WO2007074976A1

WO2007074976A1 - Feed for bee mite control and a method of its preparation

Info

Publication number: WO2007074976A1
Application number: PCT/KR2006/005130
Authority: WO
Inventors: Hoh-Gyu Hahn; Kee Dal Nam; Jin Dong Kim
Original assignee: Korea Institute Of Science And Technology; Iljinfnp Co., Ltd.
Priority date: 2005-12-28
Filing date: 2006-11-30
Publication date: 2007-07-05
Also published as: KR100755027B1

Abstract

The present invention relates to a feed for controlling bee mites and a method of its preparation. In the present invention, the feed is prepared by mixing cymiazole with pollen or pollen substitute with a predetermined ratio. The feed prepared as a result is shown to have excellent activity in controlling bee mites, and also applicable to young honey bees, to which the conventional acaricides have not been applicable, thus eliminating problems raised with the conventional acaricides while capable of breeding and nurturing of young honey bees..

Description

FEED FOR BEE MITE CONTROL AND A METHOD OF ITS PREPARATION

Technical Field

The present invention relates to a feed for controlling bee mites and a method of

its preparation. In the present invention, the feed is prepared by mixing cymiazole

with pollen or pollen substitute with a predetermined ratio. The feed prepared has

shown to have excellent effect in controlling bee mites, and also applicable to young

honey bees, to which the conventional acaricides have not been applicable, thus

eliminating problems raised with the conventional acaricides while capable of

breeding and nurturing of young honey bees.

Background Art

Generally, apiculture (bee farming) is considered a field of industry aiming at

profits to be earned by producing high level of health food with good nutrition by

means of good management in breeding, proliferation and production, distribution

of products, strategies for controlling diseases and harmful insects, improvement of

bee farming instruments, establishment and utilization of bee plants. Honey is the representative health to be obtained from bee farming. Honey is a

monosaccharide comprising glucose and fructose as active ingredients, which can be

directly absorbed through the intestinal walls and consumed in muscle cells in each

part of a body thereby providing energy necessary for physical activities while the

remainders are stored in the liver in the form of glycogen. Further, honey is a

complete health food containing inorganic substances necessary for a body to

promote physiological activities and assist with the complete development of bone

tissues.

Bee farming can produce a variety of products and contribute much to the

increase in household income due to the demand on the rapid increase. Further, it

can be used as a pollen mediator of various kinds of farming crops to improve their

products. Therefore, it has been adopted in the world as one of the major

agricultural policies and has been continuously developed.

For the development of bee farming industry it is essential to protect honey bees

from infections by bacteria, viruses, fungi, parasites, etc, in particular, from harmful

insects such as bee mites.

The bee mites, being parasites to honey bees, pass the winter by adhering

themselves to the bodies of honey bees. They lay 3-4 eggs in each hive which grow

into young mites having four pairs of legs 2 days after oviposition. Then, the young mites acquire nutrient from the grown pupa of honey bees by attaching themselves

thereto as parasites, and grow together with the honey bees, thus preventing the

normal development and activities of the honey bees.

There are four methods that have been used for the control of bee mites as

follows.

First, there is a method to control bee mites by an intermittent treatment of

beehives with insecticides during the period of from the end of February to the end

of April. This is a strip treatment method where beehives are treated therein with an

insecticide, which is then instantly dissipated and evaporated, adhered to the body

of bee mites thereby capable of controlling bee mites.

Second, there is a method to control bee mites by a smoking treatment using a

smoke generated when the insecticide is burned.

Third, there is a method to control bee mites by spraying an insecticide where

the insecticide mixed with water is sprayed in between the hives.

Fourth, there is a method to control bee mites by treating them with an

insecticide which is mixed in a sugar solution (a breeding solution) to be fed to the

parasitic bee mites.

The conventional insecticides which have been used for the control of bee mites

and the methods of their uses are shown in Table 1. [Table 1]

The first and the third methods above are shown effective in controlling bee

mites via direct sublimation, burning or spraying of the insecticide. However, they

are disadvantageous in that the insecticide remains in the products of bee farming

after the treatment thereby contaminating the beehives, which becomes a stress for

honey bees. The present invention is the one that improved the fourth method

above.

The fourth method relates to feed honey bees with a culturing solution

containing the insecticide, which is then delivered to bee mites parasitic to the honey bees, thereby stopping metabolism of the bee mites and causing them to death. The

bee mites which obtain the body fluid from the honey bees to which they are

parasitic are destined to die within 1-2 days. However, this method has drawbacks

that old or adult honey bees tend to stir up the environment due to the stress

induced by the insecticide.

Further, while adult honey bees are capable of obtaining the culturing solution

and may be able to control bee mites, young honey bees cannot intake the culturing

solution and are thus hardly able to control bee mites, and they may become

disabled even though they intake the culturing solution.

Therefore, the inventors of the present invention have made extensive efforts to

solve the above problems. Based on the fact that the nurturing period of honey bees

and the bee mite control period, the inventors offered honey bees a pollen feed,

which is optimized by adding to pollen cymiazole, a chemical component having an

effect of controlling bee mites, to provide pollen as their nutrient source for their

proliferation, i.e., nurturing their offsprings, while allowing bee mites to suck the

body fluids of honey bees which are then destined to death by intaking the

cymiazole contained in the above pollen feed, thereby completing the present

invention. Therefore, an object of the present invention is to provide a feed for controlling

bee mites prepared by fermentation of a mixture comprising cymiazole and pollen

or cymiazole and pollen substitute and a method of its preparation.

Disclosure

In an embodiment, the present invention provides a feed for bee mite control

prepared by fermentation of a mixture comprising cymiazole and pollen or

cymiazole and pollen substitute.

In another embodiment, the present invention provides a method of preparing a

feed for bee mite control comprising:

1) mixing sugar to a ground pollen or its substitute;

2) adding cymizole to distilled water and dissolving it at 20 - 32 °C for 10 - 15

minutes to obtain an aqueous cymiazole solution;

3) adding dropwisely the aqueous cymiazole solution to the mixture in step 1)

for from 30 minutes to 4 hours, kneading the mixture, and molding it to obtain a

pollen feed; and

4) fermenting the pollen feed in a mellow room maintained at 25 - 32 ⁰C for 3 -

7days. Preferred embodiments of the present invention are set forth herein below.

with pollen or pollen substitute with a predetermined ratio. The feed prepared as a

result is shown to have excellent activity in controlling bee mites, and also applicable

to young honey bees, to which the conventional acaricides have not been applicable,

thus eliminating problems raised with the conventional acaricides while capable of

breeding and nurturing of honey bees.

The present invention relates to a feed for controlling bee mites which is

prepared by adding cymiazole to pollen or pollen substitute followed by

fermentation.

The pollen used in this invention is essential to sustain the lives of adult honey

bees as well as to nurture young honey bees. Sufficient pollen should be supplied

for the breeding of honey bees, and artificial pollen or pollen substitute may be

provided if the pollen is not sufficiently supplied. Pollen or fermented substitute

pollen can be provided by feeding into a brood box used during the winter season.

In a further embodiment of the present invention, the feed is added with sugar so

that honey bees are in favor of intaking the feed. Preferable, the sugar is added with a mixed ratio of 2:8 - 8:2 relative to the weight of pollen or pollen substitute. If the

amount of sugar exceeds the above amount, it would cause a problem in molding

the product into a certain shape, the product may be flown down, and deteriorate

the nutritional effect. In contrast, if the pollen exceeds the above amount, it would

greatly reduce preference, and may cause problems in aging and fermentation.

The effect of controlling bee mites in the present invention has been optimized

to have the dual effect of pollen as a nutrient and a drug for controlling insects by

adding cymiazole to pollen so that the body fluid of honey bees, which contains the

cymiazole as well as pollen, is transferred to the bee mites through the body fluid of

honey bees being suck thereby leading the bee mites to death.

In particular, the feed of the present invention contains a predetermined

amount of cymiazole, a drug for controlling bee mites, in addition to pollen to be

used as a nutrient for honey bees. Preferably, cymiazole is contained 0.001 - 1.0

weight % relative to the total 100 weight % of the feed. If the amount of cymiazole is

less than 0.0001 weight %, the feed would not have excellent control effect of bee

mites. Meanwhile, if the amount of cymiazole exceeds 1.0 weight %, honey bees tend

to avoid to intake the feed and their preference is greatly reduced. In a still further embodiment of the present invention, there is provided a

method for preparing a feed for bee mite control as set forth hereunder.

In the first step, pollen or pollen substitute is pulverized and then added with

sugar, wherein the sugar is preferably mixed with pollen or pollen substitute with a

mixed ratio of 2:8 - 8:2 relative to the weight of 2:8 - 8:2.

In the second step, cymiazole is prepared by dissolving cymiazole at 20 - 32 ⁰C

for 10 - 15 minutes to obtain cymiazole at a concentration of 0.01 - 1.0%.

Here, the cymiazole solution should contain a suitable amount of distilled water

so that it can perform a suitable effect of aging fermentation. If the concentration of

the aqueous cymiazole solution is less than 0.01%, the effect of the insecticide in

controlling bee mites becomes much reduced. Meanwhile, if the concentration of the

aqueous cymiazole solution is greater than 1.0%, it is not favored by honey bees due

to decreased palatability and thus it may not be used as a feed for honey bees.

Further, if the temperature is too low, dissolution may not be proceeded properly

thus making it difficult to evenly disperse cymiazole in the pollen feed. Meanwhile,

if the temperature is too high, it worsens processability and is also likely to

decompose the cymiazole. In case a sugar solution of 0.03 - 3.0% is added to the

above aqueous cymiazole solution, it may cause honey bees to avoid the resulting

feed due to reduced palatability. In the third step, the mixture obtained in step 1) is dropwisely added with the

aqueous cymiazole solution in step 2) and kneaded to form a pollen feed. The

dropwise adding of the aqueous cymiazole solution is proceeded slowly for a period

of 30 minutes to 4 hours so that it can be evenly and easily absorbed into the pollen

feed. The pollen feed can be molded into a certain shape using a conventional

method.

In the fourth step, the pollen feed prepared in step 3) is aged at 25 - 32 ⁰C for 3-7

days. If the temperature is outside the above range, aging becomes deteriorated or

the effects of the active nutrients may be decreased.

Thus obtained cymiazole is contained 0.001 - 1.0 weight % relative to the total

weight of the feed. The feed of the present invention was compared with those of

control groups - a group treated with a conventional pollen (control 1) and a group

treated with a drug (control 2) - and the results are shown in the following Table 2.

[Table 2]

[Table 3]

Further, the feed prepared according to the present invention, as shown in the

experimental example, has advantages that it can exhibit its maximal drug effect

because the cymiazole contained in the feed is not decomposed, the drug content is

maintained at a constant level, and provides good palatability.

The advantages of the feed may be summarized as follows.

(1) The feed is surely able to control bee mites.

The pollen feed can nurture young honey bees while blocking the proliferation

of bee mites as well as their control.

(2) There is no risk resulted from the residue of a drug.

There is almost no risk a drug remnant as is the case with when the feed is

supplied in a sugar solution. Further, there is no need to use additional drug for

treatment and thus the feed is free from contamination of bee farming products.

(3) The feed can greatly reduce drug resistance.

(4) The feed does not give any stress to honey bees.

When honey bees are under stress, they shake their bodies vigorously, do not

hold their wings to their bodies but fly with their wings wide open, take aggressive

actions. The above behavior of honey bees are often observed when they are placed

under stressful conditions, however, they were observed to remain calm when they

were fed with the feed prepared using pollen. (5) The feed can greatly reduce labor requirement.

The feeding including pollen prepared by in this invention does not require

treatment of a drug with either a feeding solution or the drug itself separately.

Therefore, the labor cost can be greatly reduced.

(6) The feed provide a good palatability.

Pollen is a food to be essentially provided to honey bees during spring, fall and

other no-honey-producing seasons, and thus it can provide a good palatability.

(7) The feed can be evenly shared by honey bees.

When the feed is provided using a sugar solution it is hard to control the supply

rate because it is fed by means of a water supply device. Further, honey bees with

relatively high activity tend to eat more than those with less activity, thus the

intended drug effect of controlling bee mites is not effective. In contrast, when the

feed prepared using pollen is provided to honey bees, it can be fed evenly both to

the honey bees with less activity as well as young honey bees thus capable of

maximizing the controlling effect.

(8) The feed does not necessitate the treatment with additional controlling drug.

In spring, feed should be given to honey bees and thus there is no need to treat

them with a drug for controlling bee mites. Description of Drawings

Fig. 1 shows pictures of pollen feeds prepared according to Example 1, wherein (a)

shows a picture of a pollen feed before fermentation and (b) shows a picture of a

pollen feed after fermentation.

Fig. 2 shows a HPLC spectrum on the cymiazol extracted from the feed prepared

according to Example 1.

Fig. 3 shows a chemical structure of cymiazol HCl salt used in preparing the feed of

Example 1.

Fig. 4 shows ¹H NMR spectrum of cymiazol extracted from the feed prepared

according to Example 1.

Fig. 5 shows ¹³C NMR spectrum of cymiazol extracted from the feed prepared

according to Example 1.

Fig. 6 shows a picture of bee mites controlled by using feed prepared according to

Example 1.

Best Mode

The present invention is described further with reference to the following

examples but they should not be construed as limiting the scope of the present

invention. Example 1: Preparation of pollen feed containing 0.025% of cymiazole

1) 40 kg of pollen or pollen substitute was pulverized to 40 - 80 mesh and then

completely mixed with 30 kg of sugar by stirring.

2) 20 g of cymiazole HCl and 60 g of sugar were mixed, and then the mixture

was completely dissolved in 10 kg of distilled water at 27 - 30 °C for 10 - 15 minutes

to obtain an aqueous cymiazole solution.

3) The mixture obtained in step 1) was mixed and kneaded while dropwisely

adding it with the aqueous cymiazole solution obtained in step 2) for 30 minutes to 1

hour, and finally obtained 80 kg of a pollen feed in a rectangular form as shown in

Fig. l(b).

4) The above pollen feed was fermented in an aging room at 30 ⁰C for 3-7 days

(fermentation may be continued even after the pollen feed is shaped into a certain

form).

5) The above pollen feed was packed into a product.

Example 2: Preparation of pollen feed containing 0.020% of cymiazole

1) 4 kg of pulverized pollen was completely mixed with 3 kg of sugar by

stirring. 2) 1.6 g of cymiazole HCl and 50 g of sugar were mixed, and then the mixture

was completely dissolved in 1 kg of distilled water at 20 ⁰C for 10 - 15 minutes to

obtain an aqueous cymiazole solution.

3) The mixture obtained in step 1) was mixed and kneaded while dropwisely

adding it with the aqueous cymiazole solution obtained in step 2) for 20 minutes,

and finally obtained 8 kg of a pollen feed in an oval form to be placed on top of

standard bee hives.

4) The above pollen feed was fermented in an aging room at 25 ⁰C for 10 days.

5) The above pollen feed was packed into a product.

Example 3: Preparation of pollen feed containing 0.0178% of cymiazole

1) 40 kg of pollen or pollen substitute was pulverized to 40 - 80 mesh and then

completely mixed with 30 kg of sugar in a mixer.

2) 14.24 g of cymiazole HCl and 65.76 g of sugar were mixed, and then the

mixture was completely dissolved in 10 kg of distilled water at 27 - 30 ⁰C for 10 - 15

minutes to obtain an aqueous cymiazole solution.

3) The mixture obtained in step 1) was mixed and kneaded while dropwisely

adding it with the aqueous cymiazole solution obtained in step 2) for 30 minutes,

and finally obtained 80 kg of a pollen feed in a rectangular form. 4) The above pollen feed was fermented in an aging room at 30 ⁰C for 3-7 days.

5) The above pollen feed was packed into a product.

Test Example 1: Evaluation of Palatability

Ten 1 kg packs of the feed prepared in Example 1 were sent to 5 selected bee farms

[Singi-myeon, Samcheok-si, Gangwon-do, Korea; Imok-ri, Namseong-myeon,

Cheongwon-gun, Chungcheongbuk-do, Korea; Bongnyong-dong, Sangju-si,

Gyeongsangbuk-do, Korea; Ogok-2 ri, Buk-myeon, Cheonan-si,

Chungcheongnam-do, Korea; Gilmyeong-2 ri, Ildong-myeon, Pocheon-si,

Gyeonggi-do, Korea], and the total of 50 packs were evaluated for their palatability

over two week period as compared with those of pollen substitute.

1) excellent palatability : 7 bee hives

2) good palatability : 28 bee hives

3) fair palatability : 15 bee hives

4) poor palatability: 0 bee hive

5) very poor palatability: 0 bee hive Test Example 2: Evaluation of Storage Stability

1 kg each of the feed prepared in Examples 1-3 was extracted 3 times with 2,500

mL of ethyl alcohol, dried with anhydrous magnesium sulfate, and then the organic

solution was evaporated under reduced pressure and finally obtained a light brown

oil-phased solid. The amount of cymiazole contained in the solid obtained thereof

was different from that used in the Examples because the cymiazole was adsorbed to

the pores of the pollen.

In order to evaluate the storage stability of the feed prepared according to the

present invention, the cymiazole content contained in the feed at the stage of

immediately after its preparation, those of l-,3-, 6-,9-, and 12 months after its

preparation, respectively, were compared. The results are shown in Table 4. The

cymiazole content was analyzed by HPLC using the soluble substance extracted

from the above solid substance with methylene chloride.

lTable 4]

Cymiazole content extracted from the feed; Evaluation via HPLC on storage stability

As shown in the above Table 4, the results of the storage stability test revealed

that the cymiazole contained in the feeds prepared in Examples 1-3 are very stable during the period of immediately after its preparation to 12 months after its

preparation and thus it can be concluded that it is stable during the process of

fermentation, aging and storage (see Fig. 2).

Test Example 3: Analysis of Water Content

The water content of the feed prepared in the present invention is closely

associated with fermentation, aging, palatability, etc., and thus the water content of

the feed was analyzed after its preparation.

The feeds prepared in Examples 1-3 were analyzed at regular intervals from

immediately after its preparation to 12 months after its preparation. The theoretical

water content of the feed is 12.5% [equation : (10/80) X 100 = 12.5%].

The feeds prepared in Examples 1-3 were removed of their water and volatile

components by placing them at 105 ⁰C for 3 hours, and their average water content

is shown in Table 5. The water content of the feeds was shown higher than that of

the theoretical value because of the water content contained in the pollen itself.

[Table 5] Water content of the feed of the present invention

As shown in the above Table 5, the water content of the feeds prepared in

Examples 1-3 is 13-14%. If the water content is less than 10% it is difficult to make a

suitable pollen feed. Meanwhile, if the water content is greater than 30% it prevents

a proper molding of the pollen feed. Therefore, it is preferred that the water content

of a pollen feed be in the range of 10-20% to be suitable for fermentation, aging and

palatability.

Test Example 4: Identification of Components

This test is to confirm that cymiazole, contained in the feed after preparation, is

not decomposed during the process of aging, storage, and distribution.

As shown in Example 1, 1 kg of the feed (pollen feed) was extracted with ethyl

alcohol after storing it at room temperature for 1 year, and then the organic solution

was evaporated under reduced pressure to obtain 0.14 g of a solid on brown oil

phase. The resultant was dissolved in methylene chloride, and then separated by

thin film chromatography through a silica gel 230 mesh using ethyl acetate-hexane

(1:1, v /v) as a developing solution

The resulting separated fraction was dried using anhydrous magnesium sulfate,

and then the solvent was removed by distillation under reduced pressure. Then the resultant was dissolved in a deuterated chloroform and then the structure of

cymiazole was identified using ¹H NMR and ¹³C NMR spectra (see Fig. 3).

1) ¹H NMR Spectrum (300 MHz, Varian NMR) (see Fig. 4)

¹H NMR (CDCl₃) δ 2.28(s, 3H, CH₃), 2.31(s, 3H, CH₃), 4.10(s, 3H, CH₃), 6.67(d,

J=4.44.Hz, IH, 4-vinyl CH), 7.02-7.26(m, 3H, ArH), 7.28(d, J=4.44.Hz, IH, 4-vinyl

CH), 12.8 (br. s, IH, HCl salt).

2) ⁱ³C NMR Spectrum (75.5 MHz, Varian NMR) (see Fig. 5)

¹³C NMR (CDCl₃) δ 17.41, 20.89, 37.70, 107.57, 126.31, 128.19, 131.98, 132.33,

134.18, 134.48, 139.28, 169.30.

3) Analysis Chart of cymiazole HCl

Beilstein test(+) : A sample was applied to a copper wire and performed a flame

reaction. The result showed the presence of a blue flame, which is a sign that it

contains chloride, a halogen atom, thus confirming it is present in the form of HCl

salt. Test Example 5: Evaluation of the Effect of Bee Mite Control

The honey bees infected with bee mites were administrated with the raw

cymiazole and the feed prepared in Example 1, respectively, and their effect were

examined.

- test period : August 5, 2005 - end of August, 2005

- test place : bee farm in Singi-myeon, Samcheok-si, Gangwon-do, Korea

Eight bee hives, each with 3 honey bees, were used in the experiment. Four bee

hives were treated with cymiazole while the other four were treated with the feed

prepared in Example 1.

Cymiazole was administered by using a 500 mL of a culturing solution

containing 247 mg of cymiazole about 6 pm right before honey bees returned to their

bee hives.

The feed in Example 1 was prepared to have a shape of a flat rectangle with 1 kg

of weight containing 247 mg of cymiazole, respectively. The feed was given to honey

bees through the top of hives for about 2 weeks.

The control effect of the feed on bee mites was observed for 24 days at 3 day

intervals. The number of dead bee mites resulted from the treatment of the drug or

the feed prepared in Example 1, respectively, was counted a total of 8 times for 24 days at 3 day intervals starting from the above treatment. A white sheet was placed

on the bottom of each bee hive before counting.

The number of bee mites, which were not controlled by the treatment of the

drug itself or the feed prepared in Example 1, was counted by separating them from

honey bees by anesthesizing the honey bees by spraying them with about 100 mL of

ethyl ether for 5-10 minutes, followed by placing them into 95% ethanol (see Fig. 6).

[Table 6] Comparison of bee mites control effects between cymiazole and the feed of the present invention and behavior of honey bees therefrom (3 honey bees/ bee hive)

Industrial Applicability

As stated above, the feed for controlling bee mites prepared according to the

present invention has advantages of having superior control effect applicable even

to young honey bees while capable of proliferation and nurture of honey bees.

Further, the drug for controlling bee mites is contained in the pollen feed to be

fed to honey bees. Therefore, it can prevent the bee farming products and bee hives

from being contaminated by the drug remaining after treatment of the drug. In

addition, the drug containing feed is fed to be evenly shared by honey bees thus

capable of preventing generation of drug resistance and saving labor work.

Finally, the feed is supplied along with pollen with good palatability. Therefore,

a certain amount of the drug can be continuously fed to honey bees thereby

improving the effect of controlling bee mites while reducing the stress that normally

resulted when an excessive amount of drug is used.

Claims

1. A feed for bee mite control prepared by fermentation of a mixture

comprising cymiazole and pollen or cymiazole and pollen substitute.

2. In claim 1, said cymiazole is contained in the amount of 0.001 - 1.0 weight %

°1 relative to the total weight of the feed 0.001 - 1.0 weight %.

3. In claim 1 or claim 2, sugar is contained in the amount of 2:8 - 8:2 weight ratio

with relative to the weight of said pollen or pollen substitute.

4. A method of preparing a feed for bee mite control comprising:

1) mixing sugar to a ground pollen or ground pollen substitute;

minutes to obtain an aqueous cymiazole solution;

3) drop wisely adding said aqueous cymiazole solution to said mixture in step 1)

pollen feed; and

4) fermenting said pollen feed in an aging room kept at 25 - 32 ⁰C for 3 - 7days.

5. In claim 4, the concentration of said aqueous cymiazole solution is 0.01 -

1.0 %.

6. In claim 4, sugar solution with a concentration of 0.03 - 3.0% is added to said

aqueous cymiazole solution.

7. In claim 4, said cymiazole is contained 0.001 - 1.0 weight % relative to the total

weight of the feed.

8. In claim 4, said sugar is contained in a molar ratio of 2:8 - 8:2 relative to the

weight of pollen or pollen substitute.