September 2010, 31(5) 865-871 (2010)
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Antibacterial activity of house fly-maggot extracts against MRSA (Methicillin-resistant
Staphylococcus aureus) and VRE (Vancomycin-resistant enterococci)
Sang O Park1, Jae Hong Shin1, Won Keun Choi1, Byung Sung Park*1, Jin Seok Oh2 and Aera Jang3
1

2

College of Animal Life Science, Kangwon National University, Chuncheon - 200 701, South Korea
College of Agriculture Life Science, Kangwon National University, Chuncheon - 200 701, South Korea
3
National Institute of Animal Science, RDA, Suwon - 441 706, South Korea(Received: September 10, 2009; Revised received: Jan 27, 2010; Accepted: February 27, 2010)

Abstract: The principal objective of this study was to determine the in vitro antibacterial activity of the water-soluble protein enzymatichydrolysates and the ethanol (EtOH)-extracted fraction obtained from fly-maggots (Musca domestica L.) against MRSA (methicillinresistant Staphylococcus aureus) strains and VRE (Vancomycin-resistant enterococci) 5117 strain. The water soluble protein enzymatichydrolysates were prepared via 4 or 8 hr of incubation after mixing with the water-soluble protein extracted from the fly-maggots plus
thermolysin. The EtOH-extracts A was prepared by homogenizing after mixing with the fly-maggots plus pure EtOH at room temperature.
The EtOH-extracts B and C was obtained via filtering after stationing for 24 hr at 4-20oC using the EtOH-extracts A, respectively. The
growth inhibition curves for MRSA strain 3595 and VRE strain 5117 in the water-soluble protein enzymatic hydrolysates were increased andevidenced concentration-dependent inhibition in the 8-hr hydrolysate as compared with the 4-hr hydrolysate (p<0.05). The growth inhibition
curves for MRSA and VRE strains in the EtOH-extracted fraction obtained from the fly-maggots were high in EtOH-extract C as comparedwith EtOH-extracts A and B (p<0.05). The minimum inhibitory concentrations (MIC) in the EtOH-extracts C, in which the growth inhibitionof MRSA and VRE strains was increased, were determined to be 40, 50, 50, 60, 40 and 60 µg ml-1 in MRSA strains 3598, 3595, 3601, 3589,
3597 and 3595, respectively. While the butanol fraction obtained from EtOH extract C evidenced profound antibacterial activity against the
MRSA and VRE strains, the antibacterial activity of the hexane, ethyl acetate, and water layers could not be adequately confirmed.
Key words: Maggot extracts, MRSA, Antibacterial activity, Minimum inhibitory concentration
PDF of full length paper is available online

Introduction
Abuse and misuse of antibiotics in humans and livestock has
spurred the evolutionary development of bacteria that have selective
resistance to antibiotics. The horizontal transfer of antibiotic resistancegenes in the environment (Shakibaie et al., 2009), the discovery of
resistant bacteria in fish (Matyar et al., 2004) and livestock farm
products (Toroglu et al., 2009; Jones et al., 2002), water
environments (Toroglu and Toroglu, 2009), as well as the clinicalappearance and global spread of methicillin-resistant Staphylococcus
aureus (MRSA) and Vancomycin-resistant enterococci (VRE)(Cosgrove et al., 2005) have become medical and social issues ofgreat concern (Toroglu et al., 2005; Perl, 1999). With the increasing
popularity of antibiotic-free organic livestock farming in the past decade,the European Union ruled that feeds used for the production oforganic livestock products be completely organic and that the use ofantibiotics be no longer permitted (Commission Regulation EC 2277,
2003). Following this lead, regulations concerning the use of antibiotics
in environmentally-sensitive settings and in organic livestock farming
are being implemented in countries around the globe, including
Korea. Therefore, it is imperative to develop new alternative
substances to replace antibiotics to minimize the damage or loss thatplant-intensive livestock farming industry may suffer by the exclusion* Corresponding author: bspark@kangwon.ac.krof antibiotics from animal feed, and to insure continuous improvementof livestock productivity (Dibner and Richards, 2005).
The development of active antibacterial substances from fly
maggots (Musca domestica L.) that do not promote acquisition ofbacterial resistance as a replacement in animal feed has become an
active area of research. Biotherapy using fly maggots has beenused for the treatment of chronic osteomyelitis and purulent
inflammation in soft tissues since the early years of the twentiethcentury, and has enjoyed a renaissance in the wake of thedevelopment of bacterial antibiotic resistance (Sherman et al., 2000).
Of particular interest, secreted and extracted material from fly maggots
displays potential antibacterial activity against MRSA (Bexfield et al.,2004). The basic therapeutic mechanism of fly maggots is a synergy
involving several compounds, the core being the aforementioned
antibacterial activity of the secreted/extracted materials (Bexfield etal., 2004; Vistnes et al., 1981).
The main therapeutic benefits of fly maggots in biotherapy ofpatients with burns, pressure sores, or postoperative MRSA
inflammation include dissolution of necrostic and infected surface
tissue in the wound, sterilization of the wound by killing the infecting
bacteria, and resultant accelerated healing. Treatment of both chronic
wounds that do not respond to conventional remedy and antibiotic
Journal of Environmental Biology  September, 2010 866
resistance bacteria are amenable to maggot therapy, with secreted
and extracted products being more effective than the maggots
themselves (Jaklic et al., 2008; Sherman et al., 2000). The proteolytic
enzymes contained in fly maggot secretion aid in healing bydecomposing and metamorphosing the general extra cellular matrixes
in the wound, which allows new tissue to form (Horobin et al., 2005;
Lerch et al., 2003).
Studies pertaining to the therapeutic effects of fly maggots
and their obtained products have involved five alkaline and neutral
active antibacterial maggot-derived substances (Wang and Sun,
1997), a 20–22 kDa protein with antibacterial activity against
Staphylococcus aureus and Bacillus subtilis (An et al., 2004),
separation of a 10 kDa peptide that is active against Gram-positive
and Gram-negative bacteria, cloning of the gene for the antibacterial
compound cecropin, and the action of an antibacterial peptide
expressed in Escherichia coli (Liang et al., 2006). Previously, we
reported on a 5 kDa peptide present in the water-soluble proteinhydrolysates obtained from fly maggots that inhibits MRSA (Yoon etal., 2008; Jang et al., 2007). Although there has been great interest
and research concerning the anti-MRSA action of fly maggots, until
the present study there has been no report on the antibacterial
actions of ethanol (EtOH) extracts.
The main purpose of this study was to investigate theantibacterial actions of the water-soluble protein hydrolysates and
EtOH extract from fly maggots against MRSA and VRE. The aim of
the study was to obtain basic data that would facilitate the use of fly
maggots or their products as an antibacterial supplement in animal
feed for organic livestock farming.

Park et al.
EtOH extraction and solvent fractionation: Frozen fly maggots
and pure ethanol (EtOH, 99.5%) were mixed in a 1:4 ratio prior tohomogenization for 3 min using a Ultra-Turrax T25 homogenizer(IKL-Labortechnik, Staufen, Germany) followed either immediatelyby the extraction process (extract A), 24 hr after storage at 4oC(extract B), and after storage at -20oC (extract C). The upper layerof the solution was separated by centrifugation for 15 min in an
automatic refrigerated centrifuge at 20,000 ×g, and then concentrated
at using a N-100 rotary vacuum evaporator (EYELA. Tokyo, Japan),and freeze-dried and stored at -20oC. When it was used, it was first
diluted to an appropriate level of concentration (Galbraith et al.,1969).
To evaluate the antibacterial potency of the EtOH extracts, asolvent fraction was prepared using silica gel column
chromatography. Hexane was added to the activated 230–400 meshsilica gel (Merck, Whitehouse Station, NJ, USA) to produce a slurrythat was loaded into a glass column (50×65 mm, 120 ml) and washedwith four volumes of hexane. 400 mg of extract C was loaded onto acolumn and n-hexane, ethyl acetate and butanol equal to four volumesof the silica gel were sequentially added. The sequential increase inpolarity achieved sequential fractionation. The resulting fraction was
decompressed and concentrated, organic solvents were removed
using nitrogen gas, and the product was stored at -20oC after freezedrying (Yasuda, 1997).

Materials and Methods
Production of water-soluble protein hydrolysates: Test
samples and fly maggots were supplied by Medilavatec (Seoul,
Korea). Frozen fly maggots were washed with 4 (phosphate bufferedsaline (0.01 mole, pH 7.4) and transferred to sterile distilled water.
Approximately 100 fly maggots and 100 ml of sterie distilled water
were cultured for 24 hr at room temperature (approx. 20oC). The
solution layer containing fly maggot secretion was recovered using
a pipette and centrifuged at 20,000 ×g for 15 min in a RC-3 automaticrefrigerated centrifuge (RC-3, Sorvall, Pittsburgh, PA, USA). The
upper layer of the solution was sterilized using a 0.20 µm membranefilter (Advantec MFS, Dublin, CA, USA) and freeze-dried using a(BenchTop 3.3 series vacuum freeze dryer (VirTis, Gardiner, NY,
USA) before use as a water-soluble protein sample (An et al.,2004).

Growth curve assay: Six MRSA strains 3589, 3595, 3596, 3597,
3598, and 3601, and VRE strain 5117 were obtained from the
Culture Collection of Antimicrobial Resistant Microbes (CCARM;
Seoul, Korea). Growth curve assays were performed for MRSA
3595 and VRE 5117 using the water-soluble protein hydrolysates.
For each strain, a culture solution was prepared using 10 ml MullerHinton broth (Difco, Detroit, MI, USA) and three subcultures weremade at 37oC (McCarthy et al., 1979). Using a model UV mini-1240
spectrophotometer (Shimadzu, Kyoto, Japan), the optical density(O.D.) of the culture was adjusted to 0.4 (106 colony forming units(CFU) ml-1) at 650 nm and the suspension was divided into 100 µl
aliquots that were aseptically dispensed into wells of a 96-well plate(SPL, Seoul, Korea) after dilution to 108. The water-soluble proteinhydrolysates was diluted with sterile distilled water to 100, 200 and
300 µg ml-1. A hydrolysates-free sample (0 µg ml-1) was used as acontrol. Each suspension was sterilized and filtered using a 0.20 µm
membrane filter and 100 µl was added to wells and cultured for 48 hr
at 37oC. The growth promotion and inhibition curve for each strain
was determined by O.D 650 nm using a microplate reader (Bio-Tek
Instruments, Winooski, VT, USA) (Stubbings et al., 2004) .

The water-soluble protein extract was mixed with thermolysin(Sigma-Aldrich, St. Louis, MO, USA) (100:1 w/v, pH 7.4) and thencultured for 4 or 8 hr at 37oC to produce two hydrolysates. Eachwater-soluble protein hydrolysates was heated for 5 min to interrupt
the enzymatic hydrolysis of protein, and then freeze-dried and store
until used for the growth curve assay for the particular strain(Vercruysse et al., 2005).

Measurement of minimum inhibitory concentration (MIC):
MIC of the EtOH extracts against the selected MRSA strains wasassessed using the modified broth microdilution method (Amster,1996). A culture of each MRSA strain was adjusted to an O.D 650 nm
of 0.4 (corresponding to 106 CFU ml-1) and 100 µl aliquots wereaseptically dispensed in wells of a 96-well plate. EtOH extract C was
diluted with sterile distilled water to 20, 40, 50, 60, 70 and 100 µg ml-1. An

Journal of Environmental Biology  September, 2010 Anti-MRSA activity of maggot extract

867

extract-free control (0 µg ml-1) was also included. Each suspension
was sterilized and filtered using a 0.20 µm membrane filter, and 100µl was added to wells and cultured for 48 hr at 37oC. The effect ofeach EtOH extract on MRSA and VRE proliferation was determinedby O.D at 650 nm using a microplate reader.

Table - 1: Antibicrobial activity of ethanol extracts from house fly-maggots
against MRSA and VRE1 (Inhibition zone diameter: mm)

Antibacterial action assay: Antibacterial action for each EtOH
extract and solvent fraction was assessed as previously described(Murry et al., 1999). After adjusting the O.D (650 nm) of each MRSA
and VRE culture to 0.4 (corresponding to 106 CFU ml-1), an aliquot
was coated evenly on 4-5 mm thick Muller-Hinton agar (Difco, USA)using a cotton bud by rolling the bud on the medium. 50 µl of extracts
A, B, and C and the solvent fraction, were diluted with sterile distilledwater to 4 mg ml-1 prior to absorption onto an individual fixed 8 mmdiameter paper disk (Advantec, Toyo Roshi Kaisha, Ltd., Japan).
Each strain was cultured for 48 hr at 37oC before assessing
antimicrobial action. Antimicrobial action was indicated by the diameterof inhibition zone that was measured with a CD-15CPX diamatic
caliper (Mitutoyo, Kawasaki, Japan).

StrainABCMRSA 3589
MRSA 3595
MRSA 3596
MRSA 3597
MRSA 3598
MRSA 3601
VRE 5117

0.5
-2
1.2
3.5

5.0
5.0
2.0
1.0
3.5
3.0
13.5

5.9
5.3
7.2
9.8
12.0
6.3
6.7

1

Extract A: extracted by 99.5% ethanol at room temperature within 30 min
Extract B: extracted by 99.5% ethanol at 4oC for 24 hr
Extract C: extracted by 99.5% ethanol at -20oC for 24 hr
2
No inhibition zone against MRSA and VRE

the zone processed with 200 µg ml-1 and from 42 hr in the zone
processed with 300 µg ml-1 compared with the control zone. These
processed zones showed significant (p<0.05) growth inhibition of
VRE 5117 compared with the control zone.

Statistical analysis: Variation analysis (ANOVA) was performed
using the GLM procedure of SAS Software (SAS, Cary, NC, USA).
Statistical significance was tested based on p<0.05 for all the datausing Duncan’s multiple range test (SAS, 2004).

The results of the growth curve assay for the hydrolysates
produced after 8 hr of enzymatic hydrolysis of water-soluble protein
for each culture time for MRSA 3595 and VRE 5117 are shown in
Fig. 3,4. For MRSA 3595, the zone process with enzymatic hydrolysis
showed a stronger and concentration-dependent growth inhibition
effect over 48 hr and this effect showed a significant (p<0.05)
continuous trend until the end of 40 hr of culture. This result was
clearly different from the result shown in the Fig. 1, which is considered
to have been the result of the residue that inhibited the growth of
MRSA 3595 during the 8 hr exposure to enzymatic hydrolysis of thewater-soluble protein. On the other hand, the growth rate of VRE
5117 was strongly inhibited from 18-48 hr in the zone processedwith 300 µg ml-1. In the zone processed with 200 µg ml-1, growth
inhibition began from 42 hr of culture. These process zones showeda significant (p<0.05) difference compared with the control zone.
These results may have been caused by the active antibacterial 5
kDa peptide previously identified in the extract of fly maggots (Yoonet al., 2008; Jang et al., 2007), which is also supported by previous
descriptions of antibacterial action of fly maggot secretions and extracts(Jaklic et al., 2008; Hou et al., 2007; Bexfield et al., 2004; Thomas
et al., 1999). MRSA is a bacterium responsible for several difficult-totreat infections in humans. MRSA is especially troublesome in hospitalassociated (nosocomial) infections. Wyllie et al. (2006) report a death

Results and Discussion
The results of the growth curve assay for the hydrolysates
produced after 4 hr of enzymatic hydrolysis of water-soluble protein
for each culture time for MRSA 3595 and VRE 5117 are shown in
Fig. 1, 2. For MRSA 3595, the zone processed with enzymatichydrolysates was similar to the control zone at the beginning of
culture. However, the enzymatic hydrolysates processed zone wasindicative of faster growth than the control zone from 18 hr onward.
The growth curves of both strains displayed a concentrationdependent effect. The zones processed with 200 and 300 µg ml-1
showed a significant (p<0.05) increase from 24-36 hr comparedwith the control zone. Therefore, the hydrolysates produced after 4
hr of enzymatic hydrolysis likely exerted a growth stimulatory effectrather than acting as an inhibitor. On the other hand, with VRE 5117
there was no substantial difference between the enzymatichydrolysates process zone and the control zone until 30 hr, but aslow decrease was observed from 36 hr onward. The growth
inhibition effect on VRE 5117 was clearly pronounced after 48 hr inTable - 2: Minimum inhibitory concentration (MIC) of the ethanol extracts from house fly-maggots against MRSA1
Concentration of ethanol extracts (µµg ml-1)

StrainMRSA 3595
MRSA 3596
MRSA 3597
MRSA 3601
MRSA 3589
MRSA 3598

MIC

0

20

40

50

60

70

100

(µ
µg ml-1)

+2
+
+
+
+
+

+
+
+
+
+
+

+
+
+
+
-

+
+
-

_
_
-

-

-

60
50
40
50
60
40

1

Extracted by 99.5% ethanol at -20 for 24 hr 2, Degree of clarity of inhibition zone by growth inhibition, + growth, - no growth
Journal of Environmental Biology  September, 2010 

868Park et al.

Table - 3: Antimicrobial activity of the partition layers of ethanol extracts fromhouse fly-maggots against MRSA and VRE1 (200 µg disk-1)
Inhibition zone on plate (mm)StrainMRSA 3595
MRSA 3596
MRSA 3597
MRSA 3601
MRSA 3589
MRSA 3598
VRE 5117

HexaneEthylacetate

Butanol

water

-

-

12.8
12.3
9.0
11.0
9.8
15.3
14.3

10.0
10.5
11.5

1

Extracted by 99.5% ethanol at -20oC for 24 hr, 2No inhibition zone wasformed, Data represents mean values, n=3

rate of 34% within 30 days among patients infected withMRSA, a rate similar to the death rate of 27% seen among MSSAinfected patients. VRE is the name given to a group of bacterial
species of the genus Enterococcus that is resistant to the antibiotic
vancomycin. VRE species have an enhanced ability to pass resistantgenes to other bacteria. While infection of healthy individuals is
uncommon, it is possible that they could be colonized with newlyresistant bacteria (Mascini et al., 2006).
The results of the assessment of antibacterial action of freezedried fly maggots and pure EtOH extracts against MRSA and VRE
are shown in Table 1. Extract A produced inhibition zone diametersindicative of growth inhibition of MRSA 3589, MRSA 3598, and VRE
5117 (respective diameters of 0.5, 1.2 and 3.5 mm). However, no

1.4

Optical density (650 nm)

1.2
1.0
0.8
0.6
0.4
0.2
0
0

6

12

18

24
30
Incubation time (hr)

36

42

48

Fig. 1: Effect of water soluble protein hydrolysate from house fly-maggots for 4 hr on the growth of MRSA 3595, The concentration of water soluble proteinhydrolysate used was 0(), 100(), 200() and 300() µg ml-1, Data represents mean values, n=3

1.4

Optical density (650 nm)

1.2
1.0
0.8
0.6
0.4
0.2
0
0

6

12

18

24
30
Incubation time (hr)

36

42

48

Fig. 2: Effect of water soluble protein hydrolysate from house fly-maggots for 4 hr on the growth of VRE 517, The concentration of water soluble proteinhydrolysate used was 0(), 100(), 200() and 300() µg ml-1, Data represents mean values, n=3

Journal of Environmental Biology  September, 2010 Anti-MRSA activity of maggot extract

869

1.6
Optical density (650 nm)

1.4
1.2
1.0
0.8
0.4
0.2
0

0

6

12

18

24
30
Incubation time (hr)

36

42

48

Fig. 3: Effect of water soluble protein hydrolysate from house fly-maggots for 8 hr on the growth of MRSA 3595. The concentration of water soluble proteinhydrolysate used was 0(), 100(), 200() and 300() µg ml-1. Data represents mean values, n=3

1.6
Optical density (650 nm)

1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
0

6

12

18

24
30
Incubation time (hr)

36

42

48

Fig. 4: Effect of water soluble protein hydrolysate from house fly-maggots for 8 hr on the growth of VRE 5117, The concentration of water soluble proteinhydrolysate used was 0(), 100(), 200() and 300() µg ml-1, Data represents mean values, n=3

growth inhibition effect was observed in the other tested strains. Withextract B, the diameter of the growth inhibition zone of MRSA strains
ranged from 1.0 mm (MRSA 3597) to 5.0 mm (MRSA 3589 and3595) with only minor differences in the inhibition (MRSA 3595 both)with some differences in the inhibition against different MRSA strains
being evident. The inhibition zone of VRE 5117 strain was 13.5 mm
in diameter, indicative of more potent inhibition. With the extract C, theinhibition of MRSA varied according to strain, with the diameter ofinhibition zones ranging in diameter from 5.3 mm (MRSA 3595) to
12.0 mm (MRSA 3598).The diameter of the extract C inhibition zone
for VRE 5117 was 6.7 mm, indicative of a more potent inhibition effectthan achieved with extract A, but less potent than that exerted byextract B.
The data of Table 1 indicate that EtOH extract C displayed
the most potent inhibitory effect of the three extracts. This extract ispotentially useful as a natural antibacterial growth compound inanimal feed, especially as it is not artificially synthesized but, instead,can be extracted from millet. Nonetheless, the antibacterial effect of
extract C may have been tempered by residual fat existing in the
fly maggots. Although the supporting data is not presently shown,
we could confirm that each strain was not completely eliminatedwhen cultured with the homogenized mixture of living fly maggots
and distilled water. This may indicate that high concentration of fat
in the fly maggots was not used as the source of nourishment
needed by the strains (Hwangbo et al., 2009). Fly larval fat
represents almost one-quarter of the larval weight; isolation andrefinement of the antibacterial substance from fly maggots will likely
require complete removal of the fat. Improved separation andrefining of the antibacterial peptide from fly maggots and enhanced
potential of the EtOH extracts for use as an alternative to antibiotics
in animal feed in organic livestock farming are needed. Fly maggot
Journal of Environmental Biology  September, 2010 870
extract obtained by sterile water washing, rapid freezing withliquidized nitrogen gas, homogenization with ammonium nitrate,and centrifugation-mediated separation is inhibitory to bacteria (Houet al., 2007), although the precise nature of the inhibitory compounds
remained unidentified. But, fly maggot extracts are likely notuniversally applicable. Jaklic et al. (2008) reported that fly maggot
secreted and excreted material obtained upon submersion in distilledwater has an excellent healing effect on wounds infected with
Gram-positive bacteria such as Staphylococcus aureus, while it
not being effective in the treatment of wounds infected with Gramnegative including Proteus spp. and Pseudomonas spp., withbacteria in the genus Vagococcus being highly refractory to maggotderived compounds.
To extend our observations concerning the antibacterial
prowess of EtOH extract C, a successive experiment was performed
in which the MIC of extract C for the MRSA strains was determinedat different extract concentrations (Table 2). MICs were 40 µg ml-1
for MRSA 3597 and 3598, 50 µg ml-1 for MRSA 3601 and 3596, and60 µg ml-1 for MRSA 3589 and 3595. The sensitivity of the tested
MRSA strains to the extract is consistent with previous descriptions of
the efficacy of fly maggot secretions/excretions (Jaklic et al., 2008;
2008; Kerridge et al., 2005; Bexfield et al., 2004), even with thedichotomous methods used in the studies.
The results of the assessment of the antibacterial effect of thesolvent fraction of EtOH extract C on MRSA and VRE strains areshown in Table 3. Inhibition zones were evident using hexane,
ethylacetate, and water fractions only for MRSA 3597 and MRSA
3601, and VRE 5117, and no antibacterial effect could be confirmedin the solvent layers. On the other hand, inhibition zones were
produced by the butanol fraction when tested against MRSA; theinhibition zone varied with strain, being 9 mm MRSA 3597 and 15.3mm for MRSA 3598. The antibacterial effect of the other solvent
fractions was very low, likely reflecting the difference of antibacterial
substance in the different organic solvents. Compared with previous
results of natural antimicrobial extracts (Choi et al., 2009; Abu-Shanab
et al., 2006; Aqil et al., 2005; Cai et al., 2002; Park et al., 2001), theEtOH extract from fly maggots appears to represent and excellent
antibacterial agent.
Choi et al. (2009) reported that among the fractions ofrosemary (Rosmarinus officinalis L.), methanol and hexane fractions
showed the highest inhibitory action against MRSA. Their report
differs from the present observations in that these solvent fractions
inhibited the expression of MecA (methicillin resistant determinant, 52kDa), the protein resistance determinant of MRSA KCCM 40511,
and penicillinase (34 kDa; a cell wall protein synthesized by MecA),thereby inhibiting highly resistant bacteria. This difference may be
because of the different polarity of the organic solvents using different
natural sources.
In conclusion, EtOH extracts from fly maggots can potently
inhibit MRSA and VRE strains, with a ready supply of the inhibitory
compound being recovered in the separated butanol fraction. This
Journal of Environmental Biology  September, 2010 Park et al.
finding is expected to be helpful for the provision of basic data related
to the development of natural antibacterial growth agents for use inanimal feed in organic livestock farming.
Acknowledgments
This study was supported by Technology Development
Program for Agriculture and Forestry, Ministry for Food, Agriculture,
Forestry and Fisheries, Republic of Korea, 2009. We also
acknowledge the support in part by Institute of Animal Resources
Kangwon National University, Republic of Korea.
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Journal of Environmental Biology  September, 2010