WO2020206821A1 - 一种高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法 - Google Patents

一种高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法 Download PDF

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WO2020206821A1
WO2020206821A1 PCT/CN2019/088357 CN2019088357W WO2020206821A1 WO 2020206821 A1 WO2020206821 A1 WO 2020206821A1 CN 2019088357 W CN2019088357 W CN 2019088357W WO 2020206821 A1 WO2020206821 A1 WO 2020206821A1
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thin
bioluminescence
layer plate
layer
tea
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French (fr)
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陈益胜
黄彩虹
舒蓝萍
徐学明
金征宇
龙杰
田耀旗
柏玉香
杨哪
金亚美
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江南大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G01N30/94—Development
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
    • A23F3/00—Tea; Tea substitutes; Preparations thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G01N30/95—Detectors specially adapted therefor; Signal analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/02—Food
    • G01N33/14—Beverages
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/90—Plate chromatography, e.g. thin layer or paper chromatography
    • G01N30/94—Development
    • G01N2030/945—Application of reagents to undeveloped plate
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2570/00—Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes

Definitions

  • the invention relates to a method for screening the adulteration of fibrates lipid-lowering chemical drugs in tea with high-efficiency thin-layer chromatography combined with bioluminescence, and specifically relates to an organic fusion of HPTLC high-throughput separation and bioluminescence suppression imaging detection, and application
  • a high-throughput screening method for adding adulteration of illegal chemical drugs in different tea products belongs to the technical field of food testing.
  • hyperlipidemia is one of the most common diseases of wealth. Hyperlipidemia not only directly has a serious negative impact on human health, but also easily leads to complications such as coronary heart disease, high blood pressure, cerebral thrombosis and atherosclerosis. It has become a modern society The “invisible killer” of human health.
  • Tea is one of the three traditional beverages in the world. In addition to its widely accepted taste, the health benefits of tea are increasingly being valued by food technology researchers. More and more studies have proved that tea is rich in polysaccharides, flavonoids and polyphenols and other biologically active substances, which have a good effect on regulating blood lipid metabolism and alleviating hyperlipidemia.
  • the principle of its efficacy is to accelerate the decomposition of lipoproteins by enhancing the activity of lipoprotein lipase, and at the same time Reduce the synthesis of lipoproteins in the liver, thereby achieving the effect of reducing blood lipids.
  • Fibrates are effective in lowering blood lipids and have less side effects. They are easily used for adulteration of health tea. These behaviors are not only suspected of commercial fraud, but also because the addition of chemical drugs has the characteristics of large doses and randomness, consumers will often take it for a long time because of temporary and significant effects without knowing it, causing serious poisons and side effects, or even life threatening.
  • there is no special detection project for the adulteration of illegal chemical drugs in tea Therefore, it is of great significance to establish a screening detection method for the adulteration of tea lipid-lowering chemical drugs.
  • Luminescent bacteria refers to a type of microorganisms that can emit visible light under normal physiological conditions.
  • the luminescence principle of this type of bacteria is that under the action of luciferase catalysis and molecular oxygen, long-chain fatty aldehydes and reduced flavin mononucleotides (FMNH 2 ) are oxidized to long-chain fatty acids and oxidized flavin mononucleotides (FMN) , And at the same time emit blue-green light with wavelengths of 450-490nm. Based on this feature, luminescent bacteria are often used as optical sensing elements of biological detectors.
  • the biggest advantage of luminescent bacteria biosensing lies in the non-targeting of detection ability: its sensing principle is not based on the recognition of specific chemical structures, but on the degree of bioluminescence inhibition to characterize the toxicity of the target . More precisely, when there is interference from toxic substances in the external environment, the physiological process or cellular respiration of the luminescent bacteria is affected, resulting in the inhibition of the luminescence reaction, and the content of toxic substances is related to the degree of weakening of the luminous intensity of the bacteria. .
  • the luminescent bacteria method Compared with the toxicity test methods of zebrafish, nematodes and other model organisms with complex preparations and long cycles, the luminescent bacteria method has the advantages of simple operation (use directly after the lyophilized powder of bacteria is recovered) and fast and efficient (within a few minutes at the fastest) Result), so it has a broader application prospect.
  • analysis methods based on luminescent bacteria have played an important role in the EU drinking water safety and early warning system.
  • luminescent bacteria detection technology has played an important role in water quality emergency assurance in the "5.12 Wenchuan earthquake" disaster area and Taihu Lake water quality monitoring.
  • an optical densitometer or a microplate reader with luminescence analysis function is the instrument carrier for the application of the luminescent bacteria method.
  • the analyst only needs to mix the bacterial solution with the sample, and after a period of reaction, quantitatively detect the change in luminescence intensity.
  • this model is simple and efficient, it is not suitable for the detection of illegal addition of Chinese and Western medicines in tea, because it has the following two serious shortcomings: 1The background interference is serious. 2 Lack of selectivity to multiple targets.
  • HPTLC is currently the only chromatography tool that can be used directly with cell-based biosensors. Different from the traditional test tube method, the combination with HPTLC can fundamentally solve the problem of strong background interference and poor selectivity of the luminescent bacteria method.
  • HPTLC separation allows the originally mixed targets to move to different positions on the thin-layer plate according to the difference in molecular structure to form physical isolation; subsequently, the luminescent bacteria coupled with the thin-layer plate can be easily achieved by immersing the multiple targets in the sample Simultaneous detection. Therefore, the analytical method based on the combination of the two has the advantages of high selectivity and good versatility, and has become an emerging hot frontier of analytical chemistry, playing an important role in many fields such as environmental monitoring and natural product analysis.
  • the purpose of the present invention is to overcome the above shortcomings and provide a method for screening the adulteration of fibrates in tea with high-efficiency thin-layer chromatography combined with bioluminescence.
  • the technical scheme of the present invention is a method for screening the adulteration of fibrates lipid-lowering chemical drugs in tea by high-efficiency thin-layer chromatography combined with bioluminescence.
  • the steps are: preparing bezafibrate and ciprofibrate standard solutions, Prepare tea samples; pre-wash the thin-layer plate, and then perform HPTLC spotting; it is directly separated by HPTLC so that the originally mixed target can be moved to different positions on the thin-layer plate according to the difference in molecular structure to form physical isolation; then, The luminescent bacteria coupled with the thin-layer plate can be easily detected simultaneously for multiple targets in the sample by dipping.
  • HPTLC spotting and chromatographic conditions first use a 100 ⁇ L spotting needle to manually draw the sample solution, and use a semi-automatic thin-layer spotting instrument to purge it to a position 10cm away from the bottom of the thin-layer plate with the assistance of a 0.5MPa nitrogen stream. Flow purge speed 100 ⁇ L/s, pre-discharge volume 0.2 ⁇ L, strip width 6mm, distance from both sides at least 15mm; spot each sample prepared by steps (1) and (2), and one sample is finished. After that, manually take out the sample needle, clean it with methanol three times, and perform the next sample spotting; after all samples are spotted, take out the thin-layer plate and heat it with a hair dryer for 1 min. The purpose is to make the residual methanol attached to the spotting position Volatilize
  • the chromatographic separation was carried out in a fully automatic thin-layer developing instrument, the mobile phase ratio was 9/1, the volume ratio of ethyl acetate/methanol was 9/1, and the spreading distance was 60mm; chromatographic separation conditions: the relative humidity in the spreading cylinder was controlled by bubbling saturated magnesium chloride solution for 3 minutes , Adjust the relative humidity to 35%, pre-equilibrate the thin-layer plate for 10 minutes; when the mobile phase front reaches the predetermined height, the system automatically ends, take the thin-layer plate out, put it on the thin-layer heater, and bake for 5 minutes at 80°C to obtain bright, Low-noise bioluminescence imaging background;
  • Bioluminescence imaging detection Use an automatic dipping device to immerse the unfolded and dried thin-layer plate in the working luminescent suspension, the immersion speed is 1mm/s, the residence time is 2s, and then the thin-layer plate immersed in the working luminescent suspension is placed Enter the bioluminescence imager for imaging detection, the imaging exposure time is 40s, the interval is 2min, and 15 pictures are taken continuously;
  • step (2) the luminescent bacterial strains in step (2) are preserved using the agar plate method, which is specifically as follows:
  • a. Preparation of luminescent bacteria culture solution Take 10g agar, 30g NaCl, 5g Na 2 HPO 4 , 5g KH 2 PO 4 , 3mL glycerol, 5g peptone and 5g yeast extract, add 1L ultrapure water and stir to dissolve, then use 1mol /L sodium hydroxide aqueous solution to adjust the pH value of the prepared liquid medium to 7.5 ⁇ 0.2, and then sterilize it with an autoclave at 121°C for 15min; the sterilized medium is cooled to 60°C, while it is hot Invert the plate in a petri dish with a diameter of 10 cm;
  • step (2) the absorbance of the medium used for 600nm incident light is used as an indicator for determining the density of the bacteria; the fresh simulated seawater liquid medium is used as a reference, and the culture is monitored by a spectrophotometer during the culture process The absorbance OD 600 of the liquid to 600 nm incident light, and the culture liquid is selected as the bacterial mother liquid when the OD 600 value reaches 0.7.
  • the thin-layer board in step (3) needs to be pre-washed; the specific steps are as follows: first, pour 10 mL of methanol into the clean unfolding tank, put the blank thin-layer board, and expand it to the top, in order to minimize impurities After being cleaned, the thin-layer board needs to be kept for 5 minutes after unfolding to the top. After taking it out, the thin-layer board is dried on a thin-layer board heater at 100°C for 5 minutes to volatilize the residual organic solvent. The dried thin-layer board uses aluminum foil paper Package spare.
  • the thin layer material of the thin layer plate is ordinary silica gel.
  • step (5) the specific process of step (5) is as follows: the photos taken by the bioluminescence imager are saved in CPF, Black/white linear format, and then opened with Videosscan software, the pixel gray scale in the picture is digitized, and the integration parameters are set And conditions for quantitative analysis.
  • the luminescent strain is specifically a type of microorganism capable of emitting visible light under normal physiological conditions.
  • the luminescence principle of this type of bacteria is that under the action of luciferase catalysis and molecular oxygen, long-chain fatty aldehydes and reduced flavin mononucleotides (FMNH 2 ) are oxidized to long-chain fatty acids and oxidized flavin mononucleotides (FMN) , And at the same time emit blue-green light with wavelengths of 450-490nm. Based on this feature, luminescent bacteria are often used as optical sensing elements of biological detectors.
  • the biggest advantage of luminescent bacteria biosensing lies in the non-targeting of detection ability: its sensing principle is not based on the recognition of specific chemical structures, but on the degree of bioluminescence inhibition to characterize the toxicity of the target .
  • the beneficial effects of the present invention establishes a high-efficiency thin-layer chromatography combined with bioluminescence detection method for rapidly quantifying blood lipid-lowering chemicals in tea, which has the advantages of economy, speed and simplicity.
  • Fig. 1 is an imaging diagram of the separation result under the conditions of Example 1-3 bacterial liquid immersion-bioluminescence inhibition (false color mode).
  • Spotting track 1, BZF, 2, CPF, 3, lotus leaf, 4, lotus leaf + standard product, 5, ginkgo leaf, 6, ginkgo leaf + standard product.
  • Table 1 is the method to evaluate the quantitative detection ability.
  • Table 2 is the method quantitative accuracy assessment.
  • the analytical standards of ciprofibrate ( ⁇ 99%, HPLC) and bezafibrate ( ⁇ 96.0%, HPLC) were purchased from Aladdin (Shanghai, China).
  • Analytical pure NaCl, Na 2 SO 4 , NaNO 3 and other chemical reagents were purchased from Sigma-Aldrich.
  • the high-efficiency thin-layer silica gel plate (analytical type, specification 10 ⁇ 20 cm, batch number 1.05729.0001) of the glass substrate was purchased from Merck (Darmstadt, Germany). The thin-layer plate was washed once with methanol before use. Blank tea samples were purchased from a local supermarket.
  • the luminescent bacteria described in the following examples is Vibrio fischeri (Aliivibrio fischeri, strain collection number DSM 507), and the strain was purchased from the General Microbiology Center of the China Microbial Culture Collection Management Committee, referred to as CGMCC China Common Microbial Culture Center.
  • Preparation of simulated seawater liquid and solid medium Prepare simulated seawater liquid medium according to the following formula: 30g/L NaCl, 5g/L Na 2 HPO 4 , 5g/L KH 2 PO 4 , 3ml/L glycerol, 5g/ L peptone, and 5g/L yeast extract. After adding 1L of ultrapure water and stirring to dissolve, adjust the pH value of the prepared liquid medium to 7.5 ⁇ 0.2 with 1mol/L sodium hydroxide aqueous solution, and then sterilize it with an autoclave at 121°C for 15min. The prepared liquid culture medium is packaged and stored in the refrigerator for later use, and can be stored for 7 days at 4°C when not in use.
  • the luminescent bacterial strains are preserved using the agar plate method, and the nutrient agar plate preparation method: 10g agar, 30g NaCl, 5g Na 2 HPO 4 , 5g KH 2 PO 4 , 3mL glycerol, 5g peptone and 5g yeast extract.
  • 10g agar, 30g NaCl, 5g Na 2 HPO 4 , 5g KH 2 PO 4 , 3mL glycerol, 5g peptone and 5g yeast extract After adding 1L of ultrapure water and stirring to dissolve, adjust the pH value of the prepared liquid medium to 7.5 ⁇ 0.2 with 1mol/L sodium hydroxide aqueous solution, and then sterilize it with an autoclave at 121°C for 15min.
  • the culture medium to be sterilized is cooled to about 60°C and poured into a petri dish with a diameter of 10 cm while it is hot.
  • Thin-layer plate pre-washing The trace organic residues in the thin-layer plate stationary phase will have a significant negative impact on the bioluminescence of the bacteria, so the thin-layer plate needs to be pre-cleaned with methanol before use.
  • the dried thin-layer board is wrapped in aluminum foil for use.
  • HPTLC spotting and chromatographic conditions first use a 100 ⁇ L spotting needle to manually draw an appropriate amount of the sample solution, and use a semi-automatic thin layer spotting instrument with the help of 0.5MPa nitrogen flow to sweep to the distance to the thin layer prepared in step (4)
  • the bottom of the plate is 10cm
  • the flow rate is 100 ⁇ L/s
  • the pre-discharge volume is 0.2 ⁇ L
  • the strip width is 6mm
  • the distance between the two sides is at least 15mm.
  • the strip spacing software automatically calculates.
  • Each sample prepared in step (3) is spotted sequentially. After spotting one sample, manually take out the spotting needle, wash it with methanol three times, and then spot the next sample. After all samples are spotted, take out the thin-layer plate and heat it with a hair dryer for 1 min. The purpose is to volatilize the residual methanol attached to the spotting position.
  • the chromatographic separation is carried out in a fully automatic thin-layer spreader.
  • the mobile phase ratio is ethyl acetate/methanol (9/1, v/v), and the spreading distance is 60mm.
  • Chromatographic separation conditions Control the relative humidity in the unfolding cylinder by bubbling saturated magnesium chloride solution for 3 min (adjusting RH to about 35% by bubbling saturated magnesium chloride solution), pre-equilibration of the thin layer plate for 10 min, and the entire unfolding process takes about 25 min.
  • the mobile phase front reaches the predetermined height, the system ends automatically. Take out the thin-layer plate and bake it on a thin-layer heater at 80°C for 5 minutes to volatilize the residual organic solvent to obtain a bright and low-noise bioluminescence imaging background.
  • Bioluminescence imaging detection Use an automatic dipping device to immerse the unfolded and dried thin-layer plate into the working luminescence suspension at a dipping speed of 1mm/s and a residence time of 2s. Subsequently, the thin-layer plate immersed in the working luminescent bacteria suspension was put into the bioluminescence imager for imaging detection, the imaging exposure time was 40s, the interval was 2min, and 15 pictures were taken continuously.
  • Preparation of simulated seawater liquid and solid medium Prepare simulated seawater liquid medium according to the following formula: 30g/L NaCl, 5g/L Na 2 HPO 4 , 5g/L KH 2 PO 4 , 3ml/L glycerol, 5g/ L peptone, and 5g/L yeast extract. After adding 1L of ultrapure water and stirring to dissolve, adjust the pH value of the prepared liquid medium to 7.5 ⁇ 0.2 with 1mol/L sodium hydroxide aqueous solution, and then sterilize it with an autoclave at 121°C for 15min. The prepared liquid culture medium is packaged and stored in the refrigerator for later use, and can be stored for 7 days at 4°C when not in use.
  • the luminescent bacterial strains are preserved using the agar plate method, and the nutrient agar plate preparation method: 10g agar, 30g NaCl, 5g Na 2 HPO 4 , 5g KH 2 PO 4 , 3mL glycerol, 5g peptone and 5g yeast extract.
  • 10g agar, 30g NaCl, 5g Na 2 HPO 4 , 5g KH 2 PO 4 , 3mL glycerol, 5g peptone and 5g yeast extract After adding 1L of ultrapure water and stirring to dissolve, adjust the pH value of the prepared liquid medium to 7.5 ⁇ 0.2 with 1mol/L sodium hydroxide aqueous solution, and then sterilize it with an autoclave at 121°C for 15min.
  • the culture medium to be sterilized is cooled to about 60°C, and poured into a petri dish with a diameter of 10 cm while it is hot.
  • Pre-washing of the thin-layer plate The small amount of organic residues in the fixed phase of the thin-layer plate will have a significant negative impact on the bioluminescence of the bacteria, so the thin-layer plate needs to be pre-cleaned with methanol before use.
  • the dried thin-layer board is wrapped in aluminum foil for use.
  • HPTLC spotting and chromatographic conditions first use a 100 ⁇ L spotting needle to manually draw an appropriate amount of the sample solution, and use a semi-automatic thin layer spotting instrument with the help of 0.5MPa nitrogen flow to sweep to the distance to the thin layer prepared in step (5)
  • the bottom of the plate is 10cm
  • the flow rate is 100 ⁇ L/s
  • the pre-discharge volume is 0.2 ⁇ L
  • the strip width is 6mm
  • the distance between the two sides is at least 15mm.
  • the strip spacing software automatically calculates.
  • Each sample prepared by steps (3) and (4) is spotted in sequence. After spotting one sample, manually take out the spotting needle, wash it with methanol three times, and spot the next sample. After all samples are spotted, take out the thin-layer plate and heat it with a hair dryer for 1 min. The purpose is to volatilize the residual methanol attached to the spotting position.
  • the chromatographic separation is carried out in a fully automatic thin-layer spreader.
  • the mobile phase ratio is ethyl acetate/methanol (9/1, v/v), and the spreading distance is 60mm.
  • Chromatographic separation conditions control the relative humidity in the unfolding cylinder by bubbling saturated magnesium chloride solution for 3 minutes, adjust the relative humidity to 35%, and pre-equilibrate the TLC for 10 minutes. The entire unfolding process takes about 25 minutes.
  • the mobile phase front reaches the predetermined height, the system ends automatically. Take out the thin-layer plate and bake it on a thin-layer heater at 80°C for 5 minutes to volatilize the residual organic solvent to obtain a bright and low-noise bioluminescence imaging background.
  • Bioluminescence imaging detection Use an automatic dipping device to immerse the unfolded and dried thin-layer plate into the working luminescence suspension at a dipping speed of 1mm/s and a residence time of 2s. Subsequently, the thin-layer plate immersed in the suspension of luminescent bacteria was put into the bioluminescence imager for imaging detection, the imaging exposure time was 40s, the interval was 2min, and 15 pictures were taken continuously.
  • simulated seawater liquid and solid medium Configure simulated seawater liquid medium according to the following formula: 30g/L NaCl, 5g/L Na 2 HPO 4 , 5g/L KH 2 PO 4 , 3ml/L glycerol, 5g/ L peptone, and 5g/L yeast extract. After adding 1L of ultrapure water and stirring to dissolve, adjust the pH value of the prepared liquid medium to 7.5 ⁇ 0.2 with 1mol/L sodium hydroxide aqueous solution, and then sterilize it with an autoclave at 121°C for 15min. The prepared liquid culture medium is packaged and stored in the refrigerator for later use, and can be stored for 7 days at 4°C when not in use.
  • the luminescent bacterial strains are preserved by the agar plate method.
  • the nutrient agar plate preparation method 10g agar, 30g NaCl, 5g Na 2 HPO 4 , 5g KH 2 PO 4 , 3ml glycerol, 5g peptone and 5g yeast extract.
  • After adding 1L of ultrapure water and stirring to dissolve adjust the pH value of the prepared liquid medium to 7.5 ⁇ 0.2 with 1mol/L sodium hydroxide aqueous solution, and then sterilize it with an autoclave at 121°C for 15min.
  • the culture medium to be sterilized is cooled to about 60°C, and poured into a petri dish with a diameter of 10 cm while it is hot.
  • Pre-washing of the thin-layer plate The small amount of organic residues in the fixed phase of the thin-layer plate will have a significant negative impact on the bioluminescence of the bacteria, so the thin-layer plate needs to be pre-cleaned with methanol before use.
  • the dried thin-layer board is wrapped in aluminum foil for use.
  • HPTLC spotting and chromatographic conditions first use a 100 ⁇ L spotting needle to manually draw an appropriate amount of the sample solution, and use a semi-automatic thin layer spotting instrument with the help of 0.5MPa nitrogen flow to sweep to the distance to the thin layer prepared in step (5)
  • the bottom of the plate is 10cm
  • the flow rate is 100 ⁇ L/s
  • the pre-discharge volume is 0.2 ⁇ L
  • the strip width is 6mm
  • the distance between the two sides is at least 15mm.
  • the strip spacing software automatically calculates.
  • Each sample prepared in steps (3) and (4) is spotted in sequence. After spotting one sample, manually take out the spotting needle, wash it with methanol three times, and spot the next sample. After all samples are spotted, take out the thin-layer plate and heat it with a hair dryer for 1 min. The purpose is to volatilize the residual methanol attached to the spotting position.
  • the chromatographic separation is carried out in a fully automatic thin-layer spreader.
  • the mobile phase ratio is ethyl acetate/methanol (9/1, v/v), and the spreading distance is 60mm.
  • Chromatographic separation conditions control the relative humidity in the unfolding cylinder by bubbling saturated magnesium chloride solution for 3 minutes and adjust the relative humidity to 35% (adjusted by bubbling saturated magnesium chloride solution, pre-equilibration of the thin-layer plate for 10 min, and the entire unfolding process takes about 25 min.
  • the mobile phase front reaches the predetermined height, the system automatically ends.
  • the thin-layer plate is taken out and baked on a thin-layer heater at 80°C for 5 minutes to volatilize the residual organic solvent to obtain a bright, low-noise bioluminescence imaging background.
  • Bioluminescence imaging detection Use an automatic dipping device to immerse the unfolded and dried thin-layer plate into the working luminescence suspension at a dipping speed of 1mm/s and a residence time of 2s. Subsequently, the thin-layer plate immersed in the suspension of luminescent bacteria was put into the bioluminescence imager for imaging detection, the imaging exposure time was 40s, the interval was 2min, and 15 pictures were taken continuously.
  • Apocynum venetum was used as the sample in step (4), and the other steps were the same as in Example 3.

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Abstract

一种高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，属于食品检测技术领域。方法步骤为：配置苯扎贝特和环丙贝特标准品溶液和制备茶叶样品；对薄层板进行预洗，随后进行HPTLC点样；通过HPTLC分离使原本混在一起的目标物按分子结构的差异移动到薄层板上不同的位置形成物理隔离；随后，通过浸渍方式与薄层板耦合的发光细菌可以方便地实现对样品中多元目标的同时检测。该检测方法具有经济、快速、简便的优点。

Description

一种高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法 技术领域
本发明涉及一种高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，具体涉及一种将HPTLC高通量分离与生物发光抑制成像检测有机融合，应用于不同茶叶产品中非法化学药添加掺伪的高通量筛检的方法，属于食品检测技术领域。
背景技术
日常膳食不科学引发的“富贵病”已经取代致病微生物感染成为人类健康最重要的威胁。其中高血脂是最常见的“富贵病”之一，高血脂不仅直接对人体健康产生严重负面影响，还容易导致例如冠心病、高血压、脑血栓和动脉粥样硬化等并发症，已成为现代社会人类健康的“隐形杀手”。
茶是世界三大传统饮料之一。除了其被广泛接受的口感，茶的保健功效日益受到食品科技研究者的重视。越来越多的研究证明，茶中含有丰富的多糖、黄酮和多酚等生物活性物质，对调控血脂代谢，缓解高血脂症有良好的效果。
在此背景下，具有良好降血脂功效的保健茶产品的市场空间快速扩张。特别是一些源于药食两用原料的保健茶甚至成为“网红产品”，但是随之而来的食品药品安全问题也井喷式发生。根据媒体和食药监部门披露的案例，一些不法分子为实现产品广告宣传中所声称的降血脂效果，在茶叶中非法添加类似功效且价格低廉的化学药物。苯扎贝特(Benzafibrate,BZF)和环丙贝特(Ciprofibrate,CPF)属于苯氧芳酸类降血脂化学药，其药效原理是通过增强脂蛋白脂酶的活性加速脂蛋白的分解，同时减少肝脏中脂蛋白的合成，从而达到降低血脂的效果。贝特类降血脂化学药功效良好且副作用较小，极易被用于保健茶的掺伪。这些行为不仅涉嫌商业欺诈，而且由于化学药物的添加具有大剂量和随意性的特点，消费者在不知情的前提下，往往会因为暂时的显著效果而长期服用，引起严重的毒害和副作用，甚至危及生命。目前国内还没有针对茶叶中非法化学药掺伪的专门检测项目，因此建立针对茶叶降脂化学药物掺伪的筛查检测方法具有重要的意义。
发光细菌是指在正常生理条件下能够发射可见光的一类微生物。该类细菌的发光原理是在荧光素酶催化和分子氧的作用下，长链脂肪醛和还原型黄素单核苷酸(FMNH 2)被氧化为 长链脂肪酸和氧化型黄素单核苷酸(FMN)，同时释放出450-490nm波长的蓝绿光。基于这一特性，发光细菌常被用来作为生物检测器的光学感应元件。较之常见的理化检测手段，发光细菌生物传感最大的优势在于检测能力的非靶向性：其传感原理并非基于对特定化学结构的识别，而是通过生物发光抑制程度表征目标物毒性高低。更确切地说，当外界环境中存在毒性物质的干扰时，发光细菌的生理过程或细胞呼吸受到影响，导致发光反应受到抑制，而且有毒物质的含量与菌体发光强度变弱的程度呈相关性。
相对于准备工作复杂、周期冗长的斑马鱼、线虫等模式生物的毒性测试方法，发光细菌法的优点在于操作简单(菌体冻干粉复苏后直接使用)和快速高效(最快数分钟之内出结果)，因此具有更广阔的应用前景。目前，基于发光细菌的分析方法已经在欧盟饮用水安全和预警体系中扮演重要的角色。在我国，发光细菌检测技术在“5.12汶川地震”灾区水质应急保障和太湖水质监控等工作中起到了重要作用。
通常，光密度计或者具有发光分析功能的酶标仪是发光细菌法应用的仪器载体。分析人员只需要将菌液与样品混合，经过一段时间的反应后定量检测发光强度变化。这一模式虽然简单高效，但不适合茶叶中西药非法添加检测，因为其存在以下两个严重的缺陷：①背景干扰严重。②缺乏对多元目标物的选择性。
HPTLC是目前唯一能与细胞基生物传感器直接联用的色谱工具。和传统的试管法测试不同，与HPTLC的联用可以从根本上解决发光细菌法样品背景干扰强和选择性差的问题。HPTLC分离使原本混在一起的目标物按分子结构的差异移动到薄层板上不同的位置形成物理隔离；随后，通过浸渍方式使与薄层板耦合的发光细菌可以方便地实现对样品中多元目标的同时检测。因此，建立在二者联用基础上的分析方法兼备选择性高和通用性好的优点，已经成为分析化学的一个新兴热点前沿，在环境监测和天然产物分析等多个领域扮演重要的角色。
发明内容
本发明的目的在于克服上述不足之处，提供一种高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法。
本发明的技术方案，一种高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，步骤为：配置苯扎贝特和环丙贝特标准品溶液，制备茶叶样品；对薄层板进行预洗，随后进行HPTLC点样；其直接通过HPTLC分离使原本混在一起的目标物按分子结构的差异移动到薄层板上不同的位置形成物理隔离；随后，通过浸渍方式使与薄层板耦合的发光细菌可以方便地实现对样品中多元目标的同时检测。
具体步骤如下：
(1)标准溶液配制：用电子天平分别精确称量10mg的苯扎贝特和10mg的环丙贝特标准品，分别置于10mL容量瓶，甲醇定容，得到浓度为1mg/mL的标准储备液；将标准储备液平时避光静置在4℃环境，待临近分析工作开始，取出0.2mL置于10mL容量瓶，甲醇定容稀释，得到浓度为0.02mg/mL的标准工作溶液；标准工作溶液随用随配；
(2)分别制备茶叶样品溶液：取荷叶、罗布麻、银杏叶样品先分别用电动粉碎机均匀粉碎，再称取1.0g粉碎样品加入10mL甲醇，25℃超声水浴萃取30min，取出后5000r/min离心5min；离心后取上层5mL清液装入注射器，压缩压杆使之通过0.45μm尼龙滤膜，由此制得的样品清液可以直接用于HPTLC点样；
(3)HPTLC点样和色谱条件：首先用100μL点样针手动吸取样品溶液，通过半自动薄层点样仪在0.5MPa氮气流的协助下吹扫到距离薄层板底端10cm的位置，液流吹扫速度100μL/s，预排体积0.2μL，条带宽度6mm，距离两侧边缘至少15mm；对由步骤(1)、(2)制备的每个样品进行点样，一个样品点样结束后，手动取出点样针，用甲醇清洗三次后，进行下一个样品点样；全部样品点样结束后，取出薄层板，用电吹风加热1min，目的是使附着在点样位置的残留甲醇挥发；
色谱分离在全自动薄层展开仪中进行，流动相配比乙酸乙酯/甲醇体积比为9/1，展开距离60mm；色谱分离条件：通过饱和氯化镁溶液鼓泡控制展开缸内相对湿度，持续3min，调节相对湿度至35％，薄层板预平衡10min；待流动相前沿到达预定高度，系统自动结束，将薄层板取出，放在薄层加热器上80℃烘烤5min，以便获得明亮、低噪音的生物发光成像背景；
(4)生物发光成像检测：使用自动浸渍装置将展开、干燥后的薄层板浸入工作发光悬浮液，浸渍速度1mm/s，停留时间2s，随后将浸有工作发光悬浮液的薄层板放入生物发光成像仪中成像检测，成像曝光时间40s，间距2min，连续拍摄15张；
(5)分析：将通过生物发光成像仪拍摄的照片保存，然后用Videoscan软件打开，对图片中的像素灰度进行数字化，然后设定积分参数和条件进行定量分析。
进一步的，工作发光悬浮液的制备过程如下：
(1)模拟海水液体配制：
按照以下配方配制模拟海水液体培养基：30g/L NaCl，5g/L Na 2HPO 4，5g/L KH 2PO 4，3mL/L甘油，5g/L蛋白胨和5g/L酵母提取物，加入1L超纯水搅拌溶解；用1mol/L的氢氧化钠溶液将配好的液体培养基pH值调节至7.3-7.7，再使用高压蒸汽灭菌锅121℃灭菌处理15min，配制好的液体培养基封装置于冰箱中冷藏备用，不用时在4℃环境下可保存7天；
(2)发光菌株的培养和保藏：将用甘油冷冻保藏的发光细菌接种到盛有步骤(1)制备的100mL 液体培养基的三角瓶中；瓶口用灭菌的四层折叠锡箔纸包裹瓶口，确保培养过程中外界氧气能够进入瓶中，在25℃环境中100r/min摇瓶培养，得到细菌母液；然后在成熟的细菌母液中加入等体积的新鲜液体培养基，制得工作发光悬浮液；工作发光悬浮液不用时，可在4℃环境中保存3天。
进一步的，步骤(2)所述发光细菌菌种使用琼脂平板法进行保藏，具体如下：
a、发光细菌培养液的制备：取10g琼脂，30g NaCl，5g Na 2HPO 4，5g KH 2PO 4,3mL甘油,5g蛋白胨和5g酵母提取物，加入1L超纯水搅拌溶解后，用1mol/L氢氧化钠水溶液将配好的液体培养基pH值调节至7.5±0.2，再使用高压蒸汽灭菌锅121℃灭菌处理15min；待灭菌后的培养基冷却到60℃，趁热在直径10cm的培养皿中倒平板；
b、接种：首先将灭菌后的接种环浸入成熟的发光细菌培养液中，然后在营养琼脂表面划斜线，反复多次；接种后的营养琼脂置于25℃环境避光培养48h，待出观察到明显菌落后，得到细菌母液，转移到-4℃环境避光保藏。
进一步的，步骤(2)中，将所用培养基对600nm入射光的吸收度作为测定菌体密度的指标；以新鲜模拟海水液体培养基清液为参比，培养过程中用分光光度计监测培养液对600nm入射光的吸光度OD 600，选取OD 600数值达到0.7时培养液作为细菌母液。
进一步的，步骤(3)所述薄层板需要进行预洗；具体步骤如下：首先将10mL甲醇倒入洁净展开缸中，放入空白薄层板，使其展开到顶端，为使杂质尽可能被清洗干净，薄层板展开至顶端后需保持5min，取出后将薄层板放在薄层板加热器上100℃烘干5min，挥发残留有机溶剂，烘干后的薄层板用铝箔纸包裹备用。
进一步的，所述薄层板的薄层材料是普通硅胶。
进一步的，步骤(5)具体过程如下：通过生物发光成像仪拍摄的照片保存为CPF，Black/white linear格式，然后用Videoscan软件打开，对图片中的像素灰度进行数字化，然后设定积分参数和条件进行定量分析。
进一步的，所述发光菌株具体为在正常生理条件下能够发射可见光的一类微生物。该类细菌的发光原理是在荧光素酶催化和分子氧的作用下，长链脂肪醛和还原型黄素单核苷酸(FMNH 2)被氧化为长链脂肪酸和氧化型黄素单核苷酸(FMN)，同时释放出450-490nm波长的蓝绿光。基于这一特性，发光细菌常被用来作为生物检测器的光学感应元件。较之常见的理化检测手段，发光细菌生物传感最大的优势在于检测能力的非靶向性：其传感原理并非基于对特定化学结构的识别，而是通过生物发光抑制程度表征目标物毒性高低。
本发明的有益效果：本发明建立了一种快速定量茶叶中降血脂化学药的高效薄层色谱联用生物发光法的检测方法，具有经济、快速、简便的优点。
附图说明
图1是实施例1-3菌液浸渍-生物发光抑制(伪色彩模式)条件下的分离结果成像图。
点样轨道：1、BZF，2、CPF，3、荷叶，4、荷叶+标准品，5、银杏叶，6、银杏叶+标准品。
图2BZF的标准曲线。
图3CPF的标准曲线。
表1是方法定量检测能力评估。
表2是方法定量准确性评估。
具体实施方式
以下实例中，环丙贝特(≥99％,HPLC)和苯扎贝特(≥96.0％,HPLC)的分析标准品购自Aladdin(上海，中国)。分析纯NaCl,Na 2SO 4,NaNO 3和其他化学试剂购自Sigma-Aldrich。玻璃基板的高效薄层硅胶板(分析型，规格10×20cm，批次号1.05729.0001)购自Merck(Darmstadt,Germany)。薄层板在使用之前用甲醇清洗一次。空白茶叶样品购自当地超市。
以下实施例所述发光细菌为费氏弧菌(Aliivibrio fischeri，菌种保藏编号DSM 507)，菌株购自中国微生物菌种保藏管理委员会普通微生物中心，简称CGMCC中国普通微生物菌种中心。
实施例1
(1)模拟海水液体和固体培养基配制：按照以下配方配制模拟海水液体培养基：30g/L NaCl,5g/L Na 2HPO 4，5g/L KH 2PO 4,3ml/L甘油,5g/L蛋白胨,和5g/L酵母提取物。加入1L超纯水搅拌溶解后，用1mol/L氢氧化钠水溶液将配好的液体培养基pH值调节至7.5±0.2，再使用高压蒸汽灭菌锅121℃灭菌处理15min。配制好的液体培养基封装置于冰箱中冷藏备用，不用时在4℃环境下可保存7天。
(2)发光菌株的培养和保藏：将用甘油冷冻保藏的发光细菌接种到盛有步骤(1)制备的100mL液体培养基的三角瓶中。瓶口用灭菌的四层折叠锡箔纸包裹瓶口，确保培养过程中外界氧气能够进入瓶中。按照Chen等描述的方法在25℃环境中100r/min摇瓶培养。然后在成熟的细菌母液中加入等体积的新鲜液体培养基，制得工作发光悬浮液。工作发光悬浮液不用时，可在4℃环境中保存3天。
发光细菌菌种使用琼脂平板法进行保藏，其营养琼脂平板制备方法：10g琼脂，30g NaCl，5g Na 2HPO 4，5g KH 2PO 4,3mL甘油,5g蛋白胨和5g酵母提取物。加入1L超纯水搅拌溶解后，用1mol/L氢氧化钠水溶液将配好的液体培养基pH值调节至7.5±0.2，再使用高压蒸汽灭菌锅121℃灭菌处理15min。待灭菌后的培养基冷却到60℃左右，趁热在直径10cm 的培养皿中倒平板。接种时，首先将灭菌后的接种环浸入成熟的发光细菌培养液中，然后在营养琼脂表面划斜线，反复多次。接种后的营养琼脂置于25℃环境避光培养48h，待观察到明显菌落后，转移到-4℃环境避光保藏。
(3)标准溶液配制：用电子天平分别精确称量10±0.1mg的苯扎贝特(Benzafibrate,BZF)和环丙贝特(Ciprofibrate,CPF)标准品，置于10mL容量瓶，甲醇定容，得到浓度为1mg/mL的标准储备液。标准储备液平时避光静置在4℃环境，待临近分析工作开始，取出0.2mL置于10mL容量瓶，甲醇定容稀释，得到浓度为0.02mg/mL的标准工作溶液。标准工作溶液随用随配。
BZF的标准曲线如图2所示，CPF的标准曲线如图3所示。
(4)薄层板预洗：薄层板固定相中微量的有机物残留会对细菌的生物发光产生显著的负面影响，因此在薄层板使用前需要用甲醇对其进行预清洗。首先将10mL甲醇倒入洁净展开缸中，放入空白薄层板，使其展开到顶端，为使杂质尽可能被清洗干净，薄层板展开至顶端后需保持5min。取出后将薄层板放在薄层板加热器上100℃烘干5min，挥发残留有机溶剂。烘干后的薄层板用铝箔纸包裹备用。
(5)HPTLC点样和色谱条件：首先用100μL点样针手动吸取样品适量溶液，通过半自动薄层点样仪在0.5MPa氮气流的协助下吹扫到距离由步骤(4)制备的薄层板底端10cm的位置，液流吹扫速度100μL/s，预排体积0.2μL，条带宽度6mm，距离两侧边缘至少15mm，条带间距软件自动计算。由步骤(3)制备的每个样品依次进行点样，一个样品点样结束后，手动取出点样针，用甲醇清洗三次后，再进行下一个样品点样。全部样品点样结束后，取出薄层板，用电吹风加热1min，目的是使附着在点样位置的残留甲醇挥发。
色谱分离在全自动薄层展开仪中进行。流动相配比乙酸乙酯/甲醇(9/1,v/v)，展开距离60mm。色谱分离条件：通过饱和氯化镁溶液鼓泡控制展开缸内相对湿度，持续3min(饱和氯化镁溶液鼓泡调节RH至35％左右)，薄层板预平衡10min，整个展开过程耗时约25min。待流动相前沿到达预定高度，系统自动结束，将薄层板取出，放在薄层加热器上80℃烘烤5min，以挥发残留的有机溶剂，以便获得明亮、低噪音的生物发光成像背景。
(6)生物发光成像检测：使用自动浸渍装置将展开、干燥后的薄层板浸入工作发光悬浮液，浸渍速度1mm/s，停留时间2s。随后将浸有工作发光细菌悬浮液的薄层板放入生物发光成像仪中成像检测，成像曝光时间40s，间距2min，连续拍摄15张。
(7)将通过生物发光成像仪拍摄的照片保存为CPF(Black/white linear)格式，然后用Videoscan软件打开，对图片中的像素灰度进行数字化，然后设定积分参数和条件进行定量分析。
菌液浸渍-生物发光抑制(伪色彩模式)条件下的分离结果成像图如图1所示。
实施例2
(1)模拟海水液体和固体培养基配制：按照以下配方配制模拟海水液体培养基：30g/L NaCl,5g/L Na 2HPO 4，5g/L KH 2PO 4,3ml/L甘油,5g/L蛋白胨,和5g/L酵母提取物。加入1L超纯水搅拌溶解后，用1mol/L氢氧化钠水溶液将配好的液体培养基pH值调节至7.5±0.2，再使用高压蒸汽灭菌锅121℃灭菌处理15min。配制好的液体培养基封装置于冰箱中冷藏备用，不用时在4℃环境下可保存7天。
(2)发光菌株的培养和保藏：将用甘油冷冻保藏的发光细菌接种到盛有步骤(1)制备的100mL液体培养基的三角瓶中。瓶口用灭菌的四层折叠锡箔纸包裹瓶口，确保培养过程中外界氧气能够进入瓶中。按照Chen等描述的方法在25℃环境中100r/min摇瓶培养。然后在成熟的细菌母液中加入等体积的新鲜液体培养基，制得工作发光悬浮液。工作发光悬浮液不用时，可在4℃环境中保存3天。
发光细菌菌种使用琼脂平板法进行保藏，其营养琼脂平板制备方法：10g琼脂，30g NaCl，5g Na 2HPO 4，5g KH 2PO 4,3mL甘油,5g蛋白胨和5g酵母提取物。加入1L超纯水搅拌溶解后，用1mol/L氢氧化钠水溶液将配好的液体培养基pH值调节至7.5±0.2，再使用高压蒸汽灭菌锅121℃灭菌处理15min。待灭菌后的培养基冷却到60℃左右，趁热在直径10cm的培养皿中倒平板。接种时，首先将灭菌后的接种环浸入成熟的发光细菌培养液中，然后在营养琼脂表面划斜线，反复多次。接种后的营养琼脂置于25℃环境避光培养48h，待出观察到明显菌落后，转移到-4℃环境避光保藏。
(3)标准溶液配制：用电子天平分别精确称量10±0.1mg的苯扎贝特(Benzafibrate,BZF)和环丙贝特(Ciprofibrate,CPF)标准品，置于10mL容量瓶，甲醇定容，得到浓度为1mg/mL的标准储备液。标准储备液平时避光静置在4℃环境，待临近分析工作开始，取出0.2mL置于10mL容量瓶，甲醇定容稀释，得到浓度为0.02mg/mL的标准工作溶液。标准工作溶液随用随配。
BZF的标准曲线如图2所示，CPF的标准曲线如图3所示。
(4)样品制备：荷叶样品先用电动粉碎机均匀粉碎，再称取1.0g粉碎样品加入10mL甲醇，25℃超声水浴萃取30min，取出后5000r/min离心5min。离心后取上层约5mL清液装入注射器，压缩压杆使之通过0.45μm尼龙滤膜，由此制得的样品清液可以直接用于HPTLC点样。
(5)薄层板预洗：薄层板固定相中微量的有机物残留会对细菌的生物发光产生显著的负面影响，因此在薄层板使用前需要用甲醇对其进行预清洗。首先将10mL甲醇倒入洁净 展开缸中，放入空白薄层板，使其展开到顶端，为使杂质尽可能被清洗干净，薄层板展开至顶端后需保持5min。取出后将薄层板放在薄层板加热器上100℃烘干5min，挥发残留有机溶剂。烘干后的薄层板用铝箔纸包裹备用。
(6)HPTLC点样和色谱条件：首先用100μL点样针手动吸取样品适量溶液，通过半自动薄层点样仪在0.5MPa氮气流的协助下吹扫到距离由步骤(5)制备的薄层板底端10cm的位置，液流吹扫速度100μL/s，预排体积0.2μL，条带宽度6mm，距离两侧边缘至少15mm，条带间距软件自动计算。由步骤(3)、(4)制备的每个样品依次进行点样，一个样品点样结束后，手动取出点样针，用甲醇清洗三次后，进行下一个样品点样。全部样品点样结束后，取出薄层板，用电吹风加热1min，目的是使附着在点样位置的残留甲醇挥发。
色谱分离在全自动薄层展开仪中进行。流动相配比乙酸乙酯/甲醇(9/1,v/v)，展开距离60mm。色谱分离条件：通过饱和氯化镁溶液鼓泡控制展开缸内相对湿度，持续3min，调节相对湿度至35％，薄层板预平衡10min，整个展开过程耗时约25min。待流动相前沿到达预定高度，系统自动结束，将薄层板取出，放在薄层加热器上80℃烘烤5min，以挥发残留的有机溶剂，以便获得明亮、低噪音的生物发光成像背景。
(7)生物发光成像检测：使用自动浸渍装置将展开、干燥后的薄层板浸入工作发光悬浮液，浸渍速度1mm/s，停留时间2s。随后将浸有发光细菌悬浮液的薄层板放入生物发光成像仪中成像检测，成像曝光时间40s，间距2min，连续拍摄15张。
(8)将通过生物发光成像仪拍摄的照片保存为CPF(Black/white linear)格式，然后用Videoscan软件打开，对图片中的像素灰度进行数字化，然后设定积分参数和条件进行定量分析。
菌液浸渍-生物发光抑制(伪色彩模式)条件下的分离结果成像图如图1所示。
实施例3
(1)模拟海水液体和固体培养基配制：按照以下配方配置模拟海水液体培养基：30g/L NaCl,5g/L Na 2HPO 4，5g/L KH 2PO 4,3ml/L甘油,5g/L蛋白胨,和5g/L酵母提取物。加入1L超纯水搅拌溶解后，用1mol/L氢氧化钠水溶液将配好的液体培养基pH值调节至7.5±0.2，再使用高压蒸汽灭菌锅121℃灭菌处理15min。配制好的液体培养基封装置于冰箱中冷藏备用，不用时在4℃环境下可保存7天。
(2)发光菌株的培养和保藏：将用甘油冷冻保藏的发光细菌接种到盛有步骤(1)制备的100mL液体培养基的三角瓶中。瓶口用灭菌的四层折叠锡箔纸包裹瓶口，确保培养过程中外界氧气能够进入瓶中。按照Chen等描述的方法在25℃环境中100r/min摇瓶培养。然后在成熟的细菌母液中加入等体积的新鲜液体培养基，制得工作发光悬浮液。工作发光悬浮 液不用时，可在4℃环境中保存3天。
发光细菌菌种使用琼脂平板法进行保藏，其营养琼脂平板制备方法：10g琼脂，30g NaCl，5g Na 2HPO 4，5g KH 2PO 4,3ml甘油,5g蛋白胨和5g酵母提取物。加入1L超纯水搅拌溶解后，用1mol/L氢氧化钠水溶液将配好的液体培养基pH值调节至7.5±0.2，再使用高压蒸汽灭菌锅121℃灭菌处理15min。待灭菌后的培养基冷却到60℃左右，趁热在直径10cm的培养皿中倒平板。接种时，首先将灭菌后的接种环浸入成熟的发光细菌培养液中，然后在营养琼脂表面划斜线，反复多次。接种后的营养琼脂置于25℃环境避光培养48h，待出观察到明显菌落后，转移到-4℃环境避光保藏。
(3)标准溶液配制：用电子天平分别精确称量10±0.1mg的苯扎贝特(Benzafibrate,BZF)和环丙贝特(Ciprofibrate,CPF)标准品，置于10mL容量瓶，甲醇定容，得到浓度为1mg/mL的标准储备液。标准储备液平时避光静置在4℃环境，待临近分析工作开始，取出0.2mL置于10mL容量瓶，甲醇定容稀释，得到浓度为0.02mg/mL的标准工作溶液。标准工作溶液随用随配。
BZF的标准曲线如图2所示，CPF的标准曲线如图3所示。
(4)样品制备：银杏叶样品先用电动粉碎机均匀粉碎，再称取1.0g粉碎样品加入10mL甲醇，25℃超声水浴萃取30min，取出后5000r/min离心5min。离心后取上层约5mL清液装入注射器，压缩压杆使之通过0.45μm尼龙滤膜，由此制得的样品清液可以直接用于HPTLC点样。
(5)薄层板预洗：薄层板固定相中微量的有机物残留会对细菌的生物发光产生显著的负面影响，因此在薄层板使用前需要用甲醇对其进行预清洗。首先将10mL甲醇倒入洁净展开缸中，放入空白薄层板，使其展开到顶端，为使杂质尽可能被清洗干净，薄层板展开至顶端后需保持5min。取出后将薄层板放在薄层板加热器上100℃烘干5min，挥发残留有机溶剂。烘干后的薄层板用铝箔纸包裹备用。
(6)HPTLC点样和色谱条件：首先用100μL点样针手动吸取样品适量溶液，通过半自动薄层点样仪在0.5MPa氮气流的协助下吹扫到距离由步骤(5)制备的薄层板底端10cm的位置，液流吹扫速度100μL/s，预排体积0.2μL，条带宽度6mm，距离两侧边缘至少15mm，条带间距软件自动计算。由步骤(3)、(4)制备的每个样品依次点样，一个样品点样结束后，手动取出点样针，用甲醇清洗三次后，进行下一个样品点样。全部样品点样结束后，取出薄层板，用电吹风加热1min，目的是使附着在点样位置的残留甲醇挥发。
色谱分离在全自动薄层展开仪中进行。流动相配比乙酸乙酯/甲醇(9/1,v/v)，展开距离60mm。色谱分离条件：通过饱和氯化镁溶液鼓泡控制展开缸内相对湿度，持续3min，调 节相对湿度至35％(饱和氯化镁溶液鼓泡调节，薄层板预平衡10min，整个展开过程耗时约25min。待流动相前沿到达预定高度，系统自动结束，将薄层板取出，放在薄层加热器上80℃烘烤5min，以挥发残留的有机溶剂，以便获得明亮、低噪音的生物发光成像背景。
(7)生物发光成像检测：使用自动浸渍装置将展开、干燥后的薄层板浸入工作发光悬浮液，浸渍速度1mm/s，停留时间2s。随后将浸有发光细菌悬浮液的薄层板放入生物发光成像仪中成像检测，成像曝光时间40s，间距2min，连续拍摄15张。
(8)将通过生物发光成像仪拍摄的照片保存为CPF(Black/white linear)格式，然后用Videoscan软件打开，对图片中的像素灰度进行数字化，然后设定积分参数和条件进行定量分析。
菌液浸渍-生物发光抑制(伪色彩模式)条件下的分离结果成像图如图1所示。
实施例4
步骤(4)中所述样品选用罗布麻，其余步骤同实施例3。
实施例5
对实施例1-4所述方法检测能力进行评估，定量检测能力评估具体结果如表1所示，定量准确性评估如表2所示。
表1方法定量检测能力评估
Figure PCTCN2019088357-appb-000001
表2方法定量准确性评估
Figure PCTCN2019088357-appb-000002
Figure PCTCN2019088357-appb-000003

Claims (9)

  1. 一种高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，其特征在于步骤为：配置苯扎贝特和环丙贝特标准品溶液，制备茶叶样品；对薄层板进行预洗，随后进行HPTLC点样；通过HPTLC分离使原本混在一起的目标物按分子结构的差异移动到薄层板上不同的位置形成物理隔离；随后，通过浸渍方式使与薄层板耦合的发光菌株方便地实现对样品中多元目标的同时检测。
  2. 根据权利要求1所述高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，其特征在于具体步骤如下：
    (1)标准溶液配制：用电子天平分别精确称量10mg的苯扎贝特和10mg的环丙贝特标准品，分别置于10mL容量瓶，甲醇定容，得到浓度为1mg/mL的标准储备液；将标准储备液平时避光静置在4℃环境，待临近分析工作开始，取出0.2mL置于10mL容量瓶，甲醇定容稀释，得到浓度为0.02mg/mL的标准工作溶液；标准工作溶液随用随配；
    (2)分别制备茶叶样品溶液：取荷叶、罗布麻、银杏叶样品先分别用电动粉碎机均匀粉碎，再称取1.0g粉碎样品加入10mL甲醇，25℃超声水浴萃取30min，取出后5000r/min离心5min；离心后取上层5mL清液装入注射器，压缩压杆使之通过0.45μm尼龙滤膜，由此制得的样品清液直接用于HPTLC点样；
    (3)HPTLC点样和色谱条件：首先用100μL点样针手动吸取样品溶液，通过半自动薄层点样仪在0.5MPa氮气流的协助下吹扫到距离薄层板底端10cm的位置，液流吹扫速度100μL/s，预排体积0.2μL，条带宽度6mm，距离两侧边缘至少15mm；对由步骤(1)、(2)制备的每个样品进行点样，一个样品点样结束后手动取出点样针，用甲醇清洗三次后，进行下一个样品点样；全部样品点样结束后，取出薄层板，用电吹风加热1min，目的是使附着在点样位置的残留甲醇挥发；
    色谱分离在全自动薄层展开仪中进行，流动相配比乙酸乙酯/甲醇体积比为9/1，展开距离60mm；色谱分离条件：通过饱和氯化镁溶液鼓泡控制展开缸内相对湿度，持续3min，调节相对湿度至35％，薄层板预平衡10min；待流动相前沿到达预定高度，系统自动结束，将薄层板取出，放在薄层加热器上80℃烘烤5min，以便获得明亮、低噪音的生物发光成像背景；
    (4)生物发光成像检测：使用自动浸渍装置将展开、干燥后的薄层板浸入工作发光悬浮液，浸渍速度1mm/s，停留时间2s，随后将浸有工作发光悬浮液的薄层板放入生物发光成像仪中成像检测，成像曝光时间40s，间距2min，连续拍摄15张；
    (5)分析：将通过生物发光成像仪拍摄的照片保存，然后用Videoscan软件打开，对图片中 的像素灰度进行数字化，然后设定积分参数和条件进行定量分析。
  3. 根据权利要求2所述高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，其特征在于工作发光悬浮液的制备过程如下：
    (1)模拟海水液体配制：
    按照以下配方配制模拟海水液体培养基：30g/L NaCl，5g/L Na 2HPO 4，5g/L KH 2PO 4，3mL/L甘油，5g/L蛋白胨和5g/L酵母提取物，加入1L超纯水搅拌溶解；用1mol/L的氢氧化钠溶液将配好的液体培养基pH值调节至7.3-7.7，再使用高压蒸汽灭菌锅121℃灭菌处理15min，配制好的液体培养基封装置于冰箱中冷藏备用，不用时在4℃环境下可保存7天；
    (2)发光菌株的培养和保藏：将用甘油冷冻保藏的发光细菌接种到盛有步骤(1)制备的100mL液体培养基的三角瓶中；瓶口用灭菌的四层折叠锡箔纸包裹瓶口，确保培养过程中外界氧气能够进入瓶中，在25℃环境中100r/min摇瓶培养，得到细菌母液；然后在成熟的细菌母液中加入等体积的新鲜液体培养基，制得工作发光悬浮液；工作发光悬浮液不用时，可在4℃环境中保存3天。
  4. 根据权利要求3所述高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，其特征在于：步骤(2)所述发光菌株使用琼脂平板法进行保藏，具体如下：
    a、发光菌株培养液的制备：取10g琼脂，30g NaCl，5g Na 2HPO 4，5g KH 2PO 4,3mL甘油,5g蛋白胨和5g酵母提取物，加入1L超纯水搅拌溶解后，用1mol/L氢氧化钠水溶液将配好的液体培养液pH值调节至7.5±0.2，再使用高压蒸汽灭菌锅121℃灭菌处理15min；待灭菌后的培养液冷却到60℃，趁热在直径10cm的培养皿中倒平板；
    b、接种：首先将灭菌后的接种环浸入成熟的发光菌株培养液中，然后在营养琼脂表面划斜线，反复多次；接种后的营养琼脂置于25℃环境避光培养48h，待观察到明显菌落后，得到细菌母液，转移到-4℃环境避光保藏。
  5. 根据权利要求3所述高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，其特征在于：步骤(2)中，将所用培养基对600nm入射光的吸收度作为测定菌体密度的指标；以新鲜模拟海水液体培养基清液为参比，培养过程中用分光光度计监测培养液对600nm入射光的吸光度OD 600，选取OD 600数值达到0.7时培养液作为细菌母液。
  6. 根据权利要求2所述高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，其特征在于：步骤(3)所述薄层板需要进行预洗；具体步骤如下：首先将10mL甲醇倒入洁净展开缸中，放入空白薄层板，使其展开到顶端，为使杂质尽可能被清洗干净，薄层板展开至顶端后需保持5min，取出后将薄层板放在薄层板加热器上100℃烘干5min，挥发残留有机溶剂，烘干后的薄层板用铝箔纸包裹备用。
  7. 根据权利要求2所述高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，其特征在于：所述薄层板的薄层材料是普通硅胶。
  8. 根据权利要求2所述高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，其特征在于：步骤(5)具体过程如下：通过生物发光成像仪拍摄的照片保存为CPF，Black/white linear格式，然后用Videoscan软件打开，对图片中的像素灰度进行数字化，然后设定积分参数和条件进行定量分析。
  9. 根据权利要求1所述高效薄层色谱联用生物发光法筛查茶叶中贝特类降脂化学药掺伪的方法，其特征在于：所述发光菌株具体为在正常生理条件下能够发射可见光的一类微生物。
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