VETERINARYACADEMY
THE FACULTY OF VETERINARY MEDICINE
Gul Shahzeb
PRIESKONIŲ, AUGINAMŲ PAKISTANE, TARŠA
MIKOTOKSINAIS IR TARŠOS MAŽINIMO GALIMYBĖS
OCCURRENCE OF MYCOTOXINS CONTAMINATION IN
SPICES FROM PAKISTAN AND PREVENTIVE MEASURES
MASTER THESIS
of Full-time Studies of Food Sciences
The supervisor: Assoc. prof., dr. Violeta Baliukonienė Department of Food Safety and Quality
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4
CONTENT
SUMMARY ... 5 SANTRAUKA ... 6 ABBREVIATIONS ... 7 INTRODUCTION ... 8 1. REVIEW OF LITERATURE ... 101.1 Spices from Pakistan ... 10
1.2 Spices Contamination with Mycotoxins... 13
1.3 Preventive measures by improving the quality of spices ... 17
1.3.1 Regulation of aflatoxins and ochratoxins in spices ... 17
1.3.2 Methods for Prevention from Mycotoxins in Spices ... 17
1.2.3 Improvement measures for quality of spices ... 21
2. RESEARCH MATERIAL AND METHODS ... 23
2.1 Research material ... 23
2.2 Research methods ... 24
2.2.1 Determination of fungi colony-forming units per sample ... 24
2.2.2 Determination fungal genera ... 24
2.2.3 Determination of mycotoxins concentrations ... 24
2.2.4 Determination antifungal properties of spices extracts ... 26
2.2.5 Statistical analysis ... 27
3. THE RESULTS OF THE RESEARCH ... 28
3.1 Spices contamination by fungi ... 28
3.3.1 Total fungal count in spices from Pakistan... 28
3.3.2 Spices contamination with fungal genera from Pakistan ... 30
3.3.3 Spices contamination with aflatoxin B1, ochratoxin A of spices from Pakistan ... 32
3.3.4 Comparison of contamination by fungi and mycotoxins (aflatoxin B1, ochratoxin A) of spices samples from Pakistani and Lithuania ... 35
3.4 A comparison of the antifungal properties of ethanolic and aqueous spices extracts on Aspergillus flavus, Aspergillus niger, Penicillium chrysogenum and Penicillium viridicatum ... 36
4. DISCUSSION OF RESULTS ... 38
CONCLUSIONS ... 42
5
SUMMARY
Occurrence of Mycotoxins Contamination in Spices from Pakistan and Preventive Measures Gul Shahzeb
The Master Thesis
The supervisor: assoc. prof., dr. Violeta Baliukonienė
Place of research:Tthe research was carried out in 2018 – 2020, in Lithuanian University of Health Sciences, Veterinary Academy, Department of Food Safety and Quality, Mycotoxicology Laboratory. Main topic: The spices: black pepper, ginger, red chilli collected from Pakistan and Lithuanian markets. The spices were divided into groups: ground packed, ground packed, not ground unpacked, dry and raw.
Extent: 48 pages. The paper contains 9 tables and 6 figures. The objective of research
Evaluate the contamination of selected different spices with mycotoxins and to determine the potential for reduction of mycotoxin contamination.
Research tasks:
1. Evaluate and compare the contamination with fungal genera and mycotoxins (aflatoxin B1, ochratoxin A). raw and dry different spices from Pakistan markets.
2. Compare contamination by fungi and aflatoxin B1, ochratoxin A of spices samples from Pakistan and Lithuanian markets.
3. Determine the antifungal properties of spices (black pepper, ginger, red chilli) extracts in vitro. 4. Evaluate the use of preventive measures by improving the quality of spices from Pakistan.
Results and conclusion: Total fungal count ranged 2.27-3.34 log CFU/g in black pepper samples and dominated Penicillium spp.; in raw ginger samples on average - 3.276±0.829 logCFU/g and dominated Aspergillus spp. In the raw red chilli samples total fungal count ranged 0-5.091 logCFU/g. In raw and dry red chilli samples dominated Aspergillus spp. and other fungal genera. AFB1 concentration was determined 1.3-7 μg/kg in black pepper samples, in ginger samples - LOD ≥1-3 μg/kg and in red chilli samples – 1-3 μg/kg, the highest AFB1 concentration was determined - 7 μg/kg in ground, unpacked black pepper sample. OTA concentration was determined ≥1-2 μg/kg in black pepper samples, in ginger samples - LOD ≥1-4 μg/kg and in red chilli samples – LOD ≥1-4 μg/kg, the highest OTA concentration was determined - 4 μg/kg in raw ginger sample and in dry, ground, packed red chilli sample. Contamination by fungi of ground, packed red cilli samples from Pakistan and Lithuanian markets was similar (p>0.05). In spices samples from Pakistan and Lithuanian markets dominated Penicillium spp. AFB1 was found in all ground, packed spices samples from Pakistan markets, OTA was not detected; in red chilli samples from Lithuanian markets was detected low concentrations of AFB1 and OTA. The ethanolic red chilli extract presented activity on the A. flavus, A. niger, P. chrysogenum and P. viridicatum, when the aqueous spice’s extracts presented slight activity on the fungi species.
The spices quality can be improved by specific strategic preventive measures.
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SANTRAUKA
Prieskonių, auginamų Pakistane, tarša mikotoksinais ir taršos mažinimo galimybės Gul Shahzeb
Magistro baigiamasis darbas Darbo vadovas: doc., dr. Violeta Baliukonienė
Tyrimo atlikimo vieta: Tyrimas buvo atliktas 2018 – 2020m. Lietuvos sveikatos mokslų universitete, Veterinarijos akademijoje, Maisto saugos ir kokybės katedroje, Mikotoksikologijos laboratorijoje. Objektas: Prieskonių mėginiai (juodieji pipirai, raudonieji pipirai, imbieras) buvo surinkti iš įvairių prekybos vietų Pakistane ir Lietuvoje, buvo suskirstyti į 4 grupes: smulkintus supakuotus, smulkintus nesupakuotus, džiovintus nesmulkintus nesupakuotus ir šviežius neapdorotus.
Apimtis: 48 puslapiai. Darbą sudaro 9 lentelės ir 6 paveikslai.
Darbo tikslas: Įvertinti pasirinktų skirtingų prieskonių užterštumą mikotoksinais ir jų sumažinimo galimybes.
Darbo uždaviniai:
1. Įvertinti ir palyginti iš įvairių Pakistano prekybos vietų surinktų šviežių ir džiovintų prieskonių mėginių užterštumą pelėsiniais grybais ir mielėmis ir mikotoksinais (Aflatoksinas B1, Ochratoksinas A).
2. Palyginti iš Pakistano prekybos vietų surinktų prieskonių mėginių užterštumą pelėsiniais grybais ir mikotoksinais (Aflatoksinas B1, Ochratoksinas A) su Lietuvos prekybos vietose surinktų prieskonių mėginiais.
3. Nustatyti prieskonių (juodieji pipirai, raudonieji pipirai, imbieras) ekstraktų priešgrybelines savybes in vitro.
4. Įvertinti prevencinių priemonių panaudojimą norint pagerinti Pakistane pagamintų prieskonių kokybę.
Rezultatai ir išvados: Bendras pelėsinių grybų ir mielių skaičius juodųjų pipirų mėginiuose svyravo nuo 2,27 iki 3,34 logksv/g ir vyravo Penicillium spp., o neapdoroto imbiero mėginiuose nuo 3,276 iki 0,829 logksv/g ir vyravo Aspergillus spp. Šviežiuose neapdorotuose raudonųjų paprikų mėginiuose pelėsinių grybų ir mielių skaičius svyravo nuo 0 iki 5,091 log ksv/g. Neapdorotuose džiovintuose raudonųjų paprikų mėginiuose vyravo Aspergillus spp. ir kitų genčių pelėsiniai grybai. Juodųjų pipirų mėginiuose AFB1 koncentracija svyravo nuo 1,3 iki 7 μg/kg, imbiero mėginiuose nuo ≥1-3 μg/kg, raudonųjų pipirų mėginiuose nuo 1 iki 3 μg/kg. Didžiausia AFB1 koncentracija – 7 μg/kg buvo nustatyta smulkintame nesupakuotame juodųjų pipirų mėginyje. Juodųjų pipirų mėginiuose OTA koncentracija buvo nustatyta ≥1-2 μg/kg, imbiero mėginiuose - ≥1-4 μg/kg, raudonųjų pipirų mėginiuose - ≥1-4 μg/kg. Didžiausia OTA koncentracija – 4 μg/kg buvo nustatyta šviežiame neapdorotame imbiero ir smulkintame supakuotame raudonosios paprikos mėginyje. Smulkintų supakuotų raudonųjų pipirų mėginių užterštumas pelėsiniais grybais buvo panašus ir statistiškai patikimas tiek iš Pakistano tiek iš Lietuvos prekybos vietų (p>0,05), prieskonių mėginiuose vyravo Penicillium spp.. AFB1 buvo nustatytas visuose maltuose supakuotuose prieskonių mėginiuose iš Pakistano, o OTA nebuvo aptikta. Raudonųjų paprikų mėginiuose iš įvairių Lietuvos prekybos vietų buvo nustatytos nedidelės AFB1 ir OTA koncentracijos. Etanolinis raudonosios paprikos ekstraktas efektyviausiai sumažino A. flavus, A. niger, P. chrysogenum ir P. viridicatum pelėsinius grybus, o vandeninio ekstrakto poveikis nebuvo toks efektyvus.
Prieskonių kokybę galima pagerinti įgyvendinat pasirinktas specialiąsias prevencines priemones. Raktažodžiai: juodieji pipirai, imbieras, raudonieji pipirai, pelėsiniai grybai, Aflatoksinas B1, Ochratoksinas A
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ABBREVIATIONS
AFB1– aflatoxin B1 bw – body weight
CFU – colony forming units ºC – degrees Celsius
CV – Coefficient Variation
FAO – Food and Agriculture Organization DON – deoxynivalenol
ELISA – Enzyme Linked Immune Sorbent Assay HPLC – High Performance Liquid Chromatography LAB – Lactic Acid Bacteria
LOD – below the detection limit ND – not detected
mm – millimeters OTA – ochratoxin A
SDA – Sorbent Dextrose Agar SD – Standard Deviation SE – Standard Error
S&Q – Safety and quality spp. – species SPSS – Statistical Package for Social Sciences TLC – Thin-layer chromatography
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INTRODUCTION
The important group of agricultural vendible is the spices which are used all over the world for gastronomic purposes, that is, as an ingredient to flavor different types of prepared food items or drinks, as ingredients of medicine, perfumes, and incense, and as a condiment. They are good not only for our taste but also for our health [1]. Their contamination with fungus is one of the major problems that can affect the human well-being and also degrade the quality and taste of the spices [2].
Mycotoxins are actually referred to “poisonous substances” produced by fungi. Mycotoxins are naturally occurring toxins which are produced on the food such as cereals, nuts, dry fruits, spices and legumes. There are various types of mycotoxins such as Aflatoxins, Ochratoxins, Zearalenone Trichothecenes, Patulin and Ergot alkaloids [3]. But most important among are aflatoxins (B1, B2, G1 and G2) and ochratoxins. Most of the mycotoxins are carcinogenic and some may also have teratogenic and nephrotoxic affect. In Pakistan there are different types of spices which are red chili, green chili, black paper, ginger [1,4]. Most of spices are used for food on regular basis. But contamination with mycotoxins is a big problem. Fungi are basically a great hazard in this regard and source of contamination. In Pakistan the spices are grown in the fields on large scale in the fields and so air and soil is main inoculum source. There is also a problem of contamination during cultivation, harvesting, packaging and transportation. Due to bad handling the spices are exposed to external environment [5]. Trichothecenes and zearalenone have great tendency of contamination during hot and humid weather and from May to September the weather is suitable for such contamination due to high moisture in atmosphere [6-8]. When the spices are transported and due to refrigeration issue Alternaria has more chances to contaminate the spices [5]. Post-harvest decay is also another source of contamination of mycotoxins because there are number of species which contaminate spices during that period of time. But the fungi are not endemic to certain geographical areas and cause toxicity when the environmental conditions are suitable [9]. In order to control and prevention of contamination of mycotoxins it is important to control field infection by fungi of ginger and other spices. Also make a schedule for suitable pre-harvest, harvest and post-harvest. Storing places should be free from such mycotoxin contamination. By maintaining moisture and humidity level the chances of contamination can be reduced.
9 The objective of research
Evaluate the contamination of selected different spices with mycotoxins and to determine the potential for reduction of mycotoxin contamination.
Research tasks:
1. Evaluate and compare contamination with fungal genera and mycotoxins (aflatoxin B1, ochratoxin A) raw and dry different spices from Pakistan markets
2. Compare contamination by fungi and mycotoxins (aflatoxin B1, ochratoxin A) of spices samples from Pakistan and Lithuanian markets.
3. Determine the antifungal properties of spices (black pepper, ginger, red chilli) extracts in vitro. 4. Evaluate the use of preventive measures by improving the quality of spices from Pakistan.
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1. REVIEW OF LITERATURE
1.1 Spices from Pakistan
Spices are familiar constituents in almost all common dishes worldwide, on account of their properties such as flavor enhancer, color imparting, texture developer/improver, and palatability. According to some estimates, the global trade of spices is around USD 3 billion while the reported daily intake of spices by an adult US citizen of around 50 kg weight is 5 g [6]. Food and Agriculture Organization has reported that the annual production of spices in Pakistan is around 74.74 thousand tons [10].
Red chili is one of the main spices in Pakistan and makes about 85% of total production of country. It has mainly three types named Maxi, Desi and Nageena. The main mycotoxin contaminants for the red chili are Aflatoxin B1 [11]. Through HPLC whole (n=22) and powdered (n=22) were analyzed. Sixteen 73% and 19 (86.4%) samples of whole and powdered chilies, respectively were contaminated. Pakistan continues to remain among the top five producers in the world. Sindh is the largest producer of red chillies with annual production of 85.000 tons which amounts to 85% of the country’s produce. In, Sindh holds the position of top producer of red chillies, a spice liked for its strong pungent taste and red color [2]. It’s hot, pungent taste is owing to the presence of capsicum, a chemical substance that also quickens digestive process and possesses great medicinal value. The main varieties from Kunri are Desi, Mexi, and Nageena while Talhari, a winter variety is from Badin. Ghotki is from Ghotki and Khairpur and Sanam is cultivated in the outskirts of Karachi [1,2]. However, Dundicut or Loungi variety from Mirpur Khas which is round-shaped and is mainly used for chilli powder. The varieties grown in Pakistan are of high quality and clearly superior to other varieties grown in the region [12]. Pakistan has realized only a fraction of its potential as producer of red chillies. Despite better varieties, Pakistani red chillies fetch lower price than Indian varieties. Proper post-harvest handling and marketing of Sindhi red chillies are the need of the hour.
Green chili. In Pakistan green chili is a significant cash crop among vegetables. The crop occupies 19% of total area under vegetable cultivation [1]. And its production areas are mainly concentrated in Sindh (46917 ha) in Punjab (6400 ha), in Baluchistan (2082 ha) and in khayber pakhtun khwa (392 ha) [3,4]. Aflatoxin (B1) is main mycotoxin contaminant for chilies. Effects of winter and summer season in Pakistan on contamination of mycotoxin in chili were studied [13]. In winter, collected samples concentration was ranging from 0.00-52.30 µg/kg for pods and 0.00-74.60 µg/kg for ground samples.
11 Whereas in summer collected samples concentration was ranging from 0.00-61.50 µg/kg for pods and 0.00-95.90 µg/kg for ground samples [14]. So results reveled that aflatoxin contamination was more in summer. The crop is mostly cultivated in summer season throughout the country but in Sindh province the crop is also grown in autumn/spring season also [15]. Most of the surveys were conducted at flowering/fruiting stage. Aphids were observed during samples collection, feeding on the underside of the leaves mostly early in the morning that were identified as Aphis gossypii at Insect Pest Management Program, NARC, Islamabad. Chilli (Capsicum annuum L.) of family Solanaceae is both a vegetable and a spice crop of significant economic value in Pakistan [1]. Chilli is an important ingredient in day to day curries, pickles and chantries. It is very remunerative and brings good returns to the farmers. Chillies are produced seasonally but consumed throughout the year. The pods are marketed both in green and red forms [1,5]. There exists a great scope for its export. The pungency in chilies is due to an alkaloid capsaicin which has high medicinal value. The stem end of the pod has most of the glands that produce the capsaicin [16]. Though Pakistan exports are showing satisfactory trends, nowadays Pakistan is facing a very tough competition in the international export market as price of the Pakistan chili powder is considered high and other competing countries are providing chili at very competitive rates to the major importing countries [17]. The exports can be further improved, provided Pakistan is able to meet the strict quality demands of the international market [12]. As a leading producer of chilli crop in the world, India is also the largest exporter of chili in the world. It contributes one-fourth of the total quantity of chili exported in the world.
Ginger is one of the main spices in Pakistan and also used as herbal medicine. Firstly, ginger has been used since antiquity as food additives for the purpose of flavoring. Ginger is widely used as a food condiment or a natural supplement in Pakistan [3,4]. It can be marketed in many forms as fresh or dried product, in liquid or solid forms. Aflatoxins and ochratoxin are the main sources of contamination for ginger. Ginger is a horizontally thick underground stem (rhizome) covered by a delicate skin [17]. Considered as a native of south-east Asia, it is cultivated in Jamaica Island and other tropical areas [18]. It grows well at an elevation of 1,500 meters above the sea level. Green ginger, if dried for three days in the sun becomes ‘saunth’ on the fourth day. It is valued not only for the aromatic flavor but is also acclaimed in ayurvedic, tibbe-e-unani, allopathic, aromapathic and household remedies of our grannies in urban and rural areas [19]. Pakistan produces chillies, coriander, garlic, onion, and turmeric in commercial quantities but its ginger production is not enough to meet local requirements. Its large-scale commercial cultivation is limited to just 10 districts in upper, middle and lower Sindh [1,5]. Ginger is propagated by its stem rhizome cut into small pieces with one or two eye buds on each piece which
12 generates new shoots within 8-10 days after sowing. It can also be sown in flatbeds and on ridges, and the seed of which should not be planted deep. Ginger grows in semi-tropical and temperate zones [4]. High temperatures are desiccating and resulting in death of the seedlings. The plant requires sparse shade and can be intercropped with trees like mangoes, guavas and chikoos in fruit orchards. It takes about 10 months to remain in the field and starts showing withering signs from the 8th month which indicates that the crop is ready for digging. It is the only crop that can take a year and is not grown by small farmers [1,18]. The climatic conditions especially in Sukkur, Mirpurkhas, and Dadu are most favorable for ginger cultivation which can be further exploited by providing technology and incentives to the growers. It also contains iron, vitamin A, thiamine, riboflavin, niacin and vitamin C [19,20]. The special aroma in ginger is due to the oil present in it and the hot taste due to the resin found in the oil. Pakistan imports ginger to meet its domestic demand as the majority of people prefer cook meat, pulses and vegetables with it. In allopathic medicine, ginger is a stimulant, carminative, an expectorant, and promotes salivation. It provides relief in rheumatic pains, pulmonary, catarrhal, febrile diseases and neuralgia. Ginger relieves nausea, combats motion sickness, and reduces dizziness and flatulence.
Black paper (paper nigrum) is known as “king of spices”. And belongs to family piperaceae. It is widely used in Pakistani cuisines and also as herbal medicine. In Pakistan black paper grows at a large scale [21]. It is one of main ingredients of daily food. Mycotoxin contamination to this spice is when there is harvesting and during post-harvest period. Black pepper is produced from the still-green, unripe drupes of the pepper plant. The drupes are cooked briefly in hot water, both to clean them and to prepare them for drying [3,22]. The heat ruptures cell wall in the pepper, speeding the work of browning enzymes during drying. The drupes dry in the sun or by machine for several days, during which the pepper skin around the seed shrinks and darkens into a thin, wrinkled black layer. Once dry, the spice is called black peppercorn [22]. On some estates, the berries are separated from the stem by hand and then sun-dried without the boiling process. Once the peppercorns are dried, pepper spirit and oil can be extracted from the berries by crushing them. Pepper spirit is used in many medicinal and beauty products. Pepper oil is also used as an Ayurveda massage oil and in certain beauty and herbal treatments. Dried ground pepper has been used since antiquity both for its flavor and as a traditional medicine. Black pepper is the world's most traded spice, and is one of the most common spices added to cuisines around the world. Black pepper (Piper nigrum L.) the "King of Spices" is a universal table condiment. It is extensively used in Pakistani cuisines and herbal medicines and imported in bulk from neighboring countries [4,8]. The black pepper vine is generally cultivated by seed because other vegetative propagation methods are slow and time consuming [23]. Therefore, the tissue culture
13
technique is considered more efficient and reliable method for rapid and mass propagation of this economically important plant. The present study was initiated to develop protocol for micro-propagation of black pepper vine. The stem, leaf and shoot tip explant from mature vine were cultured on MS medium supplemented with different concentrations of plant growth regulators (2,4-D, BA, IBA) [20]. Best callus was produced on MS medium with 1.5 mg/l BA by shoot tip explant. This plant and its active component piperine can stimulate the digestive enzymes of pancreas, intestines and also increases biliary bile acid secretion when orally administrated. Some reports have been demonstrated that black pepper consumption in humans increased orofecal transit time [17,24]. Piperine prevents and minimizes diarrhea produced by various oil and chemicals and also reduces intestinal fluid accumulating in mouse intestine. The active agents of P. nigrum activates the epithelial cells in rat jejunum to permeates the uptake of various amino acids through the activation of membranes, enhance the production of proteins which are later used for the formation of cytoskeleton system due to surface adsorption property. This valuable specie also has the power to minimize different mutations like ethylcarbamte induced mutation in Drosophila. As compare to mutation, black pepper also reduced tumor formation in mice such as Ehrlich ascites tumor [ 9].
1.2 Spices contamination with mycotoxins
Aflatoxins (AF) are poisonous carcinogen that are produced by certain mold which grow in soil, decaying vegetation and grains. It is produced by Aspergillus flavus and A. parasiticus. Aflatoxins analysis on mycotoxin is not simple because of interference of high colored materials that are co-extracted with aflatoxins [20]. And currently analytical methods for determination of aflatoxins include Thin layer chromatography (TLC), High performance liquid chromatography (HPLC), and ELISA. Aflatoxins are a group of toxic metabolites produced by species of Aspergillus, specifically A. flavus, A. parasiticus and A. nomius, which were found worldwide in air and soil [13,25].
They are a significant threat to both human and animal health, because they are potent carcinogens, mutagens and teratogens. Aflatoxins are also classified as Group 1 carcinogens by the International Agency of Research on Cancer (IARC), primarily affecting liver [26]. Aflatoxins commonly found are aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), and G2 (AFG2). AFB1 is the most
potent of all aflatoxins known to date and is generally found in the highest concentration in food and animal feeds. AFB1 is the most prevalent among all aflatoxins types and represents 75% of all aflatoxins produced in fungal contaminated food commodities. Aflatoxins in various agricultural
14 products can be contaminated when drying of agricultural commodities is delayed or moisture level exceeds critical values for the mold growth during storage of the crops. Especially, spices are usually produced in countries with tropical climates that have high temperature, humidity and rainfall, length of storage and aeration conditions of storerooms [27,28].
Besides carcinogenicity, aflatoxins are also reported as teratogenic, hepatotoxic, mutagenic, growth retardant and immune suppressant [29,30].
Ochratoxins (OT) are a group of related compounds produced by Aspergillus ochraceus, Penicillium verrucosum, and other Penicillium species [31]. The most important toxin of the group is ochratoxin A (OTA). OTA has a significant economic impact on food commodities in that OTA producing fungi are found to be a contaminant in a wide variety of foodstuffs [24,32].
Temperate climate and secondly Aspergillus ochraceous which contaminate spices in tropical weather conditions like in Pakistan [33].
On analyzing OTA structure, it is found as a polyketide-derived secondary metabolite and contains a dihydrocoumarin moiety coupled to an l-β-phenylalanine (Phe), derived from the shikimic acid pathway, by an amide bond. Its chemical name is: l-phenylalanine-N-[(5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2-benzopyrane-7-yl)carbonyl]-(R)-isocoumarin [34].
It causes nephrotoxicity in all most all the mammalian species. This group contaminates spices like black papers, chilies and ginger in open fields, in dry conditions and in storage. Ochratoxin A is a kidney toxin, produced mainly by Penicillium verrucosum in temperate climates and Aspergillus ochraceus and the rare Aspergillus carbonarius in warm and tropical countries that can contaminate agricultural products prior to harvest or more commonly during storage. This compound has been shown to have nephrotoxic effects on all mammalian species and has been associated with fatal human kidney disease, referred to as Balkan Endemic Nephropathy and with an increased incidence of tumors of the upper urinary effect. OTA has immunosuppressive effect and inhibition of macromolecular synthesis, increased lipid peroxidation, and inhibition of mitochondrial respiration [31,32].
Ochratoxin A has been classified by the International Agency for Research on Cancer (IARC) as Group 2B, possibly carcinogenic to humans.
Both groups of mycotoxins can contaminate spices in the field, during drying and/or in storage. Ochratoxins have been found in a wide variety of agricultural commodities such as corn, wheat, barley, flour, coffee, rice, oats, rye, beans, peas, and mixed feeds, and are notably present in wine, grape juice, and , and are notably present in wine, grape juice and dry fruits [35].
15 OTA is a chemically stable compound; hence, ordinary food processing measures fails to substantially reduce its presence in foods and beverages.
Co-occurrence of AFs and OTA in spices have also been reported in several studies conducted in Pakistan. OTA contamination of black pepper, coriander seeds, powdered ginger and turmeric powder were estimated using indirect competitive ELISA.
All analyzed samples of ground paprika contained OTA above the maximum permissible concentration. The concentration range in the paprika samples was 2.0–19.1 μg/kg, in one of the samples the determined value was close to the highest acceptable level. In 37.5% of pepper samples (3/8) the concentration of OTA above the detection limit was determined, however, none of the samples exceeded the MPC marked OTA in 15 of 25 paprika samples (60%) at the range of 2.16–16.35 μg/kg. In the same study, 13.3% of pepper samples were contaminated with OTA in the range of 1.61– 15.85 μg/kg, while in the presented studies 3 per 8 samples (37.5%) were contaminated, in the range of 1.7–2.4 μg/kg [36]. The analysis of pepper samples carried out by Jalili and Selamat (2015) showed contamination with ochratoxin A in 40% of samples (8/20), ranging from 0.11 to 3.16 μg/kg [37]. In a study conducted by Ozbey and Kabak (2012) 17.4% of analyzed pepper samples (4/23) were contaminated with ochratoxin A ranging from 0.87 μg/kg to 3.48 μg/kg [38]. Other studies showed a contamination with ochratoxin A of 5.9 μg/kg in 1 of 20 pepper samples, where in the remaining samples, the OTA concentration was below the detection limit equal to 5.0 μg/kg [39].
Fumonisins (FUM). These are most important group of mycotoxins formed by the Fusarium verticillioides, F. proliferatum or Aspergillus niger. Such mycotoxins also contaminate spices during pre-harvest, harvest and post-harvest period. Most abundant fumonisins found in nature is Fumonisin B1 (FB1) which can cause esophageal and liver cancer. There are four main components which are FB1, FB2, FB3 and FB4 [40,41]. As the fumonisins appear to be non-genotoxic the possibility that they belong to another class of non-genotoxic carcinogens, the peroxisome proliferators, was investigated [27]. FB1 bears a clear structural similarity to the cellular sphingolipids, and this similarity has been shown to disturb the metabolism of sphingolipids by inhibiting the enzyme ceramide synthase leading to accumulation of sphinganine in cells and tissues. FB1 is neurotoxic, hepatotoxic, and nephrotoxic in animals, and it has been classified as a possible carcinogen to humans [27]. The cellular mechanisms behind FB1-induced toxicity include the induction of oxidative stress, apoptosis, and cytotoxicity, as well as alterations in cytokine expression. The effects of FB1 on different parameters vary markedly depending on what types of cells are studied or what species they originate from. These aspects are important to consider when evaluating the toxic potential of F11. Fumonisins are also occasionally
16 found in rice, black tea leaves, asparagus, pine nuts, and wine. The US Food and Drug Administration recommends that maize should not be used for human consumption at levels above 2–4 ppm total fumonisin while the European Union (EU) has a regulatory limit of 0.2–2 ppm in maize products [41].
Patulin. It is a secondary metabolite produced by a wide range of Aspergillus and Penicillium, Byssochlamys fungi. It is highly toxic following acute exposure and also has teratogenic effects. Patulin is a toxic, secondary metabolite most commonly associated with apple spoilage; however, its occurrence has been reported in a variety of fruits and agricultural products. This mycotoxin is produced by several fungal species that occasionally contaminate agricultural commodities [41].
Patulin contamination is a health risk to humans and reduces commodity values. Exposure to this mycotoxin is associated with a broad range of adverse effects, including gastrointestinal diseases and potential for carcinogenicity, and genotoxicity, immunotoxicity and neurotoxicity have been observed. Patulin is reactive under basic conditions and forms adducts through nucleophilic conjugation with thiol containing species, such as proteins and glutathione [36].
Trichothecenes may accumulate in infected crop plants, and upon ingestion, lead to the development of diseases (mycotoxicoses) in humans and animals [41]. Trichothecenes are one of the major classes of mycotoxins, causing a significant economic impact on cereal and grain crops each year. Trichothecenes are small, amphipathic molecules that can move passively across cell membranes. They are easily absorbed via the integumentary and gastrointestinal systems, allowing for a rapid effect of ingested trichothecenes on rapidly proliferating tissues. Exposure to these toxins can cause feed refusal, immunological problems, vomiting, skin dermatitis, and hemorrhagic lesions. They are also phytotoxic and can cause chlorosis, inhibition of root elongation, dwarfism, and act as a virulence factor in wheat head scab [41]. Trichothecenes are a family of over 200 toxins with a common tricyclic 1epoxytrichothec-9-ene (EPT) core structure. They have been classified into four groups (Types A, B, C, and D) based on the substitution pattern of EPT. Types A, B and C can be differentiated based on the substitution at the C-8 position. Type A trichothecenes include compounds that have a hydroxyl group at C-8 (e.g., neosolaniol), an ester function at C-8 (e.g., T-2 toxin), or no oxygen substitution at C-8 (e.g., trichodermin, 4,15-diacetoxyscirpenol, and harzianum A) [41].
17 1.3 Preventive measures by improving the quality of spices
1.3.1 Regulation of aflatoxins and ochratoxins in spices
The regulated limits for AF and AFB1 are 5–10 ng/g in most countries. In EU aflatoxins and ochratoxins (OTA) in spices regulatory limits are set at low levels (Table 1).
Table 1. Aflatoxins and ochratoxin A in spices regulatory limits
EU Commission Regulation No. 165/2010 No. 165/2010
Chillies, chilli powder, cayenne, paprika, nutmeg, ginger, turmeric, white and black pepper as well as mixtures of any of these individual spices
5 µg/kg for aflatoxin B1 and 10 µg/kg for total aflatoxins
15 µg/kg for ochratoxin A
liquorice - 20 µg/kg for ochratoxin A
The maximum permissible limit set in Pakistan for total aflatoxins in all foodstuff except milk and milk products is 20 μg/kg, while no limit is defined separately for AFB1 [42].
Exposure of consumers to these toxins can be compared with safety guidelines such as tolerable daily intakes (TDIs). The TDI of 0.11–0.19 ng/kg bw in Asia at 10–5 liver cancer risk is always referred for AF. For OTA, the TDI level is 1.2–14 ng/kg bw, as recommended by the Joint Expert Committee on Food Additives (98/53/EC) and the Scientific Committee for Food (European Commission No. 472/2002).
1.3.2 Methods for prevention from mycotoxins in spices
Physical treatment. After the crops have been harvested, drying, proper storage and suitable transportation of the commodities are of prime importance. Several favorable factors contribute to the growth of fungi, namely high moisture content, humid climate, warm temperature (25-40 °C), insect infestation and pes damage [16]. Many means and measures for the prevention of fungal contamination have been emphasized and practically done. Heating and cooking under pressure can destroy nearly 70% of aflatoxin in rice compared to under atmospheric pressure only 50% destroyed. Dry and oil roasting can reduce about 50-70% of AFB1. Ionizing radiation such as gamma-rays can stop growth of food spoilage organisms, including bacteria, molds and yeasts. It also inactivates pathogenic organisms including parasitic worms and insect pests [43]. It has been reported that gamma irradiation (5-10 M-rad) caused reduction of aflatoxins. The irradiation, however, could not completely destroy the toxin and its mutagenicity. Certain conditions such as moisture content, heat, ultraviolet or gamma irradiation, sunlight and pressure at different treatment-periods have been simultaneously combined
18 with the chemicals for the enhancement of detoxification. To design strategies for the reduction or elimination of mycotoxins, knowledge about their fungal sources are needed.
Physiological Methods
Prevention of mould growth: In stored grain, mould damage may be prevented mainly by three kinds of methods viz. drying of grain, controlled atmosphere storage and chemical treatment.
Drying of grain: It is an established fact that dry grain stores long, safe from insects and moulds because the requirements of moisture for their development are not met. The average Indian farmer perform drying of grain conventionally under direct sun light. The most widely used indigenous practice of grain drying is to spread threshed grains in thin layers on Kachcha floor with cow-dung in the open sun and stirring it by human labor till the grains are dried to safe level. Some farmers have also been used Pucca floors. Sun drying of grains on Kachcha surface was quicker as compared to Pucca surface, transparent polythene and black polythene. 5.30% loss in the moisture content of maize dried in sun-light could be achieved during 8.5 hours’ time. Exposure of aflatoxin contaminated groundnut oil to sun light has given very promising results as it destroyed about 99% of the aflatoxins. Other methods of grain drying include mechanical drying, in-bin drying, infrared, microwave or sonic and solar energy drying. Researches are being conducted to employ these methods also [36].
Controlled atmosphere storage: The significance of underground storage lies behind the philosophy of grain cooling and depleting the oxygen content to the desired level whereby the microbes and insects cannot grow. Air-tight storage also works on the same phenomenon where the depletion of oxygen by grain respiration manipulates disinfection by inhibiting aerobic fungi, elimination of mycotoxin production and conservation of desirable quality factors in the grain. Natural cooling is another effective method of preserving grain. The low temperature does not allow the microflora to grow as most of them are thermophilic [30].
Separation of infected grains: Physical separation of infected grains is an efficient and feasible method of minimizing mycotoxin contamination. This is affected either by manual operation or with the help of an electronic sorter. Fungal infection of seeds or grain usually imparts character color or other physical properties.
Detoxification: Cooking at atmospheric pressure can destroy about 50% of the toxins. Dry roasting and oil roasting of groundnut reduce aflatoxins to a significant degree. Cooking rice under 15 Ibs. pressure for 5 minutes gave maximum destruction of aflatoxins (72%) as compared to ordinary cooking or cooking with excess water. Light has also been employed successfully to destroy aflatoxin
19 in crude groundnut oil. Studies have shown that visible light is more effective than either ultra-violet or infra-red light [16,41].
Lactic Acid Bacteria (LAB): Due to their nutritional requirements, these bacteria are mainly divided into four genera: Lactococcus, Lactobacillus, Leuconostoc and Pediococcus. They are traditionally used as preservative agents to prevent spoilage and to extend the shelf life of food and feed.
According to, three mechanisms may explain the antimicrobial efficiency of LAB: the yield of organic acid, competition for nutrients and production of antagonistic compounds. Several species or subspecies such as Lactococcus lactis subsp. lactis, Lc. lactis subsp. cremoris, Lc. lactis subsp. diacetylactis, Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus curvatus are able to synthesize peptides or antimicrobial proteins known as bacteriocins, whose activity is only directed against closely taxonomically-related bacteria. Numerous studies have reported that these molecules are inactive against Gram-negative bacteria and eucaryotic microorganisms such as yeasts or mould. Moreover, the action of the antifungal properties of LAB on some mycotoxinogenic moulds have also been reported by a few authors [8]. As of this time, the main LAB recognized for their ability to prevent or limit mycotoxinogenic mould growth belong to the genera Lactococcus and Lactobacillus and, to a lesser extent, to Pediococcus and Leuconostoc. A 10-fold concentrated culture filtrate of Lb. plantarum 21B isolated from sourdough and grown in wheat flour hydrolysate was shown to possess an efficient antifungal activity against Penicillium corylophilum, Penicillium roqueforti, Penicillium expansum, Aspergilus niger, A. flavus and Fusarium graminearum [16].
Effects of Ultraviolet Radiations: The effects of two exposure times per day (6 and 16 h) of UV-A or UV-B radiation, combined with dark and dark plus light incubation periods during 7-21 days on fungal growth and mycotoxins production of Aspergillus species were studied. A. carbonarius and A. parasiticus were inoculated and media under diurnal and nocturnal temperatures choosing light photoperiod according to harvest conditions of these crops [16]. Ultraviolet irradiation had a significant effect on A. carbonarius and A. parasiticus colony size (diameter, biomass dry weight, and colony density) and mycotoxin accumulation. Inhibition of A. carbonarius fungal growth decreased when exposure time was reduced from 16 h to 6 h, but this was not always true for ochratoxin A production. OTA reduction was higher under UV-A than UV-B radiation and the reduction increased along time conversely to the aflatoxins (AFs). Aflatoxin B1 was the main toxin produced by A. parasiticus except in the UV-B light irradiated colonies which showed a higher percentage of AFG1 than AFB1 [21]. Morphological changes were observed in colonies grown under UV-B light. Ultraviolet (UV)
20 germicidal irradiation has been applied to the sterilization of agricultural products including stored grain for foodstuffs or animal feed. Although UV treatment is known to be effective for killing pathogenic moulds that contaminate the surface of grain, it remains unclear how and to what extent such irradiation is able to eliminate mycotoxins. The evaluation in vitro the effects of mild (intensity = 0.1 mW cm (-2) at 254 nm UV-C) and strong (24 mW cm (-2)) UV irradiation on two feed-contaminating mycotoxins, zearalenone (ZEN) and deoxynivalenol (DON) has been done. When exposed to mild irradiation, the levels of ZEN and DON (both 30 mg kg (-1) initially) were reduced as irradiation time increased, and became undetectable at 60 min. Strong UV irradiation also reduced the mycotoxin levels in the same time-dependent manner, but more rapidly. It was therefore confirmed in vitro that UV irradiation is effective at reducing the levels of ZEN and DON [12].
Effects of Temperature: The main environmental factors influencing grain fungi and mycotoxins are temperature and aw. Aflatoxin preharvest Heat (e.g. >25 °C) and drought dangerous but humidity
dangerous for cottonseeds. Rain at dry-down dangerous and Cool temperatures safe (e.g. <20 °C). Dry at grain fill and rain/humidity dangerous at other times and cool temperatures and low rain are safer, apart from at grain fill. For Ochratoxin A high temperate dangerous (towards 30 °C). Dry conditions problem on occasions and <21 °C at is safe. As Ergot is concerned <15 °C. Optimum day mean 25 °C, humidity 96%, Max 28 °C, night humidity 86% and sowing is between Sowing June 15 to July 14 [41].
Chemical Methods
Ammonia: This process involves ammonia and almost all the mycotoxins are degraded by ammonia at high pressure and ambient temperature. The efficacy of mycotoxins detoxification by ammonia is positively correlated with ammonia used, time, temperature and pressure.
Ozone: Practical methods to degrade mycotoxins using ozone gas (O3) have been limited due to
low O3 production capabilities of conventional systems and their associated costs. It is possible that the
rapid delivery of high concentrations of O3 will result in mycotoxin degradation in contaminated
grains-with minimal destruction of nutrients. The major objectives of such studies were to investigate the degradation and detoxification of common mycotoxins in the presence of high concentrations of O3. In
this regard, aqueous equimolar (32 μM) solutions of aflatoxins B1, AFB2, AFG1, AFG2, cyclopiazonic acid (CPA), fumonisin B1 (FB1), ochratoxin A (OA), patulin, secalonic acid D (SAD) and zearalenone IZEN) were treated with 2, 10 and/or 20 weight % O3 over a period of 5.0 min and analysed by HPLC.
Results indicated that AFB1 and AFG1 were rapidly degraded using 2% O3, while AFB2 and
AFG2 were more resistant to oxidation and required higher levels of O3 (20%) for rapid degradation.
21 detectable by HPLC. Degradation of FB1 did not correlate with detoxification, since FB1 solutions treated with O3 were still positive in two bioassay systems [43].
Nixtamalization: Traditional nixtamalization and an extrusion method for making the dough (masa) that requires using lime and hydrogen peroxide were evaluated for the detoxification of aflatoxins. The traditional nixtamalization process reduced levels of aflatoxin B1 by 94%, aflatoxin M1 (AFM1) by 90% and aflatoxin B1 -8,9-dihydrodiol (AFB1 -dihydrodiol) by 93%. The extrusion
process reduced levels of AFB1 by 46%, AFM1 by 20% and AFB1 -dihydrodiol by 53%. Extrusion treatments with 0, 0.3 and 0.5% lime reduced AFB1 levels by 46, 74 and 85%, respectively. The inactivation of AFB1, AFM1 and AFB1 -dihydrodiol in the extrusion process using lime together with hydrogen peroxide showed higher elimination of AFB1 than treatments with lime or hydrogen peroxide alone. The extrusion process with 0.3% lime and 5% hydrogen peroxide was the most effective process to detoxify aflatoxins in corn tortillas, but a high level of those reagents negatively affected the taste and aroma of the corn tortilla as compared with tortillas elaborated by the traditional nixtamalization process [43].
1.3.3 Improvement measures for quality of spices
To improve the quality of spices the whole process from cultivation to marketing should be monitored. The crops should be cultivated at proper time and the pods are well ripened and partially withered at plant itself they should have superior pungency and color retention properties. Post-harvest decay and all aspects should be monitored. Storage should be proper and temperature for keeping in stores should be maintained. Drying of the spices should be on the clean surfaces s that to void contamination by all means [16]. Refrigerated transportation is required which must be quite proper and meet the needs of temperature during transportation. For packaging new and clean bags are required which should be safe from any kind of unhygienic measures. And the most important aspect is to avoid humidity and excessive moisture because mycotoxins contaminate in hot and humid environment. Some scientific approaches like micropropagation, somaclonal variation, in vitroconservation, synseed technology, protoplast fusion, production of flavor and coloring components and development of novel transgenics have great potential in conservation, utilization and increasing the production of spices. Efficient micropropagation systems are available for many spices which are being used for propagation, conservation, safe movement and exchange of germplasm, crop improvement through soma clonal variation and transgenic pathways. Studies on the production of metabolites, flavor and coloring compounds using immobilized and transformed cell cultures are
22 being attempted. Molecular markers and maps are being generated for crop profiling, fingerprinting, identification of duplicates, and marker-assisted breeding. Transcriptome sequencing is becoming an important tool for identification, isolation and cloning of useful genes. Bio-technological approaches involving microbials (antagonists/hyper parasites and PGPRs) with broad spectrum of disease suppression and growth promotion have been found effective in crop health management in spice crops [43].
23
2. RESEARCH MATERIAL AND METHODS
2.1 Research material
The research work was conducted in 2018 – 2020, in Lithuanian University of Health Sciences, Veterinary Academy, Department of Food Safety and Quality, Mycotoxicology Laboratory.
In April-May 2019 the dry ground packed spices (black pepper, ginger, red chilli) samples (n=4) were collected from Lithuanian markets (Table 2).
Table 2. Spices from Lithuanian markets
Spice name Numbers of samples Description of sample
Black pepper 1 ground packed
Ginger 1 dry ground packed
Red chilli 2 dry ground packed
In May-August 2019 the raw and dry spices (black pepper, ginger, red chilli) samples (n=35) were collected from Pakistan markets. The samples from Pakistan markets were divided in 1) raw, 2) dry not ground unpacked, 3) dry ground unpacked and 4) dry ground packed (Table 3).
Table 3. Spices from Pakistan markets
Spice name Numbers of samples Description of sample
Black pepper
5 dry not ground
unpacked
2 ground unpacked
3 ground packed
Ginger
5 raw
2 dry ground unpacked
3 dry ground packed
Red chilli
5 raw
5 dry not ground
unpacked 3 dry ground, unpacked
2 dry ground packed
The samples were taken at random from Lithuanian and Pakistan markets. The unpacked samples from Pakistan markets (weight 30-50 g) were placed in polyethylene bags.
The samples were frozen immediately at – 20 ºC and kept until the beginning of the laboratory analyses.
24
2.2 Research methods
The following studies were performed:
• Mycological studies (fungi colony-forming units per sample, fungal genera). • Determination of mycotoxins concentrations by TLC (Romer Labs Inc. ®Method). • Determination antifungal properties of spices extracts.
2.2.1 Determination of fungi colony-forming units per sample
Dilution method according to standard Microbiology of food and animal feedings stuffs — Horizontal method for the enumeration of yeasts and moulds: Part 2: Colony count technique in products with water activity less than or equal to 0.95 (ISO 21527–2:2008) was used to determine total fungal count (CFU/g) in spice samples, in triplicates.
Ten grams of each sample were added to 90 ml portion of sterile distilled water in 250 ml Erlenmeyer flask and homogenized thoroughly on an electric shaker RS-OS 20 (Germany), at constant speed for 20 min. Tenfold serial dilutions (10-1 to 10-3) were then prepared. One milliliter from each dilution series was uniformly dispensed under the surface 15 ml Sabouraud Dextrose Agar (SDA) (Oxoid) fortified by 0.5 mg chloramphenicol/ml (Sigma) in Petri dishes and incubated at 27±1 ºC for 5–10 days and examined for the growth of fungi.
Fungi colony-forming units per sample (CFU/g) was calculated: CFU/g = (no. of colonies x dilution factor) / volume of culture plate. CFU/g were converted into log (CFU/g).
2.2.2 Determination fungal genera
The fungal morphology was studied macroscopically by observing the colony features (color, shape, size and hyphae). Fungi were identified according to morphological and microscopic characteristics [44,45].
2.2.3 Determination of mycotoxins concentrations
Concentration of aflatoxin B1 was analyzed by Thin-layer chromatography (TLC) and described by Romer Labs Inc. ®Method (Code: a/z-tl-01-00.2) and Ochratoxin A - Romer Labs Inc. ®Method (Ota_tlc_my_060307).
25 Chemicals: Acetic acid, glacial (100%, p.a.), Sulfuric acid (p.a., 95-97%), Aluminum chloride (Merck, Germany), Acetonitrile (CHROMASOLV® HPLC, >99.9%), Methanol (CHROMASOLV® HPLC, >99.9%), Acetone (CHROMASOLV®, HPLC, ≥99.8%), Toluene (CHROMASOLV® HPLC, >99.9%) (Sigma Aldrich, Germany), Deionized water.
Equipment: grinder Bosch TSM6A013B (Germany), shaker RS-OS 20 (Germany), Romer®Evap - system (Romer Labs, Inc., USA), Auto SpotterTM, Model 10 (Romer Labs, Inc., USA).
Consumables: clean-up columns MycoSep®226 AflaZon+, MycoSep®229 Ochra (Romer Labs Diagnostic GmbH, Austria), Biopure Aflatoxin B1 (2 µg/ml acetonitrile) standard (Romer Labs Diagnostic GmbH, Austria), Biopure Ochratoxin A (10 µg/ml in acetonitrile) standard (Romer Labs Diagnostic GmbH, Austria), Silica Gel 60 F254 TLC Plates (Romer Labs Diagnostic GmbH, Austria), 100 μl syringes, Whatman No 4 filter paper (Whatman, Inc., Clifton, New Jersey, USA).
All samples of spices were dried at 60 °C for 24 h in an oven and were ground.
Extract 12.5 grams of each sample with 50 mL acetonitrile/DI water (84/16, v/v) shake for 2 h. The extract was filtered through Whatman No 4 filter paper.
Determination of Aflatoxin B1 (AFB1) concentration. 45 μL of glacial acetic acid was added to 4.5 mL of the filtrate and mixed well. 4.5 mL of the filtrate was pushed through clean-up column MycoSep® 226AflaZon+ (following the methodology supplied by the manufacturer). After purified 2 mL extract was evaporated to dryness under vacuum in a 60 °C. Residue was dissolved with 300 µL toluene/acetonitrile (97/3, v/v). 80 μL of sample and AFB1 spotting standard: 0.05 μg/mL (10, 20, 40, 80 μL) were spotted onto the silica gel TLC plate with the AutoSpotter. The matrix was prepared by adding 48 μL of AFB1 spiking standard (0.625 μg/mL each of AFB1) to 9 mL of 84/16 v/v acetonitrile/water. Plate was developed in 9/1 (v/v) chloroform/acetone until solvent front was 1 cm from the top of the plate. The plate was dried in the air. The plate was viewed under a long wave of UV light. AFB1 appeared blue and has a Rf (retention factor) of approximately 0.45. Estimate toxin in samples and spikes compared to the standards. AFB1 concentration in sample was calculated by Romer Labs Inc. ®Method.
Determination of Ochratoxin A concentration. 70 μL of glacial acetic acid was added to 7 mL of the filtrate and mixed well. The filtrate was pushed through clean-up column MycoSep®229 Ochra (following the methodology supplied by the manufacturer). After purified 4 mL extract was evaporated to dryness under vacuum in a 60 °C. Residue was dissolved with 400 µl of 99/1 v/v toluene/acetic acid. 100 µL of each sample and spike along with 10, 30 and 50 µL of 1 µg/mL Ochratoxin A working standard were spotted onto a silica gel TLC plate with the AutoSpotter. Plate was developed in 18/1/1
26 v/v/v toluene/methanol/acetic acid until solvent front was 1cm from the top of the plate. The plate was dried in the air. The plate was viewed under a long wave of UV light. OTA appeared blue - green with a Rf of approx. 0.6. Estimate toxin in samples and spikes compared to the standards. OTA concentration in sample was calculated by Romer Labs Inc. ®Method.
The detection limit was 1 μg (ppb) of AFB1, OTA – 1 μg (ppb).
2.2.4 Determination antifungal properties of spices extracts Used in the study: black pepper, ginger, red chilli.
Production of plant extracts by solutions of different polarity. Extracts were prepared by maceration using aqueous and ethanol solvents. Dried ground black pepper, ginger, red chilli (30 g) were macerated with 30 mL of was purified water and ethanol (96%) for 48 hours at room temperature (24±2 °C), before material was extracted for 1 hour at 24±2 °C by mechanical shaker. The extracts were filtered through Whatman No 1 filter paper. The filtered extracts were stored at 4 °C.
Fungi Aspergillus flavus, A. niger, Penicillium chrysogenum and P. viridicatum were isolated from the tested spices samples.
Antifungal Screening Test. Antifungal activity of examined ethanolic and aqueous black pepper, ginger and red chilli extracts against A. flavus, A. niger, P. chrysogenum and P. viridicatum were measured as diameter of growth of the test fungi (mm) [46]. 1 mL of ethanolic and aqueous base plant extract was dispensed into each extract labeled Petri dish. Sabouraud Dextrose Agar (SDA) (Oxoid) cooled at 45 ºC and 15-20 mL was dispensed separately into each of the plates including the plates without plant extract and ethanol, purified water. Each plate was swirled gently in a clockwise and anticlockwise motion to mix the extracts with the SDA and allowed to solidify. A streak of the pure cultures of the test fungi were then transferred unto the Petri dishes containing the SDA and the plant extract using a sterile inoculation needle. The plates were covered and incubated at temperature at 27±2 ºC. After 2-5 days the diameter growth of the test fungi was measured using calipers and expressed in millimeters.
As positive controls using ethanol and purified water only with three plates each corresponding to each test fungi. As negative controls without any antimicrobial agent (plant extract) nor ethanol and purified water with three plates corresponding to each test fungi.
27 2.2.5 Statistical analysis
The data were analysed using the SPSS version 20.0 (IBM Corp., NY, Armonk) and “Microsoft Office Excel 2010” calculating the mean of values (X), standard error (SE) standard deviation (SD), coefficient of variation (CV). Statistical analysis of the data was carried out using the T test. The data was considered to be reliable from the statistical point of view when p<0.05.
28
3. THE RESULTS OF THE RESEARCH
3.1 Spices contamination by fungi
3.3.1 Total fungal count in spices from Pakistan
The samples of spices were analyzed to determine the contamination level of fungi: total fungal count and fungal genera.
The determination of total fungal count in spices were determined which spices samples were most infected and which were least infected, and the average level of total fungi colony-forming units per sample in all tested samples was calculated.
The samples of black pepper were divided in three groups: 1) ground packed, 2) ground unpacked, 3) dry not ground unpacked (not ground, unpacked). Data shows the total fungal count in black pepper samples. in Fig. 1.
Fig. 1 Total fungal count in black pepper from Pakistan markets
In ground packed group of black pepper total fungal count was found between 2.01-2.86 log CFU/g, on average - 2.447±0.248 logCFU/g (SD=0.43; CV=0.186). In sample no. 2 of this group was found mostly - 2.86±0.22 logCFU/g. In ground unpacked group total fungal count was found on average 2.428±0.15 log CFU/g (SD=0.212; CV=0.045). In not ground unpacked group of black pepper the tested number of samples was the highest. In this group total fungal count was found 2.881-3.691 log10 CFU/g, on average - 3.342±0.132 log CFU/g (SD=0.296; CV=0.088). In sample no. 22 of this
group was found mostly - 3.691±0.124 logCFU/g.
The total fungal count was found only 0.77% more in ground packed group of black pepper compared to ground unpacked group (p>0.05). Black pepper of ground packed group was contaminated
29 26.78% more fungal count compared to not ground unpacked group (p>0.05). Black pepper of ground unpacked group compared to not ground unpacked group the total fungal count was found 32.04% more in not ground unpacked group (p>0.05). The black pepper samples of not ground unpacked group were the most contaminated with fungi.
The samples of ginger were divided in three groups: 1) dry ground packed (ground packed), 2) dry ground unpacked (ground unpacked), 3) raw. The total fungal count in ginger samples is presented in Fig. 2.
Fig. 2 Total fungal count in ginger from Pakistan markets
In the raw ginger samples total fungal count ranged between 0-4.411 logCFU/g, on average - 3.276±0.829 logCFU/g (SD=1.856; CV=3.444). In 20% of raw ginger samples was not found fungi. In sample no. 29 of this group was found the highest total fungal count – 4.411±0.514 logCFU/g.
In ginger samples of ground packed group total fungal count ranged 1.88-2 logCFU/g, on average – 1.88±0.6 logCFU/g (SD=0.104; CV=0.01). In 66.7% samples of this group was found 1.82 log CFU/g.
In ginger samples of ground unpacked group total fungal count was found 1.82-2.12 logCFU/g, on average – 1.97±0.15 logCFU/g (SD=0.212; CV=0.045).
There are two groups which were compared. One group is ground packed and another group is ground unpacked. The ginger samples of ground packed group were contaminated by fungi 4.6% less compared to ginger samples of ground unpacked group (p>0.05). The ginger samples of ground, packed group were contaminated by fungi 42.6% less compared to raw ginger samples (p=0.002). The ginger samples of ground unpacked group were contaminated by fungi 39.9% less compared to samples of raw ginger (p=0.01).
30 The samples of red chilli were divided in four groups: 1) dry ground packed (ground packed), 2) dry ground unpacked (ground unpacked); 3) dry not ground unpacked (dry), 4) raw.
Fig. 3 Total fungal count in red chilli from Pakistan markets
In the raw red chilli samples total fungal count ranged from 0 to 5.091 logCFU/g, on average – 3.208±0.866 logCFU/g (SD=1.937; CV=3.753). The dry red chilli group samples were contaminated by fungi on average 3.368±0.142 logCFU/g (SD=0.318; CV=0.10147). In dry red chilli group were found contamination by fungi 4.7% less than in raw samples (p>0.05).
The ground, packed red chilli samples were contaminated by fungi on average 2.605±0.175 log CFU/g (SD=0.247; CV=0.061) and red chilli samples of ground unpacked were contaminated by fungi on average 2.52±0.09 logCFU/g (SD=0.155; CV=0.024). The red chilli samples of ground packed group were contaminated 3.07% more than samples of ground packed group (p>0,05). Red chilli samples of ground packed group and ground unpacked group compared to dry samples contamination 22.6% and 25.1% respectively lower were than in dry red chilli samples (p>0.05).
3.3.2 Spices contamination with fungal genera from Pakistan
Spices are very hygroscopic. Thus, after drying they need to be effectively packaged to prevent any increase in aw, which would allow mycotoxigenic fungi to become active and produce mycotoxins.
Raw and dry spices are very sensitive to contamination with mycotoxigenic fungi, so there is a need for detection of fungi genera, especially those that produce aflatoxins and ochratoxins. We assigned all the other fungi to the other fungal genera group: Rhizopus spp., Mucor spp., Cladosporium spp., Fusarium spp., Acremonium spp., Scorulopsis spp. and other.
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Fig. 4 Percentage contamination of black pepper by fungal genera from Pakistan markets
The samples of black pepper ground packed group 67±18.5% and ground unpacked group 61.1±27.8% were contaminated other genera (Fig. 4). When samples of not ground unpacked were contaminated Aspergillus spp. the highest (68.7±9.15%). Fungi of Aspergillus spp. was not detected in samples of ground packed group. Fungi of Penicillium spp. were detected in black pepper samples from all groups, the samples of ground packed group were contaminated Penicillium spp. the highest (34±19.4%).
Penicillium spp. was the most prevalent species in the examined ginger samples (Fig. 5). Penicillium spp. ranged 25.9-66.7%. In samples of ground unpacked group Penicillium spp. was detected the highest. Aspergillus spp. was detected only in raw ginger samples – 38.7±17.27%.
32 Red chilli is the second largest consumed spice throughout the world, after black pepper. Its exotic characteristics of taste, aroma, colour and pungency, as well as the multivariate forms of consumption, have made this spice widely used.
Fig. 6 Percentage contamination of red chilli by fungal genera from Pakistan markets
Comparison of contamination of raw and dry red chilli samples with Aspergillus spp. and Penicillium spp., Aspergillus spp. was found 6.11% less in dry samples (p>0,05), Penicillium spp. – 80% more in raw samples (p<0,05). Penicillium spp. was not detected in samples of ground unpacked and ground packed groups (Fig. 6).
3.3.3 Spices contamination with aflatoxin B1, ochratoxin A of spices from Pakistan
Spices have extensively been reported to be frequently contaminated by mycotoxins in different countries around the world. AF and OTA are among the most important contaminants from a consumer point of view.
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Table 4. Aflatoxin B1 concentration in spices from Pakistan markets
Spices n Positive samples n (%) Aflatoxin B1 concentration, µg/kg Range of AFB1 concentration in positive samples Mean of positive samples ± SD Black pepper ground packed 3 2 (66.7) 3 3±1.41 ground unpacked 2 2 (100) 1.7-7 4.35 ±2.65
not ground unpacked 5 1 (20) 1.3 0.26 ± 0.52
Ginger
ground packed 3 2 (66.7) 1.7-3 1.57±1.23
ground unpacked 2 0 ¹LOD≥1 ²ND
raw 5 1 (20) 1 1±0.4
Red chilli
ground packed 2 1 (50) 1.7 1.7±0.85
ground unpacked 3 1 (33.3) 3 1±1.41
dry not ground
unpacked 5 4 (80) 1.3-3 1.72±1.15
raw 5 3 (60) 1-3 1±1.09
¹LOD - below the detection limit; ²ND - not detected.
50% black pepper samples showed contamination with AFB1 out of which 10% samples displayed their toxicity level above the EU limit which is 5 μg/kg for AFB1 (Table 4). The highest AFB1 concentration was established in samples of ground unpacked. AFB1 concentration between the ground packed and ground unpacked groups was not statistically different (p>0.05). Statistically different was between ground packed and not ground unpacked groups (p=0.001) and between ground unpacked and not ground unpacked groups (p=0.001).
In 66.7% ground packed ginger samples was found AFB1. The highest concentration was 3 µg/kg in ground packed ginger sample.
In red chilli samples AFB1 was found 1-3 μg/kg and between groups was not statistically different (p>0.05).
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Table 5. Ochratoxin A concentration in spices from Pakistan markets
Spices n Positive samples n (%) Ochratoxin A concentration, µg/kg Range of OTA concentration in positive samples Mean of positive samples ± SD Black pepper
ground packed 3 0 ¹LOD≥1 ²ND
ground unpacked 2 0 ¹LOD≥1 ²ND
not ground unpacked 5 2 (40) 1-2 0.6±0.8
Ginger
ground packed 3 0 ¹LOD≥1 ²ND
ground unpacked 2 0 ¹LOD≥1 ²ND
raw 5 4 (80) 2-4 2.32±1.35
Red chilli
ground packed 2 2 (100) 1-4 2.5±1.5
ground unpacked 3 0 ¹LOD≥1 ²ND
dry not ground
unpacked 5 2 (40) 1.2-2 0.64±0.82
raw 5 0 ¹LOD≥1 ²ND
¹LOD - below the detection limit; ²ND - not detected
28.5 % spices samples showed contamination with OTA. In raw ginger samples was found 2-4 μg/kg of OTA.
