prevention of pre-harvest aflatoxin production and the effect of different harvest times on peanut (...

Prevention of pre-harvest aflatoxin production and the effect of different harvest times on peanut(Arachis hypogaea L.) fatty acids

Öner Canavara,b* and Mustafa Ali Kaynaka

aDepartment of Crop Science, Faculty of Agriculture, Adnan Menderes University, Aydın, Turkey; bDepartment of Crop Science, Facultyof Agriculture and Horticulture, Humboldt Universität, Berlin, Germany

(Received 16 December 2012; final version received 31 May 2013)

The aim of this study was to investigate the relationship between aflatoxin and fatty acids and to determine the optimumharvest time to avoid pre-harvest aflatoxin formation. It was established that harvest time had statistically significant effectson the levels of saturated fatty acids: myristic acid (C14:0), palmitic acid (C16:0), heptadecanoic acid (C17:0), stearic acid(C18:0), arachidic acid (C20:0), behenic acid (C22:0), lignoceric acid (C24:0), monounsaturated fatty acids; palmitoleicacid (C16:1), heptadecenoic acid (C17:1), oleic acid (C18:1) and gadoleic acid (C20:1); and on polyunsaturated fatty acids:linoleic acid (C18:2) and linolenic acid (C18:3). By delaying the harvest time, the ratio of saturated fatty acids decreasedand unsaturated fatty acids increased. It was shown that the longer harvesting was delayed, the greater the quantity of oleicacid that was produced. Before harvest time, if the soil moisture was 5% or higher, aflatoxin was produced by fungi. It wasfound that the weather conditions of the region were suitable for aflatoxin production. Soil moisture appears to be moreimportant than soil temperature for aflatoxin formation. The production of aflatoxin was not observed in the first and secondharvests, both of which are at early harvest times. It was found that aflatoxin B1 during harvest time was the mostsignificant of the four toxins. The third harvest time, which is the most widely used, was observed to have significantproblems due to aflatoxin formation. Therefore, it is suggested as a result of this study that the harvest of peanuts must bedone considering seed yield before the middle of September to avoid aflatoxin formation at harvest time.

Keywords: aflatoxin; fatty acids; HPLC; harvest time; peanut

Introduction

Fungi, which are very useful microorganisms for humans,are used for the production of various antibiotics, vita-mins, enzymes, organic acid, alcohol, oil and animal feedproducts, and also to assist in the ripening of certainfoodstuffs. However, along with these beneficial aspectsthey also have a harmful side. Therefore, fungi are amongthe most hotly debated microorganisms today. Secondarymetabolites, which can have carcinogenic and mutageniceffects on humans and animals, are only the beginning oftheir negative health effects. In general, these compoundsare called ‘mycotoxins’ and are formed by fungi. Theeffect of fungi on human health comes in two ways.Disease, also known as mycosis, can occur with directcontact with fungi. Diseases caused by intoxication frommycotoxins are called mycotoxicoses. Aflatoxin is amycotoxin and is very important because it has an extre-mely high toxic effect on human health.

Although they are generally characterised as post-har-vest pathogens, the fungi Aspergillus parasiticus and A.flavus act as weak pathogens that colonise their hosts andproduce aflatoxin during periods of high temperature andlow soil moisture, when their hosts are highly stressed(Diener et al. 1965; Wilson & Payne 1994). Aflatoxin,which is a secondary metabolite, is produced by A. flavus,

A. parasiticus and A. nomius both pre-harvest and duringpost-harvest storage in tropical and subtropical regions(Nakai et al. 2008). A. flavus produces aflatoxin B1(AFB1) and aflatoxin B2 (AFB2), whereas A. parasiticusproduces B toxins and aflatoxins G1 (AFG1) and G2(AFG2). A. nidulans, which is not considered to be anagricultural threat, has been used as a model geneticsystem for studies on aflatoxin biosynthesis because itproduces sterigmatocystin, a toxic aflatoxin precursor.Among all classes of aflatoxin, AFB1 is known to be themost significant in terms of animal and human healthrisks. Therefore, raw peanuts now entering the EuropeanUnion must have less than 15 μg kg−1 of total aflatoxinsand no more than 8 μg kg−1 of AFB1 (Commission of theEuropean Communities, according to Decision 165/2010in 27/02/2010).

Peanuts (Arachis hypogaea L.) can produce energydue to their high oil, protein and fibre content. Thesecharacteristics lead the nuts to become sensitive to fungalcontamination, both pre- and post-harvest. As a result ofinappropriate processing and storage conditions, peanutsand peanut products may be contaminated with microor-ganisms. Cherry et al. (1975) and Deshpande andPancholy (1979) reported that changes to the chemicalmake-up of the peanut seed, such as the amount of

*Corresponding author. Email: [email protected]

Food Additives & Contaminants: Part A, 2013Vol. 30, No. 10, 1807–1818, http://dx.doi.org/10.1080/19440049.2013.818720

© 2013 Taylor & Francis

mailto:[email protected]

proteins, oil content and fatty acid components, are inevi-table. All these are significantly changed as a result offungal infection. Although there are a number of strategiesto reduce the entry of aflatoxin into the peanut chain, thereare also chemical treatments such as acetosyringone, syr-ingaldehyde and sinapinic acid (Hua et al. 1999), β-car-otene (Wicklow et al. 1998), 3-methyl-1-butanol, nonanol,camphene and linonen (Wright et al. 2000), propionic acid(Calori-Dominguez et al. 1996), eugenol (Bullerman et al.1977; Jayashree & Subramanyam 1999), and ammoniaapplications during post-harvest to reduce both fungalgrowth and toxin production. However, the World TradeOrganization (WTO) does not allow the chemical treat-ment of raw peanut seeds. There is no peanut cultivar orgerm plasma that may be resistant to the aflatoxin pro-duced by A. flavus, A. parasiticus or any other fungi.However, significant research and strategising have beencarried out to reduce aflatoxin and to prevent it from beingproduced without using chemical treatments. Manyresearchers have identified that aflatoxin is produced inpeanut seeds by A. flavus and A. parasiticus fungi in thefield during harvesting, drying, stacking and before sto-rage. Therefore, determining the best harvest time to mini-mise aflatoxin during peanut production is the beststrategy because it is a very practical method for all farm-ers to use and it is without the aid of chemical treatment.Klich (2007) found that A. flavus, which produces afla-toxin, was isolated from some plant parts and soil in allclimate zones. Generally, it is more prevalent at 26–35°latitude than it is in cold or tropical regions. Aydin pro-vince in Turkey, which is at 37–39° latitude, is a hotregion similar to a typical Mediterranean climate. Theaflatoxin problem is increasing in peanut fields aroundthis latitude. Hill et al. (1983) reported that if the airtemperature is 33–35°C and also, according to the reportfrom Cole et al. (1985), if the soil temperature is 25.7–26.3°C in peanut field areas, then these conditions are verysuitable for aflatoxin production. The temperature, soilhumidity and climate conditions of target peanut produc-tion areas are very useful in determining the best harvesttime. Also, fatty acids constantly change according to theposition of each pod in the plant, from formation of theseed until maturation. Peanut plants have a never-endinggrowth cycle (indeterminate) and, therefore, at harvesttime the seeds on a single plant have various levels ofmaturity that consistently affect the peanut composition interms of quality and flavour (Sanders et al. 1982). Thegoal is to find which conditions have the precise effects onthe fatty acid composition of peanut oil. The specific fattyacid composition must be known in order to produce avariety of products. Maturity has a significant effect onflavour potential in peanuts (Mozingo et al. 1991; McNeill& Sanders 1996; McNeill & Sanders 1998). Harveststudies show strong relationships between maturity andoverall peanut quality (McNeill & Sanders 1996;

Timmannavar et al. 2003). McNeill and Sanders (1996)noted that mature peanut pods have greater flavour poten-tial than those from immature pods. Horn et al. (2001)found that there is oxidative deterioration in the colour andodour of oil that can occur with each fatty acid, especiallylinoleic and linolenic fatty acids, which are able to formhydroperoxidase quickly and are the most important fattyacids. The aims of this study were: (1) to quantify therelationship of fatty acid and aflatoxin concentration; (2)to determine the amount of aflatoxin concentration atdifferent harvest times; and (3) to identify practical waysto reduce aflatoxin levels by altering harvest dates.

Materials and method

NC-7, which is a Virginia-type peanut cultivar, was cho-sen for the experiment because of its high market valueand the fact that it is widely used and the most frequentlygrown type of peanut in Turkey. The NC-7 seeds wereplanted on 5 May 2008 and 7 May 2009 at the CropScience Department of the Faculty of Agriculture atAdnan Menderes University, Aydin, Turkey (37° 39′ E27° 52′ N in the West Aegean Region of Turkey). Thefield experiments were conducted in a randomised designwith four replications. Before being planted, 49.5 kg ha−1

nitrogen (N), 49.5 kg ha−1 phosphate (P2O5), and49.5 kg ha−1 potassium (K2O) fertilisation was providedby applying 330 kg ha−1 of 15–15–15 fertiliser in the field.A second dose of N 49.5 kg ha−1 was provided with150 kg ha−1 of ammonium nitrate (33%) at blooming orthe second irrigation. Irrigation and weed control wereapplied to plots during the growing period when neces-sary. A total of 240 g l–1 spiromesifen was sprayed toprevent damage from Tetranychus urticae Koch on 4 July2008 and 20 July 2009. Plots consisted of four rows 8 mlong × 2.8 m wide. The harvest area was 19.6 m2 in eachparcel. Each plot was mechanically dug, inverted andallowed to air-dry in the field for 7–13 days before har-vest. The integrity and maturity of the pods were main-tained and they were placed in mesh bags to cure withambient air until mean seed moisture was 8–10%. Peanutpod mesocarp colours change with maturation from white(most immature) to yellow, orange, brown and black (mostmature). Peanuts were harvested in both years on thefollowing dates: 3 September, 16 September, 6 October,22 October and 6 November 2008, and 11 September, 24September, 8 September, 22 October and 7 November2009. The peanuts were harvested in accordance with the‘shellout (fruit shelled method)’, which is where the col-our of the peanuts within the shell is analysed to determinewhether the mesocarp colour is more than 60% brown orblack. This method is often used to find the most suitabletime to harvest peanuts (Williams & Drexler 1981; Pattee& Young 1982; Holbrook et al. 1989; Arıoğlu 1999). Theharvest time (when the colour of the mesocarp is at least

1808 Ö. Canavar and M.A. Kaynak

60% brown or black) was defined to be the control harvesttime. All pods from several border plants were removedby hand to obtain approximately 180–220 pods beforeeach harvest time, from the last day of August to the lastharvest time in December. In both years, the percentagesof brown and black-coloured mesocarps were found to be40%, 50%, 60%, 70% and 80% for the five harvest peri-ods. In order to keep plant density constant, an extra tworows were sown to determine a more accurate harvest timefor each parcel. To determine the correct harvest time, thismethod was used over 2-day intervals from late Augustuntil December.

Conditions of soil and climate of experiment area

The properties of the soil in the experiment area are shownin Table 1. A loam texture, high level of pH and loworganic matter content were found. Aydin province has atypical Mediterranean climate (Table 1). The climate datafor 2008, 2009 and the mean of several years’ worth ofdata are shown in Table 2. The amount of precipitation(mm) generally increased from September to November inboth 2008 and 2009 and the mean of multiple years (Table2). The temperature decreased from approximately 23.9°Cto 12.0°C in both 2008 and 2009 and the mean of multipleyears (Table 2). The mean relative humidity of Novemberwas higher than the values for September and October inall years.

Measuring soil moisture and soil temperature of theexperiment area

The soil moisture content was expressed by weight as theratio of the mass of water present to the dry weight of thesoil sample. The soil samples were 5–10 cm deep. Soil

moisture content was checked over 2-day intervals foreach replication (four times) from 1 September to thelast harvest time. After the wet soil samples were weighed,they were dried to constant weight in an oven at a tem-perature of 105°C for 24 h (Figures 7 and 8).

The moisture content in dry weight was calculatedusing the following formula:

%SM: wt of wet soil þ tareð Þ � wt of dry soil þ tareð Þ=ðwt of dry soil þ tareÞ � tareð Þ

Soil temperature was measured by a Rotronic HygropalmHP28 Probe (Hauppage, NY, USA) in 2-day intervals foreach replication from 1 September to the last harvest time.

Fatty acid analysis

The fatty acids of the peanut oil were measured using aGC-2010 gas chromatograph. Firstly, 0.1 g peanut oil,2 ml n-heptane, and 0.2 ml potassium hydroxide (KOH)were weighed and mixed. After this solution was mixedfor 30 s, there was a 30-min waiting period until the oilsubsided. A total of 100 µl were taken by micro-syringefrom the upper layer and injected into the GC. Thecolumn used was 25 m × 0.25 mm ID film thickness0.10 µm Permaband FFAP (Macherey-Nagel, Duren,Germany). Helium carrier gas was used at a linear velo-city of 1 ml m–1 and the split ratio was 40:1. The initialcolumn temperature was 290°C, air flow 400 ml s–1, H2

flow 40 ml s–1 held for 10 min. The temperature rampwas 5°C min–1 until 165°C, then held for 10 min. Thecolumn temperature was then increased by 2°C min–1

until 210°C. The fatty acid composition was calculatedby the area percentage of each peak (Reed et al. 2004).

Table 1. Soil properties of the experiment area.

Saturation (%) Structure (%) Total salt (%) pH CaCO3 (%) Organic material (%)

45.2 Clay 0.01 low 8.1 1.9 low 1.5 low

Table 2. Climate data for the experiment region in 2008 and 2009, and the climate mean of long time years (LT) during the growthhabit.

Months

Precipitation (mm) Temperature (°C) Relative humidity (%)

2008 2009 LT 2008 2009 LT 2008 2009 LT

May 17.2 17.6 35.4 21.1 21.2 21.0 47.0 49.4 56.8June – – 13.3 27.4 26.9 26.1 38.2 40.4 49.4July – – 3.3 29.0 30.6 28.5 37.0 39.4 49.5August – 9.5 2.4 29.3 30.4 27.4 44.5 41.5 54.0September 21.8 36.0 11.1 23.8 23.9 23.3 53.6 54.8 56.7October 27.0 20.0 42.5 18.6 21.2 18.5 60.5 59.1 63.5November 71.0 99.3 91.0 14.9 13.4 12.0 71.5 74.4 69.4

Note: LT = 1971–2008.

Food Additives & Contaminants: Part A 1809

Aflatoxin analysis

After each harvest was complete, seeds from all pods wereimmediately taken to a laboratory to be analysed so as toprevent post-harvest accumulation of aflatoxin. A total of0.5 kg peanut kernel samples for one replication was ran-domly taken from 5 kg peanut kernels gathered in onereplication throughout the harvest. Aflatoxins were extractedfrom a 50 g subsample already ground using a methoddeveloped by the immunoaffinity column provider (R-Biopharm Rhone Ltd, Glasgow, UK) according to AOAC999.07:2000 and (Magnoli et al. 2007). A 50 g portion of afinely ground sample was added to a 250 ml Erlenmeyerflask and mixed with a 150 ml methanol (Merck, Darmstadt,Germany) and 100 ml distilled water. The mixture wasblended for 5 min to obtain a homogeneous mixture andfiltered with Whatman No. 4 filter paper to remove particu-late matter. A 5 ml aliquot of the above extract was mixedwith 10 ml of distilled water and filtered through a micro-fibre filter. A total of 20 ml of either portion was taken andpassed through the immunoaffinity column. The column waswashed with 10 ml PBS containing 0.01% Tween 20, andthen with 10 ml double distilled water. A 20 µl sample wasinjected into the HPLC (Shimadzu, Tokyo, Japan).Fluorescence was recorded at excitation and emission wave-lengths of 362 and 425 nm, respectively, connected to a pre-column (C-18, 25 cm, 4.6 mm, 5 μm particle size) andquantified using post-column derivatisation using a KobraCell, operated at 100 μA (R-Biopharm Rhone Ltd). Thecolumn temperature was 40°C. The mobile phase waspumped at 120 μl min−1 potassium bromide and 100 µl nitricacid. Acetonitrile–methanol–water (2:3:6, v/v/v) were addedat a rate of flow of 1 ml min−1. The retention times of AFB1,AFB2, AFG1 and AFG2 were 6.3, 8.5 and 9.9 min, respec-tively, in a 20 min run time. Each sample was analysed threetimes.

Assay and recovery of aflatoxins B1, B2, G1 and G2

The recoveries were ascertained with the addition ofAFB1, AFG1, AFB2 and AFG2 to clean peanut seedsamples. A sample (50 g of milled peanut seeds) withoutaflatoxin was placed in a 250 ml Erlenmeyer flask andspiked with standard solutions of 8 μg kg−1 total aflatoxincontaining equal amounts of each aflatoxin componentand the recovery rates were determined. The extractionand clean-up procedures applied to the samples weresimilarly applied. The level of aflatoxin was determinedby HPLC. The mean of the recovery rest was determinedto be 85% with an RSD of 8%.

Statistical analysis

The study was conducted as a randomised block designwith four replications over 2 years. The statistical analysis

of aflatoxin concentration was not carried out for differentharvest times in both years because there was no aflatoxincontamination in the first and second harvests. The fattyacid data were analysed using SPSS by combining bothyears of randomised block designs. Analysis of variance(ANOVA) was applied to analyse the variance of aflatoxinconcentration and how it was affected by harvest timesover the 2 years. Significant differences between themeans were tested using Fisher’s least squares difference(LSD) method. All differences referred to in the test weresignificant at 0.05.

Results

The daily maximum and minimum temperatures, dailyprecipitation and daily temperature of 5–10 cm of soilbetween 1 September and 30 November, which were theharvest times of the experiment in 2008 and 2009, areshown in Figures 1–4. The highest maximum temperaturewas approximately 35°C and occurred from 5 to 15September (times are not given) (Figure 1). In particular,after the second harvest time in 2009 (4 September 2009),the daily temperatures were above 30°C for 1 week(Figure 1). The temperatures then suddenly decreased to30°C until the third harvest time. The daily minimumtemperature was between 4 and 22°C during the harvesttimes in both years (Figure 2). The daily maximum andminimum temperatures for both years were not shown tobe parallel. The daily minimum temperature was 4°C at itslowest value, and this occurred during November in 2009(Figure 2). The maximum and minimum temperaturesgenerally decreased throughout the harvest times in bothyears. It did not rain before the first harvest, which wasbetween 3 and 6 September in both years (Figure 3). Thenumber of rainy days and precipitation in 2009 was

Figure 1. Daily maximum temperature from 1 September to 30November in both 2008 and 2009. HT, harvest time.


greater than in 2008 (Figure 3). The day with the most rainfor each year was not on the same day. The highestprecipitation amount was 32 mm on 6 November 2009(Figure 3). There was a lot of rain from late October toNovember in 2008, unlike in 2009 (Figure 3). Althoughthe temperature of the 5–10 cm soil-depth samples fluctu-ated throughout the harvest time, their temperature gener-ally decreased from 35°C to 10°C until the end of

November (Figure 4). Generally, the appearance of theregion’s climate conditions was determined by the rainfall.Also, although there were differences in terms of the dailymaximum and minimum temperatures, precipitation andthe temperatures of 5–10 cm soil depth between 2008 and2009, these values were suitable for fungal functions.

There was a significant amount of change to fatty acidcomponents at different harvest times in both years.ANOVA results showed that in both years harvest timehad a significant effect on palmitic, palmitoleic, heptade-canoic, stearic, oleic, arachidic, gadoleic, behenic andlignoceric fatty acids, but not on myristic, linoleic andlinolenic fatty acids (Tables 3 and 4). The interaction ofharvest time and year was statistically significant in termsof palmitic, palmitoleic, heptadecenoic, stearic, oleic, lino-leic, linolenic, arachidic, gadoleic, behenic and lignocericfatty acids (Tables 3 and 4). The values of myristic,palmitic and behenic fatty acids significantly decreasedduring the harvest times. In contrast, the values of palmi-toleic, heptadecanoic, heptadecenoic, stearic, oleic andlinoleic fatty acids generally increased during the harvesttimes (Table 5). The values of linolenic, arachidic, gado-leic and lignoceric fatty acids increased until the third orfourth harvests, and then decreased in the fifth (Table 5).Many differences in fatty acid composition occurredbetween 2008 and 2009. The ratios of myristic, palmito-leic, heptadecanoic, oleic, gadoleic, behenic and lignoceric

Figure 2. Daily minimum temperature from 1 September to 30November in both 2008 and 2009. HT, harvest time.

Figure 3. Daily precipitation from 1 September to 30 Novemberin both 2008 and 2009. HT, harvest time.

Figure 4. Daily soil temperature of 5–10 cm-deep soil from 1September to 30 November in both 2008 and 2009. HT, harvesttime.

Table 3. Results of years-combined variance associated with fatty acids at different harvest times.

Variation source f.d. Myristic Palmitic Palmitoleic Heptadecanoic Heptadecenoic Stearic

Year 1 10.714* 3.430 1.600 75.000** 225.000** 1165.820**Error 1 6 0.001 0.017 0.001 0.001 0.001 0.001Harvest 4 1.278 42.245** 4.145* 17.737** 38.143** 42.291**Y × H 4 2.500 3.666* 5.418** 2.368 5.000** 16.092**Error 2 24 0.001 0.033 0.001 0.001 0.001 0.004

Notes: ** and *Significant at p ≤ 0.01 and 0.05, respectively.Y, year; H, harvest; f.d., degree of freedom.


fatty acids of 2008 were higher than those of 2009 (Table5). The most notable feature of the fatty acid profiles wasthe relative contribution of oleic and linoleic acids to the

total profile (show in Figures 5 and 6 as a percentage).Figure 5 shows that the values of oleic acid in 2008 werestatistically higher than those of 2009. Especially, it can be

Table 4. The results of years combined variance associated with fatty acids in different harvest time.

Variation source f.d. Oleic Linoleic Linolenic Arachidic Gadoleic Behenic Lignoceric

Year 1 251.851** 367.849** 6.400* 82.620** 32.517** 30.810** 29.000**Error 1 6 0.142 0.081 0.001 0.001 0.001 0.022 0.001Harvest 4 6.500** 1.003 2.433 4.339** 11.973** 13.373** 5.068**Y × H 4 5.435** 12.999** 4.100* 6.103** 5.300** 8.522** 7.669**Error 2 24 0.174 0.183 0.001 0.003 0.001 0.032 0.001

Notes: ** and *Significant at p ≤ 0.01 and 0.05, respectively.Y, year; H, harvest; f.d., degree of freedom.

Table 5. Fatty acid contents of peanut oil at different harvest time in both years.

Myristic Palmitic PalmitoleicC14:0 C16:0 C16:1

Harvest times 2008 2009 Mean 2008 2009 Mean 2008 2009 Mean

1 0.028 0.023 0.026 9.958a 9.618a 9.788 0.083b 0.073b 0.0782 0.028 0.020 0.024 9.248b 9.560a 9.404 0.090ab 0.090a 0.0903 0.020 0.020 0.020 8.800c 8.923b 8.861 0.100a 0.093a 0.0964 0.023 0.020 0.021 8.743c 8.850b 8.796 0.098a 0.098a 0.0985 0.018 0.020 0.019 8.910c 9.090b 9.000 0.100a 0.098a 0.099Mean 0.023a 0.021b 9.131 9.226 0.094 0.090LSD0,05 (Year) 0.007 (Year × Harvest) 0.266 (Year × Harvest) 0.012

Heptadecanoic Heptadecenoic StearicC17:0 C17:1 C18:0

1 0.048 0.038 0.043c 0.023c 0.020b 0.021 2.383d 2.770 d 2.5762 0.050 0.040 0.045c 0.030b 0.020b 0.025 2.493c 2.858cd 2.6753 0.053 0.050 0.051b 0.033b 0.030a 0.031 2.700b 3.010ab 2.8554 0.063 0.053 0.057a 0.043a 0.030a 0.036 2.643b 3.053 a 2.8485 0.068 0.050 0.059a 0.040a 0.030a 0.035 2.978a 2.933bc 2.955Mean 0.056a 0.046b 0.034 0.026 2.639 2.924LSD0,05 (Harvest) 0.005 (Year) 0.003 (Year × Harvest) 0.004 (Year × Harvest) 0.097

Linolenic Arachidic GadoleicC18:3 C20:0 C20:1

1 0.073a 0.070c 0.071 1.398b 1.513bc 1.455 1.230b 1.168b 1.1992 0.070a 0.073bc 0.071 1.510a 1.488c 1.499 1.303a 1.188ab 1.2453 0.078a 0.078bc 0.078 1.500a 1.613a 1.556 1.268ab 1.182ab 1.2254 0.070a 0.080ab 0.075 1.398b 1.580ab 1.489 1.182c 1.223a 1.2035 0.078a 0.088a 0.083 1.483a 1.460c 1.471 1.168c 1.198ab 1.183Mean 0.074 0.078 1.458 1.531 1.230 1.192LSD0,05 (Year × Harvest) 0.009 (Year × Harvest) 0.077 (Year × Harvest) 0.042

Behenic LignocericC22:0 C24:0

1 3.600b 3.382a 3.491 0.130c 0.133b 0.1312 3.927a 3.060bc 3.494 0.203a 0.128b 0.1653 3.503b 3.308ab 3.405 0.160b 0.168a 0.1644 3.012c 3.158abc 3.085 0.158b 0.168a 0.1625 3.102c 2.923c 3.012 0.158b 0.140b 0.149Mean 3.429 3.166 0.161 0.147LSD0,05 (Year × Harvest) 0.261 (Year × Harvest) 0.026


observed that oleic acid in peanut oil in 2009 increased atharvest time (Figure 5). However, after the second harvesttime this increase was slow and the values of oleic acid inthe third, fourth and fifth harvest times remained statisti-cally in the same group as in 2009 (Figure 5). Peanutseeds showed significantly lower levels of linoleic acidat the first harvest time compared with the other harvesttimes (Figure 6). The values of linoleic acid in the peanutoil obtained from each harvest time increased until thefourth harvest time, and then decreased in both years. Inparallel, the values of linoleic acid in peanut oil remainedstable in the third, fourth and fifth harvest times, like oleicacid in both years (Figure 6). It can be observed in Figure7 that in both years while the unsaturated fatty acids in thepeanut oil significantly decreased by delaying the harvesttime, the saturated fatty acids in the peanut oil increased.The increases in saturated fatty acids and decreases inunsaturated fatty acids in peanut oil during harvest timeoccurred especially markedly by R2 = 0.93 and 0.96 in2009 (Figure 7).

Figures 8 and 9 show the soil temperature and soilmoisture of the study area, which were measured over

2-day intervals from 1 September to the last harvest timein both years. Soil temperature varied between 16 and 30°C in 2008 (Figure 8), and varied between 16 and 34°Cduring the harvest time in 2009 (Figure 9). Soil moisturevaried between 1% and 11% in 2008 (Figure 8), andbetween 2% and 19% during the harvest time in 2009(Figure 9). Figures 8 and 9 show that the highest totalaflatoxin amount, which was produced by Aspergillus ssp.,was during the third harvest (control harvest time) in bothyears. Even though total aflatoxin production in 2008 wasobserved to be 28.06 μg kg−1 during the third harvest and3.50 μg kg−1 during the fourth harvest, it increased to90.59 μg kg−1 in the third harvest, to 2.66 μg kg−1 in thefourth and to 2.86 μg kg−1 in the fifth in 2009 (Figures 8and 9; not shown in Table 6). No aflatoxin production wasfound in the first and second harvests (earlier harvest time)in either year (Table 6 and Figures 8 and 9). Aflatoxinproduction in 2009 was observed to be higher than that of2008 during late harvest times. It can be revealed thatgenerally the soil moisture of 5–10 cm soil depth wasincreased by rainfall before the harvest time includingaflatoxin formed on peanut seeds (Figures 8 and 9). Thedata presented in this study indicate that AFB1 at26.16 μg kg−1 and AFB2 at 1.90 μg kg−1 in the thirdharvest, and AFB1 at 3.32 μg kg−1 and AFB2 at0.18 μg kg−1 in the fourth harvest were observed in

1.HT 2.HT 3.HT 4.HT 5.HT2008 28.583 28.653 29.48 29.968 29.107

2009 30.163 30.482 31.298 31.472 31.028

y = 0.2363x + 28.449R2

= 0.4142

y = 0.272x + 30.073R2

= 0.6068

27

27.5

28

28.5

29

29.5

30

30.5

31

31.5

32

Lin

olei

c ac

id (

%)

cbc

aa

ab

c

c

ab

a

b

Figure 6. Relationship of linoleic acid with harvest time in both2008 and 2009. HT, harvest time.

1.HT 2.HT 3.HT 4.HT 5.HT

2008 52.478 52.39 52.287 52.598 52.508

2009 49.748 50.003 50.908 51.178 51.243

y = 0.0268x + 52.372R2 = 0.1278

y = 0.4165x + 49.367R2 = 0.9019

4848.5

4949.5

5050.5

5151.5

5252.5

53

Ole

ic a

cid

(%)

a a a a a

b

b

a

a a

Figure 5. Relationship of oleic acid with harvest time in both2008 and 2009. HT, harvest time.

1.HT 2.HT 3.HT 4.HT 5.HTSaturated (%) 17.53 17.45 16.74 16.04 16.72Unsaturated (%) 82.47 82.55 83.23 83.96 83.28

y = –0.303x + 17.805R2

= 0.6133

y = 0.303x + 82.189R2

= 0.6169

07

142128354249566370778491

(%)

2008

1.HT 2.HT 3.HT 4.HT 5.HTSaturated (%) 17.47 17.15 17.09 16.88 16.61Unsaturated (%) 82.43 82.82 82.83 83.05 83.3

y = –0.199x + 17.637R2

= 0.9659

y = 0.197x + 82.295R2

= 0.938

07

142128354249566370778491

(%)

2009

Figure 7. Relationship of saturated and unsaturated fatty acidswith harvest time in both 2008 and 2009. HT, harvest time.


2008 (Table 6). By contrast, AFB1 at 85.86 μg kg−1 andAFB2 at 4.40 μg kg−1 in the third harvest, AFB1 at2.00 μg kg−1 and AFB2 at 0.66 μg kg−1 in the fourthharvest, and AFB1 at 2.20 μg kg−1 and AFB2 at0.66 μg kg−1 in the fifth harvest were observed in 2009(Table 6). It was observed in the chromatogram of HPLCthat the highest aflatoxin kind was AFB1, especially in the

third harvest (control harvest time) for both years (Figures10 and 11).

Discussion

The average amounts of each fatty acid for the five dif-ferent harvest times reported in this study are virtually

Figure 8. (colour online). Total aflatoxin (μg kg−1), soil temperature (°C) and soil moisture (%) in different harvest times in 2008.

Figure 9. (colour online). Total aflatoxin (μg kg−1), soil temperature (°C) and soil moisture (%) in different harvest times in 2009.


identical (Gorbet & Knauft 1997). These results are alsosimilar to the fatty acid profiles reported previously(Young et al. 1972; Pancholy et al. 1978). Significantdifferences were recorded for all fatty acids except formyristic acid. However, recently it has been reported thatmonounsaturated fatty acids were as effective as polyun-saturated fatty acids in the reduction of low-density lipo-protein cholesterol in humans (Mensink & Katan 1989).For this reason, the amount of heptadecenoic acid, gado-leic acid and oleic acid in oil should be elaborated duringseed ripening. The percentage of oleic acid was alsoassociated with an increase in the ratio of unsaturated tosaturated fatty acids and the percentage of mature peanutseeds increased by ripening. There are many chemical andenzymatic reactions during fatty acid synthesis such as

regulation, hydrolysis, elongation and desaturases. Thefatty acid composition of a plant cell is largely determinedby the activities of several enzymes that use these acyl-ACPs at the termination phase of fatty acid synthesis(Ohlorogge 1997). If the relative activities of theseenzymes that regulate the products of fatty acid synthesisare destroyed by any high or cold temperature, the ratio offatty acids can be easily changed. Of course, it is verydifficult to know at which stage the changes in the synth-esis of fatty acids occur in tissue. This study revealed thatthe ratio of mature seed was increased by ripening in adelayed harvest time, thus the flow of fatty acid occurredfrom unsaturated fatty acids to saturated fatty acids. Thisstudy also showed that the most abundant fatty acids inpeanut seeds are oleic, linoleic and palmitic acids. The

Table 6. Amount of aflatoxin (μg kg−1) in different harvest times in 2008 and 2009.

Harvest times

Aflatoxin B1 Aflatoxin B2 Aflatoxin G1 Aflatoxin G2

2008 2009 2008 2009 2008 2009 2008 2009

1.HT 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.002.HT 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.003.HT 26.16 85.86 1.90 4.40 0.00 0.00 0.00 0.334.HT 3.32 2.00 0.18 0.66 0.00 0.00 0.00 0.005.HT 0.00 2.20 0.00 0.66 0.00 0.00 0.00 0.00

Figure 10. Chromatogram containing aflatoxin B1 and B2 in 3.HT in 2008.

Figure 11. Chromatogram containing aflatoxin B1 and B2 in 3.HT in 2009.


result of this study showed a similar conclusion to thefindings of Hinds (1995) and Sanders (1980), who pointedout that the ratio of oleic acid increased with the delayingof the harvest. However, this study also showed that notonly does oleic acid increase with delayed harvest time,but also do palmitoleic, heptadecanoic and stearic fattyacids. Also, the amount of linoleic acid increased until thefourth harvest in both years. It was concluded that becauseof the high amount of oil contained in a peanut, the fattyacid component can deteriorate quickly due to lipid oxida-tion, depending on a number of factors, such as the pre-sence of oxygen, light, moisture, high temperatures andthe percentage maturation of seed. According to the find-ings of Gao and Kolomiets (2009), linoleic acid (C18:2)and linolenic acid (C18:3) are the major substrates forlipoxygenase (LOXs), and primarily catalyse the incor-poration of molecular oxygen (dioxygen) into C18:2 andC18:3. In addition, the evidence of Cherry et al. (1975)and Deshpande and Pancholy (1979) was that as a resultof fungal infection, changes in the chemical content ofpeanuts were inevitable. Therefore, it could be consideredthat changes in the levels of fatty acid composition ofpeanut seed oil caused the decrease in linoleic acid inthe fifth harvest. The decrease in the percentage of linoleicacid after the fourth harvest in 2008 was higher than thedecrease at the same time in 2009. As to the reason why, itcan be considered that there was a drought period at thattime in 2008. Dwivedi et al. (1996) stated that if there is ahigh temperature at harvest time, the ratio of linoleic andbehenic acids decreases, while the ratio of oleic and stearicacids increases. This is because, as Baydar and Turgut(1995) pointed out, the activities of oleoyl-PC desaturaseand linoleoyl-PC desaturase enzymes decrease at hightemperatures. Therefore, while the synthesis of linoleicacid decreases, the amount of oleic and stearic acidsincreases. Our results were in parallel to previous resultsreported by Kim and Hung (1991), which were that thesaturated fatty acids decreased with delayed harvesttimes, while unsaturated fatty acids increased withdelayed harvest times. The reason for this is that stearicand palmitic fatty acids are raw materials for the synth-esis of unsaturated fatty acids, and therefore saturatedfatty acids are transformed into unsaturated fatty acids(Baydar & Turgut 1995). Although only a few reportshave evaluated the effect of altered fatty acid composi-tion of peanut seeds on aflatoxin production, these datasuggest that plant-derived fatty acids did not play animportant role in regulating the colonisation of seeds byfungal pathogens and controlling pathogen developmentand mycotoxin production. These researchers concludedthat the effect of the products of the lipoxygenase path-way on aflatoxin biosynthesis in vitro might not be greatenough to affect pre-harvest aflatoxin contamination inpeanuts. Holbrook et al. (2000) pointed out that a fieldstudy comparing peanut genotypes of reduced versus

normal linoleic acid composition revealed no measurableeffect of these fatty acids on pre-harvest aflatoxin con-tamination. The addition of linoleic acid to the growthmedium has been reported to increase development and/or aflatoxin production by Aspergillus in some studiesand to reduce it in others (Fabbri et al. 1983; Calvo et al.1999). As a result of this study, it may be considered thatmany reasons and factors could have an effect on linoleicacid and fatty acids. It was considered that due to the lackof any aflatoxin production by fungus in the first andsecond harvests in both years, Davis and Diener (1969)and Vidhyasekaran et al. (1972) reported that there maybe high phytoalexin production to combat the fungalinfections in immature peanut pods. In addition,Blankenship et al. (1984) and Dorner et al. (1989)expressed that even when the soil temperature was23.6°C, which is suitable for aflatoxin production, theyfound that unless the soil moisture was 5% or more atharvest time there was no aflatoxin production, unlike inthe present finding. It was determined that aflatoxin pro-duction occurred by delaying the harvest time in bothyears similar to the present findings (Cole et al. 1985;Llewellyn et al. 1988; Barros et al. 2003; Timmannavaret al. 2003; Dorner 2008). Also because of the lowtemperature, rainfall and frost levels at the later harvesttimes, peanuts pods may be easily injured (Horn et al.1995). According to Horn et al. (2000), aflatoxin can bequickly produced by fungus in damaged seeds. After thepeanut pods were dug up at each harvest time, theyremained in the field for 10–15 days until the seedhumidity levels dropped to 8–10%. During that periodthere was a lot of precipitation and sudden fluctuations intemperature on a daily basis. The peanut pods weredirectly exposed to rain, and therefore aflatoxin concen-tration was increased due to the higher soil moisturelevels. The soil moisture was 5% or greater due to therain, and therefore aflatoxin concentration was greatest atthese harvest times. The result of this study was thataflatoxin production was greatest in the third harvest,and not in the fourth or fifth harvests, which is normalfor peanut fields at 37° 39′ latitude, like Aydin province.Although the other mycotoxins such as ochratoxin, zear-alenone, patulin, etc. were not analysed in the third,fourth and fifth harvests, the moulds of many fungiwere observed in this study. Even if the aflatoxin con-centrations of the fourth and fifth harvests were lowerthan that of the third harvest in both years, the lateharvest times could not be classified as the best harvesttimes in this study because of the high amount of mouldcontaining other mycotoxins. This study suggests that theharvesting of peanut pods should be done earlier so as toavoid significant aflatoxin production.

Reducing or eliminating pre- and post-harvest afla-toxin contamination in crops is a serious challenge facingscientists today. Aflatoxin contamination can be


minimised by an early harvest time. This will contributegreatly to achieving the goal of devising novel strategiesto eliminate pre-harvest aflatoxin contamination, resultingin a safer food and feed supply, as reported by Guo et al.(2009).

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