Study on Time-Resolution Fluorescence Immunochromatography of Aflatoxin B1 in Yellow Rice Wine

1. Introduction

Yellow rice wine is a traditional liquor with unique health care functions in China. Compared with wines such as beer and wine, research on the metabolic mechanism and control technology of harmful substances in yellow rice wine started late, foreign mature research results and available experience are lacking[3]. As a secondary metabolite of related microorganisms, fungal toxin has always been a safety hazard in the food industry, AFB₁ is a highly toxic and highly carcinogenic substance, which is more toxic than potassium cyanide and arsenic. It is very easy to contaminate food crops, meat and dairy products. The melting point of aflatoxin is higher (265～269°C), the crystallization state is relatively stable under high temperature conditions[4,5,6]. The traditional yellow rice wine brewing glycochemical agent is a naturally fermented raw wheat curd, but yellow rice wine factories in various places often make their own rice, the climate, environment and room conditions vary greatly from place to place. The composition of microorganisms in the curd is very different, and aflatoxin-producing aspergillus aflatox is very likely to breed[3], and most of the yellow rice wine brewing process is carried out openly without sterilization., There are a large number of microorganisms in all kinds of raw materials and various tools. The brewing process environment and the air have the opportunity to invade beneficial and harmful microorganisms[7]. There are many researches showed that the AFB₁ in the production of distilled liquor (Daqu, fermented grains, lost rice, yellow water, base wine, finished wine) is far below 5μg·kg^-1. Especially AFB₁ is almost non-existent in base wines and finished wines[8,9,10]. Unlike liquor, the mash and yellow rice wine are nutritious medium in the brewing process of yellow rice wine, although the low pH, alcohol and mash has a certain inhibitory effect on microorganisms, there have not a distillation process, some non-cultured bacteria may also reproduce and increase possibility of aflatoxinB₁ pollution[11].26 samples of bottled yellow rice wine were determined by Ji Xiaofeng[1], although The content of AFB₁ does not exceed the limit standard of starting fermented food (5μg/kg), the detection rate is as high as 100%, and is very stable under normal conditions, especially once formed in the wine body, it is extremely stable and difficult to decompose. From this point of view, with the rapid growth of the yellow rice wine consumption market and improvement of consumers' nutritional health and safety awareness, the quality and safety issues of AFB₁ in yellow rice wine are also becoming more and more important to consumers, study on the establishment of AFB₁ with high sensitivity and high-fast detection methods are very urgent and necessary.

The existing detection methods of AFB₁ in yellow rice wine are mainly instrumental analysis and enzyme-linked immuno assay (ELISA) method. Enzyme-linked immunosorbent technology is the most commonly used in enzyme-linked immuno assay, but because it is prone to false positive results, it can only be used for preliminary screening. Instrument analysis methods such as HPLC-MS/MS and GC-MS improve the accuracy and sensitivity of analysis, but due to the high cost of equipment and the need for professional and technical personnel, the high detection cost is not conducive to carry out the detection of large-scale samples[12,13]. Immune analysis technology has the characteristics of strong specificity, high sensitivity, large analysis capacity, fast analysis and low cost, but traditional radioimmunology is limited due to the environment pollution and background fluorescence interference, The Time-resolution fluorescence immune analysis Technique (TRFIA) can mark trivalent rare earth ions which can produce fluorescence, such as Eu ( III), Tb (III), Sm (III), Use its long fluorescence decay time features of waiting for the natural fluorescence decay in the sample before measuring the fluorescence of rare earth ions can eliminate the interference of natural fluorescence. It has the advantages of high sensitivity, good anti-m matrix interference and strong stability[14,15,16,17].

Therefore, this paper is based on time-resolution fluorescence immuno assay technology, Set up rapid detection technology of AFB₁ in yellow rice wine with high resolution, high sensitivity, high security, thus filling the gap of rapid detection technology of AFB₁ in yellow rice wine.

2. Results and Discussions

2.1. Detection limit and linear range of the method

The results show that (Figure 1 and Figure 2) when the contention of AFB₁ is less than 0.8μg·kg^-1, the concentration is not linearly correlated with the detection card. The detection limit of time-resolution fluorescence immunochromatography in yellow rice wine of AFB₁ is 0.3μg·kg^-1, the linear range is 0.8-12.0μg·kg^-1. The linear equation of the corresponding standard curve is y=-0.1913x+0.5206 (R²=0.9948), the standard deviation is 0.018.

2.2. Preprocessing optimal reaction conditions

Through the recovery rate experiment (Table 1) and R extreme difference analysis (Table 2) display RB>RC>RA, indicating the influencing factors order is B > C > A, althrough intuitive analysis (Figure 3) determine that the optim combination is A2B3C2. Variance analysis (Table 3) Results show：B is significant, but A/C is not significant, FB>FC>FA, so B>C>A, consistent with the R extreme analysis results.

2.3. Accuracy and precision of the method

2.3.1. In-batch accuracy and precision of the method

Table 4 shows that the addition and recovery rate of the same batch of time-resolution fluorescent immunochromatography test strips is 77.9% - 105.7%. With the increase of the marked concentration and the increase of the recovery rate, the coefficient of variation of the measurement results becomes smaller, mainly because the difference between the signal is small, and the interference of the signal has a greater impact on the results. The coefficient of variation is in4.47% -10.26%, It shows that the detection technology has good accuracy and precision.

2.3.2. Inter-batch accuracy and precision of the method

For yellow rice wine samples, the addition and recovery rate of time-resolution fluorescence immunochromatography test strips between different batches is 75.9% - 89.8%, coefficient of variation8.10% - 13.86% (Table 5), indicating that the batch of the detection technology has good accuracy and precision.

2.4. Comparison of results on AFB₁ detection in actual sample test results between time-resolution fluorescence immunochromatographys and HPLC

By the Table 6, It can be seen that The relative error of the detection results between time-resolution fluorescence immunochromatography technology established in this study and national food safety standards [18] (GB5009.22—2016) is less than 10%, explain the test results of time-resolution fluorescence immunochromatography technology are consistent with the the standard method and meet the requirements of rapid detection of aflatoxin in agricultural products.

3.1. Establish a standard curve

3.1.1. Preparation of sample diluent

Weigh 1.0 g Sucrose,0.5 g Bovine serum albumin and 2.5g Twain20 (Comes from China National Pharmaceutical Group Chemical Reagent Beijing Co., Ltd.), mix well and use ultra-pure water (Comes fromUPH-III-10Preparation of Upu ultra-pure water manufacturing system) fixed capacity to 100.0 mL[19].

3.1.2. Selection of blank matrix solution

Choose yellow rice wine samples with few impurities and bright color (alll purchased in large local supermarkets). Adopt the third method in national of food safety standards (GB5009.22—2016 ) --high performance liquid chromatography- Post-column derivative method for testing (HX-G Photochemical post-column derivative instrument, Wuhan Hengxin Reagent Technology Co., Ltd.; Shimazu LC-20ALiquid chromatograph, Total aflatoxin immune affinity columnComes fromJiangsu Su Weiwei Biological Research Co., Ltd.), choose the sample without AFB₁ as a blank matrix solution.

3.1.3. Standard working solution preparation

Buy AFB₁ standard products (2 mg·kg^-1, Beijing Tanmo Quality Inspection Technology Co., Ltd.), use methanol (United StatesFisher) fixed capacity to 10 mL, the quality concentration of the formulated product is 200μg·kg^-1, to save the standard reserve solution at -20°C. Then dilute the reserve liquid gradient with a blank matrix solution to receive a series of standard working solutions of 0.2, 0.4, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, 12.0, 14.0 and 16.0µg·kg^-1, test from low to high concentrations, according to the detection line T Signal value and quality control line C, the ratio of signal value (T/C) and the natural logarithm of the standard solution concentration (lnc), draw standard curves.

3.2. Certain sample pretreatment optimal reaction conditions

3.2.1. Sample pretreatment and AFB₁ content determine

Use an electronic balance (CP4102Mould, Aohaus Instruments (Changzhou) Co., Ltd.) to weigh 5.0 g yellow rice wine into centrifugal tube, mix evenly in the oscillator (KB5010Type, Haimen Qilin Bell Instrument Manufacturing Co., Ltd.), High-speed centrifugation (5810RHigh-speed low-temperature centrifuge, Eppendorf) for 5 minutes (6000r/min), add 3.0mLsample diluentto 1.0mL cleansing liquid, The sample diluent is mixed evenly with an vortex meter and filtered by an organic filter membrane to obtain the liquid to be tested for later use. Open the sample cup and add 150μL sample, the sample to be tested is fully dissolved, mixed, and warmed in the heater, Remove the test strip from the test cartridge, insert it down into the sample cup according to the arrow, and chromatography. After the hardware self-test of the instrument according to the operation steps of the aflatoxin time-resolution fluorescence meter, take it out after tomography and dry the lower liquid. Use a time-resolution fluorescence reader to read signal values and determine AFB₁ content within 2 minutes (Aflatoxin time-resolution fluorescence meter, incubator, sample bottle and other accessories all by Jointly developed by the Institute of Oil Crops of the Chinese Academy of Agricultural Sciences and Shanghai U You Biotechnology Co., Ltd).

3.2.2. Pretreatment for the optimal reaction conditions

Add AFB ₁ standard working solution to the blank matrix sample of yellow rice wine, ensure the final concentration of addition is 5μg·kg ^-1, carry out three factors and three levels of orthogonal experiments according to 3.2.1. The oscillation time (A) is respectively 10min, 20min, 30min, reaction temperature (B) respectively is 28°C, 32°C, 37°C, chromatography time (C) is respectively 4min, 6min, 8min. The best reaction conditions for sample pretreatment are determined through extreme difference and variance analysis of recovery rate and variance analysis.

Table 7. Experimental factors of recovery rate.

Level Level Level Factor Factor	A	B	C
	Oscillation time (min)	Reaction temperature (°C)	Chromatography time (min)
1	10	28	4
2	20	32	6
3	30	37	8

Table 7. Experimental factors of recovery rate.

Level Level Level Factor Factor	A	B	C
	Oscillation time (min)	Reaction temperature (°C)	Chromatography time (min)
1	10	28	4
2	20	32	6
3	30	37	8

3.3. AFB₁ detection limit and linear range

The detection is limited to the minimum amount (or minimum concentration) that the object to be tested can be recognized from the background detection of the blank matrix sample. According to national standards GB/T 27404—2008 ( Riterionon Quality Control of Laboratories-Chemical Testing of Food[20], calculate the detection limit with the formula in (1). The linear range is obtained according to the recovery test of blank matrix samples.

CL=3Sb/b

(1)

Formula (1) : CL- Methodological detection limit; Sb- Standard deviation of blank value; b -Method standard curve slope

3.4. AFB₁ detection of in-batch precision

A blank matrix sample was added with a recovery test to obtain the in-batch precision of this method. Add AFB₁ standard working solution into the blank matrix sample of yellow rice wine to make the final concentration of AFB₁ is 1, 5, 10μg·kg^-1, using the same batch of aflatoxin time-resolution fluorescence immunochromatography test strips to test six times, calculate the average addition recovery rate and variation coefficient in the in-batch (CV).

3.5. AFB₁ detection of inter-batch precision

A blank matrix sample was added with a recovery test to obtain the inter-batch precision of this method. Add AFB₁ standard working solution into the blank matrix sample of yellow rice wine to make the final concentration of AFB₁ is 1, 5, 10μg·kg^-1, using different batches of aflatoxin time-resolution fluorescence immunochromatography test strips to test six times, calculate the average addition recovery rate and variation coefficient between batches (CV).

3.6. Comparison of test results from actual samples betwen time-resolution fluorescence immunochromatography and HPLC

Choose seven actual samples of yellow rice wine randomly, aflatoxin time-resolution fluorescence immunochromatography test strip method and the national food safety standard respectively (GB5009.22—2016 ) is compared and tested to compare the consistency of the two methods.

4. Conclusion

AFB₁ in yellow rice wine is a very important safety hazard, and it is very necessary to test it. In the existingdetection method, HPLC-MS/MS and GC-MS operate complexly and are not easy to popularize. Although enzyme-linked immunization methods are fast and convenient, they are prone to false positives. This article is studied about Time-resolution fluorescence immunochromatography rapid detection technology of AFB₁ in yellow rice wine for the first time. Compared with national standard method, this method has the advantages of simple operation, fast detection speed, high sensitivity, good repeatability and stability, low dosage of organic reagents, and high safety for the environment and inspectors, filling the blanks of quick and accurate detection of AFB₁ in yellow rice wine, and has broad application prospects.