Proximate composition, fungal isolation and contamination of aflatoxin B1 in chickpea seeds from the Punjab, Pakistan

Abstract Chickpea, Cicer arietinum L., is a nutrient rich crop that is widely cultivated and consumed in Pakistan. However, chickpea is highly prone to fungal growth leading to contamination with aflatoxins, the most potent carcinogen found in nature. In this study, fifty chickpea seed samples were collected from the local markets of the Punjab, Pakistan, to evaluate their nutritional quality, fungal and AFB1 contamination. Proximate analysis suggested that chickpea seeds contained 5.5–6.93% moisture, 62.24–63.24% carbohydrates, 22.75–23.44% protein, 4.99–5.4% fat, 5.62–5.84% fiber and 2.92–3.16% ash. Morphological identification techniques revealed fourteen fungal species belonging to six fungal genera from which Aspergillus flavus was the leading contaminant. AFB1 analysis revealed that sixty-two percent samples were contaminated with AFB1. All the AFB1 positive samples contained AFB1 level more than 2 ppb and 12.9% samples contain AFB1 level more than 20 ppb, exceeded the maximum limit (ML) assigned by EU and USA (FDA & FAO) respectively. The results of the present studies reported that chickpea is a highly contaminated commodity in terms of fungi and AFB1 that’s why further investigations and monitoring are required to reduce the fungal and AFB1 contamination. These baseline data are an initial step in the effort to deal with this significant food safety issue. Graphical Abstract


Introduction
Chickpea, Cicer arietinum L., is the third most important cool-season grain legume crop and second most grown crop of Pakistan with an annual production of 760,000 million tons cultivated in an area of 1.094 million hectares (Jan et al. 2020;Rafiq et al. 2020). Pakistan is the third-largest producer of chickpea in the world with a 7% share of global production (Maurya and Kumar 2018;FAOSTAT 2019). Chickpea seeds are major and inexpensive source of highly digestible nutrients such as carbohydrates, minerals, proteins, vitamins, amino acids, and fibers, especially for people in developing countries (Dhankhar et al. 2019). Besides being an excellent source of fundamental nutrients, chickpea seeds contain a diverse range of bioactive compounds that exhibit high antioxidant, anti-diabetic, anti-cancer, and anti-inflammatory properties, for improving health by decreasing the incidence of diseases (Cid-Gallegos et al. 2020;Mahbub et al. 2021).
Chickpea is an important crop in Pakistan owing to its multidimensional uses, but it is highly susceptible to pathogenic fungi that significantly affects productivity (Wu et al. 2014). Some of the contaminating fungi such as Aspergillus, Alternaria, Fusarium, and Penicillium produces several mycotoxins like aflatoxins, alternariol, ochratoxin A, patulin, fumonisins, zearalenone and nivalenol/deoxynivalenol, with aflatoxin produced by Aspergillus as the most prevalent (Enyiukwu et al. 2014;Hathout and Aly 2014). The four major aflatoxins commonly isolated from foods and feeds are aflatoxin B 1 (AFB 1 ), aflatoxin B 2 (AFB 2 ), aflatoxin G 1 (AFG 1 ), and aflatoxin G 2 (AFG 2 ), and they are categorized as group 1 human carcinogens by the International Agency for Research on Cancer (IARC) (EFSA 2020). Over half of the global population, especially in developing countries, are at threat of chronic exposure to unknown levels of aflatoxins, which can be associated with decreased growth rate and feeding efficiency, reduced liver and kidney function, and immune system suppression (Shuaib et al. 2010;Diedhiou et al. 2011). AFB 1 is the most harmful aflatoxin to humans and animals as it is carcinogenic due to its association with hepatocellular carcinoma which leads to liver cancer (Qureshi et al. 2015).
Agriculture plays a pivotal role in economic development in Pakistan by contributing a 21.8% share of the GDP, from which chickpea contributes about 4.7% share of GDP to the national economy (FAOSTAT 2019). Raw chickpea is a prominent export product used by several companies to gain money and as a result, the food sector in the industrialized world expects the highest-quality raw materials that meet regulatory aflatoxin limitations. The warm and humid conditions of Pakistan, especially in the province of Punjab, facilitates thriving mycoflora, especially A. flavus, and by extension aflatoxin production (Kumar et al. 2017). As a consequence, economic losses due to A. flavus contamination can reach up to 100% when the presence of aflatoxins is beyond acceptable levels (Bhatnagar-Mathur et al. 2015). Additionally, many Pakistani farms do not utilize proper handling practices or food storage facilities, and this poses serious threats to the health of local people. There is paucity of data on fungal and mycotoxin contamination of leguminous crops, especially chickpea in the Punjab area of Pakistan, therefore, the current study was carried out to investigate the nutritional composition, fungal and AFB 1 contamination in chickpea seeds from the Punjab area in Pakistan.

Proximate composition
In this study, fifty chickpea seed samples collected from three districts (Rawalpindi, Chakwal, and Khushab) of the Punjab Pakistan were evaluated for proximate composition. The highest moisture content was reported in the samples of Chakwal district (6.37%) followed by Khushab (5.92%) and Rawalpindi (5.5%). There were no significant (p>0.05) difference in proximate composition from all the collection sites as shown in Figure S1. Carbohydrates were reported at 63.24% in Khushab, 63.21% in Rawalpindi, and 62.24% in Chakwal. Proteins were higher in Chakwal at 23.44%, followed by Rawalpindi (22.97%) and Khushab (22.75%). Fiber contents were also higher in Chakwal at 5.84%, followed by Rawalpindi (5.83%) and Khushab (5.62%). Fat contents were higher in Rawalpindi at 5.4% followed by Chakwal (5.03%) and Khushab (4.99%). The highest ash content was found in Rawalpindi at 3.16% followed by Khushab (3.12%) and Chakwal (2.92%). Previous studies reported that chickpea seeds contained approximately 22% protein on a dry weight basis. However, these may vary slightly on the basis of plant species, growing conditions, maturity, and variety (Roy et al. 2010). Protein contents were significantly higher in chickpea than wheat and maize making it a viable alternative protein source (Gupta et al. 2017) (2004); Tsialtas et al. (2002); Sanchez-vioque et al. (1999) where protein, fat, carbohydrates, and fiber contents of chickpeas were reported. Some studies reported the nutritional profiling of chickpea seeds from Pakistan. Khattak et al. (2021) reported chickpea contained high amount of protein, ash and moisture. Zia-Ul-Haq et al. (2007) analyzed desi chickpea cultivars grown in the Punjab Province of Pakistan and reported that chickpea seeds contained sufficient amounts of macronutrient and micronutrient. The present study is in agreement with the studies of Khan et al. (1995); Khattak et al. (2007); Masood et al. (2014) where proximate composition of chickpea seeds was reported from Pakistan.

Fungal isolation
Chickpea seeds were screened for the presence of seedborne fungi. The presence of fungi were identified based on macro-morphological and micro-morphological characteristics (Table S1) and all the tested samples were positive for fungal growth. A total of fourteen fungal species viz. Aspergillus flavus, A. niger, A. oryzae, A. nidulenes, A. ochraceus, A. fumigatus, A. versicolor, Alternaria alternata, A. solani, Fusarium oxysporum, F. semitectum, Penicillium expansum, Rhizopus microsporus, and Mucor spp. belonging to six genera were reported ( Figure S2). Isolation frequency and relative density of all fungal species are presented in Table S2. The overall result revealed that Aspergillus flavus and A. niger were the leading contaminants followed by Fusarium oxysporum, Alternaria alternata, and Penicillium expansum. The least reported fungi were Rhizopus microsporus, Mucor spp, and Aspergillus fumigatus. Such a high fungal diversity reported from the present study was in agreement with the results of previous studies. In Pakistan, various studies have been reported on different agricultural commodities in terms of fungal isolation. Previous studies reported high fungal diversity in wheat, rice, maize and sesame seeds by  Ajmal et al. (2021). Unfortunately, in Pakistan, up till now limited studies are presented on the incidence of mycoflora in chickpea seeds but various studies were reported on chickpea throughout the world. Shamsi and Khatun (2016) reported various species of Aspergillus, Alternaria, Penicillium spp., Curvularia and Rhizopus from stored chickpea seeds. Menon (2017) reported Alternaria spp., Aspergillus spp., Penicillium spp. and Fusarium spp. from chickpea seeds. Sahile and Assefa (2018) reported Aspergillus niger, A. fumigatus and A. flavus from chickpea. The results of these studies indicated that chickpea is a highly contaminated commodity in terms of fungi. Jabeen et al. (2011) revealed that the chloroform fraction of the methanolic extract of Melia azedarach leaves was highly effective against Ascochyta rabiei, the cause of destructive blight disease of chickpea. Arman (2011) reported nine flavonoids from chickpeas: naringin, naringin malonate, liquiritigenin, naringenin, biochanin A, daidzein, formononetin, maackiain and medicarpin, These compounds play a vital role as phytoalexins because of their antimicrobial activity. Jabeen and Javaid (2010) reported that the aqueous and n-hexane extracts from leaves, fruit, rootbark and stembark of Syzygium cumini showed significant antifungal activity resulting in 7-64% and 12-39% reduction in fungal growth respectively. Further investigations and studies are required to reduce the fungal contamination in agriculturally important crops such as chickpea.

Aflatoxin B 1 analysis
The analytical data regarding the contamination of AFB 1 are summarized in Table S3. The results indicated that out of fifty chickpea seed samples, sixty-two percent samples were contaminated with AFB 1 with a mean concentration of about 9.32 ppb and a range of 5-29 ppb. Contamination of AFB 1 was significantly (p < 0.05) higher in Rawalpindi with the mean concentration of about 13.42 ppb and the range of 15-29 ppb. All the AFB 1 positive samples contained AFB 1 levels that were more than 2 ppb, exceeded the maximum limit (ML) (2 ppb) assigned by EU (European Union), and 12.9% of samples contained AFB 1 level more than 20 ppb, exceeded the ML (20 ppb) assigned by FDA (Food and Drug Administration) and FAO (Food and Agriculture Organization). HPLC chromatogram of AFB 1 positive sample is shown in Figure S3. Environmental factors such as temperature and relative humidity influence the fungal growth and AFB 1 production (Giorni et al. 2012). Agricultural products can be contaminated when drying of commodities are delayed or moisture level exceeds critical values for the mold growth during storage of the crops (Sahile and Assefa 2018). The high fungal diversity and AFB 1 level in chickpea seeds reported from the present study is due to the temperature range (26-41 C) of the study area which is optimum for fungal growth and AFB 1 production (Reddy et al. 2009). Previous studies reported high AFB 1 level in chickpea seeds in high humidity levels specifically when humidity level was above 65% (Villers 2014). AFB 1 was reported in 10.33% samples by Sahile and Assefa (2018). Three varieties of chickpea were evaluated for AFB 1 which showed that 100% of chickpea samples were contaminated with AFB 1 with an average amount of 167.7 ppb and a range of about 62.2-250 ppb (Mohana et al. 2016). AFB 1 was identified in 20% of the samples, with concentrations ranging from 3.03-4.24 ppb (Mushtaq et al. 2015). The results of these studies indicated that chickpea is a highly contaminated commodity, and the findings for fungal flora and AFB 1 are similar to those of the present study.

Pearson's correlation and cluster analysis
The Pearson's correlation analysis as shown in Table S4 reported a significant (p < 0.05) positive correlation in A. flavus (r ¼ 0.98), A. ochraceus (r ¼ 0.99), and Mucor spp (r ¼ 0.99) with aflatoxins. K-means cluster analysis of fungi from three districts are presented in Figure S4. The dendrogram showing four clusters. First two clusters contain fungi from Rawalpindi region and 3 rd and 4 th cluster showing the fungal isolates from both Chakwal and Khushab region. The present study is in agreement with the previous study by Borutova et al. (2012) in which a total of 1468 corn kernels were analyzed by HPLC and correlation analysis (r 2 ¼ 0.85-0.88); the highest correlation between mycotoxins was AFB 1 and FB 1 . Norlia et al. (2018) reported a moderate relationship (Pearson's r ¼ 0.425, p ¼ 0.00) between AFB 1 and A. flavus/A. parasiticus.

Principal component analysis
First principal component (PC1) is positively influenced by isolation frequency and relative density of fungi and negatively influenced by aflatoxins. The second principal component (PC2) is highly positively influenced by aflatoxins. The distribution of fungi and the seed samples is depicted in the biplot of PC1 and PC2 ( Figure S5). It was inferred that seeds collected from Rawalpindi have highly close association with aflatoxins while rest of seeds collected from Chakwal and Khushab have no strong correlation with aflatoxin. In the present study, contamination of AFB 1 was reported higher in Rawalpindi and PCA analysis reported that Rawalpindi has close association with aflatoxin production in chickpea seeds It was probably the weather conditions and high humidity level are more favorable for fungal growth and consequently AFB 1 production in Rawalpindi as compared to Chakwal and Khushab.

Experimental
Provided as supplementary file.

Conclusion
The current study showed that chickpea seeds are important and nutritious source of protein, carbohydrates, fat, fiber, and ash, but a threatened commodity due to fungi and AFB 1 contamination. Mycological profiling indicated that Aspergillus flavus and A. niger were the leading contaminants. AFB 1 analysis revealed that sixty-two percent of samples were contaminated with AFB 1 . All the AFB 1 positive samples contained AFB 1 levels exceeding the maximum limit of EU regulation which is an alarming condition in terms of food security in the local people of Pakistan as well as it may be hindrances to export to EU countries. Therefore, appropriate actions are needed to improve harvesting practices, packaging, handling, transportation, and storage conditions. Regular monitoring of food and feed samples and organizing workshops for farmers, traders, and exporters might help create awareness about the toxic nature of AFs. AFB 1 has a major effect on human health. Thus, storage conditions for chickpea seeds in Pakistan should be controlled nationwide.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
The author(s) reported there is no funding associated with the work featured in this article.