CN110618281B - Method for measuring content of biotin in insect body - Google Patents
Method for measuring content of biotin in insect body Download PDFInfo
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- CN110618281B CN110618281B CN201910850178.8A CN201910850178A CN110618281B CN 110618281 B CN110618281 B CN 110618281B CN 201910850178 A CN201910850178 A CN 201910850178A CN 110618281 B CN110618281 B CN 110618281B
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
Abstract
The invention relates to a method for measuring the content of biotin in an insect body, which comprises the following steps: (1) extracting and treating an insect sample; (2) preparing a biotin gradient solution; (3) preparing a biotin determination culture solution; (4) measuring biotin; the lactobacillus plantarum, various culture media and the like used in the detection method are common strains or consumables in the market, the cost is low, the cost of biotin detection is obviously reduced, and the culture method is simple; the detection time of the invention is 22 hours, which shortens 22-26 hours compared with the detection time of the commercial kit and improves the efficiency of the vitamin detection of insects.
Description
Technical Field
The invention relates to a determination method, in particular to a determination method of biotin content in an insect body.
Background
The B vitamins are indispensable nutrient elements for animals. The B vitamins are coenzymes of various enzymes in the animal body. Biotin (vitamin B7), a coenzyme for acetyl-coa. Is easily soluble in water, stable to acid and easily damaged by alkali. Has a crucial influence on the metabolic process of a living body. The quantitative detection of biotin in organisms is the basis for the study of the metabolism of life. At present, the detection method mainly comprises a microbiological method, a fluorescence analysis method, a spectrophotometry method, an electrochemical method, a high performance liquid chromatography method or a high performance capillary electrophoresis method and the like. However, these methods are commonly used for food and feed biotin detection.
The method for measuring the biotin in the insect body has few researches, only the bedbugs and the like adopt high performance liquid chromatography to measure the biotin content in the body, wherein the measurement result comprises biotin without physical activity, and no report is provided for measuring the biotin content in the insect by adopting a microbiological method at present. The kit for detecting biotin by using a microbiological method in the market is used for detecting biotin in food, has a small detection limit range, needs 44-48h for long detection time, is expensive, has the detection cost of about 5000 yuan each time, and is difficult to be used for physiological science research of small insects.
Disclosure of Invention
In order to make up for the defects of the prior art, the invention aims to provide a method for measuring the content of biotin in an insect body, which adopts a microbiological method to measure the content of biotin in the insect body, so that the cost of biotin detection is obviously reduced, and the detection efficiency is improved.
The invention aims to realize the following technical scheme, and the method for measuring the content of biotin in an insect body is technically characterized by comprising the following steps of:
(1) insect sample extraction process
Taking 20-50 mg insect samples, grinding the insect samples by using 100 mu L citrate buffer solution, and adding 200 mu L of 1mol/L sulfuric acid; water bath at 95 ℃ for 2h, shaking for 5 times, adjusting the pH to 7 by using a sodium hydroxide solution with the concentration of 2mol/L, and fixing the volume to 5 ml;
(2) preparing biotin gradient solution
a. Preparing a biotin standard stock solution with the concentration of 100 mug/mL;
b. preparing a biotin standard intermediate solution with the concentration of 1 mu g/mL;
c. preparing a biotin standard working solution with the concentration of 10 ng/mL;
d. preparing biotin gradient solution of 0.0-0.8 ng/mL;
(3) preparation of Biotin assay Medium
a. Preparation of microbial strains
The microorganism strain is lactobacillus plantarum (A)Lactobacillus Plantarum) (ii) a The culture medium of the lactobacillus plantarum is a lactobacillus agar culture medium and a lactobacillus broth culture medium;
the culture method of the lactobacillus plantarum strain comprises the following steps: culturing 3-5 generations of Lactobacillus plantarum strain.
The 1 st generation lactobacillus plantarum strain is subjected to streak culture on a lactobacillus agar culture medium and is cultured for 18 hours at 37 ℃;
culturing the 2 nd generation lactobacillus plantarum strain: selecting a first generation of single colony in a lactobacillus broth culture medium, and culturing for 18h at 37 ℃ to obtain a lactobacillus broth liquid;
culturing the 3 rd generation lactobacillus plantarum strain: taking the 2 nd generation lactobacillus broth, and culturing for 18h at 37 ℃ in a lactobacillus broth culture medium;
culturing the lactobacillus plantarum strain of the 4 th generation and the 5 th generation, culturing the lactobacillus plantarum broth of the previous generation in a lactobacillus broth culture medium according to the 3 rd generation culture method, and culturing at 37 ℃ for 18 h;
centrifuging the 3 rd to 5 th lactobacillus plantarum broth liquid at 2200rpm for 3 min; discarding supernatant, adding normal saline, mixing, washing bacteria for 3 times, and adjusting OD with normal saline 630nm : 0.8-0.95, obtaining the lactobacillus plantarum bacterial liquid for later use;
b. preparation of biotin measurement culture solution
Culturing free biotin in water bath at 95 deg.C for 30min, quickly cooling to room temperature, shaking once every 10 min; filtering, sterilizing and replacing; adding the lactobacillus plantarum bacterium liquid into a free biotin culture medium according to a ratio of 1:100, and uniformly mixing to obtain a biotin determination culture liquid;
(4) biotin assay
a. Sample processing
Respectively mixing the insect sample and the biotin gradient concentration solution with a biotin determination culture solution according to the mass-to-volume ratio of the insect sample or the biotin gradient concentration solution to the biotin determination culture solution =1:1, placing the treated samples in a clean microplate, culturing for 22 h at 37 ℃, and then placing the samples in the clean microplate to determine the OD value at 630 nm;
b. establishing a biotin determination standard curve
According to OD values obtained by measuring after mixing and culturing each biotin gradient concentration solution and biotin measuring culture solution, drawing a standard curve by taking a biotin content (ng) standard system as a horizontal coordinate and a 630nm optical density value as a vertical coordinate to obtain a standard curve linear equation;
c. calculation of Biotin content in insect samples
The OD value obtained by measuring the insect sample after the mixed culture of the insect sample and the biotin measuring culture solution is taken as the Y value of a biotin measuring standard curve, the corresponding biotin concentration (the x value of the standard curve) is found out on the standard curve, and the biotin content of each insect sample is calculated by the following formula:
wherein:
Nthe content of biotin in the sample is expressed in nanograms per milligram (ng/mg);
cthe concentration of biotin calculated on a standard curve of biotin assay in nanograms per milliliter (ng/mL);
v: after the sample in the step (1) is extracted, the volume is determined, and the unit is milliliter (ml);
m: insect mass in milligrams (mg);
F: and (4) diluting the volume by a constant volume after the sample is extracted.
Further, the test insects were hemipteran insects.
Further, the concentration of the biotin gradient solution in the step (2) is 0.0, 0.1, 0.2, 0.3, 0.4, 0.6 and 0.8 ng/mL in sequence.
Further, when the content of the insect biotin is measured to be more than 1.0 ng/mL by the measuring method, the insect sample in the step (1) is extracted to a constant volume for dilution, and then the step (4) is carried out; and (4) when the content of the insect biotin is lower than 0.1 ng/mL, re-extracting the insect sample or increasing the mass of the insect sample, and then performing the step (4).
The invention has the beneficial effects that:
(1) the lactobacillus plantarum and various culture media used for detection are common strains or consumables in the market, the cost is low, the culture method is simple, and the cost of biotin detection is obviously reduced.
(2) The detection time of the invention is 22 hours, which shortens 22-26 hours compared with the detection time of the commercial kit and improves the efficiency of the vitamin detection of insects.
Drawings
FIG. 1 is a standard curve for biotin determination;
FIG. 2 shows the biotin content in three insects.
Detailed Description
Embodiment 1 a method for measuring the biotin content in an insect body, comprising the steps of:
1. insect sample extraction process
The test insects were:
(1) bemisia tabaci: the population is donated by Zhejiang university in 2017 at 9 months, identified as MEAM1 hidden seed bemisia tabaci, and then a test population is established by using cotton as a host plant and continuously raised and maintained in an artificial climate chamber.
(2) The greenhouse trialeurodes vaporariorum is collected from Shenyang agriculture university tobacco in Shenyang city of Shenyang, Liaoning, in 2017, is identified as greenhouse trialeurodes vaporariorum, and then an experimental population is established by taking the tobacco as a host plant and is continuously raised and maintained in an artificial climate chamber.
(3) Brown planthopper: in 2017, the rice is donated by Zhejiang university in 9 months, and a test population is established by taking rice as a host plant and is continuously raised and maintained in an artificial climate chamber.
The climatic chamber was set to the following environmental conditions: temperature 26 ± 1 ℃, relative humidity 60 ± 10%, illumination period L: D =14 h: 10 h (light time: 06: 00-20: 00; dark time: 20: 00-06: 00).
The sample processing method comprises the following steps:
respectively taking 100 pairs (female parent and male parent =1: 1) of greenhouse whitefly and bemisia tabaci; taking 1 pair of (male female parent) brown planthoppers, weighing the brown planthoppers respectively, grinding the brown planthoppers in a 2.0 ml homogenate tube by using 100 mu L of citrate buffer solution, transferring the brown planthoppers to a 15ml centrifuge tube, and adding 200 mu L of 1mol/L sulfuric acid; water bath at 95 ℃ for 2h, shaking for 5 times during the period, and adjusting the pH to 7 by using a sodium hydroxide solution with the concentration of 2 mol/L; and the volume is adjusted to 5ml, filtered and sterilized.
2. Preparing biotin gradient solution
(1) Biotin (vitamin B7) standard stock solution (100. mu.g/mL): weighing 5mg of biotin standard substance, fixing the volume of 50% ethanol to 50ml, storing the stock solution in a refrigerator, and storing in dark place;
(2) biotin standard intermediate (1. mu.g/mL): accurately sucking 1mL of biotin standard stock solution, fixing the volume to 100mL by using pure water solution, filtering and sterilizing;
(3) biotin standard working solution (10 ng/mL): accurately sucking 500 μ L of biotin standard intermediate solution, diluting to 50mL with pure water solution, filtering, and sterilizing;
(4) the biotin standard working solution and sterile purified water were added to sterile 2mL centrifuge tubes at the volume ratios shown in the table below, 3 replicates per concentration.
TABLE 1 vitamin B7 gradient solution preparation method
| Numbering | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
| Standard working fluid | 0 | 0 | 10 | 20 | 30 | 40 | 60 | 80 |
| Sterile pure water | 1000 | 1000 | 990 | 980 | 970 | 960 | 940 | 920 |
| Concentration (ng/mL) | 0 | 0 | 0.1 | 0.2 | 0.3 | 0.4 | 0.6 | 0.8 |
3. Preparation of Biotin assay Medium
(1) Preparation of the Strain
The strain is as follows: lactobacillus plantarum: (A)L. Plantarum) ATCC No. 8014 from Tokyo Lubo technologies, Inc.
Culture medium: lactobacillus agar medium and Lactobacillus broth medium are provided by Beijing Luqiao science and technology, Inc.
Biotin (vitamin B7) standard standards were supplied by Sigma company;
ultrapure water (Wahaha);
50% of ethanol: 50mL of absolute ethyl alcohol is put into a 100mL volumetric flask, and the volume is adjusted to 100mL by using purified water.
Citric acid monohydrate buffer solution (pH 4.5, Kyosu chemical Co., Ltd.): 1.5 g of citric acid monohydrate was weighed into a 100mL beaker with magnetic stirrer, about 50mL of distilled water was added until dissolved, 0.48 g of NaOH was added, the pH was adjusted to 4.5 (with 0.1 mol/l HCl), the solution was transferred to a 100mL volumetric flask and the volume was fixed with distilled water. The buffer solution can be stored for 3 days at 2-8 ℃;
hydrochloric acid (1.0 mol/L, Kyosu chemical Co., Ltd.): 8.33mL of hydrochloric acid (37%) distilled water is added to a volume of a 100mL volumetric flask;
sulfuric acid (3%, west longgaku chemical corporation): measuring 3mL of sulfuric acid (98%), and quantitatively containing distilled water in a 100mL volumetric flask;
sodium hydroxide (2 mol/L, Kyosu chemical Co., Ltd.): 8 g NaOH was dissolved in 50mL distilled water, shaken up and made to volume in a 100mL volumetric flask.
The lactobacillus plantarum strain culture method comprises the following steps: culturing 3-5 generations of Lactobacillus plantarum strain.
The lactobacillus plantarum strain culture 3-generation method comprises the following steps:
culturing the strain of the 1 st generation:
marking 10 mu L of lactobacillus plantarum bacterial liquid on a lactobacillus agar culture medium, and culturing for 18h at 37 ℃;
culturing strains at generation 2:
picking a single colony, and culturing the single colony in a lactobacillus broth culture medium at 37 ℃ for 18 h;
culturing the 3 rd generation strain:
taking 10 μ L of 2 nd generation lactobacillus broth solution, culturing at 37 deg.C for 18h, centrifuging at 2200rpm, and centrifuging for 3 min; discarding the supernatant, adding 5mL of physiological saline, mixing uniformly, wherein the bacteria washing process aims to avoid errors caused by biotin in the culture medium to the test, centrifuging at 2200rpm for 3 min; discarding the supernatant; washing the bacteria for 3 times, and adjusting OD with physiological saline 630nm : 0.8-0.95 for standby;
(2) preparation of Biotin assay culture solution
A free biotin medium, which is a medium lacking biotin and purchased from BD company (reagent No. 241910, biotin determination medium), was used; culturing free biotin in water bath at 95 deg.C for 30min, quickly cooling to room temperature, shaking once every 10 min; filtering and sterilizing for later use; culturing the strain of the 3 rd generation to obtain a bacterial liquid according to the ratio of 1: adding 100 portions of the mixture into a free biotin culture medium, and uniformly mixing to obtain a biotin determination culture solution.
4. Biotin assay
(1) Sample processing
Treatment 1: 1mL of the insect sample obtained in the step 1 is taken and added with 1mL of the biotin determination culture solution obtained in the step 3, and the mixed solution is placed in a 2mL centrifuge tube;
and (3) treatment 2: 1mL of each vitamin B7 gradient solution obtained in the step 2 is taken, 1mL of the biotin determination culture solution obtained in the step 3 is added, and the mixed solution is placed in a 2mL centrifuge tube;
culturing the two treated samples at 37 ℃ for 22 h, respectively placing 300 mu L of each sample into a clean microporous plate, and measuring the OD value at 630 nm;
(2) establishing a standard curve for biotin determination
According to the OD value obtained in the treatment 2, a standard curve (shown in FIG. 1) is drawn by taking a standard system of the content (ng) of biotin (vitamin B7) as an abscissa and a 630nm optical density value as an ordinate; the biotin concentration is in the range of 0.2-0.8 ng, and the linear relation between the concentration and the optical density value is better.
(3) Calculation of Biotin content in insect samples
Using each OD measurement value obtained by the measurement in treatment 1 as a Y value of a biotin measurement standard curve, finding out a corresponding biotin concentration (x value of the standard curve) on the standard curve, and calculating the biotin content of each insect sample by the following formula:
wherein:
Nthe content of biotin in the sample is expressed in nanograms per milligram (ng/mg);
cthe concentration of biotin calculated on a standard curve for biotin determination, i.e., the value x in FIG. 1, in nanograms per milliliter (ng/mL);
v: constant volume for sample extractionTotal volume in milliliters; (i.e., 5ml in this example)
m: insect mass in milligrams (mg);
F: sample dilution factor (factor of dilution after sample treatment to 5 ml)
For the sake of accuracy, if the content of the insect biotin measured by the above method exceeds 1.0 ng/mL, the sample may be extracted to a constant volume for dilution, and the measuring step may be repeated, or if the content of the insect biotin measured by the above method is less than 0.1 ng/mL, the sample may be re-extracted, or the sample may be increased in mass, and the measuring step may be repeated.
(4) Measurement results
The method is adopted to detect the contents of vitamin B7 in bemisia tabaci, trialeurodes vaporariorum and nilaparvata lugens, the measurement result is shown in table 2, and the difference of the contents of the three insects is shown in figure 2.
TABLE 2 measurement of biotin content in insect test
5. The content of biotin in bemisia tabaci detected by the determination method is compared with that of biotin in standard kit method
In order to verify the accuracy and the practicability of the method, the insect samples are measured by using a kit, the culture and the measurement are carried out according to the kit specification, the calculation is carried out according to software provided by the kit, and the detection result is shown in a table 2, wherein the kit is a vitamin B7 (biotin) detection kit (purchased from Bayer in Germany).
TABLE 2 result of measuring biotin content of insect sample by using kit
Meanwhile, for the tobacco whitefly and trialeurodes vaporariorum samples, the method and the kit method are compared to calculate the recovery rate result, and the result is as follows: the P value of this test was > 0.1, indicating no significant difference between the results of the two assays.
Table 3 comparison of results of two assays for vitamin B7
Note: and (3) carrying out T test on the detection results of the two determination methods, wherein the P value is more than 0.1.
Claims (4)
1. A method for measuring the biotin content in an insect body is characterized in that: the method comprises the following steps:
(1) insect sample extraction process
Taking 20-50 mg insect samples, grinding the insect samples by using 100 mu L citrate buffer solution, and adding 200 mu L of 1mol/L sulfuric acid; water bath at 95 ℃ for 2h, shaking for 5 times, adjusting the pH to 7 by using a sodium hydroxide solution with the concentration of 2mol/L, and fixing the volume to 5 ml;
(2) preparing biotin gradient solution
a. Preparing a biotin standard stock solution with the concentration of 100 mug/mL;
b. preparing biotin standard intermediate solution with the concentration of 1 mu g/mL;
c. preparing a biotin standard working solution with the concentration of 10 ng/mL;
d. preparing biotin gradient solution with the concentration of 0.0-0.8 ng/mL;
(3) preparation of Biotin assay Medium
a. Preparation of microbial strains
The microbial strain is lactobacillus plantarum; the culture medium of the lactobacillus plantarum is a lactobacillus agar culture medium and a lactobacillus broth culture medium;
the culture method of the lactobacillus plantarum strain comprises the following steps: culturing 3-5 generation strain of Lactobacillus plantarum;
the 1 st generation lactobacillus plantarum strain is subjected to streak culture on a lactobacillus agar culture medium and is cultured for 18 hours at 37 ℃;
culturing the 2 nd generation lactobacillus plantarum strain: picking the first generation of single bacterial colony in a lactobacillus broth culture medium, and culturing for 18h at 37 ℃ to obtain a lactobacillus broth liquid;
culturing the 3 rd generation lactobacillus plantarum strain: taking the 2 nd generation lactobacillus broth, and culturing for 18h at 37 ℃ in a lactobacillus broth culture medium;
culturing the lactobacillus plantarum strain of the 4 th generation and the 5 th generation, culturing the lactobacillus plantarum broth liquid of the previous generation in a lactobacillus broth culture medium according to the 3 rd generation culture method, and culturing at 37 ℃ for 18 h;
centrifuging the 3 rd-5 th generation lactobacillus plantarum broth at 2200rpm for 3 min; discarding supernatant, adding normal saline, mixing, washing bacteria for 3 times, and adjusting OD with normal saline 630nm : 0.8-0.95 to obtain the lactobacillus plantarum bacterium liquid for later use;
b. preparation of biotin measurement culture solution
Culturing free biotin in water bath at 95 deg.C for 30min, quickly cooling to room temperature, shaking once every 10 min; filtering, sterilizing and replacing; adding the standby lactobacillus plantarum bacterial liquid into a free biotin culture medium according to the proportion of 1:100, and uniformly mixing to obtain a biotin determination culture solution;
(4) biotin assay
a. Sample processing
Respectively mixing an insect sample and a biotin gradient concentration solution with a biotin determination culture solution according to the mass-volume ratio of the insect sample or the biotin gradient concentration solution, namely the biotin determination culture solution =1:1, placing the treated samples in a clean microporous plate for culturing at 37 ℃ for 22 h, and then placing the samples in the clean microporous plate to determine an OD value at 630 nm;
b. establishing a biotin determination standard curve
According to the OD value obtained by measuring after mixing and culturing each biotin gradient concentration solution and biotin measuring culture solution, drawing a standard curve by taking a biotin content standard system as a horizontal coordinate and a 630nm optical density value as a vertical coordinate to obtain a standard curve linear equation;
c. calculation of Biotin content in insect samples
The OD value obtained by measuring the insect sample after mixed culture and the biotin measuring culture solution is taken as the Y value of a biotin measuring standard curve, the corresponding biotin concentration is checked out on the standard curve, and the biotin content of each insect sample is calculated by the following formula:
wherein:
Nthe content of biotin in the sample is nanogram per milligram;
cthe concentration of biotin obtained by calculation on a biotin determination standard curve and the unit is nanogram per milliliter;
v: after the sample in the step (1) is extracted, the volume is determined in milliliters;
m: insect mass in milligrams;
F: and (4) diluting the volume by a constant volume after the sample is extracted.
2. The method for measuring the biotin content in an insect body according to claim 1, wherein: the insects are hemipteran insects.
3. The method for measuring the biotin content in an insect body according to claim 1, wherein: and (3) the concentration of the biotin gradient solution in the step (2) is 0.0, 0.1, 0.2, 0.3, 0.4, 0.6 and 0.8 ng/mL in sequence.
4. The method according to claim 1, wherein the method comprises the steps of: when the content of the insect biotin is measured to be more than 1.0 ng/mL by the measuring method, the insect sample in the step (1) is extracted to a constant volume for dilution, and then the step (4) is carried out; and (4) when the content of the insect biotin is lower than 0.1 ng/mL, re-extracting the insect sample or increasing the mass of the insect sample, and then performing the step (4).
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