KR100828114B1 - Antibiotic feedstuff additives comprising ethanol extract of house fly larvae as active ingredient and manufacturing method thereof - Google Patents

Abstract

An antimicrobial feed additive containing an ethanol extract of house fly larvae as an effective ingredient and its production method are provided to promote the growth of probiotic bacteria and inhibit the growth of harmful bacteria, thereby obtaining the same (or more) antimicrobial effect as fructooligosaccharide. An antimicrobial feed additive containing an ethanol extract of house fly larvae as an effective ingredient is produced by the steps of: (i) drying house fly larvae by a freeze-drying process, a spray-drying process or a dryer at 80~150°C for 3~7hr, and dehydrating the dried larvae to obtain a dried sample; (ii) dissolving the dried sample in a mixed organic solvent to remove fat and obtain residue; and (iii) mixing the residue with ethanol to obtain an ethanol extract. The organic solvent is hexane or a mixed solvent of CHCl2 and MeOH, wherein the ratio of CHCl2 and MeOH is 2:1.

Description

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

1 and 2 are graphs showing the growth rate of Bifidobacterium bifidum strain

3 and 4 are graphs showing the growth rate of Bifidobacterium long gum ( B. longum ) strains

5 and 6 are graphs showing the growth rate of Bifidobacterium infantis strain

7 and 8 are graphs showing the growth rate of Lactobacillus ashdophyllus ( L. acidophilus ) strains

9 and 10 are graphs showing the growth rate of L. casei strain

11 and 12 are graphs showing the inhibitory effect on C. perfringens strains

13 and 14 are S. aureus Graph showing inhibitory effect on strains

Figure 15 and Figure 16 shows the bacteroid B. fragilis ( B. fragilis ) Graph showing inhibitory effect on strains

The present invention provides an antimicrobial feed additive comprising ethanol extract of House fly larvae as an active ingredient, and drying the house fly larva and removing moisture to obtain a building sample; Dissolving the building sample in a mixed organic solvent to remove fat and to obtain a residue; And a step of obtaining the ethanol extract by mixing the residue with ethanol.

The antimicrobial feed additive containing the ethanol extract of the housefly larva of the present invention as an active ingredient, which has a crude protein content of 50% or more, uses a housefly larva as an excellent protein source that can replace soybean meal and fish meal for broilers and laying hens. In addition, the antimicrobial effect of the same or higher than the existing fructooligosaccharide can be obtained through the effects of growth promotion on the enteric beneficial bacteria and inhibition of the growth of harmful bacteria.

Antibiotics were originally used for the prevention and treatment of diseases, but since the 1940s

It was used to promote livestock growth and increase productivity.

Recently, the emergence of super bacteria (super bugs) against antibiotics such as methicillin and vancomycin-resistant Staphylococcus aureus (MRSA, VRSA; Methicillin, Vancomycin resistant Streptococcus aureus ), Salmonella Typhimurium DT104 Efforts to find material materials continue. 40-50% of the amount of antimicrobial produced worldwide is used as a growth accelerator or infection prevention agent in livestock feed, and antimicrobial agents in these feeds act as a major factor in the transmission of resistance. Europe has banned the use of antibiotics as antimicrobial growth promoters added to feeds since January 2006. The Republic of Korea is gradually restricting the use of antibiotics. Without antibiotics, diarrhea-related diseases and enteritis can increase, especially in young pigs and chickens. Therefore, it is necessary to develop new materials that are natural and have the effect of killing harmful bacteria, such as antibiotics.

Bifidobacteria bacteria (bifidobacteria) and Lactobacillus bacteria (lactobacilli) is well known as a beneficial microorganisms in the human and animal health, and other microorganisms such as Escherichia coli (Escherichia coli), well chwigyun (Clostridium perfrigens) can be harmful. Human intestinal microbes consist of more than 400 species, and the colon is the one with the highest microbial population (10 ¹⁰ -10 ¹²⁾ . Lactobacillus accounts for 0.07-1% of intestinal microorganisms, while Bifidobacteria accounts for 25-30%. Bifidobacteria and lactobacilli are considered as important probiotics for humans. Non-degradable dietary ingredients are defined as living microorganisms added to feeds that are beneficial for the growth of colon microorganisms.

Non-degradable dietary ingredients (Prebiotics) is a newly emerging material as a feed additive and functional food material that can replace probiotics and antibiotics. Non-degradable dietary ingredients are those that do not decompose in the stomach and small intestine and are transferred to the large intestine to selectively stimulate the growth of microorganisms in the colon, particularly bifidobacterium bifidum , or selectively activate the microorganism's vitality or the limited number of microorganisms. A substance that stimulates and has a beneficial effect on people and livestock.

 The conditions of the non-degradable dietary ingredient may be mentioned as follows.

V) should not be hydrolyzed and absorbed at the top of the digestive tract.

Ii) be a selective substrate of the flora that stimulates the growth of microorganisms and is beneficial to metabolically activated colon (selective fermentation in the colon).

V) It should be possible to change the microorganisms in the colon with a healthier composition.

I) Must exhibit the effect of a luminal or organ beneficial to the host (human, animal) (metabolized by beneficial microorganisms).

Moreover, the following are mentioned as an effect of a non-degradable diet component.

Iii) The ingested non-degradable dietary component is used as a food for beneficial microbial fermentation after transfer to the colon of the colon without hydrolysis by enzymes secreted from the stomach and small intestine. Non-degradable dietary ingredients stimulate the growth of healthy Lactobacilli and Bifidobacteria, especially Pidobacterium bifidum, and the harmful bacteria Clostridia , Escherichia coli, Salmonella, Pak Inhibit growth of steroids, etc.

Ii) microbiology of nutrients such as antibiotics, vitamins and growth promoters in the large intestine

It can be used as a substrate for the production of enemies, thus improving gastrointestinal function.

Iii) enhance the bioavailability of minerals. In particular, by lowering the pH of the cecum by the microbial fermentation and increasing the concentration of short chain fatty acids has the function of promoting the absorption of calcium and iron intestine.

Iv) increase the amount of distributed snow by increasing microbial population, prevent diarrhea and immune function

Enhancement of animal growth can be promoted.

Iii) Lower body fat, reduce the number of very low density lipoprotein (VLDL) particles in the blood, prevent hyperlipidemia, and be a bio marker for cancer prevention.

Moreover, the following are mentioned as a candidate substance of a non-degradable dietary component.

I) As non-degradable carbohydrates, fructooligosaccharides (Fructooligosaccharides or Oligfructose, FOS) are widely known as representative non-degradable dietary ingredients, and Inulin, extracted from chicory and pork potatoes, has begun to be used as a functional food material in the European market. However, while oligosaccharides are an important source of energy for intestinal microorganisms, chickens and pigs are not known to be used as energy sources because they cannot enzymatically degrade oligosaccharides in the intestine.

Ii) Some peptides and proteins (milk and vegetable proteins are partially non-degradable) are of interest as candidates.

I) Lipid metabolism by colonic microorganisms of lipids (ethers and esters) is not yet known.

In 2004, Pectin in citrus peels was developed as a non-degradable dietary ingredient in pig feed by preventing diarrhea in piglets.

House fly larvae ( Musca domestica L. ) is a good source of animal protein that can replace soybean meal and fish meal for broilers and laying hens with a crude protein content of more than 50%. Houseflies have been used for a long time in Europe and elsewhere in the United Kingdom as a medical research and wound healing agent. However, since the effects of non-degradable dietary ingredients are not known, research on antimicrobial feed additives using houseflies is needed.

Therefore, the present invention has been made in view of this point, and by using the housefly larvae as an excellent protein source, it has an antimicrobial effect equivalent to or higher than that of the existing fructooligosaccharide through the effect of promoting growth against the enteric beneficial bacteria and inhibiting the growth of harmful bacteria. To provide a completely new form of feed additives to obtain.

In the present invention, to investigate the effect of non-degradable dietary components of housefly larvae, the present invention was completed by examining the utilization ability and physiological activity of housefly larvae by intestinal beneficial microorganisms, particularly Bifidobacteria and lactobacilli.

That is, the present invention comprises the antimicrobial feed additive comprising the ethanol extract of the housefly larva as an active ingredient, and drying the housefly larva and removing moisture to obtain a building sample; Dissolving the building sample in a mixed organic solvent to remove fat and to obtain a residue; And a step of obtaining the ethanol extract by mixing the residue with ethanol.

In the step of drying the housefly larvae to remove moisture, the drying temperature is 80 ~ 150 ℃, the drying time is suitable 3 ~ 7 hours, the drying is freezing (freezing) drying, spray drying or using a dryer Can be done.

As a mixed organic solvent used to remove the fat from the dry matter sample, a nucleic acid is used, or a mixed solvent of CHCL ₂ and MeOH is preferable, and the ratio is about CHCL ₂ : MeOH = 2: 1.

In addition, the ethanol extraction is also preferably used repeatedly three or more times in consideration of the purity of the product.

Hereinafter, the present invention will be described in more detail with reference to Examples and Test Examples.

Example 1 (ethanol extract sample of skim fly larvae)

200 g of live housefly larvae were dried at 100 ° C. for 5 hours using a dry oven to remove moisture. In this way, 98 g of building material from which moisture was removed was obtained. 30 g of the moisture-removed building sample was mixed with 300 ml of a mixed organic solvent (CHCL ₂ : MeOH = 2: 1) at 20 ° C. for 2 hours and then filtered using three layers of gauze. Residue was completely blown off the organic solvent in the hood for 1 hour and then re-extracted three times. The residue was collected in one place to obtain 31 g of skim fly larvae residue. 31g of skim fly larvae residue and 300ml of pure ethanol were mixed, and an ethanol extract sample was prepared using a reflux condenser. Reflux cooling extraction was carried out for 3 hours in a heating mentle at 60 ° C. with a Reflux condenser attached, and the extracts were collected by filtration using filter paper No2 and collected in one place. Re-extracted. The extract was concentrated under reduced pressure using a rotary vacuum evaporator (Eyela N-1000, Tokyo Rikakikai C0., Japan). Thus, 10 g of the ethanol extract sample of the prepared skim fly larvae was obtained, and the yield of the sample from the live fly larvae was 5%.

Test example (microbial culture)

(1) Use strain and medium

As shown in Table 1, five beneficial bacteria (three anaerobic bacteria: Bifidobacterium bifidum, Bifidobacterium infantis , and Bifidobacterium longum ., 2 types of aerobic bacteria: Lactobacillus acidophilus , Lactobacillus casei ) and three harmful bacteria (two anaerobic bacteria: Clostridum perfingens , Bactetoids fragilis ., one aerobic bacterium: Staphylococcus aureus ) were obtained from the Korea Food Research Institute and subcultured. RCM medium (reinforced clostridial medium, oxoid CM 149) was used for five anaerobic bacteria, and MRS medium (oxoid CM 359, Difco 0881) and Nutrient medium were used for three aerobic bacteria.

division Strain name KTRI NO   Utilization Badge Anaerobic Beneficial bacteria Bifidobacterium bifidum 00894 ATCC 29521 Reinforced clostridial medium (Oxoid CM 149) Bifidobacterium infantis 00974 ATCC 15697 〃 Bifidobacterium longum 00977 ATCC 15707 〃 Harmful bacteria Clostridum perfingens 00803 ATCC 13124 〃 Bactetoids fragilis 00749 ATCC 25285  〃 Aerobic Beneficial bacteria Lactobacillus acidophilus 00150   MRS medium (Oxoid CM 359; Difco 0881) Lactobacillus casei 00346 ATCC 9018 〃 Harmful bacteria Staphylococcus aureus 00219 ATCC 14458   Nutrient medium

(2) sample culture

The cultivation method for eight strains of ethanol extract samples of skim flies larvae is as follows. Samples of skim fly larvae ethanol extract were added to the liquid medium composition of the strains of the strains shown in the table by adding 0 (non-degreased fly larvae), 0.1, 0.2, 0.3% (wt / vol, broth medium) to sterilize the test medium. It was. Meanwhile, 0, 0.5% (fr / tool, broth medium) of fructooligosaccharide (FOS, fructooligosaccharide: Sigma prod. USA), which is widely known as a non-degradable dietary ingredient, was prepared and sterilized by preparing a medium for each strain. In order to investigate the selective growth promoting effect (bifidogenic effect) of intestinal bipidose bacteria of skim fly larvae extract, it was used as a reference. Each of the eight different strains was passaged using the broth medium. Anaerobic bacteria were activated by passage three times for 24 hours using an incubator at 37 ° C. under anaerobic conditions using the Gas-Pak ^R method (BBL microbiology systems, Cockeysville, MD). 0.1 ml (10 ⁹ CFU / ml) of each activated strain was inoculated into a sterile liquid test medium to which ethanol extract samples of skim fly larvae were added. Gas-Pak ^R The culture (BBL) was anaerobicly cultured at 37 ° C. for 0, 5, 10, 15, 20, 24, 36 and 48 hours under agitation. After incubation, the OD value was measured at 600 nm using a spectrophotometer (UV / Vis Spectrophoto meter UV-260, Shimadzu Co., Japan). Using the medium without the ethanol extract sample of skim fly larvae was measured OD value at 600nm as a control to compare the difference to determine the utilization of skim fly larvae extract from the growth of microorganisms.

1 and 2 are the control (0%), 0.1%, 0.2%, 0.3% and 0.5% of fructooligosaccharides without adding skim fly larva ethanol extract to Reinforced clostridial medium (RCM) broth The effect of B. bifidum on growth promotion is shown.

As shown in the figure, the OD value of the culture medium showing the growth rate and utilization of the carbon source of the intestinal Pidobacterium bifidum in the treatment group containing the skim fly larva ethanol extract using Pidobacterium bifidum, compared with the control The treatment of ethanol extract of skim fly larvae increased the most after 20 and 24 hours, and was higher as the level of ethanol extract of skim fly larvae increased. The initial growth rate of the intestinal P. bacterium bipiderum was similar to each other, but after 15 hours of cultivation, the addition of 0.3% of skim fly larva ethanol extract showed rapid growth. On the other hand, this result was higher than the treatment was added to commercially sold fructooligosaccharide 0.5% (Fig. 2).

To Figs. 3 and 4 showing the Bifidobacterium ronggeom (B. longum), Fig. 5 and 6 Bifidobacterium Infante tooth (B. infantis) strain growth, they, too, Bifidobacterium bipyridinium bushes and Similar trends were observed.

7 and 8 show the effect on the growth of Lactobacillus ashdophyllus ( L. acidophilus ), Figure 9 and Figure 10 L. casei ( L. casei ) strain.

As shown in the drawing, the resultant culture of the skim fly larva ethanol extract and fructooligosaccharide in MRS broth medium, respectively, The growth rate of the Lactobacillus ashdophyllus and Lactobacillus casei strains and the OD value of the culture medium indicating the utilization of carbon source were increased from the beginning of culture as the level of the skim fly larvae ethanol extract was increased. Lactobacillus ashdophyllus The fungus showed the highest growth rate at 10 hours of culture and maintained at a constant level after 10 hours. The initial growth rates of Lactobacillus cassia were similar to each other, but 0.2%, except for the addition of 0.1% of skim fly larva ethanol extract. The addition of 0.3% showed that the bacteria grew rapidly up to 10 hours and showed the maximum growth rate after 20 hours. Grown on MRS broth medium containing skim fly larvae ethanol extract Lactobacillus ashdophyllus Strains and The growth rate of Lactobacillus casei strain was similar to that of fructooligosaccharide.

11 and 12 are well-bacterial after control (0%), 0.1%, 0.2%, 0.3% and 0.5% of fructooligosaccharide, respectively, without adding skim fly larva ethanol extract to Reinforced clostridial medium (RCM) broth ( C. perfringens ) shows an inhibitory effect on the strain. Intestinal well-being bacteria up to 5 hours of initial culture in the treatment group containing skim fly larva ethanol extract The growth of the strain was slightly lower than that of the control, but after 10 hours, the growth rate was continuously lower than that of the control. The growth of the bacterial count at 48 hours was found to be most inhibited in the medium to which 0.3% skim larvae ethanol extract was added. This result showed a growth inhibitory effect similar to that of the strain cultured with the addition of fructooligosaccharide.

13 and 14 are S. aureus The results of the cultures were investigated after the addition of the control (0%), 0.1%, 0.2%, 0.3% and 0.5% of fructooligosaccharides without the addition of skim fly larva ethanol extracts in the growth of Nutrient broth. Intestinal yellow staphylococcus aureus up to 10 hours of initial culture in both the control and the treated groups containing the skim fly larva ethanol extract The growth of the strain was similar, but growth inhibition effect appeared from 15 hours. Staphylococcus aureus from 24 hours of culture The growth of the strain showed a sharp decrease, which was caused by S. aureus ethanol extract It was found that the growth of the strain was strongly inhibited, and in particular, the culture treatment of 0.3% addition of skim fly larva ethanol extract almost suppressed at 48 hours. This result was greater than the growth inhibitory effect of the strain cultured in fructooligosaccharide.

15 and 16 are bacteroids after addition of the control (0%), 0.1%, 0.2%, 0.3% and 0.5% of fructooligosaccharide, respectively, without addition of the skim fly larva ethanol extract in Reinforced clostridial medium (RCM) broth. B. fragilis Inhibitory effect on the strain. Intestinal Bacterial Praxilis Bacteria Treated with Ethanol Extract of Skim Fly Larvae The growth of the strain showed slightly higher trend than the control until 15 hours, but the intestinal bacteroid praxilis from 20 hours of culture. It can be seen that the growth of the strain was rapidly inhibited and the growth of the bacteroid Praxilis strain was almost stopped at 48 hours of culture. And this result was similar to the inhibitory effect of the bacteria cultured in fructooligosaccharide.
On the other hand, the present invention has been described an example of testing three species ( Clostridum perfingens, Bactetoids fragilis and Staphylococcus aureus ) as the microorganisms of the antimicrobial action of the ethanol extract , but the same inhibition against other harmful microorganisms as these are representative harmful bacteria You can expect the effect.

As described above, in the present invention, the ethanol extract of skim flies larvae is sufficient, and the effect of non-degradable dietary ingredients having selective growth promoting effect of Bifidobacteria can be used as feed for livestock that can replace antibiotics. .

As described above, according to the present invention, by using an ethanol extract of housefly larva as an excellent protein source that can replace soybean meal and fishmeal for broilers and laying hens with a crude protein content of 50% or more, it promotes growth and harmful bacteria for enteric beneficial bacteria The anti-microbial effect of growth inhibits the anti-microbial effect compared to the existing fructooligosaccharide, and can produce a new animal feed additive to replace the antibiotic.

Claims (6)

Antimicrobial Feed Additive Using Ethanol Extract of Degreased Housefly Larvae Drying the housefly larvae and removing moisture to obtain a building sample; Dissolving the building sample in a mixed organic solvent to remove fat and to obtain a residue; And Mixing the residue and ethanol to obtain an ethanol extract; The method of claim 1, characterized in that consisting of The method of claim 2, The drying method in the step of drying the housefly larvae and removing moisture to obtain a building sample, the drying temperature is 80 ~ 150 ℃, the drying time is 3 to 7 hours The method of claim 2, Drying of the housefly larva is characterized in that the use of freezing (freezing) drying, spray drying or dryer The method of claim 2, The mixed organic solvent for removing fat from the building sample and obtaining a residue is a nucleic acid or a mixed solvent of CHCL ₂ and MeOH, and the ratio is CHCL ₂ : MeOH = 2: 1. delete

Images