CN105784860B - The detection method of the aflatoxin of solid sample during a kind of liquor production - Google Patents

Abstract

The invention discloses a method for detecting aflatoxin in solid samples in the liquor production process, belonging to the technical field of food detection. In the present invention, the presence of aflatoxin in liquor distiller's grains is detected by performing relevant pretreatment on liquor distiller's grains respectively in combination with a UPLC-MS/MS detector. The invention provides a novel liquor quality and safety index detection technology, which is simple and convenient to operate, and can quickly and timely monitor the trace amount of aflatoxin that may exist in liquor distiller's grains, thereby effectively controlling the liquor quality and safety.

Description

A kind of detection method of aflatoxin in solid sample in liquor production process

technical field

The invention relates to a method for detecting aflatoxin in solid samples in the liquor production process, belonging to the technical field of food detection.

Background technique

Aflatoxins belong to mycotoxins and are classified as "Class I carcinogens" by the Cancer Research Institute of the World Health Organization. It is very easy to pollute food crops, meat and dairy products, etc., and its harmfulness is that it has a destructive effect on human and animal liver tissues. In severe cases, it can cause liver cancer or even death.

Grain crops are the main production raw materials of the liquor industry, and it is very likely that aflatoxin and toxin-producing microorganisms will be brought into liquor, threatening the safety of liquor production. Now there are many safety problems in the food industry, but there are almost no reports about the impact of aflatoxin on liquor production. Therefore, it is very necessary to verify the safety research of aflatoxin in liquor production. At the same time, with the continuous growth of the liquor market, its by-product waste is more and more used in the feed processing industry, so the detection of aflatoxin in waste is also very important.

At present, the detection objects of aflatoxins are mainly corn, rice, soybean, dairy products, edible oil, condiments, etc.; fermented alcoholic beverages such as wine and beer are also reported in the literature; detection methods mainly focus on thin-layer chromatography, high-performance liquid Phase chromatography, enzyme-linked immunoassay, liquid mass chromatography, etc. Since the liquor production process involves a variety of raw materials, microorganisms, and metabolites, solid samples in the liquor production process, such as Daqu, distiller's grains, distiller's grains, etc., are extremely rare compared with grain samples such as grains. Complicated, there are many unknown interfering substances in the detection process.

However, there are still few studies on liquor in China. Some scholars use the ELISA method to detect the content of aflatoxin B1 in Daqu, distiller's grains, yellow water, base wine, and finished wine; ELISA detection method is prone to false positives, and the detection results It is too large, and the presence of multiple toxins cannot be detected at the same time, and the experimental results still need to be verified qualitatively and quantitatively. Some scholars also use high-performance liquid chromatography to detect the content of aflatoxin B1 in sorghum, Daqu, wine grains, yellow water, and finished wine. However, this method has problems such as long detection time and insufficient sensitivity, and the aflatoxin B1 that can be detected simultaneously There are few types of aspergillus toxins.

Therefore, it is an urgent problem to be solved at present to develop a rapid, accurate, high-sensitivity, and simultaneous detection of multiple aflatoxins for solid samples in the liquor fermentation process.

Contents of the invention

In order to overcome the above problems, this method provides a method based on UPLC-MS/MS for the simultaneous detection of four aflatoxins in solid samples in the liquor production process, which can quickly and easily realize the solid samples in the liquor production process, such as distiller's grains Detection of aflatoxin in . The method of the invention is easy to operate, short in time consumption, and can be applied to the simultaneous detection of a large number of samples. The method of the invention realizes the simultaneous detection of the aflatoxins AFB1, AFB2, AFG1 and AFG2, and has high recovery rate and good stability.

The detection method of the present invention comprises the following steps:

(1) Pretreatment: take the dried solid sample and sieve it;

(2) Extraction: add a certain proportion of water and solvent A, shake and mix to obtain a mixed solution;

(3) Salting out: add salting out agent in the mixed solution, shake and mix and centrifuge;

(4) Purification: take the centrifuged supernatant in step (3) and add a purifying agent, and shake and centrifuge;

(5) Concentration: take the supernatant after centrifugation in step (4), dry it with nitrogen and redissolve it and pass through a 0.22 μm membrane;

(6) Sample injection analysis: the solution sample after passing through the membrane in step (5) is subjected to UPLC-MS/MS detection.

In one embodiment of the present invention, the solid sample is liquor distiller's grains, or other solid samples in the liquor production process, such as Daqu and fermented grains.

Specifically, the sieving in the step (1) is to pass through a 20-mesh sieve.

Preferably, the solvent A in the step (2) is acetonitrile.

Preferably, in the step (2), 10ml of water and 15ml of acetonitrile are added according to 5g of dried solid sample.

Preferably, the salting-out agent in the step (3) is to add 1g of NaCl and 4g of MgSO ₄ for every 5g of dried solid sample.

Specifically, the shaking method in the step (3) is manual shaking up and down, and the shaking time is 1 minute.

Preferably, the purifying agent in the step (4) is to add 150 mg of PSA and 900 mg of MgSO ₄ for every 5 g of the dried solid sample.

Specifically, in the step (4), the oscillation method is vortex oscillation, and the oscillation time is 30s.

Specifically, in the centrifugation step in the steps (3) and (4), the centrifugation parameter is 4000 rpm for 5 minutes.

Specifically, in the step (5), in the reconstitution step of drying with nitrogen, the reconstitution solution is the mobile phase (mobile phase A and mobile phase B are mixed at a volume ratio of 1:1), and the volume is 0.5 mL.

Specifically, the sample volume of the solution used for UPLC-MS/MS detection in the step (5) is 5 μL.

Preferably, the chromatographic condition in the UPLC-MS/MS in the step (6) is to use a C18 column, the mobile phases are A and B, A is formic acid water, B is methanol, and gradient elution is used.

Preferably, the mobile phases A and B, the concentration of mobile phase A is 0.01% (volume fraction), and the flow rate is 0.500ml/min.

In one embodiment of the present invention, the chromatographic conditions are specifically: ACQUITY UPLC BEH C18 is used as a chromatographic column; 0.01% formic acid water is used as mobile phase A, 100% methanol is used as mobile phase B, and the flow rate is 0.5 ml/min; the elution method is gradient elution, and the elution procedure is as follows: 0min, 5% mobile phase B; 7min, 60% mobile phase B; 9min, 100% mobile phase B; 11.00min, 100% mobile phase B; 11.10 min, 5% mobile phase B; 14.00 min, 5% mobile phase B.

Preferably, the mass spectrometry conditions of UPLC-MS/MS in the step (6) are as follows: the ion source is an electrospray ionization source (ESI); the spray voltage is 3000V (+); the ion source temperature is 150°C, and the desolvation temperature is 300°C.

Among them, AFB1: parent ion is 313.10; qualitative ion is 240.90, collision energy is 35V; quantitative ion is 268.70, collision energy is 32V; AFB2: parent ion is 315.10; qualitative ion is 258.90, collision energy is 35V; quantitative ion is 286.90, Collision energy is 25V; AFG1: parent ion is 329.10; qualitative ion is 242.80, collision energy is 25V; quantitative ion is 310.90, collision energy is 20V; AFG2: parent ion is 331.10; qualitative ion is 244.90, collision energy is 35V; The quantitative ion is 313.00, and the collision energy is 24V.

In one embodiment of the present invention, the method further includes drawing a standard curve with aflatoxin standard substances, thereby calculating the content of aflatoxin in distiller's grain samples.

In one embodiment of the invention, the method specifically includes:

(1) Extraction: Add 10 g of low-temperature water to 5 g of dried distiller's grain powder (wet distiller's grains dried through a 20-mesh sieve) and mix well, then vortex and shake 15 mL of acetonitrile for 30 seconds to extract the aflatoxin;

(2) Salting out: add 1g NaCl and _4g MgSO4 to the mixed solution, shake for 1min, and rotate at 4000rpm for 5min; reduce the water content in the mixed extract solution and saturate the aqueous solution to make the mixed solution stratify into an acetonitrile layer and a water layer;

(3) Purification: take the supernatant after salting out in step (2), add purifying agent (150mg PSA, 900mg MgSO ₄ ) and vortex shake for 30s, 4000rpm for 5min;

(4) Concentration: Take the purified supernatant in step (3), dry it with nitrogen, and redissolve it in 1ml methanol: 0.1% formic acid water (1:1) solution, filter through a 0.2μm membrane and take 5μL for UPLC - MS/MS quantitative detection.

Beneficial effects of the present invention:

(1) The method of the present invention is first applied to the detection and analysis of aflatoxins in distiller's grains of liquor. The method has high sensitivity and is easy to operate. The pretreatment is simple, easy to operate and time-consuming, and the detection time (14min) by liquid chromatography-mass spectrometry is shorter than that of the existing high-performance liquid chromatography (30min); the detection limit of AFB1 is 0.09μg/kg, which is much lower than The detection limit of the existing liquor sample detection; can detect a variety of aflatoxins at the same time, including: AFB1, AFB2, AFG1, AFG2.

(2) It can be applied to the detection of aflatoxins in distiller's grains and other solid samples in the liquor production system. The method of the invention uses UPLC-MS/MS to inject and analyze liquor distiller's grain samples to detect the content of aflatoxin in liquor distiller's grains, has fast analysis speed, reduces false positives at the same time, improves determination accuracy, and has strong practicability. This method has important guiding significance for the detection of aflatoxin content in liquor distiller's grains, and has a guiding role for the timely safety monitoring of liquor production system. Promoted the implementation of food safety work in liquor production.

Description of drawings

Fig. 1 is the separation effect diagram of 4 kinds of aflatoxins under the chromatographic conditions of the present invention.

Detailed ways

Experimental Materials:

Triple quadrupole mass spectrometer (waters), ACOUITY Ultra Performance LC liquid chromatograph (U.S. Waters Company), ultrapure water machine (U.S. Millipore Company), aflatoxin standard (Sigma Company), acetonitrile, methanol, formic acid Chromatographically pure, PSA (Shanghai Anpu Scientific Instrument Co., Ltd.).

Example 1: Qualitative and quantitative detection of aflatoxin in distiller's grains

Check as follows:

(1) Extraction: Add 10 g of low-temperature water to 5 g of dried distiller's grain powder (wet distiller's grains dried through a 20-mesh sieve) and mix well, then vortex and shake 15 mL of acetonitrile for 30 seconds to extract the aflatoxin;

(2) Salting out: add 1g NaCl and _4g MgSO4 to the mixed solution, shake for 1min, and rotate at 4000rpm for 5min; reduce the water content in the mixed extract solution and saturate the aqueous solution to make the mixed solution stratify into an acetonitrile layer and a water layer;

(3) Purification: take the supernatant after salting out in step (2), add purifying agent (150mg PSA, 900mg MgSO ₄ ) and vortex shake for 30s, 4000rpm for 5min;

(4) Concentration: take the purified supernatant in step (3), dry it with nitrogen, and redissolve it in 1ml of methanol: 0.1% formic acid water (1:1) solution, filter through a 0.2μm membrane, and inject 5μL into UPLC - MS/MS quantitative detection.

The chromatographic conditions of UPLC-MS/MS: ACQUITY UPLC BEH C18; 0.01% formic acid as the mobile phase A, 100% methanol as the mobile phase B, the flow rate is 0.5mL/min; according to the gradient elution method in Table 1 Carry out elution, and the elution procedure is as follows: 0min, 5% mobile phase B; 7min, 60% mobile phase B; 9min, 100% mobile phase B; 11.00min, 100% mobile phase B; 11.10min, 5% mobile phase B ; 14.00 min, 5% mobile phase B. Under the chromatographic conditions, the four aflatoxins can be completely separated and the peak shape is better (as shown in Figure 1).

Table 1 Mobile phase gradient

The mass spectrometry conditions of UPLC-MS/MS are as follows: the ion source is electrospray ionization source (ESI); the spray voltage is 3000V(+); the ion source temperature is 150°C, and the desolvation temperature is 300°C.

Among them, AFB1: parent ion is 313.10; qualitative ion is 240.90, collision energy is 35V; quantitative ion is 268.70, collision energy is 32V; AFB2: parent ion is 315.10; qualitative ion is 258.90, collision energy is 35V; quantitative ion is 286.90, Collision energy is 25V; AFG1: parent ion is 329.10; qualitative ion is 242.80, collision energy is 25V; quantitative ion is 310.90, collision energy is 20V; AFG2: parent ion is 331.10; qualitative ion is 244.90, collision energy is 35V; The quantitative ion is 313.00, and the collision energy is 24V.

After the above steps, the qualitative detection of the aflatoxin in the sample is achieved by comparing the peak time with the aflatoxin standard. In addition, by using a known amount of aflatoxin standard to draw a standard curve, and substituting the target peak area into the standard curve, the corresponding aflatoxin content in the injected sample can be obtained, and then the solids in the liquor production process can be known. The content of aflatoxin in the sample is to realize the quantitative detection of aflatoxin AFB1, AFB2, AFG1 and AFG2. In addition to distiller's grain samples, the method of the invention can also be used to detect Daqu and distiller's grain samples.

Embodiment 2: Research on the effectiveness of the quantitative detection method of the present invention

Accurately weigh 5 g of dried distiller's grains, add different volumes of standard mixed solutions, perform detection according to the method of Example 1, and prepare a corresponding standard curve. The specific concentrations are shown in Table 2 to Table 5.

Table 2 Concentration of AFB1 standard curve

Table 3 AFB2 standard curve concentration

Table 4 AFG1 standard curve concentration

Table 5 AFG2 standard curve concentration

Accurately draw each 5ul of the above-mentioned series of solution standard solutions, sample injection analysis, record the chromatographic peak area of each component to be measured, take the injection concentration as the abscissa (X), and the peak area as the ordinate (Y), carry out regression analysis, the result ( See Table 6) show that each component linear relationship is good.

Table 6 Aflatoxin B1, B2, G1, G2 regression equation and correlation coefficient

Meanwhile, the detection limit, quantitative limit, recovery rate and stability data of the method of the present invention are shown in Table 7 to Table 10. In addition to distiller's grain samples, the method of the invention is used to detect Daqu and distiller's grains samples, and the linear correlation of the standard curve is also very good, and the detection limit, quantification limit, recovery rate and stability are similar to those of the distiller's grains sample.

Table 7 Aflatoxin B1, B2, G1, G2 detection limit parameter values

Table 8 Aflatoxin B1, B2, G1, G2 limit of quantitation parameter values

Table 9 Aflatoxin B1, B2, G1, G2 recovery experimental parameter values

Table 10 Aflatoxin B1, B2, G1, G2 stability (RSD) parameter value

Embodiment 3: the influence of detection condition on detection result

The present invention also compares the influence of different processing methods on the detection effect.

(1) Influence of sample state: use wet distiller's grains instead of dry crushed distiller's grains as a sample, and other steps are consistent with Example 1. The results show that the finally detected aflatoxin content is about 35% lower than that in Example 1, the stability and recovery rate of detection are seriously affected, and the effective detection of aflatoxin cannot be realized at all.

(2) The influence of the extraction solvent: Methanol is used instead of acetonitrile as the extraction solvent, and other steps are consistent with Example 1. The results showed that the finally detected aflatoxin content was about 22% lower than that in Example 1, and the stability and recovery rate of the detection were seriously affected. In addition, the inventors have also found that when acetonitrile is directly used without adding water in the extraction solvent, the content of the finally detected aflatoxin is reduced by about 60% compared with the combination of acetonitrile and water, indicating that the combination of acetonitrile and water can be used as the extraction solvent significantly. Improve the accuracy of detection.

(3) Impact of purification: omit the purification treatment in the step (3) of embodiment 1, it is found that the content of the aflatoxin finally detected is about 43% lower than that of embodiment 1, and the stability and recovery rate of detection are received. Serious impact. This also shows that the purification treatment method of the present invention can significantly improve the accuracy of detection.

(4) The influence of the liquid phase method: using the liquid phase gradient elution program: 0min, 5% mobile phase B; 0.10min, 5% mobile phase B; 6min, 60% mobile phase B; 9min, 100% mobile phase B; 10min, 100% mobile phase B; 10.10min, 5% mobile phase B; 11min, 5% mobile phase B; the peak shape presented in the spectrum cannot completely separate the four types of aflatoxins.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (7)

1. a method for detecting 4 kinds of aflatoxins in a solid sample in the liquor production process simultaneously, is characterized in that, described method comprises the following steps: (1) Pretreatment: take the dried solid sample to be tested, and sieve, the solid sample to be tested is distiller's grains, Daqu or fermented grains; (2) Extraction: add a certain proportion of water and acetonitrile, shake and mix to obtain a mixed solution; (3) Salting out: add salting out agent in the mixed solution, shake and mix and centrifuge; (4) Purification: take the centrifuged supernatant in step (3) and add a purifying agent, and shake and centrifuge; (5) Concentration: take the supernatant after centrifugation in step (4), dry it with nitrogen and redissolve it and pass through a 0.22 μm membrane; (6) Sample injection analysis: the solution sample after passing through the membrane in step (5) is subjected to UPLC-MS/MS detection. 2. The method according to claim 1, characterized in that, the method also comprises drawing a standard curve with aflatoxin standard substance, thereby calculating the content of aflatoxin in the sample to be tested. 3. The method according to claim 1, characterized in that, in the step (2), 10ml of water and 15ml of acetonitrile are added according to the dried solid sample of 5g. 4. The method according to claim 1, characterized in that the salting-out agent is to add 1g NaCl and 4g MgSO ₄ for every 5g of dried solid sample. 5. method according to claim 1, is characterized in that, in the UPLC-MS/MS in described step (6), chromatographic condition is to adopt C18 post, mobile phase is A and B, and A is formic acid water, and B is methyl alcohol , using gradient elution. 6. The method according to claim 1, wherein the chromatographic conditions in the UPLC-MS/MS are specifically: using ACQUITY UPLC BEH C18 as a chromatographic column; using 0.01% formic acid water as the mobile phase A phase, and using 100 % Methanol is the mobile phase B, the flow rate is 0.5ml/min; the elution method is gradient elution, and the elution procedure is as follows: 0min, 5% mobile phase B; 7min, 60% mobile phase B; 9min, 100% mobile phase B; 11.00 min, 100% mobile phase B; 11.10 min, 5% mobile phase B; 14.00 min, 5% mobile phase B. 7. method according to claim 1, is characterized in that, in described step (6), UPLC-MS/MS adopts electrospray ionization source, adopts positive and negative ion mode to carry out data acquisition, and ion source voltage is 3kV, and ion source The temperature was 150°C, and the desolvation temperature was 300°C.