Solution for HPLC detection of fumonisin in Puribond feed

  Fumonisin causes and characteristics

Fumonisins are mainly secondary metabolites produced by the propagation of Fusarium oxysporum f. moniliforme and f. proliferatum under certain temperature and humidity conditions. It was not until 1988 that researchers in South Africa and the United States first isolated fumonisins b1 from moldy corn. So far, the fumonisins have been found to have 11 kinds of FA1, FA2, FB1, FB2, FB3, FB4, FC1, FC2, FC3, FC4 and FP1. Grain is susceptible to contamination by the above two fungi during processing, storage and transportation, especially when the temperature is suitable, which is more conducive to its growth and reproduction, thus producing a class of toxins with similar structural properties, of which FB1 is the main component of 60. Above %, its toxicity is also the strongest. Therefore, fumonisin can cause serious damage to animal husbandry and even human health through processes such as grain processing and feed production.

The pure fumonisin is white needle crystal, which is a kind of related polar and water-soluble metabolite. It is a diester compound of polyhydric alcohol and glycerol tricarboxylic acid. It is very stable to heat and is not easily destroyed by cooking. In most foods. The processing and processing are relatively stable.

Food contamination

The FB1 food contamination situation is widespread around the world, mainly polluting corn and corn products, and its contaminated feed is mainly corn-based feed. FB1 is a water-soluble mycotoxin that is stable to heat and is not easily destroyed by cooking. Therefore, like AFT (aflatoxin), controlling mold contamination during crop growth, harvesting and storage is still essential.

The contamination rate and pollution level of fumonisin on corn is affected by countries and regions, corn varieties and seasons. From 1995 to 1996 , Amra et al . tested 120 samples collected from different provinces of Egypt . The results showed that the FB1 pollution rate of white corn and yellow corn collected in winter was the highest. Fumonisin can also contaminate other foods and their products, and there are differences in pollution conditions in different regions.

In 1996 , China investigated the pollution of FB1 in corn, wheat and other food crops . It was found that different areas have different degrees of pollution. The contamination rate of corn fumonisin in Linxian County, a high incidence area for esophageal cancer in China, is 48% . Therefore, it is suspected that the high incidence of esophageal cancer in this area is associated with the consumption of this toxin corn. The toxin has been listed by the World Health Organization as one of several mycotoxins that were first studied in recent years.

The harm of fumonisin

1 . Horse white matter softening               
This is a neurological disorder in horses. According to the results of the South African researchers in 1988 , the horse was injected subcutaneously at a level of 0.125 mg / kg body weight per day . After about 7 days, the horse began to go crazy and mad, dying from the railing. Anatomy found that the horse's brain showed white matter softening symptoms. In 1989 , fumonisin in corn caused huge losses to agriculture and animal husbandry in many states in the United States.                
　　 2 . Porcine pulmonary edema              
　　 Scientists in the United States and South Africa in 1992 and 1994 have shown that daily intake of fumonisin above 0.4 mg / kg body weight can cause pulmonary edema in pigs, and can also cause disturbances in the reproductive system of pigs, such as premature birth, abortion, stillbirth And abnormal estrus cycles. The disease is found in the United States and other countries.
　　 3 . Mouse liver cancer               
　　 In 1991 , South African researchers conducted a toxicological test of fumonisin in mice. The results showed that fumonisin induced liver cancer. In 1998 , the rat was subjected to a fumonisin toxicology test, and the same result was obtained.                 
　　 4 . Human esophageal cancer               
As early as 1988 , South African scientists investigated areas with high and low rates of esophageal cancer. The incidence of esophageal cancer was positively correlated with the consumption of fumonisin in staple corn, and further animal experiments yielded the same results. In 1994 , Chinese scholars and Japanese scholars conducted a survey of Linxian County, Henan Province, in the high incidence area of ​​esophageal cancer, and found that the level of fumonisin in the staple corn in the region is as high as 30~50mg / kg , and the highest value of fumonisin in moldy corn is 118.4mg. / kg . The mechanism by which fumonisins cause esophageal cancer is still unclear and needs further confirmation and research.  

National limit standards for fumonisin

In 2001 , the US Food and Drug Administration (FDA) issued a maximum circular guidance for fumonisin in corn and corn products for human consumption, stipulating that the maximum limit of fumonisin in human corn is 2 mg / kg . At the same time, FDA animal husbandry The Medical Center (CVM) has also issued a maximum amount of guidance on the regulation of fumonisins in animal feed, with a limit of 1 to 50 mg / kg .

             Table 2 FDA recommended limit for fumonisin standard in animal feed (June 2000)

Detection of fumonisin

The detection of fumonisin has been carried out by thin layer chromatography, gas chromatography, enzyme-linked immunosorbent assay ( ELISA ), high performance liquid chromatography ( HPLC ), etc. The most research and application is currently purified by immunoaffinity column. Fluorescence assay and HPLC method.

Introduction of Puriban Fumarine Toxin Detection Program

(I) Immunoaffinity column - high performance liquid chromatography: in accordance with GBT 25228-2010 grain and oil test corn and its products in the determination of fumonisin content immunoaffinity column purification high performance liquid chromatography

Pribolab® uses immunoaffinity column purification, and uses HPLC and fluorescence detectors to detect HPLC assays that provide fumonisin assays. The results are accurate and reliable, and the detection limits are good. A good method for detecting fumonisin is for reference only.

1. Equipment and consumables configuration

2. Sample preparation : Preweang provides different treatment options for the feed (for detailed solutions, please contact Puribang, Puri State has developed an optimized treatment plan for a variety of feed matrices)

Sample processing:

Peanuts, corn, rice, wheat and its products and feed

---- will be 50 g The ground sample + 5 g of salt was placed in a homogenized cup.

- Add 100 mL of methanol: water ( 80 : 20 ) solution.

---- Cover the lid and mix at high speed for 5 minutes.

Centrifuge at 4000 rpm for 5 min or filter with fluted filter paper;

---- Take 10 mL of filtrate and add 40 mL of PBS solution to dilute the filtrate and mix. Filtration with glass microfiber filter paper , taking the diluted liquid to be tested;

---- The previous step of the dilution was filtered through a microfiber filter paper, and the filtrate was collected in a glass syringe barrel and weighed 10 mL .

3. Immunoaffinity column purification :

Operating procedures: in line with GB/T 25228-2010 national standard method

- Attach the immunoaffinity column to the 10 mL syringe of the pump flow operator . Accurately transfer 10 mL of sample extract into the syringe;

---- Enrichment : Connect the air pressure pump to the syringe, adjust the pressure so that the extract slowly passes through the immunoaffinity column at a flow rate of about 2-3 mL/min (1 drop / 3 seconds) until 2-3 mL of air passes through the column. ;

---- Washing : Wash with 10mL 0.1% Tween/PBS, then rinse the column twice with 10mL water, discard all the effluent, and let 2-3mL air pass through the column;

---- elution can be carried out in one of the following ways : preferably 2

---- Elution 1: Accurately add 1.0mL chromatographic grade methanol elution, incubate for more than 30 seconds (incubation, methanol soaked in the column), then flow through the column at a flow rate of 1-2mL / min, collect all the eluent for For testing purposes.

---- Elution 2: To ensure sufficient elution, it is recommended to elute in two steps with 1.5 mL of chromatographic methanol at a flow rate of 1-2 mL/min (1 drop/sec, gravity). First, add 0.5 ml of methanol to elute, gravity through the column, wait for 30 seconds after the solvent passes through the column, then add 1 ml of methanol for elution, and incubate for more than 30 seconds before the column (incubation, methanol is soaked in the column). All methanol eluents were collected for derivatization.

4. derivatization reaction

Take 50ul of purified sample solution and standard working solution respectively, place them in 1ml of each test tube, add 50ul OPA derivative solution, mix for 30s on vortex mixer, let stand for 3min, then take 20ul of derivative liquid into high efficiency liquid. Phase chromatographic analysis.

5. High performance liquid chromatography analysis

1. The favorable conditions for the growth and reproduction of fungi in cereals and feeds are mainly suitable temperature and moisture. If the grain, feed, etc. can be stored below 10 °C and the water content is kept below 10 %, the mold can be effectively prevented.

2. Personnel engaged in the research and testing of mycotoxins must pay attention to protection. For example, wear a protective coat cap . When performing fungal separation and culture work, wear a mask and try to prevent spores from flying .

3. If there is any leakage in the operation table, it should be disinfected immediately with the new 5 % sodium hypochlorite . When treated with 5 % sodium hypochlorite ( NaOCl ), aflatoxin is destroyed within a few seconds and is a commonly used disinfectant.

4. There are also reports on the application of biological methods to detoxification. The low cost of biological methods and large results may be a promising detoxification measure.

In view of the harm of mycotoxins to the human body, teachers who are struggling to fight on the front line of anti-drug must pay attention to protect themselves!  

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