New Automated Analyser for Aflatoxin in Maize

UV rays to evaluate the contamination

1 Star Ecotronics srl – Via Quarenghi 27 – 20151 Milano Tel 02 89540225 E mail info@starecotronics.it - www starecotronics.it

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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

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Aspergillus flavus: major producer of aflatoxins

Aspergillus flavus is a commensal fungus aerobic, which is spread over several food-related 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

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.

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Aspergillus flavus metabolites of interest : AFLATOXINS

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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.

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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.

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Fluorescence properties of AFLATOXINS

AFLATOXIN B1 e AFLATOXIN B2 = fluorescence in the blue

(B = blue)

AFLATOXIN G1 e AFLATOXIN G2 = flourescence in green

(G = green)

but…

The aflatoxin quantities produced are very low and the fluorescence is not detectable without special equipment (expensive, requiring highly specialized personnel).

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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.

AFLAFLESH INSTRUMENT

Fluorescence from contaminated maize kernels

The contaminated maize kernels emit yellow/green fluorescence.

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AFLAFLESH -1

IS THE FIRST INSTRUMENT DESIGNED SPECIFICALLY FOR THE NEEDS OF THE AGRO FEED INDUSTRY.

AFLAFLESH-2

IS A NEW INSTRUMENT DESIGNED BASED ON THE EXPERIENCE OF THE PREVIOUS INSTRUMENT “AFLAFLESH-1”

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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

Comparison AFLAFLESH-1 and AFLAFLESH-2

Corn

entrance

Sample

conveyor

Computer

digital

camera

Corn exit

Vibrator and

calibrator

electronic

panel UV lamp

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AFLAFLESH INSTRUMENT

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

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AFLAFLESH Software

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

AFLAFLESH Software

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.

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This is a tipical image of the software during analysis:

AFLAFLESH Software

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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.

This is a tipical view of the software while in Administrator mood.

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.

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AFLAFLESH Software

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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.

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.

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AFLAFLESH Software

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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

MINI RING TEST - 4 accreditated labs

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.0001395845

Machinery Directive: Approval to CE standards

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Thank you for your attention!

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GUINA VINJAHU Star Ecoronics srl

Via G. Quarenghi 27 – 20151 Milano Tel 02 89540225 Fax 02/45479084

E-mail:info@starecotronics,it