New Automated Analyser for Aflatoxin in Maize. UV rays to evaluate the contamination

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1 New Automated Analyser for Aflatoxin in Maize UV rays to evaluate the contamination Star Ecotronics srl Via Quarenghi Milano Tel E mail info@starecotronics.it - www starecotronics.it 1

2 Aspergillus flavus metabolites of interest : AFLATOXINS Regarding food matrices contamination, the AFLATOXINS are among the mycotoxins of most interest. AFLATOSSINA B1 AFLATOSSINA B2 AFLATOSSINA G1 AFLATOSSINA G2 2

3 Aspergillus flavus: major producer of aflatoxins Aspergillus flavus is a commensal fungus aerobic, which is spread over several foodrelated matrices. The growth, the toxin and the production of metabolites of Aspergillus flavus are influenced by many environmental parameters: temperature humidity the presence of oxygen ph substrate 3

4 Aspergillus flavus metabolites of interest : AFLATOXINS Principal contaminated matrix: cereal, coffee, dried fruits, species. Matrices for animal feed: conveyance of the toxin in products for human consumption. IMPLICATIONS: protecting the health of consumers and operators of agricultural and livestock sector. There are strict norms regarding the acceptable tolerances for AFB1. It is necessary to have quick and effective method to determine the concentration of aflatoxin on the interest matrices. 4

5 Aspergillus flavus metabolites of interest: ACID KOJIC - BGYF test This metabolite is produced mainly as a result of oxidative stress. The kojic acid molecule functions as a scavenger of radicals: it is in fact able to oxidize itself in place of other cellular components of the fungus, preventing oxidative damage. In the industry, the kojic acid is widely used in various fields, including medicine, cosmetics and food. 5

6 Biosynthesis of AFLATOXINS and KOJIC ACID Recent studies have shown that both KOJIC ACID and AFLATOXINS are produced in response to oxidative stress. The regulatory genes of this response are the same for both metabolites ANALYSIS ON METABOLITES: simultaneous production of KOJIC acid and AFLATOXINS in most cases. Metabolic regulation and molecular implications have not yet been fully clarified. The biosynthesis of aflatoxins has been studied in detail and most of the enzymatic steps and reaction intermediates are identified. The kojic acid biosynthesis, however, has not yet been fully clarified. The tests carried out have shown that kojic acid is synthesized in quantities of three orders of magnitude greater than the concentrations of aflatoxins. 6

7 Fluorescence properties of AFLATOXINS AFLATOXIN B1 e AFLATOXIN B2 = (B = blue) fluorescence in the blue AFLATOXIN G1 e AFLATOXIN G2 = (G = green) flourescence in green but The aflatoxin quantities produced are very low and the fluorescence is not detectable without special equipment (expensive, requiring highly specialized personnel). 7

8 The fluorescence properties of the KOJIC acid KOJIC acid fluorescence is known as BGYF (Bright Greenish Yellow fluorescence under long wave UV light (365nm). Thanks to the high concentrations of metabolite present on the contaminated matrices (in particular the broken corn kernels), is also easily detectable by easy to use instruments. THEREFORE The detection of the kojic acid is an optimal method for indirect determination of the aflatoxin presence in a sample. 8

9 AFLAFLESH INSTRUMENT Fluorescence from contaminated maize kernels The contaminated maize kernels emit yellow/green fluorescence. 9

10 AFLAFLESH -1 IS THE FIRST INSTRUMENT DESIGNED SPECIFICALLY FOR THE NEEDS OF THE AGRO FEED INDUSTRY. 10

11 AFLAFLESH-2 IS A NEW INSTRUMENT DESIGNED BASED ON THE EXPERIENCE OF THE PREVIOUS INSTRUMENT AFLAFLESH-1 11

12 Comparison AFLAFLESH-1 and AFLAFLESH-2 Specific AFLAFLESH-1 AFLAFLESH-2 Sample quantity 5 kg 1 kg Analysis time 20 min 10 min Optical resolution 0,35 Mpixel 3,5 Mpixel Weight 75 kg 40 kg Sample distribuition (on the belt) Freefall Through vibrating slide 12

13 AFLAFLESH INSTRUMENT Corn entrance electronic panel Computer digital camera Vibrator and calibrator UV lamp Corn exit Sample conveyor 13

14 IMAGE ACQUISITION SYSTEM The high sensitivity and high resolution camera continuously takes up photos of the corn that passes on the conveyor belt. After a predetermined movement of the conveyor belt the photos are stored so as to record the whole transit of the corn. The stored frame is then analyzed by the software to calculate the pixels of the image that represent the kernels that emit fluorescence. The analysis of each frame is carried out before a new frame is acquired. The results of all the frames are then integrated into a final report that provides the total number of pixels to be processed in ppb of Aflatoxin with the calibration curve 14

15 AFLAFLESH Software The software provides, via separate passwords both Administrator and User access. 15

16 AFLAFLESH Software This is a tipical image of the software during analysis: The lower left image shows in real time the maize kernes passing along the conveyor. The uper left image shows the photo analysed by the software in order to detect the contamination. The right hand screen - shows the number of the contaminated kernals and the relative area. 16

17 AFLAFLESH Software This is a tipical view of the software while in Administrator mood. The right part of the screen shows the image of the sample captured by the digital camera The right part of the screen shows the contaminated grains to be counted by the software. 17

18 CORNVISION SOFTWARE The Cornvision software function separating the three components of the color: green, red and blue and detect the relative intensities for each pixel of the image. Since the fluorescence emitted by Kojic acid is a bright greenish yellow fluorescence (BGYF) a software trap is implemented in order to exclude false positive that should not be taken into consideration. 18

19 AFLAFLESH Software A report is generated at the end of the analysis containing the full information of the just completed. The estimated value of AFB1 is expressed in ppb. Together with the report a.avi video format is stored at the end of each analysis. 19

20 AFLAFLESH Software RESULTS OF ANALYSIS: Number of contaminated kernels Total surfice contaminated in pixel. ppb of aflatoxin B1 DATA STORAGE Each test is also saved as.avi file for any later reference. The analysis report is saved in.pdf format and it can be printed out also later on, in a second moment. 20

21 AFLAFLESH Software MINI RING TEST - 4 accreditated labs Sample C13 Sample C18 Sample C51 LAB. 1 2,16 15,6 26,3 LAB. 2 1,1 10,5 14,2 LAB. 3 1,9 9,35 23,5 LAB. 4 1,22 8,25 22,8 MEDIA 1,6 10,93 21,7 ELISA 3,19 14,08 24,42 AFLAFLESH 3,07 8,35 23,2 21

22 Conclusion No sample preparation needed No regent involved Easy to use Fast analysis (10 min 1 kg broken maize) Non distrucive sample after analysis Patented system Nb Machinery Directive: Approval to CE standards 22

23 Thank you for your attention! GUINA VINJAHU Star Ecoronics srl Via G. Quarenghi Milano Tel Fax 02/