WO1997013526A1 - Method of improving lipid digestion - Google Patents

Method of improving lipid digestion Download PDF

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Publication number
WO1997013526A1
WO1997013526A1 PCT/DK1996/000424 DK9600424W WO9713526A1 WO 1997013526 A1 WO1997013526 A1 WO 1997013526A1 DK 9600424 W DK9600424 W DK 9600424W WO 9713526 A1 WO9713526 A1 WO 9713526A1
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WO
WIPO (PCT)
Prior art keywords
galactosidase
strain
feed
aspergillus
mono
Prior art date
Application number
PCT/DK1996/000424
Other languages
French (fr)
Inventor
Inge Helmer Knap
Anders Ohmann
Original Assignee
Novo Nordisk A/S
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 Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU72103/96A priority Critical patent/AU7210396A/en
Publication of WO1997013526A1 publication Critical patent/WO1997013526A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01022—Alpha-galactosidase (3.2.1.22)
    • 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
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/30—Feeding-stuffs specially adapted for particular animals for swines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases

Definitions

  • the present invention relates to a method of improving the lipid digestion of animal feed. More specifically, the invention provides a method of improving the digestion of lipids present in the feed supplied to mono-gastric animals, which method comprises administration of an effective amount of an ⁇ - galactosidase.
  • Microbial enzymes are well accepted as a class of feed enhancers, that by improving feed digestibility are able to increase the efficiency of the feed utilization.
  • Feed enzymes function by enhancing the digestibility of feed components. This enhancement may partly be brought about by degradation of poly- and oligosaccharides in cereals and vegetable proteins. Certain non-starch polysaccharides are responsible for an increased viscosity of the gut contents, but also for masking of readily digestible starches and proteins rendering them less accessible to endogenous digestive enzymes.
  • Oligosaccharides are not degraded in the small intestine by the digestive enzymes of mono-gastric animals, and hence do not offer their full energy potential to the animal.
  • the fermentation of these oligosaccharides has been associated with scouring in young pigs, and a condition known as black diarrhoea and sticky droppings in turkey poults.
  • black diarrhoea and sticky droppings in turkey poults have been associated with scouring in young pigs, and a condition known as black diarrhoea and sticky droppings in turkey poults.
  • the hydrolysis of these oligosaccharides are known to solve two problems, one of animal welfare and the other relating to an improved economy in production.
  • ⁇ -galactosidase is known to improve the metabolizable energy by converting raffinose and stachyose in e.g. soy and other vegetable proteins into assimilable sugars.
  • raffinose and stachyose in e.g. soy and other vegetable proteins into assimilable sugars.
  • any effect of ⁇ -galactosidase on the lipid digestion has never been reported.
  • the invention relates to the use of an ⁇ - galactosidase for improving the digestion of lipids present in the feed supplied to a mono-gastric animal, characterized by administration of an effective amount of an ⁇ -galactosidase to the mono-gastric animal. Therefore, in a first aspect, the invention provides a method of improving the digestion of lipids present in the feed supplied to mono-gastric animals, which method comprises administration of an effective amount of an ⁇ -galactosidase.
  • the invention provides, a feed enhancing additive comprising an ⁇ -galactosidase in an amount effective for improving the lipid digestion.
  • the present invention relates to the use of an ⁇ - galactosidase for improving the digestion of lipids among mono- gastric animals. More specifically the invention provides a method of improving the digestion of lipids present in the feed supplied to mono-gastric animals, which method comprises administration of an effective amount of an ⁇ -galactosidase.
  • the mono-gastric animal is a poultry, in particular a broiler chick, a layer or a turkey, a pig, in particular a piglet, or a young calf .
  • the animal feed composition comprises substantial amounts of leguminous, in particular soybean, cotton seed, lupine, peas and beans, and crucifera, in particular rapeseed.
  • the ⁇ -galactosidase administered to the mono-gastric animal preferably is an ⁇ -galactosidase with activity towards the substrates raffinose, stachyose and/or verbascose.
  • the ⁇ -galactosidase may be administered in any convenient form, and may be administered either prior to feeding the animal, or simultaneously with feeding the animal.
  • the ⁇ -galactosidase is administered simultaneously with feeding the animal, e.g. provided in the form of a feed enhancing additive.
  • an effective amount of an ⁇ -galactosidase is the amount of ⁇ -galactosidase necessary for achieving a significantly improved lipid digestion. It is at present contemplated that the amount of ⁇ -galactosidase administered should be in the range of from about 10 to about 20.000 GALU per kg of animal feed, preferably in the range of from about 100 to about 2.000 GALU per kg of animal feed.
  • the ⁇ -galactosidase administered to the mono-gastric animal may be of microbial origin, preferably of bacterial or fungal origin.
  • Bacterial ⁇ -galactosidases are available from strains of E. coli , and from strains of Bacillus, in particular a strain of Bacillus stearothermophilus or a strain of Bacillus subtilis .
  • Fungal ⁇ -galactosidases are available from strains of
  • the ⁇ -galactosidases is derived from a strain of Aspergillus oryzae, or a strain of Aspergillus ficuum, a strain of Aspergillus aculeatus, or a strain of Aspergillu ⁇ niger.
  • ⁇ -galactosidases also are available from yeast, in particular from a strain of Saccharomyces cereviciae, or from a strain of Saccharomyces oleaginosus .
  • the ⁇ -galactosidase is provided in the form of a mono-component preparation, produced by recombinant DNA technology.
  • a mono-component preparation produced by recombinant DNA technology.
  • the present invention provides a feed enhancing additive, which additive comprises an ⁇ -galactosidase in an amount effective of improving the digestion of lipids.
  • a feed enhancing additive is an enzyme preparation comprising one or more feed enhancing enzymes and suitable carriers and/or excipients, and which enzyme preparation is provided in a form that is suitable for being added to animal feed.
  • the feed enhancing additive of the invention may be prepared in accordance with methods known in the art and may be in the form of a dry or a licruid preparation.
  • the enzyme to be included in the preparation may optionally be stabilized in accordance with methods known in the art.
  • the feed enhancing additive of the invention is a granulated enzyme product which may readily be mixed with feed components, or more preferably, form a component of a pre-mix.
  • the granulated enzyme product may be coated or un ⁇ coated.
  • the particle size of the enzyme granulates preferably is compatible with that of feed and pre-mix components. This provides a safe and convenient mean of incorporating enzymes into feeds.
  • the feed enhancing additive of the invention is a stabilized licruid composition, which may be an aqueous or oil-based slurry.
  • the liquid composition may optionally be added to the animal feed after pelleting.
  • the feed enhancing additive comprises an ⁇ -galactosidase in an amount corresponding to an activity in the range of from about 50 to about 20.000 GALU/g additive, preferably of from about 200 to about 5.000 GALU/g additive.
  • the ⁇ -galactosidase activity may be determined relative to a p-nitrophenyl- ⁇ -D-galactopyranoside substrate (p-NPGal) .
  • p-NPGal p-nitrophenyl- ⁇ -D-galactopyranoside substrate
  • ⁇ - galactosidase hydrolyses the colorless substrate forming p- nitrophenol, which is yellow in alkaline solution, and which color can be monitored spectrophotometrically at 405 nm.
  • 1 GALU is defined as the amount of ⁇ -galactosidase required for hydrolyzing 1 ⁇ mol p-
  • NPGal per minute under standard conditions (i.e. using 0.80 mM substrate, after incubation for 15 minutes at pH 5.5 and at 37°C) .
  • the foodstuff under study was soy bean meal, and the inclusion level at the expense of all basal ingredients was 25%.
  • Two experiments of enzyme supplementation were carried out using a commercially available ⁇ -galactosidase preparation (NovozymeTM 696 1000 L, an Aspergillus niger ⁇ -galactosidase preparation available from Novo Nordisk A/S, Denmark) , and an experimental preparation of an ⁇ -galactosidase obtained from a strain of Aspergillus niger according to the method described in WO 94/23022 (cf. in particular Example 4), in Table 2 designated (c) . Both preparations were dosed in amounts equal to an activity of 2,000 GALU/kg soy bean meal.
  • the feed composition of the basal diet is shown Table 1, below.
  • the balance trial (21-25 days) was carried out according to the European reference method for the in vivo determination of etabolizable energy, MEn [Bourdillon et al . , Br. Poult. Sci. 1990 31 557-565] .
  • the broilers were fed the mash diets at a level of 90% of ad libi tum, and the respective excreta were quantitatively collected on a daily basis.
  • the excreta were mixed to homogeneity, freeze-dried, equilibrated and finally ground before analysis. Samples of freeze-dried excreta were analyzed for gross energy and fat.

Abstract

The present invention relates to a method of improving the lipid digestion of animal feed. More specifically, the invention provides a method of improving the digestion of lipids present in the feed supplied to mono-gastric animals, which method comprises administration of an effective amount of an α-galactosidase.

Description

METHOD OF IMPROVING LIPID DIGESTION
TECHNICAL FIELD
The present invention relates to a method of improving the lipid digestion of animal feed. More specifically, the invention provides a method of improving the digestion of lipids present in the feed supplied to mono-gastric animals, which method comprises administration of an effective amount of an α- galactosidase.
BACKGROUND ART
Microbial enzymes are well accepted as a class of feed enhancers, that by improving feed digestibility are able to increase the efficiency of the feed utilization. Feed enzymes function by enhancing the digestibility of feed components. This enhancement may partly be brought about by degradation of poly- and oligosaccharides in cereals and vegetable proteins. Certain non-starch polysaccharides are responsible for an increased viscosity of the gut contents, but also for masking of readily digestible starches and proteins rendering them less accessible to endogenous digestive enzymes.
Oligosaccharides are not degraded in the small intestine by the digestive enzymes of mono-gastric animals, and hence do not offer their full energy potential to the animal. In addition, the fermentation of these oligosaccharides has been associated with scouring in young pigs, and a condition known as black diarrhoea and sticky droppings in turkey poults. Thus the hydrolysis of these oligosaccharides are known to solve two problems, one of animal welfare and the other relating to an improved economy in production.
In this way α-galactosidase is known to improve the metabolizable energy by converting raffinose and stachyose in e.g. soy and other vegetable proteins into assimilable sugars. However, any effect of α-galactosidase on the lipid digestion has never been reported. SUMMARY OF THE INVENTION
It has now surprisingly been found that the presence of α-galactosidase improves the lipid digestion among mono-gastric animals. Accordingly, the invention relates to the use of an α- galactosidase for improving the digestion of lipids present in the feed supplied to a mono-gastric animal, characterized by administration of an effective amount of an α-galactosidase to the mono-gastric animal. Therefore, in a first aspect, the invention provides a method of improving the digestion of lipids present in the feed supplied to mono-gastric animals, which method comprises administration of an effective amount of an α-galactosidase.
In another aspect the invention provides, a feed enhancing additive comprising an α-galactosidase in an amount effective for improving the lipid digestion.
DETAILED DISCLOSURE OF THE INVENTION
The present invention relates to the use of an α- galactosidase for improving the digestion of lipids among mono- gastric animals. More specifically the invention provides a method of improving the digestion of lipids present in the feed supplied to mono-gastric animals, which method comprises administration of an effective amount of an α-galactosidase.
In a preferred embodiment of the present invention, the mono-gastric animal is a poultry, in particular a broiler chick, a layer or a turkey, a pig, in particular a piglet, or a young calf . In another preferred embodiment, the animal feed composition comprises substantial amounts of leguminous, in particular soybean, cotton seed, lupine, peas and beans, and crucifera, in particular rapeseed.
The α-galactosidase administered to the mono-gastric animal preferably is an α-galactosidase with activity towards the substrates raffinose, stachyose and/or verbascose. The α-galactosidase may be administered in any convenient form, and may be administered either prior to feeding the animal, or simultaneously with feeding the animal. Preferably the α-galactosidase is administered simultaneously with feeding the animal, e.g. provided in the form of a feed enhancing additive.
In the context of this invention, an effective amount of an α-galactosidase is the amount of α-galactosidase necessary for achieving a significantly improved lipid digestion. It is at present contemplated that the amount of α-galactosidase administered should be in the range of from about 10 to about 20.000 GALU per kg of animal feed, preferably in the range of from about 100 to about 2.000 GALU per kg of animal feed.
The α-galactosidase administered to the mono-gastric animal may be of microbial origin, preferably of bacterial or fungal origin.
Bacterial α-galactosidases are available from strains of E. coli , and from strains of Bacillus, in particular a strain of Bacillus stearothermophilus or a strain of Bacillus subtilis . Fungal α-galactosidases are available from strains of
Neurospora, Rhizopus and Aspergillus . In a preferred embodiment of the invention, the α-galactosidases is derived from a strain of Aspergillus oryzae, or a strain of Aspergillus ficuum, a strain of Aspergillus aculeatus, or a strain of Aspergilluε niger. α-galactosidases also are available from yeast, in particular from a strain of Saccharomyces cereviciae, or from a strain of Saccharomyces oleaginosus .
In a preferred embodiment, the α-galactosidase is provided in the form of a mono-component preparation, produced by recombinant DNA technology. By providing the α-galactosidase in the form of a mono-component preparation, the formation of undesirable oligosaccharides may be prevented, thereby reducing digestive discomfort.
Feed Enhancing Additives In another aspect the present invention provides a feed enhancing additive, which additive comprises an α-galactosidase in an amount effective of improving the digestion of lipids.
In the context of this invention, a feed enhancing additive is an enzyme preparation comprising one or more feed enhancing enzymes and suitable carriers and/or excipients, and which enzyme preparation is provided in a form that is suitable for being added to animal feed. The feed enhancing additive of the invention may be prepared in accordance with methods known in the art and may be in the form of a dry or a licruid preparation. The enzyme to be included in the preparation, may optionally be stabilized in accordance with methods known in the art.
In a specific embodiment the feed enhancing additive of the invention is a granulated enzyme product which may readily be mixed with feed components, or more preferably, form a component of a pre-mix. The granulated enzyme product may be coated or un¬ coated. The particle size of the enzyme granulates preferably is compatible with that of feed and pre-mix components. This provides a safe and convenient mean of incorporating enzymes into feeds.
In another specific embodiment, the feed enhancing additive of the invention is a stabilized licruid composition, which may be an aqueous or oil-based slurry. The liquid composition may optionally be added to the animal feed after pelleting.
In a preferred embodiment, the feed enhancing additive comprises an α-galactosidase in an amount corresponding to an activity in the range of from about 50 to about 20.000 GALU/g additive, preferably of from about 200 to about 5.000 GALU/g additive.
α-Galactosidase Activity (GALU)
The α-galactosidase activity may be determined relative to a p-nitrophenyl-α-D-galactopyranoside substrate (p-NPGal) . α- galactosidase hydrolyses the colorless substrate forming p- nitrophenol, which is yellow in alkaline solution, and which color can be monitored spectrophotometrically at 405 nm. In the context of this invention, 1 GALU is defined as the amount of α-galactosidase required for hydrolyzing 1 μmol p-
NPGal per minute under standard conditions (i.e. using 0.80 mM substrate, after incubation for 15 minutes at pH 5.5 and at 37°C) .
A folder, AF 204/3-GB, describing this analytical method in more detail is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.
EXAMPLES
The invention is further illustrated with reference to the following examples which are not intended to be in any way limiting to the scope of the invention as claimed.
Example 1
Effect of α-galactosidase on Energy Availability and Fat
Digestion
Male broiler chicks were obtained from a commercial hatchery. From day 1 to 16, the birds were housed on deep litter under conventional conditions for lighting, heating and ventilation. They were fed on a commercial chick starter mash diet. On day 16, the birds were weighed individually, and birds having relatively high or low body weights were discarded. Groups of four birds were then assigned randomly to each of 20 pens, housed in digestibility cages. For each treatment, five replicates were used.
The foodstuff under study was soy bean meal, and the inclusion level at the expense of all basal ingredients was 25%. Two experiments of enzyme supplementation were carried out using a commercially available α-galactosidase preparation (Novozyme™ 696 1000 L, an Aspergillus niger α-galactosidase preparation available from Novo Nordisk A/S, Denmark) , and an experimental preparation of an α-galactosidase obtained from a strain of Aspergillus niger according to the method described in WO 94/23022 (cf. in particular Example 4), in Table 2 designated (c) . Both preparations were dosed in amounts equal to an activity of 2,000 GALU/kg soy bean meal. The feed composition of the basal diet is shown Table 1, below.
Table 1
Composition of Basal Diet
Component % Inclusion rate in Diet
Sorghum 56.13
Soybean meal protein 32.45
Animal fat 6.00
Calcium carbonate 0.66
Dicalcium phosphate 2.24
Salt 0.35
DL Methionine 0.13
Vitamins/trace elements 1.00
The balance trial (21-25 days) was carried out according to the European reference method for the in vivo determination of etabolizable energy, MEn [Bourdillon et al . , Br. Poult. Sci. 1990 31 557-565] . The broilers were fed the mash diets at a level of 90% of ad libi tum, and the respective excreta were quantitatively collected on a daily basis. The excreta were mixed to homogeneity, freeze-dried, equilibrated and finally ground before analysis. Samples of freeze-dried excreta were analyzed for gross energy and fat.
The main results are presented in Table 2, below. The results of all treatments were analyzed statistically by a one factorial analysis of variance, with significant treatment differences identified by a LSD-multiple range test, according to stratgraphics version 5 (1991) . Figures followed by different letters are significantly different from each other at P=0.05. Table 2
Statistical evaluation of the main balance parameters
MEn Fat (MJ/kg) Digestibility (%)
Basal Diet (B) 12.011a 68. lb
B + 25% Soya 11.060 67.6b
B + 25% Soya + 11.216b 73.3* Novo zyme™ 696 1000L
B + 25% Soya + α- 11.191b 71.6a galactosidase exp. prep, (c)
Addition of α-galactosidase resulted in a significantly increased fat digestion in the order of 6%.

Claims

1. A method of improving the digestion of lipids present in the feed supplied to mono-gastric animals, which method comprises administration of an effective amount of an α-galactosidase.
2. The method according to claim 1, in which the α-galactosidase is of microbial origin, preferably of fungal origin.
3. The method according to claim 2, in which the α-galactosidase is a mono-component α-galactosidase.
4. The method according to any of claims 1-3, in which the α- galactosidase is derived from a strain of Aspergillus .
5. The method according to any of claims 4, in which the α- galactosidase is derived from a strain of Aspergillus aculeatus, a strain of Aspergillus niger, a strain of Aspergillus oryzae, or a strain of Aspergillus ficuum.
6. The method according to any of claims 1-5, in which the α- galactosidase is administered before or simultaneously with the animal feed.
7. The method according to any of claims 1-6, in which the α- galactosidase is added to the animal feed composition, preferably in the form of a granulate or a stabilized liquid.
8. The method according to any of claims 1-7, in which the α- galactosidase is administered in an amount corresponding to an activity in the range of from about 10 to about 20.000 GALU per kg of animal feed, preferably of from about 100 to about 2.000 GALU per kg of animal feed.
9. The method according to any of claims 1-8, in which the animal feed composition comprises substantial amounts of lipids.
10. The method according to any of claims 1-9, in which the animal feed composition comprises substantial amounts of α- galactosides, preferably stachyose raffinose and verbascose.
11. The method according to claim 10, in which the animal feed composition comprises substantial amounts of leguminous, in particular soybean, cotton seed, lupine, peas and beans, and crucifera, in particular rapeseed.
12. The method according to any of claims 1-11, in which the mono-gastric animal is a pig, a piglet, a broiler chick, a layer, a turkey, or a young calf.
13. A feed enhancing additive comprising an α-galactosidase in an amount effective for improving the lipid digestion.
14. The feed enhancing additive according to claim 13, in which the α-galactosidase is derived from a strain of Aspergillus aculeatus, a strain of Aspergillus niger, a strain of Aspergillus oryzae, or a strain of Aspergillus ficuum.
15. The feed enhancing additive according to either of claims 13- 14, in which the α-galactosidase is a mono-component α- galactosidase.
16. The feed enhancing additive according to any of claims 13-15, in which the α-galactosidase is provided in the form of a non¬ dusting granulate or a stabilized liquid.
17. The feed enhancing additive according to any of claims 13-16, comprising the α-galactosidase in an amount corresponding to an activity in the range of from about 50 to about 20.000 GALU/g additive, preferably of from about 200 to about 5.000 GALU/g additive.
PCT/DK1996/000424 1995-10-10 1996-10-08 Method of improving lipid digestion WO1997013526A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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DK113595 1995-10-10
DK1135/95 1995-10-10

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6399123B1 (en) * 2000-05-25 2002-06-04 University Of Missouri Board Of Curators Oligosaccharide removal from animal feed
WO2002081674A1 (en) * 2001-04-05 2002-10-17 Universite Laval Alpha-galactosidase as food-grade genetic marker

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990014101A1 (en) * 1989-05-16 1990-11-29 Akpharma Inc. Composition and method for reducing gastro-intestinal distress due to alpha-d-galactoside-linked/containing sugars
WO1994023022A1 (en) * 1993-03-31 1994-10-13 Novo Nordisk A/S AN α-GALACTOSIDASE ENZYME

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1990014101A1 (en) * 1989-05-16 1990-11-29 Akpharma Inc. Composition and method for reducing gastro-intestinal distress due to alpha-d-galactoside-linked/containing sugars
WO1994023022A1 (en) * 1993-03-31 1994-10-13 Novo Nordisk A/S AN α-GALACTOSIDASE ENZYME

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 155, Medline Dialog Accession No. 00798843, Medline Accession No. 83031843, GAUTAM A.K. et al., "Significance of Glycosidases in Myelin-lipid Metabolism. I. Studies in Diencephalon of Fresh Water Turtle (Lissemys Punctata Granosa)"; & J. HIRNFORSCH (GERMANY, EAST) 1982, 23 (3), *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6399123B1 (en) * 2000-05-25 2002-06-04 University Of Missouri Board Of Curators Oligosaccharide removal from animal feed
WO2002081674A1 (en) * 2001-04-05 2002-10-17 Universite Laval Alpha-galactosidase as food-grade genetic marker

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