WO2008091137A1 - Antibiotic feedstuff additives comprising ethanol extract of house fly larvae as active ingredient and manufacturing method thereof - Google Patents

Antibiotic feedstuff additives comprising ethanol extract of house fly larvae as active ingredient and manufacturing method thereof Download PDF

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Publication number
WO2008091137A1
WO2008091137A1 PCT/KR2008/000484 KR2008000484W WO2008091137A1 WO 2008091137 A1 WO2008091137 A1 WO 2008091137A1 KR 2008000484 W KR2008000484 W KR 2008000484W WO 2008091137 A1 WO2008091137 A1 WO 2008091137A1
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Prior art keywords
ethanol extract
growth
larvae
housefly larvae
antibiotic
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PCT/KR2008/000484
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French (fr)
Inventor
Kang-Jun Yoon
Heon-Tae Kim
Byung-Sung Park
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Medilarvatech Co., Ltd.
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Publication of WO2008091137A1 publication Critical patent/WO2008091137A1/en

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    • 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
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/20—Animal feeding-stuffs from material of animal origin
    • 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
    • A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
    • A23K30/20—Dehydration
    • 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
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs

Definitions

  • the present invention relates to an antibiotic feedstuff additive comprising an ethanol extract of housefly larvae as an active ingredient.
  • the present invention is also concerned with a method of preparing the feedstuff additive, comprising drying housefly larvae to remove water, dissolving the dried larvae in an organic solvent to remove fats and obtain residues, and mixing the residues with ethanol to obtain an ethanol extract.
  • the antibiotic feedstuff additive comprising an ethanol extract of housefly larvae as an active ingredient, is prepared using housefly larvae, which contain 50% or higher crude protein and are thus a good protein source, capable of replacing soybean meal and fish meal for broiler and layer chickens.
  • the feed additive promotes the growth of beneficial bacteria and inhibits the growth of harmful bacteria in the gut, thereby having prebiotic effects identical to or greater than those of conventional fructooligosaccharides.
  • Antibiotics have been primarily used to prevent and treat diseases. Since the 1940' s, they have also been utilized to promote the growth of livestock and to increase productivity.
  • Bifidobacteria and Lactobacilli are well known as bacteria that are beneficial for the health of humans and animals. Other bacteria, such as Escherichia coli and Clostridium perfringens, may be harmful .
  • Human intestinal microflora is composed of more than 400 different species of bacteria. The greatest microbial population (10 -10 ) is found in the colon. Lactobacilli accounts for 0.07-1% of intestinal microflora, whereas Bifidobacteria accounts for 25-30%.
  • Bifidobacteria and Lactobacilli are considered to be important probiotics in humans. Probiotics are defined as live microorganisms that are added to animal feed and beneficially affect the growth of colonic microorganisms.
  • Prebiotics are novel materials that are gaining increasing attention, which may be used as feed additives and in functional food instead of probiotics and antibiotics.
  • Prebiotics are non-digestible food ingredients that are not broken down in the stomach and the small intestine and migrate into the large intestine, where they beneficially affect humans and livestock by selectively stimulating the growth of colonic microorganisms, especially Bifidobacterium bifidum, or by selectively stimulating the activity or growth of a limited number of bacterial species in the colon.
  • a non-digestible food ingredient has beneficial effects as follows.
  • An ingested non-digestible food ingredient is not hydrolyzed by enzymes secreted from the stomach and the small intestine, and moves to the colon of the large intestine, where it is utilized as a fuel for the fermentation of beneficial bacteria.
  • the non-digestible food ingredient selectively stimulates the growth of beneficial species of the Bifidobacteria and Lactobacilli bacteria, especially Bifidobacterium bifiduin (bifidogenic effect), and inhibits the growth of harmful bacteria, including Clostridia, E. coli, Salmonella and Bacteroides.
  • VLDL very low density lipoprotein
  • Substances proposed as prebiotics include the following.
  • Non-digestible carbohydrates such as fructooligosaccharides (FOS; also called oligofructose)
  • FOS fructooligosaccharides
  • prebiotics are well known as prebiotics.
  • prebiotic oligosaccharides is inulins, which are usually derived from chicory and Jerusalem artichokes. Inulins are currently used as functional food materials. Oligosaccharides are important energy sources for microbial growth in the gut , but are not used as energy sources because they are not enzymatically degraded in the small intestines of chickens, pigs and other animals.
  • Pectin from orange peel was developed as a non-digestible food ingredient of swine feed because of its prebiotic effect of preventing diarrhea in animals.
  • houseflies Musca domestica L.
  • Houseflies have been used in medical studies and to heal wounds for a long time in the European Union, including England, but are little known for their prebiotic effects.
  • housefly larvae need to be studied for their application in antibiotic animal feed additives.
  • an object of the present invention is to provide a novel animal feed additive using housefly larvae as a good protein source, which stimulates the growth of beneficial bacteria and inhibits the growth of harmful bacteria in the gut, thereby having prebiotic effects identical to or greater than those of conventional fructooligosaccharides.
  • the present inventors evaluated the prebiotic effects of housefly larvae by examining the utilization of housefly larvae by intestinal beneficial bacteria, especially Bifidobacteria and Lactobacilli, and the physiological activity of the larvae, leading to the present invention.
  • the present invention provides an antibiotic feedstuff additive comprising an ethanol extract of housefly larvae as an active ingredient.
  • the present invention also provides a method of preparing the feedstuff additive, comprising drying housefly larvae to remove water, dissolving the dried larvae in an organic solvent to remove fats and obtain residues, and mixing the residues with ethanol to obtain an ethanol extract.
  • the larvae of housefly are dried to remove water at 80-150 ° C for 3-7 hours through freeze drying (lyophi lization) or spray drying or using a drying oven.
  • the dried larvae are dissolved in an organic solvent in order to remove fat components and obtain residues thereof.
  • the organic solvent is selected from among hexane and a solvent mixture of CHCL2 and MeOH.
  • the solvent mixture is preferred.
  • a 2:1 mixture of CHCL 2 and MeOH is preferable.
  • the ethanol extraction is preferably carried out three times or more in order to obtain high purity.
  • the present invention employs housefly larvae, which contain 50% or higher crude protein and are thus a good protein source capable of replacing soybean meal and fish meal for broiler and layer chickens, thereby providing a novel livestock feed additive, which promotes the growth of beneficial bacteria and inhibits the growth of harmful bacteria in the gut, thus having prebiotic effects identical to or superior to those of conventional fructooligosaccharides.
  • FIGS. 1 and 2 are graphs showing the growth rates of B. bifidum, which was dosed with an ethanol extract of housefly larvae (FIG. 1) and fructooligosaccharides (FOS) (FIG. 2);
  • FIGS. 3 and 4 are graphs showing the growth rates of B. longum, which was dosed with an ethanol extract of housefly larvae (FIG. 3) and FOS (FIG.
  • FIGS. 5 and 6 are graphs showing the growth rates of B. infantis, which was dosed with an ethanol extract of housefly larvae (FIG. 5) and FOS (FIG. 6);
  • FIGS. 7 and 8 are graphs showing the growth rates of L. acidophilus, which was dosed with an ethanol extract of housefly larvae (FIG. 7) and FOS (FIG. 8);
  • FIGS. 9 and 10 are graphs showing the growth rates of L. casei, which was dosed with an ethanol extract of housefly larvae (FIG. 9) and FOS (FIG. 10);
  • FIGS. 11 and 12 are graphs showing the growth inhibition of C. perfringens, which was dosed with an ethanol extract of housefly larvae (FIG. 11) and FOS (FIG. 12);
  • FIGS. 13 and 14 are graphs showing the growth inhibition of S. aureus, which was dosed with an ethanol extract of housefly larvae (FIG. 13) and FOS (FIG. 14); and FIGS. 15 and 16 are graphs showing the growth inhibition of B. fragilis, which was dosed with an ethanol extract of housefly larvae (FIG. 15) and FOS (FIG. 16).
  • the extraction was carried out in a heating mantle equipped with a reflux condenser at 60 ° C for 3 hrs.
  • the extracts were passed through Filter Paper No. 2 and pooled. Residues were extracted again as described above.
  • the extracts thus obtained were concentrated using a rotary vacuum evaporator (Eyela N-1000, Tokyo Rikakikai Co., Japan), thereby obtaining 10 g of an ethanol extract of non-fat housefly larvae with a yield of 5% from live housefly larvae.
  • the eight strains were dosed with the ethanol extract of non-fat housefly larvae, as follows.
  • the ethanol extract of non-fat housefly larvae was added to each broth medium at concentrations of 0%, 0.1%, 0.2% and 0.3% (wt/vol), and the prepared medium was sterilized.
  • Fructooligosaccharides (FOS; Sigma prod., USA), which are well-known for their prebiotic activity, were added to each broth medium at 0% and 0.5% (wt/vol), and sterilized.
  • FOS was used as a reference for the evaluation of the bifidogenic effect (stimulatory effect on the growth of intestinal Bifidobacterium species) of the ethanol extract of non-fat housefly larvae.
  • Each of the eight strains was sub-cultured in the prepared broth medium.
  • Anaerobic bacteria were activated by performing sub-culturing for 24 hrs three times in an incubator at 37 ° C under anaerobic conditions using a Gas-Pak method (BBL microbiology
  • cell densities of a culture grown in a medium not containing the ethanol extract of non-fat housefly larvae were measured at 600 nm.
  • the growth rates of strains grown in media supplemented with the ethanol extract were compared with those of the control .
  • FIGS. 1 and 2 show the growth rates of B. bifidum, which was dosed with the ethanol extract of housefly larvae at 0% (control), 0.1%, 0.2% and 0.3% (FIG. 1) and 0.5% fructooligosaccharides (FOS) (FIG. 2).
  • FIGS. 3 and 4 show the growth rates of B. longiun
  • FIGS. 5 and 6 show the growth rates of B. infantis. These strains displayed growth rates similar to those of B. bifidum.
  • FIGS. 7 and 8 show the growth rates of L. acidophilus
  • FIGS. 9 and 10 shows the growth rates of L. casei, wherein the growth of the strains was increased.
  • L. acidophilus and L. casei displayed an increase in OD values, representing its growth rates and carbon source utilization, from the initial stages of culture. This increase was higher as the concentrations of the ethanol extract of housefly larvae were increased.
  • L. acidophilus exhibited the highest growth rate at 10 hrs, and its growth was maintained at constant levels after 10 hrs.
  • L. casei showed similar initial growth rates, but rapidly grew after 10 hrs when dosed with 0.2% and 0.3% of the ethanol extract of housefly larvae (not in the 0.1% treatment group) and displayed the greatest growth after 20 hrs.
  • L. acidophilus and L. casei grown in an MRS broth supplemented with the ethanol extract of non-fat housefly larvae, displayed growth rates similar to those when dosed with FOS.
  • FIGS. 11 and 12 show the growth inhibition of C. perfringens, which was cultured in a reinforced clostridial medium (RCM) supplemented with the ethanol extract of housefly larvae at 0% (control), 0.1%, 0.2% and 0.3% (FIG. 11) and 0.5% FOS (FIG. 12).
  • C. perfringens which is a normal intestinal inhabitant, displayed an initial growth rate slightly lower than that of the control, and its growth was further reduced after 10 hrs. The lowest growth was observed at 48 hrs in a culture dosed with 0.3% of the ethanol extract of housefly larvae. The growth inhibitory effects of the ethanol extract of housefly larvae were similar to those of FOS.
  • FIGS. 13 and 14 show the growth inhibition of S. aureus, which was cultured in a nutrient broth supplemented with the ethanol extract of housefly larvae at 0% (control), 0.1%, 0.2% and 0.3% (FIG. 13) and 0.5% FOS (FIG. 14).
  • S. aureus which is a normal intestinal inhabitant
  • the growth of S. aureus sharply decreased after 24 hrs, indicating that the ethanol extract of non-fat housefly larvae strongly inhibited the growth of 5. aureus.
  • 5. aureus was dosed with 0.3% of the ethanol extract of non ⁇ fat housefly larvae, its growth was almost completely inhibited.
  • the growth inhibitory effects of the ethanol extract of housefly larvae were found to be greater than those of FOS.
  • FIGS. 15 and 16 show the growth inhibition of B. fragilis, which was cultured in an RCM broth supplemented with 0% (control), 0.1%, 0.2% and 0.3% of the ethanol extract of housefly larvae (FIG. 15) and 0.5% FOS (FIG. 16).
  • B. fragilis which is a normal intestinal inhabitant, displayed slightly increased growth rates until 15 hrs in the ethanol extract-containing medium, but the growth was sharply inhibited after 20 hrs, and almost stopped after 48 hrs.
  • These inhibitory effects of the ethanol extract of housefly larvae on the growth of B. fragilis were found to be similar to those of FOS.
  • the present invention examined the antibiotic activity of the ethanol extract of housefly larvae on three representative harmful bacteria ⁇ Clostridium perfringens, Bacteroides fragilis and Staphylococcus aureus), but the same growth inhibitory effects may be obtained against other harmful bacteria.
  • the ethanol extract of non-fat housefly larvae according to the present invention has significant prebiotic effects because it exhibited a bifidogenic effect, and is thus useful as a livestock feed, which is capable of replacing antibiotics.
  • the ethanol extract of housefly larvae which contain 50% or higher crude protein and are thus a good protein source capable of replacing soybean meal and fish meal for broiler and layer chickens, may be utilized to produce a novel livestock feed additive, which promotes the growth of beneficial bacteria and inhibits the growth of harmful bacteria in the gut, thereby having prebiotic effects identical to or better than those of conventional fructool igosaccharides, and is capable of replacing antibiotics.

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Abstract

Disclosed herein is an antibiotic feedstuff additive comprising an ethanol extract of housefly larvae as an active ingredient. Disclosed also is a method of preparing the feedstuff additive, comprising drying housefly larvae to remove water, dissolving the dried larvae in an organic solvent to remove fats and obtain residues, and mixing the residues with ethanol to obtain an ethanol extract. The antibiotic feedstuff additive is prepared using housefly larvae, which contain 50% or higher crude protein and are thus a good protein source capable of replacing soybean meal and fish meal for broiler and layer chickens, promotes the growth of beneficial bacteria and inhibits the growth of harmful bacteria in the gut, thereby having prebiotic effects identical to or greater than those of conventional fructooligosaccharides.

Description

[DESCRIPTION] [Invention Tit Ie]
ANTIBIOTIC FEEDSTUFF ADDITIVES COMPRISING ETHANOL EXTRACT OF HOUSEFLY LARVAE AS ACTIVE INGREDIENT AND MANUFACTURING METHOD THEREOF
[Technical Field]
The present invention relates to an antibiotic feedstuff additive comprising an ethanol extract of housefly larvae as an active ingredient. The present invention is also concerned with a method of preparing the feedstuff additive, comprising drying housefly larvae to remove water, dissolving the dried larvae in an organic solvent to remove fats and obtain residues, and mixing the residues with ethanol to obtain an ethanol extract.
The antibiotic feedstuff additive, comprising an ethanol extract of housefly larvae as an active ingredient, is prepared using housefly larvae, which contain 50% or higher crude protein and are thus a good protein source, capable of replacing soybean meal and fish meal for broiler and layer chickens. Thus, the feed additive promotes the growth of beneficial bacteria and inhibits the growth of harmful bacteria in the gut, thereby having prebiotic effects identical to or greater than those of conventional fructooligosaccharides.
[Background Art]
Antibiotics have been primarily used to prevent and treat diseases. Since the 1940' s, they have also been utilized to promote the growth of livestock and to increase productivity.
However, superbacteria (superbugs) having high resistance to antibiotics, such as methici 11 in- or vancomycin-resistant Staphylococcus aureus (MRSA or VRSA) and Salmonella typhimurium DT104, have recently been emerged. Many efforts have been made to find novel natural materials, which are able to replace conventional antibiotics. About 40-50% of antibiotics produced in the world are used as growth promoters or preventives of bacterial infections in livestock feed. Such antibiotics in animal feed are major causes of the spread of antibiotic resistance. In January of 2006, the European Union prohibited the use of antibiotics as antimicrobial growth promoters, which had been added to feedstuffs. South Korea has also gradually restricted antibiotic use in animal feed. However, the disuse of antibiotics may increase the onset of diarrhea-associated illness or enteritis in piglets or chicks. Thus, there remains a need for the development of a novel material which is naturally occurring and has an antibiotic effect of effectively killing harmful bacteria.
Bifidobacteria and Lactobacilli are well known as bacteria that are beneficial for the health of humans and animals. Other bacteria, such as Escherichia coli and Clostridium perfringens, may be harmful . Human intestinal microflora is composed of more than 400 different species of bacteria. The greatest microbial population (10 -10 ) is found in the colon. Lactobacilli accounts for 0.07-1% of intestinal microflora, whereas Bifidobacteria accounts for 25-30%. Bifidobacteria and Lactobacilli are considered to be important probiotics in humans. Probiotics are defined as live microorganisms that are added to animal feed and beneficially affect the growth of colonic microorganisms.
Prebiotics are novel materials that are gaining increasing attention, which may be used as feed additives and in functional food instead of probiotics and antibiotics. Prebiotics are non-digestible food ingredients that are not broken down in the stomach and the small intestine and migrate into the large intestine, where they beneficially affect humans and livestock by selectively stimulating the growth of colonic microorganisms, especially Bifidobacterium bifidum, or by selectively stimulating the activity or growth of a limited number of bacterial species in the colon.
For a food ingredient to be classified as a prebiotic, it must meet the following criteria:
1) it must be not hydrolyzed or absorbed in the upper digestive tract;
2) it must be a selective substrate for beneficial bacteria in the colon, thereby stimulating the bacteria to grow and become metabolically activated (selective fermentation in the colon);
3) it must be able to change the composition of the colonic microflora so that it is healthier; and
4) it must induce beneficial luminal or systemic effects within the host (humans or animals) (be metabolized by beneficial bacteria).
A non-digestible food ingredient has beneficial effects as follows.
1) An ingested non-digestible food ingredient is not hydrolyzed by enzymes secreted from the stomach and the small intestine, and moves to the colon of the large intestine, where it is utilized as a fuel for the fermentation of beneficial bacteria. The non-digestible food ingredient selectively stimulates the growth of beneficial species of the Bifidobacteria and Lactobacilli bacteria, especially Bifidobacterium bifiduin (bifidogenic effect), and inhibits the growth of harmful bacteria, including Clostridia, E. coli, Salmonella and Bacteroides.
2) It is used as a substrate for the microbial production of nutrients, such as antibiotic substances, vitamins and growth-stimulating factors, in the large intestine, thereby enhancing gastrointestinal functions.
3) It improves mineral bioavailability. In particular, it stimulates the intestinal absorption of calcium and iron by lowering cecal pH and increasing short-chain fatty acids through microbial fermentation.
4) It increases the normal, desirable microbial population in the gut, resulting in increased fecal output, and prevents diarrhea and enhances the immunity of animals, thereby promoting the growth of the host.
5) It lowers body-fat levels, reduces the number of plasma very low density lipoprotein (VLDL) particles, prevents hyperlipidemia, and has anticancer effects, reducing biomarkers for cancer risk.
Substances proposed as prebiotics include the following.
1) Non-digestible carbohydrates, such as fructooligosaccharides (FOS; also called oligofructose), are well known as prebiotics. Another example of prebiotic oligosaccharides is inulins, which are usually derived from chicory and Jerusalem artichokes. Inulins are currently used as functional food materials. Oligosaccharides are important energy sources for microbial growth in the gut , but are not used as energy sources because they are not enzymatically degraded in the small intestines of chickens, pigs and other animals.
2) Certain peptides and proteins (milk and vegetable proteins are partially non-digestible) are gaining interest as prebiotic substances.
3) The metabolism of lipids (ethers and esters) by colonic microorganisms is unknown.
4) Pectin from orange peel was developed as a non-digestible food ingredient of swine feed because of its prebiotic effect of preventing diarrhea in animals.
On the other hand, the larvae of houseflies (Musca domestica L.) contain 50% or higher crude protein and are thus a good protein source for poultry, which is capable of replacing soybean meal and fish meal for broiler and layer chickens. Houseflies have been used in medical studies and to heal wounds for a long time in the European Union, including England, but are little known for their prebiotic effects. Thus, housefly larvae need to be studied for their application in antibiotic animal feed additives.
[Disclosure]
[Technical Problem]
Accordingly, the present invention has been made keeping in mind the problems encountered in the prior art, and an object of the present invention is to provide a novel animal feed additive using housefly larvae as a good protein source, which stimulates the growth of beneficial bacteria and inhibits the growth of harmful bacteria in the gut, thereby having prebiotic effects identical to or greater than those of conventional fructooligosaccharides.
[Technical Solution]
The present inventors evaluated the prebiotic effects of housefly larvae by examining the utilization of housefly larvae by intestinal beneficial bacteria, especially Bifidobacteria and Lactobacilli, and the physiological activity of the larvae, leading to the present invention.
The present invention provides an antibiotic feedstuff additive comprising an ethanol extract of housefly larvae as an active ingredient. The present invention also provides a method of preparing the feedstuff additive, comprising drying housefly larvae to remove water, dissolving the dried larvae in an organic solvent to remove fats and obtain residues, and mixing the residues with ethanol to obtain an ethanol extract.
The larvae of housefly are dried to remove water at 80-150°C for 3-7 hours through freeze drying (lyophi lization) or spray drying or using a drying oven.
The dried larvae are dissolved in an organic solvent in order to remove fat components and obtain residues thereof. The organic solvent is selected from among hexane and a solvent mixture of CHCL2 and MeOH. The solvent mixture is preferred. A 2:1 mixture of CHCL2 and MeOH is preferable.
The ethanol extraction is preferably carried out three times or more in order to obtain high purity.
[Advantageous Effects]
In accordance with the present invention, the present invention employs housefly larvae, which contain 50% or higher crude protein and are thus a good protein source capable of replacing soybean meal and fish meal for broiler and layer chickens, thereby providing a novel livestock feed additive, which promotes the growth of beneficial bacteria and inhibits the growth of harmful bacteria in the gut, thus having prebiotic effects identical to or superior to those of conventional fructooligosaccharides.
[Description of Drawings]
FIGS. 1 and 2 are graphs showing the growth rates of B. bifidum, which was dosed with an ethanol extract of housefly larvae (FIG. 1) and fructooligosaccharides (FOS) (FIG. 2);
FIGS. 3 and 4 are graphs showing the growth rates of B. longum, which was dosed with an ethanol extract of housefly larvae (FIG. 3) and FOS (FIG.
4);
FIGS. 5 and 6 are graphs showing the growth rates of B. infantis, which was dosed with an ethanol extract of housefly larvae (FIG. 5) and FOS (FIG. 6);
FIGS. 7 and 8 are graphs showing the growth rates of L. acidophilus, which was dosed with an ethanol extract of housefly larvae (FIG. 7) and FOS (FIG. 8);
FIGS. 9 and 10 are graphs showing the growth rates of L. casei, which was dosed with an ethanol extract of housefly larvae (FIG. 9) and FOS (FIG. 10);
FIGS. 11 and 12 are graphs showing the growth inhibition of C. perfringens, which was dosed with an ethanol extract of housefly larvae (FIG. 11) and FOS (FIG. 12);
FIGS. 13 and 14 are graphs showing the growth inhibition of S. aureus, which was dosed with an ethanol extract of housefly larvae (FIG. 13) and FOS (FIG. 14); and FIGS. 15 and 16 are graphs showing the growth inhibition of B. fragilis, which was dosed with an ethanol extract of housefly larvae (FIG. 15) and FOS (FIG. 16).
[Best Mode]
[Mode for Invention]
A better understanding of the present invention may be obtained through the following example and test example, which are set forth to illustrate, but are not to be construed as the limit of the present invention.
EXAMPLE: Preparation of an ethanol extract of non-fat housefly larvae
200 g of live housefly were dried at 100°C for 5 hrs to remove water therefrom, thereby obtaining 98 g of dried larvae. 30 g of the dried larvae were dissolved in 300 ml of an organic solvent mixture of CHCL2 and MeOH (2:1) at 20°C for 2 hrs, and passed through three-layered gauze. The residues were placed in a hood for 1 hr to completely remove the organic solvents, and this process was repeated three times. The residues thus obtained were pooled, thereby obtaining 31 g of non-fat residues of housefly larvae. 31 g of the non-fat residues were mixed with 300 ml of pure ethanol, and then subjected to an extraction process using a reflux cooling apparatus to obtain an ethanol extract. The extraction was carried out in a heating mantle equipped with a reflux condenser at 60°C for 3 hrs. The extracts were passed through Filter Paper No. 2 and pooled. Residues were extracted again as described above. The extracts thus obtained were concentrated using a rotary vacuum evaporator (Eyela N-1000, Tokyo Rikakikai Co., Japan), thereby obtaining 10 g of an ethanol extract of non-fat housefly larvae with a yield of 5% from live housefly larvae.
TEST EXAMPLE: Evaluation of the effects of the ethanol extract on bacterial growth
(1) Strains and media
Five beneficial bacteria (three anaerobes: Bifidobacterium bifidum, Bifidobacterium infantis, and Bifidobacterium longum', two aerobes: Lactobacillus acidophilus, and Lactobaci1lus caseϊ) and three harmful bacteria (two anaerobes: Clostridium perfringens, and Bacteroides fragilis', one aerobe: Staphylococcus aureus), which were summarized in Table 1, below, were obtained from the Korean Food Research Institute (KFRI) and sub- cultured. Five anaerobic bacteria were cultured in a reinforced clostridial medium (RCM; oxoid CM 149), and three aerobic bacteria in a MRS medium (oxoid CM 359, Difco 0881) or a nutrient medium.
TABLE 1
Figure imgf000010_0001
(2) Culture
The eight strains were dosed with the ethanol extract of non-fat housefly larvae, as follows. The ethanol extract of non-fat housefly larvae was added to each broth medium at concentrations of 0%, 0.1%, 0.2% and 0.3% (wt/vol), and the prepared medium was sterilized. Fructooligosaccharides (FOS; Sigma prod., USA), which are well-known for their prebiotic activity, were added to each broth medium at 0% and 0.5% (wt/vol), and sterilized. FOS was used as a reference for the evaluation of the bifidogenic effect (stimulatory effect on the growth of intestinal Bifidobacterium species) of the ethanol extract of non-fat housefly larvae. Each of the eight strains was sub-cultured in the prepared broth medium. Anaerobic bacteria were activated by performing sub-culturing for 24 hrs three times in an incubator at 37°C under anaerobic conditions using a Gas-Pak method (BBL microbiology
9 systems, Cockeysville, MD). 0.1 ml (10 CFU/ml) of each activated strain was inoculated in the sterilized broth supplemented with the ethanol extract of non-fat housefly larvae. Strains were cultured anaerobically in a Gas-Pak system (BBL) at 37°C with agitation. Cells were collected at given time points (0, 5, 10, 15, 20, 24, 36 and 48 hrs). Optical densities (OD) of cultures were measured at 600 nm using a spectrophotometer (UV/Vis Spectrophotometer UV-260, Shimadzu Co., Japan). As a control, cell densities of a culture grown in a medium not containing the ethanol extract of non-fat housefly larvae were measured at 600 nm. The growth rates of strains grown in media supplemented with the ethanol extract were compared with those of the control .
FIGS. 1 and 2 show the growth rates of B. bifidum, which was dosed with the ethanol extract of housefly larvae at 0% (control), 0.1%, 0.2% and 0.3% (FIG. 1) and 0.5% fructooligosaccharides (FOS) (FIG. 2).
As shown in FIGS. 1 and 2, when B. bifidum was dosed with the ethanol extract of housefly larvae, compared to the control, the culture exhibited the highest OD values, representing its growth rates and carbon source utilization, after 20 and 24 hrs, and these OD values increased as the concentrations of the ethanol extract of housefly larvae were increased. B. bifidum, a major component of normal intestinal microflora, displayed similar growth rates at initial stages when dosed with various concentration of the ethanol extract of housefly larvae, but, after 15 hrs, the 0.3% treatment group showed the most rapid growth, which was higher than when dosed with 0.5% FOS, which is commercially available (FIG. 2).
FIGS. 3 and 4 show the growth rates of B. longiun, and FIGS. 5 and 6 show the growth rates of B. infantis. These strains displayed growth rates similar to those of B. bifidum. FIGS. 7 and 8 show the growth rates of L. acidophilus, and FIGS. 9 and 10 shows the growth rates of L. casei, wherein the growth of the strains was increased.
As shown in FIGS 7 to 10, when grown in an MRS broth supplemented with the ethanol extract of housefly larvae (FIGS. 7 and 9) or FOS (FIGS. 8 and 10), L. acidophilus and L. casei displayed an increase in OD values, representing its growth rates and carbon source utilization, from the initial stages of culture. This increase was higher as the concentrations of the ethanol extract of housefly larvae were increased. L. acidophilus exhibited the highest growth rate at 10 hrs, and its growth was maintained at constant levels after 10 hrs. L. casei showed similar initial growth rates, but rapidly grew after 10 hrs when dosed with 0.2% and 0.3% of the ethanol extract of housefly larvae (not in the 0.1% treatment group) and displayed the greatest growth after 20 hrs. L. acidophilus and L. casei, grown in an MRS broth supplemented with the ethanol extract of non-fat housefly larvae, displayed growth rates similar to those when dosed with FOS.
FIGS. 11 and 12 show the growth inhibition of C. perfringens, which was cultured in a reinforced clostridial medium (RCM) supplemented with the ethanol extract of housefly larvae at 0% (control), 0.1%, 0.2% and 0.3% (FIG. 11) and 0.5% FOS (FIG. 12). C. perfringens, which is a normal intestinal inhabitant, displayed an initial growth rate slightly lower than that of the control, and its growth was further reduced after 10 hrs. The lowest growth was observed at 48 hrs in a culture dosed with 0.3% of the ethanol extract of housefly larvae. The growth inhibitory effects of the ethanol extract of housefly larvae were similar to those of FOS.
FIGS. 13 and 14 show the growth inhibition of S. aureus, which was cultured in a nutrient broth supplemented with the ethanol extract of housefly larvae at 0% (control), 0.1%, 0.2% and 0.3% (FIG. 13) and 0.5% FOS (FIG. 14). Compared to the control, S. aureus, which is a normal intestinal inhabitant, displayed similar growth until 10 hrs in the ethanol extract- containing medium, but its growth was inhibited after 15 hrs. The growth of S. aureus sharply decreased after 24 hrs, indicating that the ethanol extract of non-fat housefly larvae strongly inhibited the growth of 5. aureus. In particular, when 5. aureus was dosed with 0.3% of the ethanol extract of non¬ fat housefly larvae, its growth was almost completely inhibited. The growth inhibitory effects of the ethanol extract of housefly larvae were found to be greater than those of FOS.
FIGS. 15 and 16 show the growth inhibition of B. fragilis, which was cultured in an RCM broth supplemented with 0% (control), 0.1%, 0.2% and 0.3% of the ethanol extract of housefly larvae (FIG. 15) and 0.5% FOS (FIG. 16). Compared to the control, B. fragilis, which is a normal intestinal inhabitant, displayed slightly increased growth rates until 15 hrs in the ethanol extract-containing medium, but the growth was sharply inhibited after 20 hrs, and almost stopped after 48 hrs. These inhibitory effects of the ethanol extract of housefly larvae on the growth of B. fragilis were found to be similar to those of FOS.
The present invention examined the antibiotic activity of the ethanol extract of housefly larvae on three representative harmful bacteria {Clostridium perfringens, Bacteroides fragilis and Staphylococcus aureus), but the same growth inhibitory effects may be obtained against other harmful bacteria.
Taken together, the ethanol extract of non-fat housefly larvae according to the present invention has significant prebiotic effects because it exhibited a bifidogenic effect, and is thus useful as a livestock feed, which is capable of replacing antibiotics.
[Industrial Applicability]
As described hereinbefore, the ethanol extract of housefly larvae, which contain 50% or higher crude protein and are thus a good protein source capable of replacing soybean meal and fish meal for broiler and layer chickens, may be utilized to produce a novel livestock feed additive, which promotes the growth of beneficial bacteria and inhibits the growth of harmful bacteria in the gut, thereby having prebiotic effects identical to or better than those of conventional fructool igosaccharides, and is capable of replacing antibiotics.

Claims

[CLAIMS] [Claim 1]
An antibiotic feedstuff additive comprising an ethanol extract of housefly larvae as an active ingredient.
[Claim 2]
A method of preparing a feedstuff additive comprising: drying housefly larvae to remove water; dissolving dried larvae in an organic solvent to remove fats and obtain residues; and mixing the residues with ethanol to obtain an ethanol extract.
[Claim 3]
The method according to claim 2, wherein the housefly larvae are dried to remove water at 80-150°C for 3-7 hours.
[Claim 4]
The method according to claim 2, wherein the housefly larvae are dried through freeze drying (lyophilization) or spray drying or using a drying oven.
[Claim 5]
The method according to claim 2, wherein the organic solvent to remove fats and obtain residues is hexane or a 2:1 mixture of CHCL2 and MeOH.
PCT/KR2008/000484 2007-01-26 2008-01-25 Antibiotic feedstuff additives comprising ethanol extract of house fly larvae as active ingredient and manufacturing method thereof WO2008091137A1 (en)

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