Study of Study of physicalphysical and and chemicalchemicalproperties of dried pistachios properties of dried pistachios as a as a sorting toolsorting tool of of aflatoxinaflatoxin

contaminated nutscontaminated nuts

M. M. GeorgiadouGeorgiadouaa, Ch. , Ch. GardeliGardeliaa, M. Komaitis, M. Komaitisaa, D.I. Tsitsigiannis , D.I. Tsitsigiannis bb, E.J. , E.J. Paplomatas Paplomatas bb and S.Yanniotis and S.Yanniotis aa

a a Agricultural University of Athens, Department of Food Science anAgricultural University of Athens, Department of Food Science and d Human NutritionHuman Nutritionb b Agricultural University of Athens, Department of Agricultural University of Athens, Department of Crop Crop ScienceScience 11

AflatoxinAflatoxin contamination in contamination in pistachiospistachios

AflatoxinAflatoxin : the most prominent group of : the most prominent group of mycotoxinsmycotoxinsNaturally produced as secondary metabolites Naturally produced as secondary metabolites of the fungi of the fungi AAspergillusspergillus sc sc FlaFlavivi4 4 substanessubstanes in pistachios: in pistachios: B1B1, B2, G1, G2, B2, G1, G2Exhibit acute and chronic toxicity, carcinogen Exhibit acute and chronic toxicity, carcinogen (affecting liver)(affecting liver)Tolerant limits have been established in Tolerant limits have been established in pistachios by authorities pistachios by authorities (Europe: 8ppb B1, 15ppb (Europe: 8ppb B1, 15ppb total)total)

22

x1000 toxic than pesticide

residues

A food safety issue A food safety issue ““beyond bordersbeyond borders”…”…

YearYear MycotoxinsMycotoxins AflatoxinAflatoxin Dried NutsDried Nuts PistachiosPistachios OriginOrigin

20020055 993993 949466 (95%)(95%) 828 828 (87%)(87%) 498 498 (60%)(60%) IranIran

20020066 877877 808011 (91%)(91%) 684 684 (86%)(86%) 225 225 (33%)(33%) IranIran

20020077 754754 705705 (94%)(94%) 556 556 (79%)(79%) 176176 (32%)(32%) Iran, Turkey, Iran, Turkey, LibanonLibanon, , U.S.A., SyriaU.S.A., Syria

20020088 931931 902902 (97%)(97%) 710710 (79%)(79%)

20092009 665665 638638 (96%)(96%) 518518 (81%)(81%) 136 136 (26%)(26%) Iran , Turkey, U.S.A.Iran , Turkey, U.S.A.

20102010 679679 649649 (96%)(96%) Iran, Turkey, U.S.A.Iran, Turkey, U.S.A.

RASFF : Border rejection notifications RASFF : Border rejection notifications

AflatoxinAflatoxin : the most : the most seriousserious chemical hazard for all chemical hazard for all pistachio pistachio produciveproducive countriescountries

AflatoxinsAflatoxins: The most : The most frequentfrequenthazard in dried nuts in Europehazard in dried nuts in Europe

mycotoxins

Source:RASFFSource:RASFF20092009

2011 2011 -- top 10 number of top 10 number of notifications notifications

HazardHazard Product categoryProduct category OriginOrigin NotificationsNotifications

11 aflatoxinsaflatoxins Feed materialsFeed materials IndiaIndia 8080

22 aflatoxinsaflatoxins Fruits & VegetablesFruits & Vegetables TurkeyTurkey 7575

33 aflatoxinsaflatoxins Nuts, nut productsNuts, nut products ChinaChina 6060

44 Salmonella Salmonella sppspp.. Fruits & VegetablesFruits & Vegetables BangladeshBangladesh 5454

55 aflatoxinsaflatoxins Nuts, nut productsNuts, nut products TurkeyTurkey 5353

66 migration of chromiummigration of chromium Food contact materialsFood contact materials ChinaChina 4848

77 migration of formaldehydemigration of formaldehyde >>>> ChinaChina 4545

88 living & dead mitesliving & dead mites Nuts, nut productsNuts, nut products UkraineUkraine 4343

99 aflatoxinsaflatoxins Herbs & SpicesHerbs & Spices IndiaIndia 4040

1010 aflatoxinsaflatoxins Nuts, nut productsNuts, nut products IranIran 3939

““ In a In a globalizedglobalized world, we all swim in the same hazardous seaworld, we all swim in the same hazardous sea……..””

PistachiaPistachia veravera var. var. AeginaAegina

Local Local greekgreek variety variety ((ChiChitzanidis, Anna, tzanidis, Anna, 2010. 2010. From From Asia to Asia to AeginaAegina, the story of the pistachio tree. XIV GREMPA Meeting on , the story of the pistachio tree. XIV GREMPA Meeting on Pistachios and Almonds, 94, 299Pistachios and Almonds, 94, 299--302)302)

Exceptional Exceptional flavourflavour and aroma due to and aroma due to ideal climate and soil compositionideal climate and soil composition

Aim to determine the production steps, conditions and /or to determine the production steps, conditions and /or handling practices that affect aflatoxin production in handling practices that affect aflatoxin production in greek pistachiosgreek pistachios

Goal to reduce aflatoxin within permitted limitsto reduce aflatoxin within permitted limits

Results

•• AflatoxinAflatoxin detected in detected in allall stages (prestages (pre--mature nuts and mature nuts and stored dried nuts)stored dried nuts)•• Above legal limits detected at Above legal limits detected at maturity stagematurity stage and and onwardonward•• Higher levelsHigher levels detected in samples coming from detected in samples coming from orchards with orchards with heavy insect damagesheavy insect damages

AflatoxinAflatoxin Reduction Strategy Reduction Strategy

G.A.PG.A.PPredictive models (climate, pest, cultivation data)Predictive models (climate, pest, cultivation data)Resistant crop linesResistant crop linesBiocontrolBiocontrol agents (e.g. nonagents (e.g. non--toxigenictoxigenic strains of strains of A. A. flavusflavus))Treatment with fungicidesTreatment with fungicides

PrePre--harvestlyharvestly: :

PostPost--harvestlyharvestly::G.H.P, G.S.PG.H.P, G.S.PDecontaminationDecontaminationDetoxificationDetoxification

SortingSorting

Sorting conceptSorting conceptOffOff--standardstandard pistachios most likely are infected pistachios most likely are infected (distorted, off(distorted, off--colored, insectcolored, insect--damaged etc.)damaged etc.)VariabilityVariability of contamination (few nuts with high of contamination (few nuts with high concentrations: 1 in 25.000 pistachios)concentrations: 1 in 25.000 pistachios)High analysis costHigh analysis costTime consuming methods Time consuming methods

Sorting methodSorting methodbased on the physical and chemical characteristics of based on the physical and chemical characteristics of

contaminated pistachios contaminated pistachios use of several technologies (P.E.F, UV, Laser, NIR, Xuse of several technologies (P.E.F, UV, Laser, NIR, X--ray ray

etc.)etc.)

Aim of the studyAim of the studyTo identify specific, distinguishable characteristics To identify specific, distinguishable characteristics that could lead to a sorting technique, applied for:that could lead to a sorting technique, applied for:

separation of healthy, intact pistachios from low separation of healthy, intact pistachios from low quality pistachios quality pistachios

rejection of rejection of aflatoxinaflatoxin contaminated or contaminated or ““suspicioussuspicious””nutsnuts

reduction of reduction of aflatoxinaflatoxin concentration in a lot within concentration in a lot within permitted legal limits permitted legal limits

Specific objectives of the studySpecific objectives of the study

Quantification of the physical and chemical Quantification of the physical and chemical properties of the local Greek variety properties of the local Greek variety Establishment of the standard values Establishment of the standard values Determination of the mentioned properties in Determination of the mentioned properties in

offoff--standardstandard and and aflatoxinaflatoxin contaminatedcontaminated samplessamplesInvestigation of any distinguishable Investigation of any distinguishable characteristics that could discriminate characteristics that could discriminate healthyhealthy, , offoff--standardstandard and and aflatoxinaflatoxin contaminatedcontaminated samplessamples

Experimental designExperimental designA. HealthyA. Healthy B. B. AflatoxinAflatoxin

contaminatedcontaminatednaturally contaminatednaturally contaminatedartificially >>artificially >>

C. Off standardC. Off standard

yellowish colored shellyellowish colored shell dark colored shelldark colored shell small distorted nutssmall distorted nutspartly hulled shellpartly hulled shell

insect hole on the shellinsect hole on the shell

AflatoxinAflatoxin analysisanalysisBoth subBoth sub--categories (naturally and categories (naturally and artificially contaminated) were analyzed for artificially contaminated) were analyzed for aflatoxinaflatoxin contamination contamination AflatoxinAflatoxin analysis conducted with HPLC analysis conducted with HPLC acc. to AOAC Official Methods for nuts acc. to AOAC Official Methods for nuts with IAC cleaning and UV with IAC cleaning and UV derivitizationderivitization..Every analysis replicated in threeEvery analysis replicated in three

526.5671.4± 64.724.3 ±6.875.0 ±3.93.5 ±0.8mean ±σNC-10

593.7352.7± 35.023.5 ± 0.9207.4 ±28.610.1 ±1.7mean ±σNC-9

831.2200.0± 40.612.6 ± 0.7320.4 ±55.15.5 ±1.5mean ±σNC-8

1047.5879.5± 77.09.7 ± 0.5158.3 ±34.3<LOQmean ±σNC-7

661.5504.1± 25.914.3 ± 7138.6 ±604.54±3.7mean ±σNC-6

774.2554.0± 49.933.4 ± 11.7197.3 ±41.99.9 ±5.5mean ±σNC-5

1143.31005.0± 29.410.9 ± 0.5127.4 ±20.6<LOQmean ±σNC-4

1015.5843.1± 4.618.8 ± 0.5152.3 ±7.31.3 ±0.2mean ±σNC-3

1187.31171.4± 26.011.6± 0.54.3 ± 1.5<LOQmean ±σAC-5

1653.21584.5± 7.060.2± 0.58.4± 0.7<LOQmean ±σAC-4

1018.61004.8± 17.77.3± 1.06.4± 1.1<LOQmean ±σAC-3

1387.51321.1 ± 56.160.6 ± 0.015.8± 0.4<LOQmean ±σAC-2

1164.31149.1±9.611.0±0.24.2 ±0.8<LOQmean ±σAC-1

600.1333.8± 7.615.7 ± 3.1244.9 ±25.15.8 ±0.1mean ±σNC-21088.7936.5± 80.96.1± 0.9146.1 ± 27.4<LOQmean ±σNC-1

TotalB1B2G1G2

Aflatoxin (μg/kg)

Sample

AflatoxinAflatoxin concentrationconcentration

NC: Naturally contaminated pistachios AC: Artificially contaminated with toxigenic strain of Aspergillus flavus

MethodologyMethodologyPhysical propertiesPhysical properties

Size Size (micrometer)(micrometer)

Weight Weight (electronic scale)(electronic scale)

Volume Volume (glass beads displacement)(glass beads displacement)

Density Density ((m/vm/v ratio) ratio)

Thermal conductivityThermal conductivity(k, (k, ΚΚDD2 2 Thermal Properties Analyzer) Thermal Properties Analyzer)

Seed textureSeed texture (compression, (compression, penetrationpenetration, , InstronInstron machinemachine))

Shell color Shell color ((colorimeter Minolta colorimeter Minolta CRCR--200200))

Chemical propertiesChemical propertiesMoisture Moisture (AOAC 925.40(AOAC 925.40))

Protein Protein (AOAC 950.48(AOAC 950.48))

Ash Ash (AOAC 923.03(AOAC 923.03))

Fat content Fat content (AOAC 948.22(AOAC 948.22))

Fatty acids Fatty acids (AFNOR, 1984(AFNOR, 1984))

Volatile profile Volatile profile (SPME, (SPME, GSGS--MSMS))

√(3)**5Artificially contaminated with non-toxigenicstrains of A.flavus

√(5)**

√(5)**

√(5)**

√(5)**

√(5)**

√(5)**

√(5)**

water content

Partly hulled nuts

Insect hole on the shell

Small distorted nuts

Dark colored shell

Yellow colored shell

Aflatoxincontaminated (naturally)

Aflatoxincontaminated (artificially)

Healthy

Sample category

√(3)**

√(3)**

√(3)**

√ (3)**√ (60-120)*

√(60)*

√(60)*

√(60)*

3

√(3)**

√(3)**

√(3)**

√ (3)**√(60)*

√(60)*

√(60)*

3

√(3)**

√(3)**

√(3)**

√ (3)**√(60)*

√(60)*

√(60)*

√(60)*

3

√(3)**

√(3)**

√(3)**

√ (3)**√(120)*

3

√(3)**

√(3)**

√(3)**

√ (3)**√(120)*

3

√(3)**

√(6)**

√(6)**

√(3**)

√(5)**

10

√(3)**

√(6)**

√(6)**

√(3)**

√(5)**

5

√(3)**

√(6)**

√(6)**

√(3)**

√(5)**

√(30)*

√(60)*

√(60)*

√(60)*

√(60)*

9

volatiles

fatty acids

fatashproteincolorbulk density

volume

weightdimensions

Chemical propertiesPhysical propertiesNumber of samples

Off-

stan

dard

* Number of pistachios per sample ** Number of replications

Statistical analysisStatistical analysisANOVA ANOVA (one(one--way and MANOVA)way and MANOVA)

Multivariate Statistical MethodsMultivariate Statistical MethodsPrincipal Component Analysis Principal Component Analysis DiscriminantDiscriminant Function Analysis Function Analysis Cluster AnalysisCluster Analysis

RESULTSRESULTS

Standard physical properties Standard physical properties var. var. AeginaAeginaSize:Size: Length = 19,6 ± 1,03 mm

Width = 10,5 ± 0,75 mmHeight = 11,2 ± 0,62 mmL/H = 1,8 ± 0,11

Weight:Weight: total= 0,97 ± 0,13 g (97 g/100 nuts)Seed= 0,5 ± 0,07 gShell = 0,4 ± 0,06 gSeed/total = 54%

VolumeVolume = 1,2 ± 0,20 mLDensityDensity = 0,8 ± 0,12 g/mLWater contentWater content = 5,8 ±0,12 g /100g

Shell color: Shell color: L= 67,6 ± 4,82C= 21,9 ± 3,17h = (83,4 ± 1,98)ο

Thermal conductivityThermal conductivity = 0,15 ± 0,02W/m oC

Texture:Texture:Penetration (F) = 109,9 ± 42,85 ΝDeformation = 0,8 ±0,4 mmModulus = 265,5 ± 53,3 ΜPa

Compression (F)= 144,2 ± 53,2 ΝDeformation =0,75 ±0,3 mmModulus = 43,7 ± 9,9 ΜPa

Moderate size pistachio acc. to Codex standardsModerate size pistachio acc. to Codex standardsOblong shapeOblong shapeDependence on water contentDependence on water contentVariability among regions of originVariability among regions of originDrying method and harvesting time affected shell Drying method and harvesting time affected shell colorcolor

Standard physical properties Standard physical properties var. var. AeginaAegina

sun driedhot-air dryerearly harvesting late harvesting

Differences between Differences between healthyhealthy and and offoff--standard standard pistachiospistachios

Partly hulled Partly hulled nutsnuts

Insect hole on Insect hole on the shell the shell

Small distorted Small distorted nutsnuts

Dark colored Dark colored shellshell

Yellow colored Yellow colored shellshell

HealthyHealthy

Sample category

aaccbb

bcbcbb

aaaab

bb

ddccbbccccaa

color (h)

bulk density

volumeweightdimensionsPhysical properties

ANOVA

Chemical propertiesChemical properties

a

a

a

a

a

a

a

ash

a

a

a

a

a

a

a

protein

Partly hulled Partly hulled nutsnuts

Insect hole on Insect hole on the shell the shell

Small distorted Small distorted nutsnuts

Dark colored Dark colored shellshell

Yellow colored Yellow colored shellshell

AflatoxinAflatoxincontaminatedcontaminated

HealthyHealthy

Sample category

abab

aa

abcabc

abcabc

bcbc

bcbc

cc

fat

Myristic C14:0Palmitic C16:0 TSPalmit-oleic C16:1 MUFAStearic C18:0 PUFAOleic C18:1 n-9 cis

Elaidic C18:1 n-9 trans MUFA/PUFALinoleic C18:2 n-9,12 TUFAA-Linolenic C18:3 n-9,12,15 TUFA/TSArahidic C20:0

Gondoic C20:1

Behenic C22:0

Fatty acids

ANOVA

Multivariate Multivariate StatisticalsStatisticalsPrincipal Component AnalysisPrincipal Component Analysis

6 components, 20 variables6 components, 20 variables

DiscriminantDiscriminant AnalysisAnalysis

MUFA, TUFA and MUFA, TUFA and linoleiclinoleic acid contributed most to acid contributed most to discriminantdiscriminant functionfunctionSamples correctly classified: 96.8%Samples correctly classified: 96.8%

aflatoxincontaminated

healthy

Volatile profileVolatile profileComparison between sample categories:Comparison between sample categories:

H (healthy)H (healthy)NC (naturally NC (naturally aflatoxinaflatoxin contaminated)contaminated)AC (artificially AC (artificially aflatoxinaflatoxin contaminated)contaminated)ANT (artificially contaminated with ANT (artificially contaminated with nonnon--toxigenictoxigenic strains of strains of A.flavusA.flavus) )

Identified volatiles:Identified volatiles: 7272

Chemical groups:Chemical groups: 12 12 (alcohols, (alcohols, ketonesketones, , aldehydesaldehydes, esters, hydrocarbons, acids, , esters, hydrocarbons, acids, monoterpenesmonoterpenes, , sesquiterpenessesquiterpenes))

MonoterpenesMonoterpenes the predominant group the predominant group (a(a--pinenepinene, a, a--terpinoleneterpinolene the main the main representatives) representatives)

Strongly related volatiles to Strongly related volatiles to aflatoxinaflatoxin contamination and contamination and A.flavusA.flavuspresence: presence:

C8 alcohols C8 alcohols –– ketonesketones -- aldehydesaldehydesSesquiterpenesSesquiterpenes ((γγ--cadinenecadinene, , bb--elemeneelemene, , ylangeneylangene))C7 C7 aldehydesaldehydes ((heptanalheptanal, heptenal_2) , heptenal_2) andand heptanolheptanol--11benzaldehydebenzaldehydebenzeneacetaldehydebenzeneacetaldehyde

DendrogramWard's Method,Squared Euclidean

0

200

400

600

800

1000D

ista

nce

H-1

H-2

H-3

H-4

H-5

H-6

H-7

H-8

NC

-1

NC

-2N

C-3

NC

-4N

C-5

NC

-6N

C-7

NC

-8

NC

-9

NC

-10

AC

-1A

C-2

AC

-3

AC

-4A

C-5

AN

T-1

AN

T-2

AN

T-3

AN

T-4

AN

T-5

Cluster AnalysisCluster Analysis Classification of 28 samples in 4 clusters

Contaminated with NonContaminated with Non--toxigenictoxigenic strains of strains of A.flavusA.flavus

aflatoxinaflatoxinContaminated Contaminated (Artificially)(Artificially)

aflatoxinaflatoxinContaminated Contaminated (Naturally)(Naturally)

HealthyHealthySample Sample categorycategory

Plot of Discriminant Functions

-5,8 -3,8 -1,8 0,2 2,2 4,2Function 1

-6

-4

-2

0

2

4

Func

tion

2

CLASSAC ANT H NC Centroids

Discrimination by the chemical group of all identified volatiles

SesquiterpenesSesquiterpenes, C8 groups and , C8 groups and monoterpenesmonoterpenes, , contributed most to contributed most to discriminantdiscriminant functionfunctionSamples correctly classified: 27 out of 28 (96,4%)Samples correctly classified: 27 out of 28 (96,4%)

(47,3%)

(32,

2%)

healthy

D

Plot of Discriminant Functions - 4 classes (C8 groups, sesquiterpenes)

Function 1 (58,75%)

Func

tion

2 (3

6,8%

)

-3,7 -1,7 0,3 2,3 4,3-1,9

0,1

2,1

4,1

6,1 CLASSAC ANT H NC Centroids

Discrimination by the C8 groups and sesquiterpenes

Samples correctly classified: 23 out of 28 (82,1%)Samples correctly classified: 23 out of 28 (82,1%)

healthy

EpilogueEpilogue

2929

separation of separation of healthyhealthy, intact pistachios from low , intact pistachios from low quality (quality (offoff--standardstandard) and rejection of ) and rejection of aflatoxinaflatoxincontaminatedcontaminated pistachios in a lot could be based on: pistachios in a lot could be based on:

Shell color (hue)Shell color (hue)Fat composition (unsaturated fatty acids, Fat composition (unsaturated fatty acids, linoleiclinoleic acid)acid)Volatile profile (Volatile profile (sesquiterpenessesquiterpenes, C8 alcohols, C8 alcohols--ketonesketones--aldehydesaldehydes))

Next steps:Next steps:Verification and validation of mentioned potentials in Verification and validation of mentioned potentials in ““realreal””market or processing conditions (rejected lots)market or processing conditions (rejected lots)Development and testing of novel technologies as sorting Development and testing of novel technologies as sorting tools (tools (e.g.labe.g.lab--onon--aa--chip sensor)chip sensor)

Thank you for your attention !Thank you for your attention !QuestionsQuestions……

ReferencesReferences[1] Smith E. J.: Aflatoxins in “Handbook of Plant and Fungal Toxicants” Ed. by J.P.Felix O’Mello, CRC Press New York , 1997, Ch.19, p.269-285. [2] Doster M.A. and Michailides T.J.: Relationship between Shell Discoloration of Pistachio Nuts and Incidence of Fungal Decay and Insect Infestation. Plant Disease, 83 (3), 1999, p.259-264.

[6] Munkvold, G.P.: Cultural and genetic approaches to managing mycotoxins in maize. Annual Review of Phytopathology 41, 2003, p.99-116.[7] Cotty, P.J. and Bayman, P.: Competitive exclusion of a toxigenic strain of Aspergillus flavus by an atoxigenic strain. Phytopathology 83, 1993, p.1283-1287.[8] Pearson T.C. and Schatzki: Machine Vision System for Automated Detection of Aflatoxin-Contaminated Pistachios, J.Agric.Food.Chem., 46, 1998, p.2248-2252.

[9] Pearson T.C., M.A.Doster, T.J.Michailides: Automated Detection of pistachio defects by machine vision” Applied Engineering in Agriculture, 17, 2001, p.81-84.[10] Magan, N. & Evans, P.: Volatiles as an indicator of fungal activity and differentiation between species and the potential use of electronic nose technology for early detection of grain spoilage. Journal of Stored Products Research, 36, 2000, p.319-340.[11] Yanniotis, S., Proshlyakov, A., Revithi, A., Georgiadou, M. & Blahovec, J.: X-ray imaging for fungal necrotic spot detection in pistachio nuts. Procedia Food Science, 1, 2011, p.379-384.

[3] Georgiadou M., Dimou A., Yanniotis, S.: Aflatoxin contamination in pistachio nuts: A farm to storage study. Food Control, 26, 2012, p. 580-586.[4] Olsen,M. 1999. Mycotoxin Prevention and Decontamination. Case study: Prevention of aflatoxins in pistachio. MYC-CONF/99/6d[5] Yu J., Payne G.A., Campbell B.C., Guo B., Cleveland T.E., Robens J.F., Keller N.P., Bennett J.W. and Nierman W.C.: MycotoxinProduction and Prevention of Aflatoxin Contamination in Food and Feed. Gustavo H.Goldman & Stephen A.Osmani «The Aspergilli», Mycology, 26, CRC Press Ch.27, 2008, p.457-472.