Food Chemistry 146 (2014) 320–326
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Occurrence of toxigenic fungi and determination of mycotoxins by HPLC-FLD in functional foods and spices in China markets Weijun Kong a,1, Riwei Wei a,b,1, Antonio F. Logrieco c, Jianhe Wei a, Jing Wen a,d, Xiaohe Xiao e, Meihua Yang a,⇑ a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, China b Guangxi University of Chinese Medicine, Nanning 530001, China c Institute of Sciences of Food Production, ISPA-CNR, Via G. Amendola, 122/O, I-70126 Bari, Italy d School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China e China Military Institute of Chinese Materia Medica, 302 Military Hospital of China, Beijing 100039, China
a r t i c l e
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Article history: Received 9 April 2013 Received in revised form 12 July 2013 Accepted 2 September 2013 Available online 13 September 2013 Keywords: Functional foods and spices Toxigenic fungi Mycotoxins HPLC-FLD Contamination occurrence
a b s t r a c t Twenty-four samples including 14 functional foods and 10 spices obtained from Chinese markets were examined for their mould profile. The mycotoxin contamination levels were also determined by an optimized HPLC-FLD method. 124 fungal isolates belonging to four different genera were recovered with Aspergillus and Penicillium as predominant fungi, with an incidence of 66.1% and 15.3%, respectively. In functional foods Aspergillus niger section (57.1%) was isolated more frequently, followed by Aspergillus flavi section (50.0%) and Aspergillus ochraceus section (21.4%), with the most contaminated samples being Coix seeds. Similar fungal presence and frequency were encountered in spice with A. niger section group (60.0%) and A. flavi section (40.0%) as main fungi. Cumin and Pricklyash peel samples showed the highest fungal contamination. Four functional foods and three spices were found to be positive at low levels for mycotoxins including aflatoxin B1 (up to 0.26 lg/kg) and ochratoxin A (OTA) (5.0 lg/kg). The more frequently detected mycotoxin was AFB1 (16.7%). Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Functional foods, a new variety of foods with significant nutritional, healthy, sensory and physiological functions in the body, represent one of the most interesting areas of study and innovation in the food industry (Sirò, Kapolna, & Kapolna, 2008). With many advantages and differences from conventional foods, the market of functional foods and related products is growing constantly all over the world. Spices, as natural food additives, contribute immensely to the taste and flavor of our foods and have been used for thousands of years. They have been reported to possess several medicinal properties (Hashem & Alamri, 2010) and are being consumed by more and more people. Although spices are present in foods in small amounts, they are among the most versatile and widely used ingredient in food preparation and processing throughout the world. Nowadays, in the markets of China, growing amounts of functional foods and spices are available and consumed every or nearly every day. ⇑ Corresponding author. Tel.: +39 080 5929357, +86 10 57833277. E-mail addresses: [email protected] (A.F. Logrieco), yangmeihua15 @hotmail.com (M. Yang). 1 These authors contributed equally to this work. 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.005
However, like many other conventional foods, agricultural products and medicinal plants (García-Cela, Ramos, & Sanchis, 2012; Liu et al., 2013), functional foods and spices may be contaminated by a wide range of toxigenic fungi, such as Aspergillus flavus section and Alternaria alternate section, as well as Penicillia and Scopulariopsisetc (Hashem & Alamri, 2010; Jalili & Jinap, 2012), from soil or plants during the procedure of growth, harvest, process, storage and transportation (de Curtis, de Felice, Ianiri, de Cicco, & Castoria, 2012; Jalili & Jinap, 2012; Romagnoli, Menna, Gruppion, & Bergamini, 2007). Taking into account the important medicinal and edible values, and with respect to the economy, safety and human health perspectives, contamination of functional foods and spices by various fungi may result in remarkably rapid deterioration in quality. As a consequence of this loss in quality, the profitability, effectiveness and safety of these matrices are considerably reduced. The rise of emerging toxigenic fungi have challenged food security and ecosystem health (García-Cela et al., 2012). Toxigenic fungi are not only considered as significant harmful pathogens to functional foods and spices, but also the principal producers of a class of toxic secondary metabolites generally termed mycotoxins, such as aflatoxins, ochratoxins, fumonisins and deoxynivalenol (Rahmani, Jinap, & Soleimany, 2009; Zhou et al., 2012). These mycotoxins have been shown to be carcinogenic, teratogenic,
W. Kong et al. / Food Chemistry 146 (2014) 320–326
tremorogenic, haemorrhagic and dermatitic to a diversity of organisms. Among these mycotoxins, aflatoxins (AFs), such as AFB1, AFB2, AFG1, AFG2 and ochratoxin A (OTA) are quite common contaminants occurring jointly in a wide range of food commodities including spices (Wangikar, Dwivedi, Sinha, Sharma, & Telang, 2005). Due to being reported as highly toxic and potential carcinogens, mutagens and teratogens, aflatoxins and OTA have been classified in Group 1 ‘as carcinogenic to humans’ and in Group 2B as ‘possibly carcinogenic to humans’ by the International Agency for Research on Cancer (IARC, 1993a; IARC, 1993b). Contamination of foods and their derived products, agricultural products and spices with mycotoxins can cause many diseases or deaths in humans and animals even when consumed in small amounts (Prouillac et al., 2012). Despite numerous studies on mycoflora and mycotoxins in agricultural products, medicinal plants, foods and related products from many countries, very limited data is available on fungal and mycotoxin contamination in functional foods and spices in Chinese markets. Consequently, the possible presence and occurrence of toxigenic fungi and mycotoxins in functional foods and spices must be monitored periodically to meet the requirements of new legislations that are continuously being revised (Mashinini & Dutton, 2006), to further ensure a healthy food and spice supply and minimizing consequences to consumers’ health. Taking all this information into consideration, the aims of the present work were: (i) to investigate the association of mycoflora with 14 functional foods and 10 spices collected from Chinese markets; (ii) to determine the incidence of mycotoxins with special reference to AFB1, AFB2, AFG1, AFG2 and OTA in these substrates by an optimized high performance liquid chromatography (HPLC) method using a on-line post-column photochemical derivatisation (PCD) and fluorescence detection (FLD). The results will provide some meaningful references for highlighting the risk assessment and investigating the quality of tested functional foods and spices with respect to fungal contamination and their potential in producing toxigenic mycotoxins. 2. Materials and methods 2.1. Sampling Twenty-four samples of functional foods and spices, composed of 17 different plant species were chosen on the basis of commercial availability and popularity, and were collected randomly from Chinese markets in Beijing city, Hebei, Ningxia, Anhui, Fujian and Sichuan provinces, China. The seeds, fruit, bark, rhizome, pericarp and leaves of all plant species were used in testing. All samples were identified by Prof. Bengang Zhang, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. Samples (40–200 g/sample) were collected in sterilized polyethylene bags and stored at 4 °C until use. The names, families and the plant parts used of 14 functional foods and 10 spices were presented in Table 1. 2.2. Materials Pentachloronitrobenzene (PCNB) agar mediums were bought from Sigma-Aldrich chemie Gmbh (Steinheim, Germany), while malt extract agar (MEA), czapek-dox agar (CDA), potato dextrose agar (PDA) and dichloran rose bengal chlorotetracycline (DRBC) mediums were from Oxoid Ltd., England. All other chemicals and reagents were analytical or HPLC grades. Methanol (MeOH) for chromatographic analysis was chromatographically pure. 2.3. Mycotoxin standards and preparation The standards of alfatoxins B1, B2, G1 and G2 were purchased from SUPELCO (Bellafonte, PA, USA), while ochratoxin A (OTA)
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standard was obtained from Alexis Corporation (Lausen, Switzerland). Because these mycotoxins are subject to light degradation, the standard solutions were prepared in a dark vial by dissolving the pure AFB1, AFB2, AFG1, AFG2 and OTA in methanol:water (1:1, v/v) to yield 250.0 ng/ml of AFB2, AFG2 and OTA, 75.0 ng/ml of AFB1 and AFG1, respectively. The solutions were stored at 4 °C away from light before use. 2.4. Mycoflora isolation and mycological analyses Functional foods and spices were surface sterilized for 2–3 min with Sodium-hypochloride (NaOCl, 50–100 ml 5% Sodium-hypochloride solution was diluted with water to 250 ml in a volumetric flask and mixed homogeneously) in distilled water, rinsed in 4 changes of sterile distilled water and air dried under aseptic conditions. Then one hundred plant pieces or seeds (ten for each plate) for each sample were aseptically plated in Petri dishes onto DRBC agar (Pitt & Hocking, 1997) and PCNB as selected media for Aspergillus and Fusarium species, respectively. All plates were incubated for 7 days (12 h/12 h light/darkness) at 25 °C and examined daily. After incubation of the plant parts/seeds, fungal infection was recorded and the incidence of fungal genus/section for each sample was determined. Fungal colonies were sub-cultured both on PDA for (Potato Dextrose Agar, 39.0 g/l, Difco), MEA and CDA mediums containing 100.0 mg/l of streptomycin and 50.0 mg/l of neomycin sulfate as antibacterial agents. When the reproductive structures were differentiated the strains were identified to the genus/section level by culture and morphological characteristics using a Heerbrugg stereoscope (Leica, Switzerland) (Leslie & Summerell, 2006; Raper & Fennel, 1977). 2.5. Incidence of fungal species The incidence of different fungal species in tested functional foods and spices was assessed by calculating the percentage relative frequency and relative density. The relative frequency (%, No. of contaminated samples/No. of analysed samples) is defined here as the percentage of samples within which a given fungal species or mycotoxin was found at least once. The relative density (%, No. of the isolates of certain genus or specie or mycotoxin/No. of the total isolates of all genera or species or mycotoxins) is related to the number of isolates of genus or specie or mycotoxin observed to occur in the samples analyzed. Their values were obtained according to other reports (Gautam, Sharma, & Bhadauria, 2009). 2.6. Extraction and clean up of AFB1, AFB2, AFG1, AFG2 and OTA from tested samples The crude materials of all samples were finely ground to a homogenous size by a mill and sized through a No. 24 mesh sieve. Fifteen grams of tested sample was mixed with 1 g sodium chloride and extracted by ultrasonication with 90 ml methanol:water (80:20, v/v) in a clean ultrasonic bath for 20 min and then filtered through a Whatman 113 V paper. Then, 10 ml of clear filtrate was diluted by adding 70 ml of phosphate-buffered saline (PBS) solution containing 2% tween-20 at pH7.0 and then filtered through the GF/A glass microfibre filter. Following the cartridge instructions, 48 ml of the sample extract was loaded on the AflaOchra HPLC™ immunoaffinity column (IAC) from VICAM (Waterworn, MA, USA). The IA column was then rinsed with 20 ml water (3.0 ml/min) and dried by passing air through it. After the column was washed with 1 ml MeOH (0.5–1.0 ml/min), the investigated mycotoxins were eluted with 1.5 ml MeOH (0.5–1.0 ml/min) and collected in a dark flask. The eluate was concentrated to 0.2 ml under a stream of N2 to evaporate the methanol at 45 °C and was
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Table 1 Names, families, the plant parts used and origins of studied functional foods and spices. Sample Functional foods
Label
English name
Latin name
Plant taxon
Family
Plant part used
Origin
Coix seed Coix seed Coix seed Coix seed Mulberry fruit Mume fruit Lotus seed Barbary wolfberry fruit Hawthorn fruit
Smeen coicis Smeen coicis Smeen coicis Smeen coicis Fructus mori Fructus mume Semen nelumbinis Fructus lycii Fructus crataegi
Gramineae Gramineae Gramineae Gramineae Moraceae Rosaceae Nymphaeaceae Solanaceae Rosaceae
Seed Seed Seed Seed Fruit-spike Fruit Seed Fruit Fruit
Beijing Beijing Beijing Beijing Beijing Beijing Beijing Ningxia Beijing
10
Bitter apricot seed
Rosaceae
Seed
Hebei
11 12 13
Ginkgo seed Common yam rhizome Common floweringquince fruit Zingiber
Semen armeniacae amarum Semen ginkgo Rhizoma dioscoreae Fructus chaenomelis
Coix lacryma-jobi L. var. ma-yuen Coix lacryma-jobi L. var. ma-yuen Coix lacryma-jobi L. var. ma-yuen Coix lacryma-jobi L. var. ma-yuen Morus alba L. Prunus mume (sieb.) Sieb. et Zucc Nelumbo nucifera Gaertn. Lycium barbarum L. Crataegus pinnatifida Bge. var. major N. E. Br. Prunus armeniaca L. var. ansu Maxim. Ginkgo biloba L. Dioscorea opposite Thunb. Chaenomeles speciosa Nakai
Ginkgoaceae Dioscoreaceae Rosaceae
Seed Rhizome Fruit
Beijing Beijing Anhui
Rhizoma Zingiberis
Zingiber officinale Rosc.
Zingiberaceae
Rhizome
Sichuan
Fructus anisi stellati Fructus anisi stellati Fructus anisi stellati Fructus foeniculi Fructus foeniculi Cortex cinnamomi Cortex cinnamomi Cuminum cyminum Pericarpium zanthoxyli Lindera communis
Illicium verum Hook. f. Illicium verum Hook. f. Illicium verum Hook. f. Foeniculum vulgare Mill. Foeniculum vulgare Mill. Cinnamomum cassia Presl. Cinnamomum cassia Presl. Cuminum cyminum L. Zanthoxylum schinifolium Sieb. et Zucc. Geraniaceae Pelargonium graveolens L.
Magnoliaceae Magnoliaceae Magnoliaceae Umbelliferae Umbelliferae Lauraceae Lauraceae Umbelliferae Rutaceae Geraniaceae
Fruit Fruit Fruit Fruit Fruit Bark Bark Fruit Pericarp Leaf
Fujian Beijing Beijing Hebei Hebei Beijing Beijing Beijing Beijing Beijing
1 2 3 4 5 6 7 8 9
14 Spices
15 16 17 18 19 20 21 22 23 24
Star anise Star anise Star anise Fennel Fennel Cassia bark Cassia bark Cumin Pricklyash peel Pelargonium graveolens L’ herit
re-dissolved in methanol–water (50:50, v/v) to yield 1 ml of solution. The solution containing investigated mycotoxins was vortexed for 30 s and filtered through a 0.45 lm filter for chromatographic detection.
2.8. Statistical analysis The statistical analysis of data was performed using student’s ttest with statistical package SPSS (version 18.0). The p-value < 0.05 was considered significant.
2.7. Detection of AFB1, AFB2, AFG1, AFG2 and OTA by HPLC-PCD-FLD The presence of AFB1, AFB2, AFG1, AFG2 and OTA was detected by a high performance liquid chromatography (HPLC) method using a on-line post-column photochemical derivatisation (PCD) with an AURA Industries reactor (AURA Industries, NY, USA) and a RF-10AXL fluorescence detector (FLD) (Shimadzu, Kyoto, Japan). HPLC analysis was performed on an Shimadzu LC-20AT HPLC system consisting of two LC-20 AD pumps, an SIL-20AC autosampler and a CTO-20A column oven. A reverse phase Unimicro Technologies C18 (4.6 250 mm, 5.0 lm, Pleasanton, CA, USA) column was used for chromatographic separation and the column temperature was set at 30 °C. The mobile phase consisted of (A) methanol and (B) 0.5% aqueous acetic acid at a flow rate of 1.0 ml/min, freshly prepared every day. The optimized gradient elution procedure was as follows: 0–12 min (52–68%, A), 12–35 min (68%, A). The injection volume was 50 ll. The maximum emission wavelength of the PCD reactor was set at 254 nm. The eluate was monitored by a fluorescence detector. During the first 15 min of analysis, fluorescence conditions were optimized for AFs (excitation 360 nm and emission 440 nm wavelengths), and after that for OTA (excitation 333 nm and emission 460 nm wavelengths). The system was interfaced via an LC solution ChemStation software (Shimadzu Kyoto, Japan) to a personal computer for controlling instruments and data acquisition and processing. To confirm the presence of AFB1 and AFG1, the derivatisation reactor was disconnected and fluorescence detector was directly connected to the HPLC pump. By this way, the peaks of AFB1 and AFG1 almost could not be seen on the chromatogram because of their low fluorescent response.
3. Results and discussion 3.1. Fungal isolates from tested samples The distribution of tested samples (seeds, fruits, barks, rhizomes, pericarps and leaves) on the medium was shown in Fig. 1A. After incubating the samples for 7 days at 25 °C, most of the samples became mouldy to different extents (Fig. 1B) and a total of 124 fungal isolates (Table 2) belonging to four different genera, such as Aspergillus (82), Penicillium (19), Fusarium (13) and Eurotium (10) were identified and recorded from the 24 functional foods and spices.
Fig. 1. (A) Distribution of tested Coix seed samples on the medium; (B) Display of mould on tested Coix seed samples after incubation.
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W. Kong et al. / Food Chemistry 146 (2014) 320–326 Table 2 Fungal isolates and their incidence in tested functional foods and spices in China market. Sample
Label
Strains of fungal isolates
Functional foods
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus
15 16 17 18 19 20 21 22 23 24
Penicillium (1) Penicillium (2) Aspergillus (4) Fusarium (2) Aspergillus (4), Fusarium (3) Aspergillus (3) Aspergillus (13), Eurotium (1) Aspergillus (12), Fusarium (1) Aspergillus (3), Eurotium (1)
Spices
Total a
No. of isolates
(3), (8), (7), (3), (1),
Penicillium (2) Penicillium (1) Penicillium (1) Penicillium (2), Fusarium (4) Eurotium (1), Penicillium (1), Fusarium (2)
(5), (1), (2), (4) (5), (1), (1), (2),
Penicillium (2) Penicillium (3) Penicillium (1) Penicillium (1) Penicillium (2), Fusarium (1) Eurotium (3) Eurotium (4)
Aspergillus (82), Penicillium (19), Fusarium (13), Eurotium (10)
Rd (%)a
5 9 8 9 5 0 7 4 3 4 6 4 4 6
4.0 7.3 6.5 7.3 4.0 0 5.7 3.2 2.4 3.2 4.8 3.2 3.2 4.8
1 2 0 4 2 7 3 14 13 4
0.8 1.6 0 3.2 1.6 5.7 2.4 11.4 10.5 3.2
124
100
Relative density (%) = No. of the isolates of a certain species/No. of total isolates of all strains.
Fig. 2. Incidences of (A) identified strains in all fungal isolates, and (B) fungal isolates in functional foods and spices.
The incidence of samples colonized by Aspergillus and Penicillium was significantly predominant from the 124 strains isolated, 82 belong to Aspergillus and 19 belong to Penicillium, with an incidence of 66.1% and 15.3%, respectively. The numbers of Fusarium and Eurotium isolates were 13 and 10, respectively, representing 10.5% and 8.1% of the total number of isolates (Fig. 2A). With respect to the tested samples, the incidence of the five different fungal isolates in 14 functional foods (59.6%) was almost 1.5 times that in the 10 spices tested (40.4%) (Fig. 2B). However, student’s t-test analysis of the incidence as relative density (%) (Table 2) did not show statistically significant variability in the means for functional foods and spices (t = 0.179, p > 0.05). The above results indicated that both the tested functional foods and spices were prone to be contaminated by the identified fungal mycoflora,
especially the genera Aspergillus and Penicillium. This observation was greatly in agreement with other investigators who dealt with mycoflora of spices and medicinal plants (Romagnoli et al., 2007). Table 2 shows that all the seed samples were infected by Aspergillus and Penicillium except sample 10 (Bitter apricot seed). Similarly, all the rhizome samples including samples 12 and 14, as well as all bark samples including samples 20 and 21 showed Aspergillus infection. The fruit samples of different matrices were found to be infected by a large number of fungi, including Aspergillus, Penicillium, Fusarium and Eurotium. With regard to functional foods, Coix seeds were found to be the most heavily contaminated samples, especially sample 4, followed by Lotus seed, Ginkgo seed and Zingiber. Mume fruit expressed no fungal isolates, which might be a result of the fruits antifungal activity, or due to its abundant organic acids that inhibit the growth of moulds. For the tested spices, Cumin and Pricklyash peel contained the most fungal isolates. In addition, the other spice samples show little contamination, especially Star anise. One sample of Star anise showed no fungal infection, which maybe due to the differences of processing technologies or storage conditions. In summary, these various results might partly elucidate that matrices of the different plant parts tested had various sensitivities to fungi and would be contaminated by different fungal isolates during their processing, storage and transportation processes. 3.2. Occurrence of potential mycotoxin-producing fungi in tested samples On the studied medium, many colonies of the genera have been identified from the 24 functional foods and spices with Aspergillus dominating. Then, three potential mycotoxin-producing fungi including A. flavus section, Aspergillus niger section and Aspergillus ochraceus section were further isolated and identified, which have been shown in Table 3. It can be shown that the highest relative frequency (%) occurrence was recovered in A. niger section (58.3%, 14/24) followed by A. flavus section (45.8%, 11/24) and A. ochraceus section (12.5%, 3/24). The relative frequency of A. niger section was significantly higher (p < 0.05) than that of the other two fungal species. Similar findings were revealed for the 14
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Table 3 Occurrence of potential mycotoxin-producing fungi in tested samples. Label
A. flavus section
A. niger section
A. ochraceus section
Functional foods
1 2 3 4 5 6 7 8 9 10 11 12 13 14
18.9 3.4 3.2 8.1 ND ND 89.7 0.7 ND 0.6 ND ND ND ND 124.6 50.0 (7/14)
ND c 11.4 8.3 1.5 ND ND 20.5 ND 5.6 3.5 20.0 ND ND 2.5 73.3 57.1 (8/14)
ND 3.4 17.3 ND ND ND 2.6 ND ND ND ND ND ND ND 23.3 21.4 (3/14)
15 16 17 18 19 20 21 22 23 24
ND ND ND 1.8 ND 13.3 10.7 ND 0.5 ND 26.3 40.0 (4/10)
ND ND ND 0.6 ND 10.0 10.7 15.2 38.9 37.8 113.2 60.0 (6/10)
ND ND ND ND ND ND ND ND ND ND 0 (0/10)
Total (%) Rf (%)b Spices
Total (%) Rf (%) a b c
Rd (%)a of potential mycotoxin-producing fungi
Sample
Relative density (%) = No. of the identified fungi of certain species/No. of all species. Relative frequency (%) = No. of contaminated samples with a mycotoxin-producing fungi/No. of all analyzed functional foods or spices samples. Not detected.
functional foods with the relative frequency (%) occurrence in the order of A. niger section (57.1%, 8/14), A. flavus section (50.0%, 7/ 14) and A. ochraceus section (21.4%, 3/14), as well as for the 10 spices of A. niger section (60.0%, 6/10), A. flavus section (40.0%, 4/ 10) and A. ochraceus section (0, 0/10). A. ochraceus section was detected in very few samples of the functional foods and in none of the spices. In addition, the highest occurrence of the three fungi presented as relative frequency (%) was found in seed samples, followed by bark, pericarp, leaf, fruit and rhizome samples. Three (samples 8, 9 and 18) out of the eleven fruit samples were infected by one of the three fungi, among which, only one spice-fruit sample (sample 22) was infected by A. niger section. None of the two rhizome samples were infected by any fungi. Two Coix seed (samples 2 and 3) and one Lotus seed (sample 7) samples were simultaneously contaminated by the three fungi. The above results indicated that the occurrence of A. niger section was significantly predominant among the three detected potential mycotoxin-producing fungi in the tested functional foods and spices, also elucidated that the matrices of studied samples were more sensitive to A. niger section than the other two fungi. The incidence presented as relative density (%) for the three detected fungi in tested samples are also listed in Table 3. For the 14 functional foods, the highest total incidence was founded in A. flavus section (124.6%) followed by A. niger section (73.3%) and A. ochraceus section (23.3%), while for the 10 spices, A. niger section revealed the highest total incidence of 113.2%, A. flavus section expressed low incidence of 26.3% and no A. ochraceus section was detected. These findings showed that A. flavus section and A. niger section might be the predominant fungi infecting functional foods and spices, respectively. On the other hand, among the functional foods, the highest incidence of A. flavus section, A. niger section and A. ochraceus section was found in Lotus seed (sample 7) collected from Beijing city. This might be related to the environmental
conditions, as it grows exposed to water and air. Among the spices, a high incidence of A. flavus section and/or A. niger section was observed in both Pricklyash peel and Pelargonium graveolens L’Herit samples. A. flavus section, A. niger section and A. ochraceus section were common fungi that could grow in different substrates. The above results have elucidated the high relative frequency and density of the three detected fungi in the functional foods and spices tested. The results also show the different susceptibilities of these matrices to the three fungi, which were in accordance with the reports of Raper and Fennel (Raper & Fennel, 1977). 3.3. Occurrence and levels of some mycotoxins in tested samples It has been reported, under favorable conditions, some fungal species can synthesize toxic metabolites (mycotoxins), and most of the identified fungi, such as A. flavus section, A. niger section and A. ochraceus section produce mycotoxins, namely aflatoxins (AFB1, AFB2, AFG1 and AFG2) and ochratoxins (OTA) (Romagnoli et al., 2007). Our previous studies have reported some mycotoxin contamination, especially AFB1, AFB2, AFG1, AFG2 and OTA in spices, aromatic and medicinal herbs (Liu et al., 2013). Here, the occurrence and levels of AFB1, AFB2, AFG1, AFG2 and OTA in the 24 samples tested were analyzed by the developed HPLC-PCD-FLD method (Wei, Yang, Qiu, Yang, & Qin, 2011), which has been validated in agreement with requirements established by Regulation (EC) No. 401/2006 (European Commission, 2006). This developed method gave good selectivity, linearity (0.2–50.0 ng/ml for AFB1, 0.06–15.0 ng/ml for AFB2, 0.3–50.0 ng/ml for AFG1, 0.09–15.0 ng/ml for AFG2 and 1.0–50.0 ng/ml for OTA), sensitivity (expressed as limits of detection (LOD) of 0.04, 0.02, 0.08, 0.03, 0.3 lg/kg and limits of quantification (LOQs) of 0.15, 0.06, 0.25, 0.1, 1.0 lg/kg for AFB1, AFB2, AFG1, AFG2, OTA,
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W. Kong et al. / Food Chemistry 146 (2014) 320–326 Table 4 Occurrence (%) of positive samples and contamination level (lg/kg) of mycotoxins in tested functional foods and spices. Samples
Functional foods
Label
AFB1
AFB2
AFG1
AFG2
OTA
1 4 5 9
0.2 0.15 – 0.26 21.4 (3/14)
– – –
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Occurrence of toxigenic fungi and determination of mycotoxins by HPLC-FLD in functional foods and spices in China markets Weijun Kong a,1, Riwei Wei a,b,1, Antonio F. Logrieco c, Jianhe Wei a, Jing Wen a,d, Xiaohe Xiao e, Meihua Yang a,⇑ a Key Laboratory of Bioactive Substances and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100193, China b Guangxi University of Chinese Medicine, Nanning 530001, China c Institute of Sciences of Food Production, ISPA-CNR, Via G. Amendola, 122/O, I-70126 Bari, Italy d School of Pharmacy, Chengdu University of Traditional Chinese Medicine, Chengdu 611137, China e China Military Institute of Chinese Materia Medica, 302 Military Hospital of China, Beijing 100039, China
a r t i c l e
i n f o
Article history: Received 9 April 2013 Received in revised form 12 July 2013 Accepted 2 September 2013 Available online 13 September 2013 Keywords: Functional foods and spices Toxigenic fungi Mycotoxins HPLC-FLD Contamination occurrence
a b s t r a c t Twenty-four samples including 14 functional foods and 10 spices obtained from Chinese markets were examined for their mould profile. The mycotoxin contamination levels were also determined by an optimized HPLC-FLD method. 124 fungal isolates belonging to four different genera were recovered with Aspergillus and Penicillium as predominant fungi, with an incidence of 66.1% and 15.3%, respectively. In functional foods Aspergillus niger section (57.1%) was isolated more frequently, followed by Aspergillus flavi section (50.0%) and Aspergillus ochraceus section (21.4%), with the most contaminated samples being Coix seeds. Similar fungal presence and frequency were encountered in spice with A. niger section group (60.0%) and A. flavi section (40.0%) as main fungi. Cumin and Pricklyash peel samples showed the highest fungal contamination. Four functional foods and three spices were found to be positive at low levels for mycotoxins including aflatoxin B1 (up to 0.26 lg/kg) and ochratoxin A (OTA) (5.0 lg/kg). The more frequently detected mycotoxin was AFB1 (16.7%). Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction Functional foods, a new variety of foods with significant nutritional, healthy, sensory and physiological functions in the body, represent one of the most interesting areas of study and innovation in the food industry (Sirò, Kapolna, & Kapolna, 2008). With many advantages and differences from conventional foods, the market of functional foods and related products is growing constantly all over the world. Spices, as natural food additives, contribute immensely to the taste and flavor of our foods and have been used for thousands of years. They have been reported to possess several medicinal properties (Hashem & Alamri, 2010) and are being consumed by more and more people. Although spices are present in foods in small amounts, they are among the most versatile and widely used ingredient in food preparation and processing throughout the world. Nowadays, in the markets of China, growing amounts of functional foods and spices are available and consumed every or nearly every day. ⇑ Corresponding author. Tel.: +39 080 5929357, +86 10 57833277. E-mail addresses: [email protected] (A.F. Logrieco), yangmeihua15 @hotmail.com (M. Yang). 1 These authors contributed equally to this work. 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.09.005
However, like many other conventional foods, agricultural products and medicinal plants (García-Cela, Ramos, & Sanchis, 2012; Liu et al., 2013), functional foods and spices may be contaminated by a wide range of toxigenic fungi, such as Aspergillus flavus section and Alternaria alternate section, as well as Penicillia and Scopulariopsisetc (Hashem & Alamri, 2010; Jalili & Jinap, 2012), from soil or plants during the procedure of growth, harvest, process, storage and transportation (de Curtis, de Felice, Ianiri, de Cicco, & Castoria, 2012; Jalili & Jinap, 2012; Romagnoli, Menna, Gruppion, & Bergamini, 2007). Taking into account the important medicinal and edible values, and with respect to the economy, safety and human health perspectives, contamination of functional foods and spices by various fungi may result in remarkably rapid deterioration in quality. As a consequence of this loss in quality, the profitability, effectiveness and safety of these matrices are considerably reduced. The rise of emerging toxigenic fungi have challenged food security and ecosystem health (García-Cela et al., 2012). Toxigenic fungi are not only considered as significant harmful pathogens to functional foods and spices, but also the principal producers of a class of toxic secondary metabolites generally termed mycotoxins, such as aflatoxins, ochratoxins, fumonisins and deoxynivalenol (Rahmani, Jinap, & Soleimany, 2009; Zhou et al., 2012). These mycotoxins have been shown to be carcinogenic, teratogenic,
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tremorogenic, haemorrhagic and dermatitic to a diversity of organisms. Among these mycotoxins, aflatoxins (AFs), such as AFB1, AFB2, AFG1, AFG2 and ochratoxin A (OTA) are quite common contaminants occurring jointly in a wide range of food commodities including spices (Wangikar, Dwivedi, Sinha, Sharma, & Telang, 2005). Due to being reported as highly toxic and potential carcinogens, mutagens and teratogens, aflatoxins and OTA have been classified in Group 1 ‘as carcinogenic to humans’ and in Group 2B as ‘possibly carcinogenic to humans’ by the International Agency for Research on Cancer (IARC, 1993a; IARC, 1993b). Contamination of foods and their derived products, agricultural products and spices with mycotoxins can cause many diseases or deaths in humans and animals even when consumed in small amounts (Prouillac et al., 2012). Despite numerous studies on mycoflora and mycotoxins in agricultural products, medicinal plants, foods and related products from many countries, very limited data is available on fungal and mycotoxin contamination in functional foods and spices in Chinese markets. Consequently, the possible presence and occurrence of toxigenic fungi and mycotoxins in functional foods and spices must be monitored periodically to meet the requirements of new legislations that are continuously being revised (Mashinini & Dutton, 2006), to further ensure a healthy food and spice supply and minimizing consequences to consumers’ health. Taking all this information into consideration, the aims of the present work were: (i) to investigate the association of mycoflora with 14 functional foods and 10 spices collected from Chinese markets; (ii) to determine the incidence of mycotoxins with special reference to AFB1, AFB2, AFG1, AFG2 and OTA in these substrates by an optimized high performance liquid chromatography (HPLC) method using a on-line post-column photochemical derivatisation (PCD) and fluorescence detection (FLD). The results will provide some meaningful references for highlighting the risk assessment and investigating the quality of tested functional foods and spices with respect to fungal contamination and their potential in producing toxigenic mycotoxins. 2. Materials and methods 2.1. Sampling Twenty-four samples of functional foods and spices, composed of 17 different plant species were chosen on the basis of commercial availability and popularity, and were collected randomly from Chinese markets in Beijing city, Hebei, Ningxia, Anhui, Fujian and Sichuan provinces, China. The seeds, fruit, bark, rhizome, pericarp and leaves of all plant species were used in testing. All samples were identified by Prof. Bengang Zhang, Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, China. Samples (40–200 g/sample) were collected in sterilized polyethylene bags and stored at 4 °C until use. The names, families and the plant parts used of 14 functional foods and 10 spices were presented in Table 1. 2.2. Materials Pentachloronitrobenzene (PCNB) agar mediums were bought from Sigma-Aldrich chemie Gmbh (Steinheim, Germany), while malt extract agar (MEA), czapek-dox agar (CDA), potato dextrose agar (PDA) and dichloran rose bengal chlorotetracycline (DRBC) mediums were from Oxoid Ltd., England. All other chemicals and reagents were analytical or HPLC grades. Methanol (MeOH) for chromatographic analysis was chromatographically pure. 2.3. Mycotoxin standards and preparation The standards of alfatoxins B1, B2, G1 and G2 were purchased from SUPELCO (Bellafonte, PA, USA), while ochratoxin A (OTA)
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standard was obtained from Alexis Corporation (Lausen, Switzerland). Because these mycotoxins are subject to light degradation, the standard solutions were prepared in a dark vial by dissolving the pure AFB1, AFB2, AFG1, AFG2 and OTA in methanol:water (1:1, v/v) to yield 250.0 ng/ml of AFB2, AFG2 and OTA, 75.0 ng/ml of AFB1 and AFG1, respectively. The solutions were stored at 4 °C away from light before use. 2.4. Mycoflora isolation and mycological analyses Functional foods and spices were surface sterilized for 2–3 min with Sodium-hypochloride (NaOCl, 50–100 ml 5% Sodium-hypochloride solution was diluted with water to 250 ml in a volumetric flask and mixed homogeneously) in distilled water, rinsed in 4 changes of sterile distilled water and air dried under aseptic conditions. Then one hundred plant pieces or seeds (ten for each plate) for each sample were aseptically plated in Petri dishes onto DRBC agar (Pitt & Hocking, 1997) and PCNB as selected media for Aspergillus and Fusarium species, respectively. All plates were incubated for 7 days (12 h/12 h light/darkness) at 25 °C and examined daily. After incubation of the plant parts/seeds, fungal infection was recorded and the incidence of fungal genus/section for each sample was determined. Fungal colonies were sub-cultured both on PDA for (Potato Dextrose Agar, 39.0 g/l, Difco), MEA and CDA mediums containing 100.0 mg/l of streptomycin and 50.0 mg/l of neomycin sulfate as antibacterial agents. When the reproductive structures were differentiated the strains were identified to the genus/section level by culture and morphological characteristics using a Heerbrugg stereoscope (Leica, Switzerland) (Leslie & Summerell, 2006; Raper & Fennel, 1977). 2.5. Incidence of fungal species The incidence of different fungal species in tested functional foods and spices was assessed by calculating the percentage relative frequency and relative density. The relative frequency (%, No. of contaminated samples/No. of analysed samples) is defined here as the percentage of samples within which a given fungal species or mycotoxin was found at least once. The relative density (%, No. of the isolates of certain genus or specie or mycotoxin/No. of the total isolates of all genera or species or mycotoxins) is related to the number of isolates of genus or specie or mycotoxin observed to occur in the samples analyzed. Their values were obtained according to other reports (Gautam, Sharma, & Bhadauria, 2009). 2.6. Extraction and clean up of AFB1, AFB2, AFG1, AFG2 and OTA from tested samples The crude materials of all samples were finely ground to a homogenous size by a mill and sized through a No. 24 mesh sieve. Fifteen grams of tested sample was mixed with 1 g sodium chloride and extracted by ultrasonication with 90 ml methanol:water (80:20, v/v) in a clean ultrasonic bath for 20 min and then filtered through a Whatman 113 V paper. Then, 10 ml of clear filtrate was diluted by adding 70 ml of phosphate-buffered saline (PBS) solution containing 2% tween-20 at pH7.0 and then filtered through the GF/A glass microfibre filter. Following the cartridge instructions, 48 ml of the sample extract was loaded on the AflaOchra HPLC™ immunoaffinity column (IAC) from VICAM (Waterworn, MA, USA). The IA column was then rinsed with 20 ml water (3.0 ml/min) and dried by passing air through it. After the column was washed with 1 ml MeOH (0.5–1.0 ml/min), the investigated mycotoxins were eluted with 1.5 ml MeOH (0.5–1.0 ml/min) and collected in a dark flask. The eluate was concentrated to 0.2 ml under a stream of N2 to evaporate the methanol at 45 °C and was
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Table 1 Names, families, the plant parts used and origins of studied functional foods and spices. Sample Functional foods
Label
English name
Latin name
Plant taxon
Family
Plant part used
Origin
Coix seed Coix seed Coix seed Coix seed Mulberry fruit Mume fruit Lotus seed Barbary wolfberry fruit Hawthorn fruit
Smeen coicis Smeen coicis Smeen coicis Smeen coicis Fructus mori Fructus mume Semen nelumbinis Fructus lycii Fructus crataegi
Gramineae Gramineae Gramineae Gramineae Moraceae Rosaceae Nymphaeaceae Solanaceae Rosaceae
Seed Seed Seed Seed Fruit-spike Fruit Seed Fruit Fruit
Beijing Beijing Beijing Beijing Beijing Beijing Beijing Ningxia Beijing
10
Bitter apricot seed
Rosaceae
Seed
Hebei
11 12 13
Ginkgo seed Common yam rhizome Common floweringquince fruit Zingiber
Semen armeniacae amarum Semen ginkgo Rhizoma dioscoreae Fructus chaenomelis
Coix lacryma-jobi L. var. ma-yuen Coix lacryma-jobi L. var. ma-yuen Coix lacryma-jobi L. var. ma-yuen Coix lacryma-jobi L. var. ma-yuen Morus alba L. Prunus mume (sieb.) Sieb. et Zucc Nelumbo nucifera Gaertn. Lycium barbarum L. Crataegus pinnatifida Bge. var. major N. E. Br. Prunus armeniaca L. var. ansu Maxim. Ginkgo biloba L. Dioscorea opposite Thunb. Chaenomeles speciosa Nakai
Ginkgoaceae Dioscoreaceae Rosaceae
Seed Rhizome Fruit
Beijing Beijing Anhui
Rhizoma Zingiberis
Zingiber officinale Rosc.
Zingiberaceae
Rhizome
Sichuan
Fructus anisi stellati Fructus anisi stellati Fructus anisi stellati Fructus foeniculi Fructus foeniculi Cortex cinnamomi Cortex cinnamomi Cuminum cyminum Pericarpium zanthoxyli Lindera communis
Illicium verum Hook. f. Illicium verum Hook. f. Illicium verum Hook. f. Foeniculum vulgare Mill. Foeniculum vulgare Mill. Cinnamomum cassia Presl. Cinnamomum cassia Presl. Cuminum cyminum L. Zanthoxylum schinifolium Sieb. et Zucc. Geraniaceae Pelargonium graveolens L.
Magnoliaceae Magnoliaceae Magnoliaceae Umbelliferae Umbelliferae Lauraceae Lauraceae Umbelliferae Rutaceae Geraniaceae
Fruit Fruit Fruit Fruit Fruit Bark Bark Fruit Pericarp Leaf
Fujian Beijing Beijing Hebei Hebei Beijing Beijing Beijing Beijing Beijing
1 2 3 4 5 6 7 8 9
14 Spices
15 16 17 18 19 20 21 22 23 24
Star anise Star anise Star anise Fennel Fennel Cassia bark Cassia bark Cumin Pricklyash peel Pelargonium graveolens L’ herit
re-dissolved in methanol–water (50:50, v/v) to yield 1 ml of solution. The solution containing investigated mycotoxins was vortexed for 30 s and filtered through a 0.45 lm filter for chromatographic detection.
2.8. Statistical analysis The statistical analysis of data was performed using student’s ttest with statistical package SPSS (version 18.0). The p-value < 0.05 was considered significant.
2.7. Detection of AFB1, AFB2, AFG1, AFG2 and OTA by HPLC-PCD-FLD The presence of AFB1, AFB2, AFG1, AFG2 and OTA was detected by a high performance liquid chromatography (HPLC) method using a on-line post-column photochemical derivatisation (PCD) with an AURA Industries reactor (AURA Industries, NY, USA) and a RF-10AXL fluorescence detector (FLD) (Shimadzu, Kyoto, Japan). HPLC analysis was performed on an Shimadzu LC-20AT HPLC system consisting of two LC-20 AD pumps, an SIL-20AC autosampler and a CTO-20A column oven. A reverse phase Unimicro Technologies C18 (4.6 250 mm, 5.0 lm, Pleasanton, CA, USA) column was used for chromatographic separation and the column temperature was set at 30 °C. The mobile phase consisted of (A) methanol and (B) 0.5% aqueous acetic acid at a flow rate of 1.0 ml/min, freshly prepared every day. The optimized gradient elution procedure was as follows: 0–12 min (52–68%, A), 12–35 min (68%, A). The injection volume was 50 ll. The maximum emission wavelength of the PCD reactor was set at 254 nm. The eluate was monitored by a fluorescence detector. During the first 15 min of analysis, fluorescence conditions were optimized for AFs (excitation 360 nm and emission 440 nm wavelengths), and after that for OTA (excitation 333 nm and emission 460 nm wavelengths). The system was interfaced via an LC solution ChemStation software (Shimadzu Kyoto, Japan) to a personal computer for controlling instruments and data acquisition and processing. To confirm the presence of AFB1 and AFG1, the derivatisation reactor was disconnected and fluorescence detector was directly connected to the HPLC pump. By this way, the peaks of AFB1 and AFG1 almost could not be seen on the chromatogram because of their low fluorescent response.
3. Results and discussion 3.1. Fungal isolates from tested samples The distribution of tested samples (seeds, fruits, barks, rhizomes, pericarps and leaves) on the medium was shown in Fig. 1A. After incubating the samples for 7 days at 25 °C, most of the samples became mouldy to different extents (Fig. 1B) and a total of 124 fungal isolates (Table 2) belonging to four different genera, such as Aspergillus (82), Penicillium (19), Fusarium (13) and Eurotium (10) were identified and recorded from the 24 functional foods and spices.
Fig. 1. (A) Distribution of tested Coix seed samples on the medium; (B) Display of mould on tested Coix seed samples after incubation.
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W. Kong et al. / Food Chemistry 146 (2014) 320–326 Table 2 Fungal isolates and their incidence in tested functional foods and spices in China market. Sample
Label
Strains of fungal isolates
Functional foods
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus Aspergillus
15 16 17 18 19 20 21 22 23 24
Penicillium (1) Penicillium (2) Aspergillus (4) Fusarium (2) Aspergillus (4), Fusarium (3) Aspergillus (3) Aspergillus (13), Eurotium (1) Aspergillus (12), Fusarium (1) Aspergillus (3), Eurotium (1)
Spices
Total a
No. of isolates
(3), (8), (7), (3), (1),
Penicillium (2) Penicillium (1) Penicillium (1) Penicillium (2), Fusarium (4) Eurotium (1), Penicillium (1), Fusarium (2)
(5), (1), (2), (4) (5), (1), (1), (2),
Penicillium (2) Penicillium (3) Penicillium (1) Penicillium (1) Penicillium (2), Fusarium (1) Eurotium (3) Eurotium (4)
Aspergillus (82), Penicillium (19), Fusarium (13), Eurotium (10)
Rd (%)a
5 9 8 9 5 0 7 4 3 4 6 4 4 6
4.0 7.3 6.5 7.3 4.0 0 5.7 3.2 2.4 3.2 4.8 3.2 3.2 4.8
1 2 0 4 2 7 3 14 13 4
0.8 1.6 0 3.2 1.6 5.7 2.4 11.4 10.5 3.2
124
100
Relative density (%) = No. of the isolates of a certain species/No. of total isolates of all strains.
Fig. 2. Incidences of (A) identified strains in all fungal isolates, and (B) fungal isolates in functional foods and spices.
The incidence of samples colonized by Aspergillus and Penicillium was significantly predominant from the 124 strains isolated, 82 belong to Aspergillus and 19 belong to Penicillium, with an incidence of 66.1% and 15.3%, respectively. The numbers of Fusarium and Eurotium isolates were 13 and 10, respectively, representing 10.5% and 8.1% of the total number of isolates (Fig. 2A). With respect to the tested samples, the incidence of the five different fungal isolates in 14 functional foods (59.6%) was almost 1.5 times that in the 10 spices tested (40.4%) (Fig. 2B). However, student’s t-test analysis of the incidence as relative density (%) (Table 2) did not show statistically significant variability in the means for functional foods and spices (t = 0.179, p > 0.05). The above results indicated that both the tested functional foods and spices were prone to be contaminated by the identified fungal mycoflora,
especially the genera Aspergillus and Penicillium. This observation was greatly in agreement with other investigators who dealt with mycoflora of spices and medicinal plants (Romagnoli et al., 2007). Table 2 shows that all the seed samples were infected by Aspergillus and Penicillium except sample 10 (Bitter apricot seed). Similarly, all the rhizome samples including samples 12 and 14, as well as all bark samples including samples 20 and 21 showed Aspergillus infection. The fruit samples of different matrices were found to be infected by a large number of fungi, including Aspergillus, Penicillium, Fusarium and Eurotium. With regard to functional foods, Coix seeds were found to be the most heavily contaminated samples, especially sample 4, followed by Lotus seed, Ginkgo seed and Zingiber. Mume fruit expressed no fungal isolates, which might be a result of the fruits antifungal activity, or due to its abundant organic acids that inhibit the growth of moulds. For the tested spices, Cumin and Pricklyash peel contained the most fungal isolates. In addition, the other spice samples show little contamination, especially Star anise. One sample of Star anise showed no fungal infection, which maybe due to the differences of processing technologies or storage conditions. In summary, these various results might partly elucidate that matrices of the different plant parts tested had various sensitivities to fungi and would be contaminated by different fungal isolates during their processing, storage and transportation processes. 3.2. Occurrence of potential mycotoxin-producing fungi in tested samples On the studied medium, many colonies of the genera have been identified from the 24 functional foods and spices with Aspergillus dominating. Then, three potential mycotoxin-producing fungi including A. flavus section, Aspergillus niger section and Aspergillus ochraceus section were further isolated and identified, which have been shown in Table 3. It can be shown that the highest relative frequency (%) occurrence was recovered in A. niger section (58.3%, 14/24) followed by A. flavus section (45.8%, 11/24) and A. ochraceus section (12.5%, 3/24). The relative frequency of A. niger section was significantly higher (p < 0.05) than that of the other two fungal species. Similar findings were revealed for the 14
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Table 3 Occurrence of potential mycotoxin-producing fungi in tested samples. Label
A. flavus section
A. niger section
A. ochraceus section
Functional foods
1 2 3 4 5 6 7 8 9 10 11 12 13 14
18.9 3.4 3.2 8.1 ND ND 89.7 0.7 ND 0.6 ND ND ND ND 124.6 50.0 (7/14)
ND c 11.4 8.3 1.5 ND ND 20.5 ND 5.6 3.5 20.0 ND ND 2.5 73.3 57.1 (8/14)
ND 3.4 17.3 ND ND ND 2.6 ND ND ND ND ND ND ND 23.3 21.4 (3/14)
15 16 17 18 19 20 21 22 23 24
ND ND ND 1.8 ND 13.3 10.7 ND 0.5 ND 26.3 40.0 (4/10)
ND ND ND 0.6 ND 10.0 10.7 15.2 38.9 37.8 113.2 60.0 (6/10)
ND ND ND ND ND ND ND ND ND ND 0 (0/10)
Total (%) Rf (%)b Spices
Total (%) Rf (%) a b c
Rd (%)a of potential mycotoxin-producing fungi
Sample
Relative density (%) = No. of the identified fungi of certain species/No. of all species. Relative frequency (%) = No. of contaminated samples with a mycotoxin-producing fungi/No. of all analyzed functional foods or spices samples. Not detected.
functional foods with the relative frequency (%) occurrence in the order of A. niger section (57.1%, 8/14), A. flavus section (50.0%, 7/ 14) and A. ochraceus section (21.4%, 3/14), as well as for the 10 spices of A. niger section (60.0%, 6/10), A. flavus section (40.0%, 4/ 10) and A. ochraceus section (0, 0/10). A. ochraceus section was detected in very few samples of the functional foods and in none of the spices. In addition, the highest occurrence of the three fungi presented as relative frequency (%) was found in seed samples, followed by bark, pericarp, leaf, fruit and rhizome samples. Three (samples 8, 9 and 18) out of the eleven fruit samples were infected by one of the three fungi, among which, only one spice-fruit sample (sample 22) was infected by A. niger section. None of the two rhizome samples were infected by any fungi. Two Coix seed (samples 2 and 3) and one Lotus seed (sample 7) samples were simultaneously contaminated by the three fungi. The above results indicated that the occurrence of A. niger section was significantly predominant among the three detected potential mycotoxin-producing fungi in the tested functional foods and spices, also elucidated that the matrices of studied samples were more sensitive to A. niger section than the other two fungi. The incidence presented as relative density (%) for the three detected fungi in tested samples are also listed in Table 3. For the 14 functional foods, the highest total incidence was founded in A. flavus section (124.6%) followed by A. niger section (73.3%) and A. ochraceus section (23.3%), while for the 10 spices, A. niger section revealed the highest total incidence of 113.2%, A. flavus section expressed low incidence of 26.3% and no A. ochraceus section was detected. These findings showed that A. flavus section and A. niger section might be the predominant fungi infecting functional foods and spices, respectively. On the other hand, among the functional foods, the highest incidence of A. flavus section, A. niger section and A. ochraceus section was found in Lotus seed (sample 7) collected from Beijing city. This might be related to the environmental
conditions, as it grows exposed to water and air. Among the spices, a high incidence of A. flavus section and/or A. niger section was observed in both Pricklyash peel and Pelargonium graveolens L’Herit samples. A. flavus section, A. niger section and A. ochraceus section were common fungi that could grow in different substrates. The above results have elucidated the high relative frequency and density of the three detected fungi in the functional foods and spices tested. The results also show the different susceptibilities of these matrices to the three fungi, which were in accordance with the reports of Raper and Fennel (Raper & Fennel, 1977). 3.3. Occurrence and levels of some mycotoxins in tested samples It has been reported, under favorable conditions, some fungal species can synthesize toxic metabolites (mycotoxins), and most of the identified fungi, such as A. flavus section, A. niger section and A. ochraceus section produce mycotoxins, namely aflatoxins (AFB1, AFB2, AFG1 and AFG2) and ochratoxins (OTA) (Romagnoli et al., 2007). Our previous studies have reported some mycotoxin contamination, especially AFB1, AFB2, AFG1, AFG2 and OTA in spices, aromatic and medicinal herbs (Liu et al., 2013). Here, the occurrence and levels of AFB1, AFB2, AFG1, AFG2 and OTA in the 24 samples tested were analyzed by the developed HPLC-PCD-FLD method (Wei, Yang, Qiu, Yang, & Qin, 2011), which has been validated in agreement with requirements established by Regulation (EC) No. 401/2006 (European Commission, 2006). This developed method gave good selectivity, linearity (0.2–50.0 ng/ml for AFB1, 0.06–15.0 ng/ml for AFB2, 0.3–50.0 ng/ml for AFG1, 0.09–15.0 ng/ml for AFG2 and 1.0–50.0 ng/ml for OTA), sensitivity (expressed as limits of detection (LOD) of 0.04, 0.02, 0.08, 0.03, 0.3 lg/kg and limits of quantification (LOQs) of 0.15, 0.06, 0.25, 0.1, 1.0 lg/kg for AFB1, AFB2, AFG1, AFG2, OTA,
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W. Kong et al. / Food Chemistry 146 (2014) 320–326 Table 4 Occurrence (%) of positive samples and contamination level (lg/kg) of mycotoxins in tested functional foods and spices. Samples
Functional foods
Label
AFB1
AFB2
AFG1
AFG2
OTA
1 4 5 9
0.2 0.15 – 0.26 21.4 (3/14)
– – –
