater efficiency in the production of animal products such as meat and eggs. Feed enzymes can play a role in minimizing the environmental impact of increased animal production.
  • the term feed enzymes includes enzymes as defined by European Union Regulation 1831/2003 annex 1 , section 4.
  • the feed enzyme is selected from the group consisting of amylase, beta- glucanase, cellulase, cellobiohydrolase (CBH), endoglucanase, glucoamylase, lipase, pectinase, phytase, protease, xylanase and combinations thereof.
  • the feed enzyme may be selected from the group consisting of amylase, beta- glucanase, phytase, protease and xylanase or combinations thereof.
  • the feed enzyme (iii) may be selected from the group consisting of amylase, cellulase, lipase, phytase, protease, xylanase and combinations thereof.
  • the feed enzyme may be a combination of amylase, protease and xylanase.
  • the feed enzyme may be a combination of phytase and xylanase.
  • the feed enzyme may be xylanase.
  • the feed additive includes a feed enzyme
  • the compound may include a mixture, such as cinnamaldehyde and at least one other of the listed compounds.
  • the animal feed additive may include cinnamaldehyde, thymol and a feed enzyme.
  • the feed enzyme may be xylanase.
  • the animal feed additive may comprise further components such as coccidiostats.
  • coccidiostats may be selected from Monensin sodium, Lasalocid, Amprolium, Salinomycin and mixtures thereof.
  • European Union registered/ approved coccidiostats include Decoquinate E756, Monensin sodium E757, Robenidine E758, Lasalocid sodium E763, Halofuginone E764, Narasin E765, Salinomycin sodium E766; product examples are Avatec (Lasalocid; Alpharma AS), Sacox (Salinomycin, Intervet International BV), Elancoban (Monensin sodium, EIi Lilly).
  • the ratio of wateremulsion stabilizer on a weight/weight basis is from 250:1 to 2.5:1; preferably from 100:1 to 5:1; preferably from 50:1 to 10:1; preferably 35:1 to 15:1.
  • the ratio of wateremulsion stabilizer may suitably be about 25:1.
  • the ratio of emulsion stabilizer: physiologically acceptable carrier on a weight/weight basis is from 1 :200 to 1:1 ; preferably from 1 :100 to 1 :2; preferably from 1 :50 to 1 :5; preferably from 1 :30 to 1 :10.
  • the ratio of emulsion stabilize ⁇ physiologically acceptable carrier may suitably be about 1 :18 or about 1 :19.
  • the ratio of water: physiologically acceptable carrier on a weight/weight basis is from 250:19 to 25:190; preferably from 10:1 to 1 :5; preferably from 5:1 to 1 :2, preferably from 5:1 to 1 :1 , preferably from 3:1 to 2:1.
  • the ratio of water: physiologically acceptable carrier may suitably be about 25:19.
  • the ratio of gum solution:oil blend on a weight/weight basis is from 90:1 to 1 :9; preferably from 50:1 to 1 :5; preferably 20:1 to 1 :2; preferably from 10:1 to 1 :1 , preferably from 9:1 to 2:1 , preferable from 8:1 to 3:1 , preferable from 7:1 to 4:1 , preferably from 6:1 to 5:1.
  • the ratio of gum solution:oil blend may suitably be about 9:1 .
  • the dosage regime may vary over the life of the animal. For example, broilers may be fed a ration with an animal feed additive containing a coccidiostat until the last week before slaughtering.
  • the broiler may be fed a ration that no longer includes the coccidiostat.
  • mature chickens may develop a resistance to coccidiosis if allowed to contract a mild infection of the disease.
  • Birds raised for placement in the laying flocks may be fed a feed containing a coccidiostat until about 16 weeks of age. This medicated feed is then replaced with a non- medicated feed.
  • the physiologically acceptable carrier Is preferably selected from maltodextrin, limestone, cyclodextrin, wheat, and mixtures thereof.
  • the animal feed additive may comprise more than one physiologically acceptable carrier.
  • the animal feed additive may comprises a feed enzyme with a first carrier, and a compound with a second carrier.
  • the carrier is wheat.
  • the animal feed additive may be made into a premix for feed using the methods detailed in US 5,314,692 the disclosures of which are incorporated by reference.
  • the animal feed additive may be an additive for a monogastric animal for example, a poultry animal, or swine.
  • the animal feed additive may suitable be a poultry feed additive.
  • the animal feed additive may comprise at least 0.1% by weight of components (i), (ii) and carvacol.
  • the animal feed additive may comprise at least 0.5%; at least 1%; at least 5%; at least 10%; at least 15%; at least 20% by weight of components (i), (ii) and carvacol.
  • the animal feed additive may comprise at least 1 % by weight of feed enzymes.
  • the animal feed additive may comprise at least 5%; at least 10%; at least 15%; at least 20%; at least 25% by weight of feed enzymes.
  • the one or more feed enzymes may be added as a formulated feed enzyme product.
  • Such feed enzyme products may comprise a single enzyme activity, or may comprise a blend of two or more enzyme activities.
  • feed enzyme products may also comprise other components, such as stabilizing agents.
  • the animal feed additive may be thermally stable to heat treatment up to 70 °C; up to 85 0 C; up to 95°C.
  • the heat treatment may be performed for up to 1 minute; up to 5 minutes; up to 10 minutes; up to 30 minutes; up to 60 minutes.
  • thermally stable means that at least 75% of the components (i), (ii) and carvacol that were present in the additive before heating to the specified temperature are still present after it cools to room temperature.
  • at least 80% of the components (i), (ii) and carvacol that were present in the additive before heating to the specified temperature are still present after it cools to room temperature.
  • the animal feed additive may have a shelf-life of greater than 30 weeks; greater than 40 weeks; greater than 50 weeks; greater than 1 year; greater than 1.5 years.
  • the shelf-life means that at least 80% of the components (i), (ii) and carvacol that were present in the additive when it was prepared are still present.
  • the animal feed additive has a dustiness of less than 0.5%, such as, e.g. less than 0.1%, less than 0.05% as determined by the Heubach-method.
  • the Heubach-method may be carried out as described in the Association of Manufacturers and Formulators of Enzyme Products (AMFEP) and the Federation of European Union Manufacturers and Suppliers of Ingredients to Bakery, Confectionery and Patisserie Industries (FEDIMA) brochure "Measuring of Dust Levels in Bakery Ingredients Using the Heubach Method", published in November 2003.
  • AFEP Association of Manufacturers and Formulators of Enzyme Products
  • FEDIMA European Union Manufacturers and Suppliers of Ingredients to Bakery, Confectionery and Patisserie Industries
  • the animal feed may comprise a feed substance comprising wheat, barley, triticale, rye, corn, tapioca, sorghum, and/ or any of the by-products, as well as protein rich components like soybean meal, rape seed meal , canola meal, sunflower seed meal and mixtures thereof.
  • the animal feed may be an animal feed for a monogastric animal, such as poultry (for example, broiler, layer, broiler breeders, turkey, duck, geese, water fowl) and swine (all age categories), pet (for example dogs, cats) and fish.
  • the animal feed may comprise at least 1 g of cinnamaldehyde per 1000 kg of animal feed, at least 2 g of cinnamaldehyde per 1000 kg of animal feed, at least 3 g of cinnamaldehyde per 10000 kg of animal feed, at least 4 g of cinnamaldehyde per 1000 kg of animal feed, at least 5 g of cinnamaldehyde per 1000 kg of animal feed.
  • the animal feed may comprise at least 1 mg of cinnamaldehyde per kg of animal feed, at least 2 mg of cinnamaldehyde per kg of animal feed, at least 3 mg of cinnamaldehyde per kg of animal feed, at least 4 mg of cinnamaldehyde per kg of animal feed, at least 5 mg of cinnamaldehyde per kg of animal feed.
  • the animal feed may comprise less than 6 g of cinnamaldehyde per 1000 kg of the animal feed; such as, e.g., less than 5.9 g of cinnamaldehyde.
  • the animal feed may comprise less than 18 g of cinnamaldehyde per 1000 kg of animal feed, such as, e.g. less than 17 g of cinnamaldehyde per 1000 kg of animal feed, less than 16 g of cinnamaldehyde per 1000 kg of animal feed, less than 15 g of cinnamaldehyde per 1000 kg of animal feed, less than 14 g of cinnamaldehyde per 1000 kg of animal feed.
  • the animal feed may comprise at least 1 g of thymol per 1000 kg of animal feed, at least 2 g of thymol per 1000 kg of animal feed, at least 3 g of thymol per 1000 kg of animal feed, at least 4 g of thymol per 1000 kg of animal feed, at least 5 g of thymol per 1000 kg of animal feed, at least 6 g of thymol per 1000 kg of animal feed, at least 7 g of thymol per 1000 kg of animal feed, at least 8 g of thymol per 1000 kg of animal feed, at least 9 g of thymol per 1000 kg of animal feed, at least 10 g of thymol per 1000 kg of animal feed, at least 11 g of thymol per 1000 kg of animal feed, at least 12 g of thymol per 1000 kg of animal feed, at least 13 g of thymol per 1000 kg of animal feed, at least 14 g of thymol per 1000 kg of animal feed, at least
  • the animal feed may comprise at least 0.00001% by weight of components (i), (ii) and carvacol.
  • the animal feed may comprise at least 0.00005%; at least 0.00010%; at least 0.00020%; at least 0.00025%; at least 0.00050%; at least 0.00100%; at least 0.00200% by weight of components (i), (ii) and carvacol.
  • the animal feed may comprise at least 0.0001% by weight of the feed enzymes.
  • the animal feed may comprise at least 0.0005%; at least 0.0010%; at least 0.0020%; at least 0.0025%; at least 0.0050%; at least 0.0100% by weight of the feed enzyme.
  • the animal feed may comprise at least 0.001% by weight of the animal feed additive.
  • the animal feed may comprise at least 0.005%; at least 0.010%; at least 0.020%; at least 0.100%; at least 0.200%; at least 0.250%; at least 0.500% by weight of the animal feed additive.
  • the method of inhibiting the growth, or reducing the population, of pathogenic bacteria in an animal may suitably be used against gram positive bacteria, or gram negative bacteria.
  • the method may be used against gram negative bacteria.
  • the method may be used for a range of bacteria, for example, the method may inhibit the growth, or reduce the population, of pathogenic bacteria in an animal wherein the bacteria is selected from the group consisting of Campylobacter, Clostridium, Lawsonia, Listeria, Staphyloccus, Salmonella and Escherichia.
  • the bacteria may be suitable selected from the group consisting of Campylobacter jejuni, C. perfringens, S. epidermis, S. infantis, S. enteritidis, S. typhimurium and E. coli.
  • the animal may be a monogastric animal, for example, a poultry animal, or swine.
  • the animal is a poultry animal.
  • the emulsion stabilizer may be a modified starch, such as starch sodium octenyl succinate.
  • the method of preparing an animal feed additive has a mixing step (c) that comprises homogenizing under pressure.
  • the term homogenizing under pressure means homogenizing under a pressure of greater than atmospheric pressure.
  • this step (c) comprises homogenizing the emulsion under a pressure of greater than 10 bar.
  • Step (c) may comprise two stages.
  • the first stage may comprise homogenizing the emulsion under a pressure of from 150 to 400 bar.
  • the second stage may comprise homogenizing the emulsion under a pressure of from 20 to 40 bar.
  • the mixture is homogenized to form an emulsion with an average droplet size of less than 5 micron; preferably less than 4 micron; preferably less than 3 micron; preferably less than 2 micron; preferably less than 1 micron.
  • the method of preparing an animal feed additive may comprises the further step of: (e) sieving the powder.
  • the powder may be suitably sieved to obtain a particle size of less than 500 micron, less than 400 micron, less than 300 micron, or less than 200 micron.
  • the method of preparing an animal feed additive may also comprise the further step of pelleting the powder.
  • the powder may be mixed with other components known in the art.
  • the powder, or mixture comprising the powder may be is forced through a die and the resulting strands are cut into suitable pellets of variable length.
  • the pelleting step may include a steam treatment, or conditioning stage, prior to formation of the pellets.
  • the mixture comprising the powder from step (e) may be placed in a conditioner, e.g. a mixer with steam injection.
  • the mixture is heated in the conditioner up to a specified temperature, such as from 60-100 0 C, typical temperatures would be 70 0 C, 85°C, 90 0 C or 95°C.
  • the residence time can be variable from seconds to minutes and even hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1 minutes 2 minutes., 5 minutes, 10 minutes, 15 minutes, 30 minutes and 1 hour.
  • the present invention provides an animal feed additive comprising: a feed enzyme; and a compound selected from the group consisting of cinnamaldehyde, carvacol, citral, eugenol, limonene, thymol, vanillin, and mixtures thereof.
  • the present invention provides an animal feed additive consisting essentially of:
  • the animal feed additive may consist essentially of the specified components, that is they may include the specified components and those that do not materially affect the basic and novel characteristics of the animal feed additive.
  • the term "consisting essentially of is taken to exclude from the animal feed additive any further non-specified components of essential oils such as, for example, capsaicin.
  • this term is taken to exclude from the animal feed additive any further non-specified enzymes.
  • this term is taken to exclude from the animal feed additive components such as organic acids (for example, organic acids derived from citric, fumaric, fulvic and humic acid).
  • the present invention provides the use of an animal feed additive as described herein, or an animal feed as described herein, in a method of medical treatment of an animal.
  • Suitable bacterial infections include those caused by Salmonella and Escherichia.
  • an animal feed additive as described herein, or an animal feed as described herein, for the treatment of a disease or condition in poultry selected from necrotic enteritis, pullorum disease, fowl typhoid, paratyphoid, colibacillosis, omphalitis, fowl cholera, erysipelas, avian vibrionic hepatitis, gastro-intestinal diseases and diseases of the respiratory and urinary tract.
  • the present invention provides a method of combating pests at a locus which comprises applying to said locus a pesticidally effective amount of an animal feed additive as described herein.
  • the locus may be any location that it is desired to be free of pests.
  • the locus may be a location where animal feed and/or animal feed additives are stored.
  • the locus may comprise cereal, grains, animal feed, vegetables, fruit, spices and combinations thereof.
  • the locus comprises comprise wheat, triticale, corn or a combination thereof.
  • the pest may be an insect.
  • the present invention provides a method of combating pests at a locus which comprises applying to said locus a pesticidally effective amount of an oil blend as described herein.
  • a pesticidally effective amount of an oil blend as described herein.
  • the pest may be an insect.
  • the locus may be a physiologically acceptable carrier as described herein.
  • the present invention provides a method of repelling pests at a locus from which it is desired to exclude such pests which comprises applying to said locus an effective amount of a repellent composition wherein the repellent composition is an animal feed additive as described herein.
  • the locus may be a location where animal feed and/or animal feed additives are stored.
  • the locus may comprise cereal, grains, animal feed, vegetables, fruit, spices and combinations thereof.
  • the locus comprises comprise wheat, triticale, corn or a combination thereof.
  • the pest may be an insect.
  • the present invention provides a method of repelling at a locus which comprises applying to said locus an effective amount of a repellent composition wherein the repellent composition is an oil blend as described herein.
  • the pest may be an insect.
  • Suitably may be a physiologically acceptable carrier as described herein.
  • Suitable the insect to be combated or repelled may be selected from a group consisting of Tribolium castaneum, Tribolium confusum, Rhyzopertha dominica, Oryzaephilus surinamensis and Cryptolestes ferrugineus.
  • the present invention provides a method of improving the epithelial cell integrity of an animal which comprises a step of orally administering to said animal an effective amount of an animal feed additive as described herein, or an animal feed as described herein.
  • FIG 1 shows effect of treatments T7 or T8 (minus and plus enzyme) on the caecal colonisation by Campylobacter jejuni in broilers (combined trials);
  • FIG. 2 shows effect of treatments T8 (minus and plus enzyme) on the caecal colonisation by Campylobacter jejuni in broilers (combined trials);
  • Figure 3 shows the levels of biogenic amines in a model for putrefaction in broiler chicks
  • Figure 4 compares the effects of chitosan and two emulsifiers on the production of biogenic amines
  • Figure 5 shows the production of individual volatile fatty acids, and individual biogenic amines
  • Figure 6 shows the response of biogenic amines and volatile fatty acids to treatment with a selection of essential oils
  • Figure 7 shows a graph of the number of microbes in the ileal digesta at 21 and 42 days
  • Figure 8 shows a graph of the total volatile fatty acid content in caecal digesta at 21 and 42 days
  • Figure 9 shows the content of biogenic amines in caecal digesta at 21 days
  • Figure 10 shows the proportion of bifidobacteria as a percentage of total microbes in caecal digesta at 21 and 42 days;
  • FIG 11 shows Ileal immunoglobulin A (IgA) concentration
  • Figure 12 shows caecal IgA concentration
  • Figure 13 shows the increase in IgA concentration expressed as a percentage for 21 days old broiler chicks
  • Figure 14 shows the increase in caecal IgA concentration expressed as a percentage for 35 days old broiler chicks;
  • Figure 15 shows the in vitro relative growth inhibition (%) of gram+ and gram- bacteria by additive EO (which is T8) and Avilamycin applied at commercial concentrations, compared with Control;
  • Figure 16 shows the effect on broiler performance for C. perfringens (caecal digesta) of addition of EO (T8) to the feed. An approximately 1 log ( ⁇ 90 %) reduction in for C. perfringens (caecal digesta) is observed compared to a control;
  • Figure 17 shows how EO (T8) increases the ileal lactate concentration indicating higher lactobacilli number / activity
  • Figure 18 shows how EO (T8) stimulates Bifidobacteria in caecum
  • FIG 19 shows that an additive (designated Flavoden H in the Figure, and T8 in Example 1) increases the corrected energy utilization (Apparent Metabolizing Energy
  • Figure 20 shows the improvement in mortality, foot pad score and gait scores of using an animal feed additive T8;
  • Figure 21 shows the improvement in weight gain development (Average Daily Gain
  • ADG feed conversion ratio
  • FCR feed conversion ratio
  • Figure 22 shows the improvement in weight gain development and feed conversion of a male broiler fed wheat based diets supplemented with additive T8, feed enzymes and cocidiostats;
  • Figure 23 shows the shelf life stability of additive T8 under various conditions
  • Figure 24 shows the heat stability of T8 in product pelleting studies
  • Figure 25 shows the in feed (pelleted) recovery as a percentage of mash
  • Figure 26 shows a sprayed dried product at a magnification of 2Ox
  • Figure 27 shows a sprayed dried product at a magnification of 78x
  • Figure 28 shows individual %G+C profiles of chicken caecum samples at 41 days as discussed in Example 6;
  • Figure 29 shows the average %G+C profiles of chicken caecum samples at 41 days as discussed in Example 6;
  • Figure 30 shows individual %G+C profiles of chicken caecum samples at 28 days as discussed in Example 11 ;
  • Figure 31 shows the average %G+C profiles of chicken caecum samples at 28 days as discussed in Example 11;
  • Figure 32 shows individual %G+C profiles of chicken ileum samples at 28 days as discussed in Example 11 ;
  • Figure 33 shows the average %G+C profiles of chicken ileum samples at 28 days as discussed in Example 11 ;
  • Figure 34 shows a strain of Oryzaephilus surinamensis with a size of 2.5-3.5 mm
  • Figure 35 shows a strain of Tribolium castaneum with a size of 2.3-4.4mm
  • Figure 36 shows the jars used for the infest ability tests in Example 15;
  • Figure 37 shows the arena used for the no choice test in Example 15;
  • Figure 38 shows the arena used for the choice test in Example 15.
  • Figure 39 shows an epithelial cell model including tight junctions.
  • Figure 40(a) and (b) show diagrammatic representations of a transepithelial electrical resistance (TEER) experiments with (a) low TEER values and (b) high TEER values.
  • TEER transepithelial electrical resistance
  • Figure 41 shows % change in TEER values with treatment with essential oils.
  • Figure 42 shows the production of volatile fatty acids in response to treatment with various essential oils and essential oil combinations.
  • Figure 43 shows the production of biogenic amines in response to treatment with various essential oils and essential oil combinations.
  • Figure 44 shows the production of 2-methylbutyric acid in response to treatment with various essential oils and essential oil combinations.
  • Figure 45 shows the effect of product formulation on cinnamaldehyde shelf life stability.
  • Figure 46 shows the effect of product formulation on thymol shelf life stability.
  • the emulsion was homogenized using a two stage homogenizer, the first stage at 250 bar, and the second stage at 30 bar to produce an emulsion with a droplet size of less than 1 micron and a viscosity of less than 70 centipoise.
  • the droplet size was assessed under a microscope (40Ox magnification) using a calibrated graticule.
  • the emulsion was fed via a spray dry feed at an inlet temperature of approximately 195 0 C and an outlet temperature of approximately 95-100 0 C to a spray drier with a fluid bed facility. This produced a spray dried powder that is free flowing and typically has a moisture content of 2.5-3.0 % m/m.
  • the spray dried powder was sieved using a vibratory sieve to produce a particle size of approximately 200 micron. The sieved powder was filled out to 1000 kg.
  • FIG. 26 and 27 An example of a spray dried product is shown in Figures 26 and 27.
  • This method produces a white, fine, water soluble agglomerated spray dried powder of irregular shaped particles.
  • the powder has good flowability and is not dusty.
  • T7 and T8 Two different blends of essential oils T7 and T8 were provided by Danisco/Cultor Animal Nutrition, Wiltshire, UK.
  • T7 consisting of cinnamaldehyde, thymol, eugenol and limonene in the ratio of 1 :3:2:6 respectively.
  • T8 consisting of cinnamaldehyde and thymol in the ratio of 1:3 respectively.
  • Eighteen diets were used, based on wheat, prepared by Target Feeds Limited, Shropshire, UK. (Table 1). Of eight diets, two of each was supplemented with 0.012%; 0.024%; 0.120% or 0.240% of T7, with one of each concentration having the enzyme added. A further two of each were supplemented with 0.005%; 0.01%; 0.05% or 0.1 % of T8, with one of each concentration having the enzyme added. The two remaining diets were un-supplemented wheat-based diet with or without the enzyme. No diet contained coccidiostats.
  • the contents of the caeca were sampled aseptically. Serial 10-fold dilutions (to 10 ⁇ 5 ) were then prepared in sterile tubes using MRD broth as diluent. Then 0.1 ml of the appropriate dilutions were used to inoculate in duplicate onto Campylobacter blood- free selective agar (Oxoid CM 739) with cefoperazone selective supplement (CCDA; Oxoid SR 155) further supplemented with 50 ⁇ g/ml nalidixic acid (CCDA + Na) to allow only the growth of the marked strain of C. jejuni. Plates were then incubated under microaerophilic conditions (85% N, 10% CO 2 and 5% O 2 ) at 42 0 C for 2 days.
  • Campylobacters were identified on the basis of colonial morphology, Gram stain, motility, catalase and oxidase reactions and then enumerated for further statistical analysis.
  • Campylobacter Enrichment Broth (Bolton Broth, Lab M, Lab 135, Bury, United Kingdom) and incubated at 42 0 C for 2 days under microaerophilic conditions. Broths were then sub-cultured onto CCDA and CCDA + Na agar plates. Incubation of plates and the presence of Campylobacters were determined as above.
  • Results were evaluated using the SAS statistical analysis package version 9.1.
  • the analysis initially omitted the controls to obtain a balanced design for comparison of the four treatments by means of orthogonal comparisons.
  • the controls were then linked first to T7 and then to T8, and individual doses of each treatment were compared with their appropriate controls by Dunnett's test. In all analyses probabilities less than 0.05 were considered statistically significant.
  • Blend T8 produced a zone of inhibition of 4mm at the highest concentration (1.1%) tested, compared with 1.5mm for T7 at the same concentration. No or very slight inhibition was observed with 0.4% for T8, compared with 0.7% for T7.
  • the results of this example show that the wheat-based diet supplemented only with an enzyme, reduced the C. jejuni levels, since the control group without either essential oils or enzyme had the highest average caecal pathogen counts. Furthermore, the addition of essential oils blends to a wheat-based diet reduced C. jejuni counts in the intestinal tract of broilers. The protective effect of both blends was enhanced by the addition of enzyme at a concentration of 0.1%. The greatest degree of reduction was observed with the cinnamaldehyde and thymol (T8) blend at the highest dosage. An apparently dose-related trend was observed as greater reduction was generally associated with increased dose levels.
  • VFA volatile fatty acid
  • Pheasant 1TE provided the following (mg/kg of diet): Limestone Trucal 270 FL 733; vitamin A 500 TFL 12.8; vitamin D3 500 2.4; vitamin B1 1.2; vitamin B12 6.0; copper sulphate 25% 16.0; biotin 2% 2.5; nicotinic acid 24.0; vitamin B2 80% 5.0; vitamin B6 1.2; vitamin K 44% 4.6; cal. pan. 98% 6.1; folic acid 0.6; iron sulphate 20% TFL 40.0; manganese oxide 62% 64.5; zinc oxide 74% feedgrade 43.240; calcium iodate 62% 0.6; selenium premix 1.0% TFL 8.0; cobalt BMP 5% 8.0; sodium molybdate 0.5.
  • the diet contains no anticoccidial agents.
  • T8 plus enzyme 48 7.9 0.128 aMean numbers (Log-io CFU/g) of C. jejuni. Probabilities less than 0.05 are considered significant. * Significant difference from T7 plus enzyme. "Significant difference from T8 plus enzyme.
  • Probabilities less than 0.05 are considered significant. * Significant difference from T7 plus enzyme. * Significant difference from T8 plus enzyme.
  • Method Heptylamine was added to the sample as an internal standard and the biogenic amines were extracted with 0.4 M perchloric acid. After centrifugation the biogenic amines in the supernatant were derivatised with dansyl chloride. The derivatives were separated on a C18 reversed phase column using mixture of ammonium acetate and acetonitrile as eluent and detected with fluorescence detector.
  • Biogenic amines Methylamine hydrochloride, Sigma M-0505, Ethylamine Sigma E-3754, Propylamine, Sigma P-5893, Butylamine, Sigma B-2266, Agmatine sulfate salt, Sigma A-7127, ⁇ -phenylethylamine hydrochloride, Sigma P-6513, 2- methylbutylamine, Aldrich 220523, Piperidine, Fluka 80642, Tryptamine hydrochloride, SigmaT-9628, Putrescine dihydrochloride, Sigma P-7505, Cadaverine dihydrochloride, Sigma C-5659, Histamine dihydrochloride, Sigma H-7250, Tyramine hydrochloride, Sigma T-2879), Spermidine trihydrochloride, Sigma S-2501 ,spermine tetrahydrochloride, Sigma S-2876, Heptylamine, Sigma H-3750 (internal standard), Derivatisation reagent: 1 % dansyl chloride in
  • Mobile phase A 0.02 M ammonium acetate (CH 3 COONH 4 ) in water (70%) - acetonitrile (30%).
  • Mobile phase B 0.02 M ammonium acetate in water (15%) - acetonitrile (85%).
  • ISTD stock solution 10 ml of 0.4 M HCIO4 was added into a 100 ml measuring flask. About 100 mg of heptylamine was added and the flask was filled to volume with 0.4 M HCIO4.
  • ileal digesta About 1 g of ileal digesta, 0.5 g caecal digesta or 1 ml of colon simulator sample was weighed into a test tube. 1 ml of internal standard (ISTD) 1 -solution was added followed by 2 ml 0.4 M HCIO 4 . Samples were shaken for 5 min and centrifuged for 5 min at maximum speed (10 ml plastic tubes max about 7000 rpm).
  • ISD internal standard
  • the growth medium used in digesta simulations for intestinal microbes was supplemented with a double amount of proteins (peptone, tryptone, casein and yeast extract; total 55g/l extra proteins).
  • Fresh caecal inoculum of broiler chicks was mixed with the growth medium.
  • Thymol and carvacrol were added at 100 mg/l and 1000 mg/l.
  • the anaerobic fermentation was conducted in test tubes at 37°C.
  • the levels of biogenic amines in Table 6 are illustrated graphically in Figure 3.
  • the total biogenic amines showed that the protein supplementation to the basal growth medium enhanced the putrefactive metabolism of the caecal microbial community.
  • the level of total biogenic amines in the negative control is at the level of healthy broiler chicks.
  • the protein-supplemented control sample showed the level of biogenic amines that is commonly found in E/mer/a-infected broiler chicks.
  • Thymol and carvarcrol at 100 mg/l decreased the concentration of biogenic amines slightly, compared to the control.
  • the same botanicals at 1000 mg/l suppressed the putrefaction back to the level of the negative control.
  • the protein supplementation to the growth medium did not alter microbial numbers.
  • the effect of botanicals is likely to be caused more by functional rather than numerical changes in the microbe community.
  • chitosan potentiates the growth inhibitory effect of botanicals, and that cinnamaldehyde in combination with any other botanical tends to effectively inhibit the growth of Salmonella. These effects were studied in the putrefaction model. Chitosan, a substance that breaks the outer layer of Gram negative bacteria, was dissolved into acetic acid and included at 500 mg/l.
  • the essential oils were tested singly and in combination with cinnamaldehyde.
  • the single botanicals were included at 100 mg/l (no replicates), and the combinations at 50+50 mg/l (two replicates/treatment).
  • the controls were replicated twice. Sampling at 24 hours for the measurement of biogenic amines and volatile fatty acid.
  • the concentrations of the major volatile fatty acids, acetic-, propionic-, and butyric acids remained also at the level of the protein-supplemented controls. This can be considered a beneficial effect, since these organic acids are an energy source to the animal. Moreover, they decrease the intestinal pH and may thus reduce the growth of harmful microbes, like Clostridia.
  • the total concentration of biogenic amines was more than two times higher in the protein-supplemented control group than in the negative control group (2829 vs. 8528 nmol/g).
  • the individual botanicals candidates reduced the production of biogenic amines by an average of 24%, and the cinnamaldehyde combinations by 32%, compared with the control.
  • the most effective combinations were cinnamaldehyde + cresol, cinnamaldehyde + thymol, and cinnamaldehyde + thyme oil (see Figure 43).
  • the reduction of biogenic amines by the botanicals can be regarded as a result towards the right direction.
  • the number of microbes was counted with a flow cytometer from a few samples. The result indicates that the botanicals increase rather than decrease the number of microbe cells in the putrefaction trial setting. The suppression of some metabolites thus reflects altered metabolic function of the microbial population.
  • a 48-hour fermentation with chitosan was made for one set of samples, in which the botanicals were added at lower levels.
  • the trial setting is presented in the table below.
  • the emulsifier were Grinsted MONO-DI HV 40 9771 (Grinsted; Danisco A/S), and Dimodan HR 10760 (Dimodan; Danisco A/S). Biogenic amines and volatile fatty acids were measured.
  • the botanicals candidates were included singly (100 mg/l) and in combinations with two (50+50 mg/l) or three (50+50+50 mg/l) component compounds of essential oils. In the combinations, cinnamaldehyde was always present. Caecal digesta inoculum was collected from eight 4-wk old broiler chicks raised on corn-soy -based diet. The fermentation tubes were sampled at 24 hours for the measurement of biogenic amines and volatile fatty acids.
  • the total concentration of biogenic amines was significantly higher than in the previous trials. In contrast, the volatile fatty acid concentrations stayed at about the same level in all the experiments. The discrepancy may be explained by differences in the caecal inoculum. The quantity and quality of the bacteria and the nutrients coming from the caecal batch may have differed between the trials, as chicks of different ages were used as donors of the caecal inocula. The higher overall production of biogenic amines may limit the capacity of the essential oils to modulate bacterial metabolism.
  • test articles were mixtures of essential oils spray dried on a maltodextrin carrier.
  • the combinations used were: carvacrol, cinnamaldehyde, citral, eugenol, limonene and thymol at a high concentration; carvacrol, cinnamaldehyde, citral, eugenol, limonene and thymol at a low concentration; cinnamaldehyde, eugenol, limonene and thymol; cinnamaldehyde, eugenol and thymol; and cinnamaldehyde and thymol.
  • the experimental diets were manufactured by ADAS Gleadthorpe, to a commercial specification and were fed in a mash form (see Tables 15, 16 and 17 for diet formulations and crude protein contents).
  • the feed was supplied in marked bags, which displayed the relevant treatment code.
  • the bags were check weighed before use. A sample of each batch of diet was taken from the feed bags at the time of feeding and retained under refrigeration following the end of the study.
  • Husbandry and management Each pen contained one tube feeder and birds had free access to water via bell drinkers (one per pen). The heights of the drinkers were adjusted regularly to keep level with the birds' backs and checks were made to ensure that the water flow was adequate.
  • the litter was provided in the form of clean woodshavings to a depth of 5cm. Minimum ventilation rate was automatically calculated and this rate was supplied by one 610mm fan controlled by a Stick digital control panel.
  • Feed usage was measured for the periods 0-21 and 21-42 days. The feed in each plot was measured by weighing back the amount of feed remaining at the end of the period and deducting it from the quantity offered. A starter ration was fed until 21 days and a finisher from 21 to 42 days.
  • the birds were weighed in their pen groups at day old. At 21 and 42 days all the birds in all of the pens were weighed in batches. The total weight of all the birds in each pen was determined.
  • the %G+C profiling was performed as described previously (Apajalahti et a/. 1998; Holben, W.E., Jansson, J. K., Chelm, B.K., and Tiedje, J. M., (1988) "DNA probe method for the detection of specific microorganisms in the soil bacterial community". Appl. Environ. Microbiol. 54, 703-711).
  • the method yields a profile of the total microbiota of the sample based on the characteristic %G+C content of the DNA of the bacterial components. Briefly, 100 ⁇ g of isolated caecal DNA was subjected to 1 ⁇ g of AT-dependent DNA binding dye bisbenzimidazole in 40ml cesium chloride.
  • the %G+C profiles were divided into twelve 5% increments. The relative abundance (%) of the two treatment groups within each 5% and 1 % increment was compared using t-test. The results ( Figures 28 and 29) indicate that at 41 d the profiles were different. Within the range of 46 to 50 (52) %GC, the control group had higher relative abundance of microbes compared to treatment group (Table 13). Moreover, between 70 to 78 %G+C, the control group had lower relative abundance of microbes compared to treatment group.
  • Table 13 Statistical differences between the treatment group and the control group at 41 d analyzed as 5% and 1% G+C increments.
  • the birds fed treatment 3/J, treatment 5/L and treatment 6/M had significantly higher bodyweights than birds fed the control treatment 1/G. Birds fed treatment 6/M had the highest bodyweight at 2.69 kg and birds fed the control treatment 1/G had the lowest bodyweight at 2.57 kg.
  • the birds During the first half of the study the birds ate on average 58.7 g feed per bird per day and during the second half of the study they ate on average 184.7 g feed per bird per day. For the study overall, the birds ate on average 119.8 g feed per bird per day. Throughout the study there were no significant differences between the treatments for feed intake.
  • FCE was higher in the first half of the study, with an average FCE of 0.565, compared to the second half of the study, with an average FCE of 0.485.
  • FCE was higher in the first half of the study, with an average FCE of 0.565, compared to the second half of the study, with an average FCE of 0.485.
  • Feed conversion ratio (FCR)
  • FCR was better in the first half of the study, with an average FCR of 1.79, compared to the second half of the study, with an average FCR of 2.09.
  • FCR was better in the first half of the study, with an average FCR of 1.79, compared to the second half of the study, with an average FCR of 2.09.
  • Table 14 Percent of %G+C content which accommodate 80% of the species within a given genus according to Apajalahti JH, Sarkilahti LK, Maki BR, Heikkinen JP, Nurminen PH, Holben WE. "Effective recovery of bacterial DNA and percent- guanine-plus-cytosine-based analysis of community structure in the gastrointestinal tract of broiler chickens". Appl. Environ. Microbiol. 1998; 64:4084-8.
  • Rapeseed meal 5.0 6.0
  • the antimicrobial activity of 10 compounds that are found in essential oils, and 3 extracts of essential oils were tested in vitro at concentrations of 5, 50 and 500 ⁇ g/ml.
  • the antibiotic avilamycin was used at therapeutic dose of 5 ⁇ g/ml.
  • E. coli, Salmonella, C. perfringens were used as potential pathogens and Lactobacillus and Bidobacterium species as representatives of potential beneficial microbiota members.
  • microbes were grown anaerobically at 37°C in medium supplemented with 5, 50 or 500 ⁇ g/ml EO. Growth was followed by measuring optical density at 600 nm for 24 h.
  • Results are expressed as the minimal concentration tested that reduced the growth of the target microbes (see Table 18).
  • the tested antibiotic was less selective, not affecting E. coli and Salmonella but affecting both Clostridium and beneficial members of the intestinal microbiota.
  • the diet specifications were as shown in Table 15. Five blends of compounds that are found in essential oils (carvacrol, cinnamaldehyde, citral, eugenol, limonene, thymol), varying in composition and inclusion level were studied.
  • the feed intake (g/bird/day) is shown in Table 20.
  • the feed conversion efficiency (FCE) is shown in Table 21
  • feed additives comprising blends of compounds that are found in essential oils can improve broiler performance parameter, reduce the microbial population measured in ileal digesta and change microbial composition and activity in the caecum.
  • feed conversion appeared to be significantly influenced by the addition of essential oils in certain combinations and strengths.
  • mortality appeared to be influenced by the addition of essential oils in certain combinations and strengths.
  • the performance was measured for the starter and grower diet periods (days 1-21 and 21-42).
  • the concentration of IgA (ELISA) in ileal and caeca! digesta was measured in the ileum and caecum at days 21 and 42.
  • the IgA levels increased from day 21 to 42, and were higher in caecum than in ileum ( Figures 11 and 12).
  • feed additives comprising blends of compounds that are found in essential oils may improve intestinal immunocompetence in broiler chicks.
  • Earlier immunomaturation may increase chick resistance against infectious diseases and improve bird performance in challenging environment.
  • Foot pad lesions were scored on a scale of 0 (no legions) to 2 in fifteen 36-day-old broiler per pen, using the left foot. Total scores per pen were calculated (minimum 0, maximum 30).
  • the reduction in mortality is calculated from the number of dead birds of theT ⁇ treatment over the number of dead birds of the control treatment.
  • Essential oil composition T8 was found to reduce mortality, and improve foot pad and gait scores by approximately 20 % (see Figure 20).
  • Granular Avizyme® 1300 was used as a feed enzyme for wheat/triticale based diets and dosed as 1000 g/ton.
  • ROSS 308 Seven thousand and eighty sexed broilers (ROSS 308) were supplied by Cobroed, Lievelde, the Netherlands. Female and male broilers were housed in floor pens (surface area: 10 m 2 ) with wood shavings. Each treatment consisted of eight replicates (pens): four replicates with 160 males and four replicates with 160 females. The birds were given feed and water for ad libitum intake. The animal facility was lit for 23 h per day. Ambient temperature was lowered stepwise (32 0 C on day 0 to 18-20 0C on day 37, from day 5 onwards minus one degree according to normal practice). Used litter underneath the bell drinkers as well as draught were used, as stress factors to the boilers.
  • NBD Newcastle Disease
  • Feed intake from day 0 to 14, day 15 to 29 and day 30 to 37.
  • FCR Feed conversion ratio
  • Meckels diverticulum and the ileo-caecal junction and caecal samples were taken from both caeca.
  • Example 5 except that the analysis was carried out at day 28 ( Figure 30).
  • Example 5 except that the analysis was carried out at day 28 ( Figure 32).
  • Diets were manufactured by double mixing. First a batch of the basal control diet was prepared per phase, sufficient for all treatments. The basal diet was split into six portions to which no (control) or the two essential oils were added with or without Avizyme 1300 according to the treatment schedule (Table 27). This procedure was repeated for the three phase diets. All diets were pelleted with steam addition (max. pellet temperature 55 0 C) through a 2.5 mm die. After passing through a cooler, the pelleted diets were bagged and labelled.
  • Healthy one-day-old broilers were used with an average body weight of 42 grams.
  • the birds were challenged using a wheat/triticale diet, which was given from day 3 onwards. During the first two days a normal starter diet was fed. Used litter was used as a challenge factor under the bell drinkers. Litter was also moistened around the drinkers. Draught was used for 1.5 h at days 10, 13 and 21 as an additional challenge factor. Birds looked somewhat slow at day 20, possibly as a result of the challenge factors. Total mortality was 5.9% for the male and 3.8% for the female chickens.
  • Essential oil blend T8 gave in the starter period a clear reduction in FCR compared to the control group, whereas the effect of T7 was intermediate (not significantly different from the control and the T8 diet). From 15 to 29 days of age both BWG and Fl were reduced, whereas FCR remained numerically lower (2.2 to 3.8 points) on the essential oil supplemented diets compared to the control diet, however essential oil blend effects were dependent on the presence of enzyme in the diet: No significant effect on BWG in the diet without enzyme was found, whereas BWG was reduced on the enzyme-supplemented diet. Cumulative feed intakes up to 29 and 37 days of age were significantly lower in the groups supplemented with either one of the essential oil blends.
  • Feed conversion ratio on the other hand was numerically improved (P>0.10) by dietary essential oil supplementation to the control diet, and not affected on the enzyme supplemented diet.
  • Essential oil blend T7 significantly improved litter quality score. Effect of enzyme or essential oil blends were not affected by broiler sex.
  • LSD is the least significant difference.
  • the %G+C profiles were divided into twelve 5% increments. The relative abundance (%) of the two treatment groups within each 5% and 1% increment was compared using t-test (Table 32).
  • the T8+Avizyme treatment resulted in significantly higher (than control) relative abundance within the %G+C range of 37 to 40 (p ⁇ 0.05; trend between 31 and 41).
  • the T8+Avizyme treatment resulted in lower relative abundance between the %GC increments 53 to 60 (p ⁇ 0.05; trend between 49 and 60).
  • Table 32 Statistical differences between caecum samples at 28 days of the treatment group and the control group analyzed as 5% and 1 % G+C increments.
  • the %G+C profiles were divided into twelve 5% increments. The relative abundance (%) of the two treatment groups within each 5% and 1 % increment was compared using t-test (Table 33).
  • the T8+Avizyme treatment resulted in significantly lower (than control) relative abundance within the %G+C range of 29 to 33 (p ⁇ 0.05; trend between 27 and 34).
  • the T8+Avizyme treatment resulted in higher relative abundance between the %GC increments 43 to 46 (p ⁇ 0.05; trend between 42 and 48).
  • Table 33 Statistical differences between the ileum samples at 28 days of the treatment group and the control group analyzed as 5% and 1% G+C increments.
  • T8+Avizyme treatment increased the relative abundance of bacteria with %G+C between 31 to 40%.
  • This %G+C range is typical of many different genuses, including Lactobacillus, Clostridium, Peptostreptococcus, Bacteroides, Enterococcus and Eubacterium.
  • the T8+Avizyme treatment also resulted in lower relative abundance of bacteria with %G+C between 48 to 60%. This %G+C range is typical of Escherichia and Salmonella.
  • T8+Avizyme treatment decreased the relative abundance of bacteria with %G+C between 27 to 34%. Within this range several genus exist, most notably Clostridium, Peptostreptococcus, Campylobacterium and Eubacterium. Conversely, the treatment increased the relative abundance of bacteria with %G+C between 42 to 48%. This range is typical of Lactobacillus, Bacteroides and Eubacterium.
  • T8+Avizyme can beneficially modulate chicken caecum and ileum microbiota.
  • This experiment comprised four experimental treatments.
  • a wheat-based control diet was supplemented with feed enzymes, and fed as such or supplemented with one of three essential oil blends (T7, T7v or T8).
  • T7v comprises cinnamaldehyde:thymol:eugenol:vanillin in the ratio of 1 :3:2:6 respectively.
  • Each treatment consisted of twelve replicate pens; six replicates with male and six replicates with female broilers. One pen was an experimental unit. An overview of the treatments in given in Table 34.
  • the concentration of the test substances is given in Table 35.
  • ROSS308 Four thousand and eight hundred male broilers (ROSS308) were supplied by Cobroed, Lievelde, the Netherlands.
  • the broilers were housed in floor pens (surface area: 10 m 2 ) with wood shavings. Each treatment consisted of sox replacates pens with 200 male broilers. Diets were fed for ad libitum intake to the broilers from 0 to 41 days of age. Water intake was supplied for ad libitum intake until 23 days of age. Subsequently, a water withdrawal period was introduced for 4 h per 24 h period, because of wet litter problems. The water supply periods were further reduced from day 35 onwards.
  • Feed conversion ratio was calculated during all aforementioned periods per pen.
  • a diet with a high indigestible protein level was used to stimulate protein fermentation in the intestinal tract.
  • Digestible amino acid levels in the diet were adequate according to the CVB recommendation (2004) for broilers in the starter, grower and finisher phase. Diets contained a chemical coccidiostat during the starter as well as the grower phase.
  • Granular Avizyme® 1302 was used as a feed enzyme and dosed at 300 g/ton. The diet composition is given in Table 37.
  • Foot pad lesions were evaluated on 21 and 28 days of age. In the current experiment no lesions were found. Due to very poor litter quality (wet) in all experiment units (values 4 to 5), no differences in litter quality could be observed.
  • Vitamin mix (0.1/1000 1 water) retinolpalmitate(vit. A) 12 500 I.E.; cholecalciferol (vit. D3) 5 000 I.E.; thiamine-HCI (vit. B1) 5 mg; riboflavin-5-sodium phosphate (vit. B2) 2,5 mg ; dexpenthenol (vit. B3) 12,5 mg ; pyridoxine-HCi (vit. B6) 4 mg; cyanocobalamin (vit. B12) 0,1 mg; ascorbic acid (vit. C) 40 mg ; ⁇ -tocoferolacetate (vit. E) 20 mg ; menadiolsodiumphosphate (vit. K3) 6 mg ; nicotinamid 40 mg ; biotin 0,2 mg ; cholinechlorid 50 mg 2 2 kg/ 1000 1
  • Body weight gain (BWG) in the starter phase (0-16 days) was significantly improved with T7 blend and this was due to increased feed intake.
  • BWG Body weight gain
  • composition of the mineral/vitamin premix (per kg of premix): Ca, 185 g; Na, 80 g; Cl 100 g; Cu, 1.200 mg; Fe, 4.500 mg; Mn, 7.000 mg; Zn, 3.700 mg; I, 100 mg; Se, 15 mg; vit. A, 1.000.000 IE; vit. D3, 200.000 IE; vit E, 2.500 IE; vit B1, 50mg; vit B2, 500 mg; Panthotenic acid 800 mg; Niacin, 4.000 mg; vit.
  • Cinnamaldehyde 100 96 95 97 95 96 81 81 86 90
  • the cinnamaldehyde and thymol are extracted from the product with a mixture of water, ethanol (96%), acetone and carvacrol (as an internal standard solution.
  • the extract is analysed by gas chromatography with flame ionisation detector.
  • Capillary column HP Innowax, 30m x 0.32 mm ID, phase thickness 0.5 ⁇ m.
  • Carrier gas helium
  • Thymol 100 100 1 2/25 80
  • the T8 Spray Dried product was prepared in accordance with the General Method.
  • the emulsion of the T8 essential oil blend was prepared in accordance with the emulsion as described for the spray dried General Method. The resulting mixture was mixed, dried and cooled. The temperature of the mixture was then raised to 4O 0 C and the mixture was crushed. The T8 mixture was then dried on a wheat carrier and then blended. The blended mixture was sieved. Any oversize particles underwent a crumbling step before being sieved again. The sieved mixture was then bagged.
  • Feed conversion ratio calculated during all aforementioned periods.
  • Jejunal contents were sampled from three birds per pen and pooled per pen on 31 days of age and underwent analysis for microflora composition.
  • the analyzed contents for moisture, crude protein and fat are presented in Table 46. Diets were pelleted over a 2.5 mm (diameter) x 2.5 cm (thickness) dye. Pelleting temperatures are presented in Table 44. Pellet quality of the experimental diets was poor for the starter diet and good for the grower and finisher diets. The starter diets were erroneously pelleted without steam addition.
  • Table 41 Calculated and analyzed contents for moisture, crude protein and fat as g/kg feed and g/kg dm (in italics)
  • the WFR in the grower phase was similar for all treatments.
  • T8 Spray Dried showed a higher efficacy than T8 Wheat Dried in the starter phase, whereas T8 Wheat Dried was more effective in the grower and finisher phase.
  • Such an age dependent effect might be used in poultry feed by changing the composition of the essential oil blends during ageing of the broilers.
  • Essential oil blends T8 Spray Dried and T8 Wheat Dried improved feed intake and body weight up to 31 days of age (i.e. during the starter and the grower phase), when added to a wheat/triticale-based broiler diet that was supplemented with a xylanase. Feed conversion ratio was not affected by dietary treatments compared to the control, probably because of the presence of coccidiostats in the starter and grower phases. Body weight gain from 31 to 37 days of age on T8 Spray Dried was similar to the control, whereas BWG on T8 Wheat Dried was 72 g higher during this last period. During the entire 37-day experimental period BWG of broilers on treatment T8 Wheat Dried was approx. 13O g higher (6.5%) compared to the control. Feed intakes of both T8 supplemented groups were 4.6% higher than the control. The numerically lower WFR in the T8 supplemented groups was consistent up to 31 days of age.
  • coccidiostat premix contained Clinacox in the starter and Stenerol in the grower phase.
  • T8 Wheat Dried 1 were obtained from Danisco Animal Nutrition.
  • Avizyme 1502 comprises amylase, protease and xylanase and was stored in a freezer prior to testing to ensure any insects were killed.
  • the main constituents of the EO product T8 Wheat Dried are thymol and cinnamaldehyde in the ratio of 3:1 respectively.
  • the T8 Wheat Dried was prepared as described in Example 13. In the following EO and T8 Wheat Dried are used interchangeable.
  • the enzyme activities of Avizyme 1502 are 600 U/g xylanase, 8000 U/g protease 800- U/g alpha- amylase.
  • For T8 Wheat Dried there are 5 g of cinnamaldehyde and 15 g of thymol in 65O g.
  • Standard laboratory diets were provided for each species, which were used as a check for insect viability on diets known to be suitable for insect development. These consisted of wholemeal flour and yeast (20:1) for T. castaneum and rolled oats, wheatgerm and i o yeast (5 : 5 : 1 ) f or O. surinamensis.
  • CE Controlled environment
  • Standard laboratory diets for each test species 300 g
  • 5 3.
  • Wheat diet + Avizyme 1502 (reference sample) 300 g
  • Jars containing test commodities 3 and 4 were rotated on a mechanical tumbler for 15 minutes to ensure the components were thoroughly mixed together. All the jars were closed with a filter paper lid and left in the experimental conditions for the duration of the experiment.
  • the adult beetles were removed and the commodities returned to the jars and incubated in the experimental conditions for periods of time long enough to include the passing of at least one generation. After sufficient time the numbers of next generation adults in each jar were counted.
  • insects were confined in arenas consisting of an aluminium ring (21 cm in diameter) sealed on to a white filter paper (27 cm in diameter) ( Figure 37).
  • the inner surface of the rings used to contain O. suhnamensis was coated with 'fluon' (PTFE) to prevent escapes.
  • a refuge consisting of electrical conduit (25 x 16 x 100 mm), containing about 2 g of a single commodity was placed into the centre of each arena ( Figure 37).
  • Five replicate arenas were prepared for each commodity and test species. In addition five arenas were also set up with refuges containing no commodities. Batches of 25 insects of each species were then removed from the laboratory cultures and placed into separate arenas. After 24 hours the numbers of insects outside and inside the refuge were counted.
  • Mean numbers of F 1 insects in the laboratory diet ranged from 1052 - 1144, indicating no problems with insect development on a familiar diet.
  • Mean numbers in the Avizyme 1502 alone ranged from 221 - 292 indicating that the insects were capable of breeding on the commodity but that development was reduced compared to the laboratory diet. This may be expected since the insects have been bred on the standard laboratory diet for many years and may have found a change in diet detrimental. No insects were found in the Avizyme and EO mixtures and the EO alone indicating that no development had taken place, presumably due to the rapid death of the parent beetles.
  • the strength of the repellency was assessed by subjecting the data (number of insects observed within the refuges as a proportion of the total in the arena) to statistical analysis (Logistic regression; Generalised linear model with logit link, Genastat 9). Pairwise comparisons were made between the proportions of insects in the refuges containing Avizyme 1502 only and those in the refuges containing the other commodities.
  • Table 47 shows the mean % of insects inside and outside the refuges containing the different commodities.
  • Table 48 shows the mean % of insects inside and outside both refuges containing the different commodities.
  • insects in the repellency bioassays were found outside the refuges. This may have been due to the relatively short exposure period (24 hours). With a longer exposure period more insects may have ventured into the refuges as the need to find food increased. The close proximity of the EO in the refuge may also have increased insect activity within the arena.
  • the object of this study was to evaluate the effect of blends of essential oils on carcass characteristics and flavour of breast meat.
  • a 3-phase feeding programme (starter, grower and finisher) as per standard commercial practice was used.
  • the diets were formulated based on wheat, maize, soybean meal and full-fat soybeans to meet standard specifications used by the industry.
  • the composition of the control diets is shown in Table 50.
  • the enzymes and essential oil blends were added in powder form and, following diet mixing, the diets were cold- pelleted (65-70 C).
  • the same diets were used in the AME evaluation study (Cohort cage study). Diet samples, prior to and after pelleting, were submitted to Danisco Innovation, Finland for analysis of essential oils.
  • Chicks were assigned on the basis of body weight to the floor pens on litter in an environmentally controlled room. The temperature was maintained at 31 0 C at the first week and then gradually reduced to 22 0 C at 35 days of age. Each diet was fed to six pens (30 birds/pen) from a day old to 35 days of age. The birds received constant fluorescent illumination and, were allowed free access to the diets and water.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Polymers & Plastics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Food Science & Technology (AREA)
  • Animal Husbandry (AREA)
  • Birds (AREA)
  • Fodder In General (AREA)

Abstract

La présente invention concerne un additif pour produit destiné à l'alimentation animale pouvant être obtenu par un procédé consistant à (a) mélanger de l'eau, un stabilisateur d'émulsion et un support acceptable d'un point de vue physiologique pour produire une solution de gomme; (b) à ajouter à la solution de gomme, isolément ou de façon combinée, un mélange huileux comprenant (i) du cinnamaldéhyde et (ii) un composé choisi dans le groupe constitué du citral, de l'eugénol, du limonène, du thymol, de la vanilline et des combinaisons de ceux-ci; (c) à mélanger la solution de gomme et le mélange huileux et à homogénéiser ce mélange sous pression pour produire une émulsion; et (d) à sécher par pulvérisation et/ou à agglomérer le mélange pour obtenir une poudre.
EP08762394A 2007-06-18 2008-06-17 Produit destiné à l'alimentation animale Withdrawn EP2170100A1 (fr)

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