Application of 3-methyl-1-butanol in preparation of blood-sucking midge repellent

Abstract

The invention belongs to the technical field of microbial resource development, and particularly relates to application of 3-methyl-1-butanol in preparation of a blood sucking midge repellent. By detecting the EAG reaction and behavioral reaction of the Chinese Taiwan Lasiohelea to the 3-methyl-1-butanol, it is proved that the 3-methyl-1-butanol has a good repelling effect on the Chinese Taiwan Lasiohelea, and when the concentration of the 3-methyl-1-butanol is 10%, the 3-methyl-1-butanol has the effect of repelling the Chinese Taiwan Lasiohelea. The 3-methyl-1-butanol serving as a monomeric compound is simple in component and environment-friendly, and can be expected to be developed into a series of products for preventing and treating blood-sucking midges.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial resource development, and particularly relates to application of 3-methyl-1-butanol in preparing a blood-sucking midge repellent. Background Art

[0002] Insects have a sensitive olfactory system to perceive various odor molecules in the environment, which leads to a series of behaviors such as foraging, finding mates, and choosing egg-laying sites. Whether blood-sucking insects can find the target host is related to the type and content of odor substances released by the latter. For example, Aedes aegypti has a stimulating response to 3-methyl-1-butanol, 6-methyl-5-heptene-2-one, geranyl acetone and nonanal. Although existing technologies have conducted relevant research on some blood-sucking insects, no research has been conducted on the host selection of midges.

[0003] Bacteria can produce unique volatile odor substances, and these volatile substances show diversity and complexity. At present, the research on volatile substances produced by bacteria mainly focuses on the production of odors in food fermentation, corruption process, building materials, etc., while there are fewer studies on the role of bacteria in producing volatile substances. At present, there are about 600 known volatile components of human skin organic matter. Studies have found that some human body odor can affect the search for hosts by blood-sucking insects. Body odor is produced by skin-resident bacteria. These bacteria convert metabolites secreted in sweat into odorants, giving the human body a unique odor. Many studies have shown that microorganisms play an important role in the host positioning of blood-sucking insects. Therefore, in-depth research on host odor substances will not only help develop attractants and repellents for midges, but also help to achieve effective monitoring and control of midges.

[0004] In recent years, with the development and large-scale use of insect attractants, real-time monitoring, large-scale trapping or killing of specific harmful insects have been achieved, and insect density has been reduced, reducing their harassment and harm to humans. At present, there is no specific repellent for blood-sucking midges, which is extremely unfavorable for the prevention and control of important harassing and blood-sucking midges such as midges in Taiwan Province, China. It is urgent to develop repellents for characteristic groups of blood-sucking midges to facilitate large-scale trapping and killing of blood-sucking midges, reduce adverse reactions caused by their harassment and blood-sucking, and avoid unnecessary economic losses. Therefore, the development of a repellent for blood-sucking midges has important economic and social benefits for the effective control of blood-sucking midges. Summary of the invention

[0005] In order to reduce the density of blood-sucking midges and alleviate the harm caused by them, the present invention provides an application of 3-methyl-1-butanol in preparing a blood-sucking midge repellent.

[0006] The technical solution adopted by the present invention is:

[0007] The present invention provides an application of 3-methyl-1-butanol in the preparation of a blood-sucking midge repellent.

[0008] Preferably, the blood-sucking midge repellent is used to repel at least one of Lasiohelea taiwana and Forcipomyia taiwana in Taiwan Province, China.

[0009] The present invention provides a blood-sucking midge repellent, and the blood-sucking midge repellent uses the 3-methyl-1-butanol described in claim 1 as the sole active ingredient.

[0010] Preferably, the blood-sucking midge repellent is composed of the 3-methyl-1-butanol described in claim 1 and a known repellent, and the volume fraction of 3-methyl-1-butanol in the blood-sucking midge repellent is ≥10%; the known repellent is at least one of geranyl acetone, nonanal, 6-methyl-5-hepten-2-one, heptanal, and nonanal.

[0011] Preferably, the blood-sucking midge repellent further includes a pharmaceutically acceptable excipient.

[0012] Preferably, the pharmaceutically acceptable excipient is one or several of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickening agent, an emulsifier, a preservative, and a stabilizer.

[0013] Preferably, the diluent includes any one of starch, lactose, sucrose, and mannitol.

[0014] Preferably, the disintegrant includes any one of starch, microcrystalline cellulose, and low-substituted hydroxypropyl cellulose.

[0015] Preferably, the precipitation inhibitor includes any one of sodium dodecyl sulfate, Tween-80, polyvinylpyrrolidone, and hydroxypropyl methylcellulose.

[0016] Preferably, the glidant includes any one of cationic polyacrylamide, poly(diallyldimethylammonium chloride), and cationic starch.

[0017] Preferably, the binder includes any one of starch paste, hydroxypropyl methylcellulose, and polyvinylpyrrolidone.

[0018] Preferably, the dispersant includes any one of sodium dodecyl sulfate, polyvinylpyrrolidone, and sodium carboxymethylcellulose.

[0019] Preferably, the suspending agent includes any one of gum arabic, tragacanth gum, sodium carboxymethylcellulose, and hydroxypropyl methylcellulose.

[0020] Preferably, the isotonic agent includes any one of sodium chloride, glucose, and mannitol.

[0021] Preferably, the thickener includes any one of gum arabic, xanthan gum, and sodium carboxymethyl cellulose.

[0022] Preferably, the emulsifier includes any one of sodium lauryl sulfate, benzalkonium chloride, and sorbitan fatty acid esters.

[0023] Preferably, the preservative includes any one of benzoic acid, sorbic acid, methyl p-hydroxybenzoate, and benzalkonium bromide.

[0024] Preferably, the stabilizer includes any one of sodium sulfite, sodium bisulfite, tocopherol, and disodium ethylenediaminetetraacetate.

[0025] Preferably, the acceptable dosage forms of the blood-sucking midge repellent include one of liquid, tablet, capsule, granule, pill, powder, and ointment.

[0026] Preferably, the auxiliary material used in the liquid is absolute ethanol.

[0027] Preferably, the method for screening the 3-methyl-1-butanol includes the following steps:

[0028] Collect human epidermal bacterial samples, culture, isolate, and purify them to obtain single colonies;

[0029] Amplify the 16S rRNA gene of the single colony and identify the type of bacteria by 16S rRNA gene sequencing;

[0030] Detect and collect the volatiles produced by the bacteria;

[0031] Screen the volatiles with electrophysiological stimulating activity against insects;

[0032] Verify the repellent effect of the 3-methyl-1-butanol against blood-sucking midges through behavioral experiments.

[0033] Preferably, the collection sites of the human epidermal bacterial samples include at least one of the hand, forehead, cubital fossa, and popliteal fossa.

[0034] Preferably, the culture medium for obtaining the single colonies is at least one of LB solid culture medium and R2A solid culture medium.

[0035] The beneficial effects of the present invention are:

[0036] The present invention provides an application of 3-methyl-1-butanol in preparing a blood-sucking midge repellent. The present invention verifies that 3-methyl-1-butanol has a good repellent effect on Taiwan Province of China midges by detecting the EAG reaction and behavioral reaction of Taiwan Province of China midges to 3-methyl-1-butanol, and has a repellent effect on Taiwan Province of China midges at a concentration of 10%. As a monomer compound, 3-methyl-1-butanol has simple ingredients and is environmentally friendly, and is expected to be developed into a series of products for controlling blood-sucking midges.

[0037] The present invention adds 16S rRNA sequencing technology to the traditional method to conduct a comprehensive analysis of epidermal microorganisms, thereby improving the accuracy of bacterial identification. The present invention combines the insect olfactory nerve potential recording EAG technology to screen compounds that are highly specific to target insects, thereby improving the accuracy of active substance screening.

[0038] The present invention provides a new method for screening microbial germplasm resources that produce specific attractant or repellent volatiles from human epidermal bacteria, constructs a system for screening and producing specific attractant or repellent volatiles from human epidermal bacteria, and is suitable for the production and function research of human epidermal bacterial volatiles.

[0039] The method of the present invention can realize modular and process-based operation, can meet the requirements of screening strains producing specific volatiles with attracting or repelling effects from human epidermal bacteria, and provides a powerful technical means for mining bacterial strain resources producing active volatiles.

[0040] The method of the invention is rapid, simple, and has important theoretical research and production practice values, and is worthy of promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The volatile substances adsorbed in the gauze of 8 volunteers.

[0042] Figure 2 The test results are behavioral responses.

[0043] Figure 3 Schematic diagram of the Y-type olfactometer. DETAILED DESCRIPTION

[0044] The present invention is further described below by specific examples, but the scope of the present invention is not limited thereto. The details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications or replacements all fall within the protection scope of the present invention.

[0045] The inventive concept of the present invention is as follows:

[0046] At present, there is no specific repellent for blood-sucking midges, which is extremely disadvantageous for the control of important nuisance and blood-sucking midges such as Lasiohelea in Taiwan Province, China. It is urgent to develop repellents for characteristic groups of blood-sucking midges to facilitate the large-scale trapping and killing of blood-sucking midges, reduce adverse reactions caused by their harassment, blood-sucking, etc., and avoid unnecessary economic losses. Therefore, the development of a repellent for blood-sucking midges has important economic and social benefits for the effective control of blood-sucking midges.

[0047] Based on this, the present invention provides the use of 3-methyl-1-butanol in the preparation of a blood-sucking midge repellent.

[0048] In order to enable those skilled in the art to better understand and implement the technical solutions of the present invention, the present invention will be further described below in conjunction with specific embodiments and drawings. In the description of the present invention, unless otherwise specified, the reagents used are commercially available, and the methods used are conventional techniques in the art.

[0049] 1. The EAG reaction method of the present invention is as follows:

[0050] First, 3-methyl-1-butanol is diluted with absolute ethanol to 3-methyl-1-butanol solutions with volume fractions of 10%, 1%, 0.1%, 0.01%, 0.001%, 0.0001%, 0.00001%, and 0.000001% respectively for standby measurement.

[0051] (1) Select healthy and active adult Lasiohelea in Taiwan Province, China. Take 1 antenna and cut it along the base of the single antenna, with a little scalp, to ensure a stable baseline.

[0052] (2) Cut off a little of the tip of the antenna. Two glass capillaries are filled with physiological saline. One sucks the base of the antenna and inserts it into the reference electrode, and the other is inserted into the measuring electrode. Operate to connect the tip of the glass capillary of the measuring electrode to the tip of the antenna, exposing more antennal sensilla.

[0053] (3) The way of attaching the antenna is the glass electrode connection method.

[0054] (4) Close the shielding cabinet and check whether the baseline is stable.

[0055] (5) During the test, 15 μL of the test substance was dropped onto a 4 cm × 0.5 cm filter paper strip and placed into a 1 mL blue pipette tip. The test was started 20 seconds later, with the pipette tip 1 cm away from the antennae. The air flow rate was 600 mL / min, the stimulus gas flow rate was 500 mL / min, and the stimulation was performed once every 1 minute, with each stimulation lasting 0.5 seconds. All concentrations of decanal were tested on each antennae. The order of determination of the test substances was as follows: blank group - low concentration test substance - high concentration test substance - blank group. Each concentration of the test substance was repeated 10 times with 10 adult midges from Taiwan Province, China. Only one antenna was taken from each adult midge from Taiwan Province, China, and anhydrous ethanol was used as the blank group.

[0056] (6) After the baseline is stable, place the object to be tested on the small hole on the taste tube and start the test recording.

[0057] (7) Calculate the relative value of EAG response.

[0058] 2. The behavioral response method of midges from Taiwan Province, China to 3-methyl-1-butanol is as follows:

[0059] The midges to be tested in Taiwan Province of China were transferred to the test room to adapt to the environment for about 1 hour. The experiment used natural light and behavioral tests were performed on the midges in Taiwan Province of China at fixed times. During the measurement, 5 replicates were made for each concentration of the test object. The Y-type olfactometer is assembled from an air pump, a gas washing bottle, a drying tower filled with activated carbon, a sample bottle, a flow meter, a silicone tube, a Y-type tube, and an insect collector. See the schematic diagram of the Y-type olfactometer for details. Figure 3 .

[0060] During the measurement, the gas flow rate was 0.4L / min. The 2.5cm×5cm filter paper with 100μL of the test object was recorded as the experimental group, and the 2.5cm×5cm filter paper with the same volume of anhydrous ethanol was recorded as the control group. After the test object and anhydrous ethanol were evaporated, they were placed in the left and right sample bottles respectively. 20 Taiwan midges were placed in the straight tube mouth of the Y-tube to observe their first choice within 5 minutes. If they did not choose within 5 minutes, they were recorded as non-reaction. The filter paper was replaced every time an experiment was conducted. After repeating the measurement twice, the direction of the two arms of the Y-tube was swapped to eliminate the influence of the geometric position on the tendency behavior of Taiwan midges. After each experiment, it was washed with distilled water, the Y-tube was wiped with alcohol, and the next group of experiments were carried out after drying, and the selection rate was counted.

[0061] Example 1

[0062] A use of 3-methyl-1-butanol in preparing a blood-sucking midge repellent is as follows:

[0063] S1. Collect human epidermal bacterial samples, culture, isolate, and purify them to obtain single colonies; amplify the 16S rRNA genes of the single colonies, and identify the bacterial species through 16S rRNA gene sequencing; detect and collect the volatile substances produced by the bacteria.

[0064] Eight volunteers with different degrees of being bitten by Lasiohelea taiwana (Shiraki) in their daily lives were selected from 285 investigators. The hands, foreheads, cubital fossae, and popliteal fossae of the eight volunteers, which are the four parts often bitten by Lasiohelea taiwana (Shiraki), were wiped with sterile swabs, and the wiping range was 5 cm. 2 , and immediately after wiping, the swabs were placed in an EP tube containing 2 mL of sterile ddH2O for shaking. After being evenly pipetted, the bacterial liquid was serially diluted to 10 -2 , 10 -3 , 10 -4 , and 10 -5 . 100 μL of the serially diluted bacterial liquid was pipetted onto LB solid medium and R2A solid medium, which are common bacterial media, for plate coating. The plates were incubated at 37 °C for 48 h, and then the bacterial colonies were picked. After six times of purification into single colonies, morphological preliminary identification was carried out. The bacteria screened by morphological preliminary identification were used for subsequent experiments. The results of morphological preliminary identification are shown in Table 1.

[0065] Table 1 Results of preliminary morphological identification of bacteria

[0066]

[0067] The purified bacteria were subjected to preliminary morphological identification, and 35 bacteria were screened for molecular biology identification. Subsequently, the 16S rRNA genes of these 35 bacteria were amplified to obtain a single band with a length of about 1500 bp, and then it was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing.

[0068] The sequenced 16S rRNA genes were assembled using the seqman software, the sequences were viewed using Editseq, and sequence alignment was performed in EzBiocloud. The alignment results are shown in Table 2, and the sequence alignment similarity is greater than 98%.

[0069] Table 2 Alignment results of 16S rRNA sequences in the EzBiocloud database

[0070]

[0071]

[0072] Meanwhile, scrape 1 g of each colony from the solid medium and place it in the sampling bottle of the solid-phase microextractor to collect the odor using headspace solid-phase microextraction. Scrape 1 g of the uncontaminated medium as a blank control. Detect the volatiles of the above bacteria. Among them, 3-methyl-1-butanol is mainly produced by Staphylococcus hominissubsp. novobiosepticus R2A01-07, up to 81.898%; the proportion of 3-methyl-1-butanol in the volatiles of Microbacterium saccharophilum R2A02-01 and Staphylococcus cohnii LB01-04 is also relatively large. Geranyl acetone is produced by multiple bacteria such as Micrococcus luteus LB02-02 and Staphylococcus haemolyticus R2A01-08, but the relative content is small. Nonanal is produced by 4 bacteria such as Micrococcus endophyticus R2A02-07, and the relative content is small. 6-Methyl-5-hepten-2-one is produced by multiple bacteria such as Micrococcus endophyticus R2A02-07 and Micrococcus luteus LB02-02, and the relative content is small. As shown in Table 3, a total of 37 main volatiles are produced. It is found from Table 13 that Micrococcus luteus LB02-02 and Micrococcus endophyticus R2A02-07 produce more types of volatiles. 3-Methyl-1-butanol is produced by 5 bacteria, 6-methyl-5-hepten-2-one is produced by 12 bacteria, nonanal is produced by 4 bacteria, and geranyl acetone is produced by 6 bacteria.

[0073] Table 3 Analysis and Identification Results of Bacterial Volatile Components

[0074]

[0075]

[0076]

[0077] S2. Screen the volatiles with electrophysiological stimulating activity against blood-sucking midges.

[0078] The gauze was soaked in methanol for 24 h, dried, and then soaked in absolute ethanol for 24 h. After detecting that there were no other organic volatiles by headspace solid-phase microextraction gas chromatography-mass spectrometry, the gauze was wound around the whole body of 8 volunteers. The volunteers were required to follow their daily lives, but during this period, they did not use deodorants, perfumes, and lotions and did not take baths. After 48 h, the volatiles in the sterile gauze were extracted by the headspace collection method. The volatile components were determined by the Nist20 and Wiley275 standard mass spectra, and the relative mass fractions of each component were determined by the peak area normalization method.

[0079] In the present invention, by detecting the volatiles adsorbed in the gauze of 8 volunteers, a total of 25 compounds with relatively high relative contents were identified, including ketones, aldehydes, alcohols, acids, etc. As Figure 2 shown, the relative content of the volatiles contained in the 8th volunteer was relatively high, reaching 54.06%, and among them, the relative content of 6-methyl-5-hepten-2-one was the highest, reaching 14.62%. In contrast, the relative content of the volatiles of the 3rd volunteer was relatively low, only 12.57%, and among them, the relative content of 6-methyl-5-hepten-2-one was the highest, 2.59%. The results are shown in Figure 1 . Generally speaking, the types of volatiles on the skin surfaces of different people are similar, but only the relative contents of each compound are different. Among them, nonanal, 6-methyl-5-hepten-2-one, and decanal exist in the skin volatiles of 8 volunteers, are relatively stable and have relatively high relative contents.

[0080] The results of the EAG reaction showed that when the concentrations of 6-methyl-5-hepten-2-one, geranyl acetone, 3-methyl-1-butanol, and heptanal were 10%, the stimulatory responses to Lasiohelea taiwana in Taiwan Province, China were the greatest; the stimulatory responses of 1% decanal and nonanal to Lasiohelea taiwana in Taiwan Province, China were the strongest; overall, the stimulatory responses of Lasiohelea taiwana in Taiwan Province, China showed an upward trend with the increase of the volatile concentration, but the upward trends of the stimulatory responses of 3-methyl-1-butanol and heptanal to Lasiohelea taiwana in Taiwan Province, China were not obvious. The relative response value of Lasiohelea taiwana in Taiwan Province, China to 3-methyl-1-butanol with a concentration of 0.000001% was relatively high, indicating that 3-methyl-1-butanol is suitable as a long-distance stimulant in the wild. The relative response value of Lasiohelea taiwana in Taiwan Province, China to geranyl acetone with a concentration of 10% was relatively high, indicating that geranyl acetone is suitable as a short-distance stimulant.

[0081] S3. Verify the repellent effect of the volatiles on blood-sucking midges through behavioral experiments.

[0082] Based on the above results, 3-methyl-1-butanol was selected to conduct behavioral experiments on Lasiohelea taiwana in Taiwan Province, China for verification. The behavioral experiment verification was completed using a Y-tube olfactometer. The Y-tube olfactometer is shown in Figure 2 . The results are shown in Figure 3As shown in Table 4, with the increase in the concentration of 3-methyl-1-butanol, the upward trend of the stimulatory response to Forcipomyia taiwana (Shiraki) in Taiwan Province of China is not obvious. The repellent rate of 10% 3-methyl-1-butanol to Forcipomyia taiwana (Shiraki) in Taiwan Province of China is the highest, reaching 70.7%.

[0083] Table 4 Attraction rate of 3-methyl-1-butanol to Forcipomyia taiwana (Shiraki) in Taiwan Province of China

[0084]

[0085]

[0086] Note: In Table 3, "*" indicates P < 0.05.

[0087] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope recorded in this specification.

[0088] The above-described embodiments only represent several implementation manners of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.

Claims

1. Use of 3-methyl-1-butanol in preparing a blood-sucking midge repellent.

2. The use according to claim 1, characterized in that The blood-sucking midge repellent is used to repel at least one of the blood-sucking midges from Taiwan Province, China and the cutting midges from Taiwan Province, China.

3. A blood-sucking midge repellent, characterized in that: The blood-sucking midge repellent contains the 3-methyl-1-butanol described in claim 1 as the only active ingredient.

4. The blood-sucking midge repellent according to claim 3, characterized in that: The blood-sucking midge repellent is composed of the 3-methyl-1-butanol according to claim 1 and a known repellent, and the volume fraction of the 3-methyl-1-butanol in the blood-sucking midge repellent is ≥10%; The known repellent is at least one of geranyl acetone, nonanal, 6-methyl-5-hepten-2-one, heptanal and nonanal.

5. The blood-sucking midge repellent according to claim 3, characterized in that: The blood-sucking midge repellent further comprises pharmaceutically acceptable excipients.

6. The blood-sucking midge repellent according to claim 5, characterized in that: The pharmaceutically acceptable excipient is one or more of a diluent, a disintegrant, a precipitation inhibitor, a glidant, a binder, a dispersant, a suspending agent, an isotonic agent, a thickener, an emulsifier, a preservative and a stabilizer.

7. The blood-sucking midge repellent according to claim 3, characterized in that: The acceptable dosage form of the blood-sucking midge repellent includes one of liquid, tablet, capsule, granule, pill, powder and ointment.

8. The use according to claim 1, characterized in that The method for screening the 3-methyl-1-butanol comprises the following steps: Collect human epidermal bacterial samples, culture, isolate and purify them to obtain single colonies; Amplify the 16S rRNA gene of a single colony and identify the bacterial species by 16S rRNA gene sequencing; detecting and collecting volatiles produced by the bacteria; Screening of volatiles with electrophysiological stimulatory activity on insects; The repellent effect of 3-methyl-1-butanol on blood-sucking midges was verified by behavioral experiments.

9. The use according to claim 8, characterized in that The collection site of the human epidermal bacterial sample includes at least one of the hands, forehead, cubital fossa and popliteal fossa.

10. The use according to claim 8, characterized in that The culture medium used to obtain the single colony is at least one of LB solid culture and R2A solid culture medium.

Images