SUNFLOWER SEED FOR HUMAN CONSUMPTION AS A SUBSTRATE FOR THE GROWTH OF MYCOPOPULATIONS

Transcription

1 APTEFF, 43, (2012) UDK: ]: SUNFLOWER SEED FOR HUMAN CONSUMPTION AS A SUBSTRATE FOR THE GROWTH OF MYCOPOPULATIONS Marija M. Škrinjar*, Žarko M. Petrović, Nevena T. Blagojev and Vladislava M. Šošo University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, Novi Sad, Serbia These mycological investigations are implicating samples of protein sunflower seed from regular cultivation in the Vojvodina Province. Samples are examined in different stages of production: reception in the silo, separation of massive fraction on peeler and then peeling, kernel after peeling, hull, final product, i.e. kernels separated from visible impurities on conveyor bel, that are later manually divided in two fractions a) seemingly whole, undamaged kernels, without change of colour, and b) seemingly damaged kernels, broken, with change of colour. For the determination of viable count of moulds and their isolation, two different media are used in parallel: Sabouraud maltose agar (SMA) and malt/yeast extract with 50% of glucose (MY50G), favourable for growth of xerophilic moulds. All samples tested were contaminated with fungi. Total viable mould count per seed varied from 1.6 (SMA) respecting 1.3 (MY50G) on reception, to 5.6 (SMA) and 7.5 (MY50G) cfu/seed in visually damaged sunflower kernels (final product). From seeds, kernels and hull, numerous moulds were isolated, belonging to 8 genera and 13 species (Alternaria alternata, Arthrinium phaeospermum, Aspergillus candidus, A. flavus, A. niger, A. ochraceus, A. versicolor, A. wentii, Cladosporium cladosporioides, Eurotium herbariorum, Penicillium aurantiogriseum, Rhizopus stolonifer and Trichoderma harzianum). Alternaria alternata, Aspergillus flavus, A.ochraceus, A. versicolor and Eurotium herbariorum were isolated on both media. Aspergillus candidus, A. versicolor, C. Cladosporioides, P. aurantiogriseum and T. harzianum were isolated only on SMA, while A. niger, A. wentii and R. stolonifer were exclusively isolated on MY50G. Most ubiquitous species is A. alternata, which is isolated from all tested samples, while A. candidus, C. cladosporioides and T. harzianum were isolated from sunflower seed on reception in silo, using SMA medium. KEY WORDS: sunflower, mycopopulations, xerophilic moulds, SMA, MYG50 * Corresponding author: Marija Škrinjar, University of Novi Sad, Faculty of Technology, Bulevar cara Lazara 1, Novi Sad, Serbia, [email protected] 115

2 APTEFF, 43, (2012) UDK: ]: INTRODUCTION Sunflower (Helianthus annus L.) is an annual plant native to the North America, transferred to Europe in the 16th century. In the Vojvodina Province, its cultivation started during the World War I, but more intensive production dates from The highest dispersion of sunflower in this area reached when new Soviet sorts were imported after the World War II, and later, by implementation of domestic hybrids with high oil content (1). Based on the content of the main parts of sunflower seed (kernel/hull), oil and proteins, two main types of sunflower can be distinguished: oil type and protein type. Oil type is aimed for industrial production of oil and usually contains 20-30% of hull and more than 40% of oil (referring on seeds with moisture content of 10%), so this type of sunflower belongs to the oil seeds with high oil content. Considering the kernel size, this sunflower type belongs to the group of small-size seeds. Protein type of sunflower is used for industrial production of protein-based sunflower products (hulled kernels, protein flour, butter sunflower kernel cream, etc.), or it can be used directly for consumption, which is why it has been called confection sunflower. This type contains 30% of oil and 40-45% of hull, while the protein content in novel sorts is about 29%, and in hybrid sorts more than 32%. Considering the kernel size, this type belongs to large-size seeds. Crop plants, including oil seeds, are constantly, although at different extents, very susceptible to contamination by different kinds of moulds (2-7). The level of mould contamination depends on diverse factors, such as: species, sort, hybrid, cultural practices during the growing and maturation phases, conditions during harvest, transport and storage, as well as climatic conditions during growing, maturation ad harvest. Sunflower is attacked by a number of diseases caused by various microorganisms, including fungi. Of the fungal foliar diseases, leaf spot caused by Alternaria helianthi, Septoria helianthi and other fungal pathogens are relatively important (8, 9). Fusarium wilt is caused by different species of the genus Fusarium (F. solani, F. oxysporum, F. helianthi, F. moniliforme and others). Sclerotinia wilt and head rot of sunflower are caused by Sclerotinia sclerotiorum (10, 11). According to many data (3, 12, 13), sunflower seeds are highly contaminated with fungi which attack the plants at different stages of development, harvesting and storage. In this respect, important role play Aspergillus and Fusarium species, zigomycete and species from the group Dematiaceous Hyphomycetes (Alternaria spp., Cladosporium spp.). A lot of fungal species that contaminate sunflower seeds are toxigenic and under proper conditions produce toxic metabolites mycotoxins, harmful for human and animal health. Considering the aforementioned, the aim of this work was to investigate the mycological properties of the protein type of sunflower seeds aimed for the human consumption, during storage and further processing to the final product, with the special focus on the level of the fungal contamination, presence of the specific species and their moiety in isolated mycopopulation. 116

3 APTEFF, 43, (2012) UDK: ]: MATERIALS AND METHODS The sunflower samples used for mycological investigation were protein hybrids, randomly sampled from one small enterprise for raw hulled sunflower seeds production in the Vojvodina Province. The following samples were used: seeds after the reception in the silo, massive seed fraction before the peeling, kernels after the peeling, hull, and final product kernels cleaned from the impurities on the conveyer, and than manually divided into two fractions: a) seemingly whole undamaged kernels without discoloration and b) seemingly damaged kernels, broken or with present discoloration. Determination of the total mould count per kernel/g, their isolation and identification. For the determination of the total mould count per kernel, the following method was used: 10 g of the sample was treated with 100 ml of 4% sodium-hypochlorite, in order to remove all moulds possibly present on the seed surface. Erlenmeyer flasks (300 ml) were shaken on the rotary shaker for 3 minutes, and then all samples were rinsed with sterile distilled water (2 x 100 ml). On the surface of the solidified media containing antibiotics (1 ml of chloramphenicol and 1 ml of oxytetracyclin per 100 ml of the medium), 8 kernels were aseptically placed. Inoculated Petri dishes were incubated for 7 days at 25 C. For the determination of the total mould count per 1g of hull, the dilution method was used, under the same incubating conditions. For the both methods, two different media were used: Sabouraud-maltose agar (SMA) and the medium containing yeast and malt extracts and 50% of glycose (MYG50: malt extract 10g; yeast extract 2.5g; glycose 500g; agar 10g; distilled water 1000ml), which has been suggested by Pitt and Hocking (14) for isolation of xerophilic moulds. All tests were conducted in triplicates. The mould growth was observed each day during 7 days, and the results were expressed as the average per kernel or gram. Identification of isolated species was done according to Ellis and Samson et al. (15,16). RESULTS AND DISCUSSION As it was mentioned above, for total mould count determination, two different media were used: SMA, general mycological medium, and MYG50, named for isolation of the xerophilic species. The moisture content of sunflower samples used in this investigation varied from 7.43% in kernel, 9.55% in seed, to 13.93% in hull, which is the reason why the medium suitable for xerophilic moulds was used. After the incubation period, it was observed that all tested samples were contaminated with moulds. In all samples, except in hull, the higher contamination was observed on SMA, comparing to MYG50 (Table 1). On the SMA medium, total mould count ranged from 1.5/g (hull) to 5.6/kernel (seemingly damaged kernels). On the MYG50 medium, total mould count varied from 1.2/kernel (massive seeds before the peeling) to 7.5/kernel (seemingly damaged kernels). 117

4 APTEFF, 43, (2012) UDK: ]: Table 1. Total viable count of moulds per sunflower seed/kernel and 1g of hull determined by using SMA and MYG50 media Sample Total viable count of moulds SMA MYG50 Seed after the reception in the silo Massive seed fraction before the peeling Kernels after the peeling Hull Seemingly whole undamaged kernels Seemingly damaged broken kernels The moulds isolated from tested samples belong to 8 genera and 13 species (Table 2), viz. Alternaria alternata, Arthrinium phaeospermum, Aspergillus candidus, A. flavus, A. niger, A. ochraceus, A. versicolor, A. wentii, Cladosporium cladosporioides, Eurotium herbariorum, Penicillium aurantiogriseum, Rhizopus stolonifer and Trichoderma harzianum. From Table 2 it could be seen that the genus Aspergillus was presented with 6 different species (A. candidus, A. flavus, A. niger, A. ochraceus, A. versicolor and A. wentii), while all other genera were presented with the one species each. Table 2. Fungal species isolated from sunflower seeds, kernels and hull by SMA and MYG50 media Species SMA MYG50 Alternaria alternata (Fr.) Keissler Arthrinium phaeospermum (Corda) M.B. Ellis Aspergillus candidus Link flavus Link niger van Tieghem ochraceus Wilhelm versicolor (Vuill.) Tiraboschi wentii Wehmer Cladosporium cladosporioides (Fres.) de Vries Eurotium herbariorum (Wiggers) Link Penicillium aurantiogriseum Direckx Rhizopus stolonifer (Ehrenb.) Lind. Trichoderma harzianum Rifai It should be mentioned that some species, such as Alternaria alternata, Aspergillus flavus, A.ochraceus, A. versicolor and Eurotium herbariorum, were isolated from specific samples by using both media, while the others were isolated by using either one of them (Table 3). The highest frequency among isolated mycopopulations had the species Alternaria alternata and Eurotium herbariorum. Alternaria alternata was the most ubiquitously found species, it was isolated from all tested samples and on both media, except from the seeds from silo, where it was isolated only on SMA. On the other hand, Eurotium herbariorum was mostly isolated by using MYG50 medium, which was expectable, since it is classified as typical xerophilic species. The species A. candidus, A. versicolor, C. cladosporium and T. harzianum were found in just one of the tested samples.

5 APTEFF, 43, (2012) UDK: ]: Table 3. Occurrence of fungal species on investigated samples determined by SMA and MYG50 From the total of 13 isolated species, even 10 (A. alternata, A. candidus, A. flavus, A. niger, A. ochraceus, A. versicolor, A. wentii, E. herbariorum, P. aurantiogriseum and R. stolonifer) have been reported to produce some of the toxic metabolites (16). Table 4 lists the toxic metabolites that this species produces under specific conditions. Table 4. The most important toxic metabolites produced by moulds isolated from sunflower seeds/kernels/hull Fungal species Alternaria alternata Aspergillus candidus A.flavus Toxins Alternariol, alterotoxin, tenuazonic acid Candidulin, terphenyllin, xanthoascin Aflatoxins, aflatrem, aflavinin, aspergillic acid, cyclopiazonic acid, 3-nitropropionic acid, paspalinin A. niger Malformin, naphthoquinones, nigragillin A. ochraceus Ochratoxin A. versicolor Sterigmatocystin, nidulotoxin A. wentii Emodin, kojic acid, 3-nitropropionic acid, wentilacton, physicon Eurotium herbariorum Penicillium aurantiogriseum Rhizopus stolonifer Sterigmatocystin Ochratoxin A, penicillic acid, xanthomegnin, viomellein, viridicatin (terrestric acid, penitrem A) Toxic cyclic peptide CONCLUSION The protein type of sunflower was tested on its mycological properties, at various processing stages. The obtained results showed that all samples were significantly contaminated with moulds. The total of 13 species, belonging to 8 genera, were isolated. Two 119

6 APTEFF, 43, (2012) UDK: ]: different media were used for the mould isolation, one general medium for mould growth, and other aimed particularly for the growth of xerophilic moulds. The mould growth was observed on the both media, but the higher total mould count was detected using general medium SMA. Also, the higher number of different species were isolated on SMA comparing to MYG50. About 76% of all isolated species are reported to produce different toxic metabolites. REFERENCE 1. Vrebalov, T. and Škorić, D.: Površine, prinosi i privredni značaj suncokreta u svetu i u našoj zemlji, in Suncokret. Ed. Milošević S., Nolit, Beograd (1988) Tabuc, C. and Stefan, G.: Assessment of mycologic and mycotoxicologic contamination of soybean, sunflower and rape seeds and meals during Archiva Zootechnica 8 (2005) Škrinjar M., Bandu, M., Dimić, E., Bjelobaba, K. and Romanić, R.: Infekcija semena suncokreta žetve 2006 aflatoksigenim gljivama. Uljarstvo 38, 1-2 (2007) Deabes M. and Al-Habib, R.: Toxigenic fungi and aflatoxin associated to nuts in Saudi Arabia. Journal of American Science 7, 8 (2011) Krnjaja V., Lević, J., Stanković, S. and Stepanić, A.: Fusarium species and their mycotoxins in wheat grain. Proc. Nat. Sci, Matica Srpska, Novi Sad 120 (2011) Kungulovski Dz., Avramovski, O., Atanasova, N., Pancevska, I. and Kungulovski, I.: Mycotoxigenic molds in spices from macedonian stores. Proc. Nat. Sci, Matica Srpska, Novi Sad 120 (2011) Luttfullah G. and Hussain, A.: Studies on contamination level of aflatoxins in some dried fruits and nuts of Pakistan. Food Control 22 (2011) Achbani, E.H., Lamrhari, A., Laamaraf, N., Bahsine M.H., Serrhini M.N., Douira A. and de Labrouche, D.T.: Downy mildew (Plasmopara halstedii): Importance and geographical distribution on sunflower in Marocco. Phytopath. Medit. 39, 2 (2000) Maširević S. and Jasnić S.: Leaf and stem spot of sunflower. Biljni lekar 34, 4-5 (2006) El-Deeb A.A., Abdallah, S.M., Mosa, A.A. and Ibrahim, M.M.: Sclerotinia diseases of sunflower in Egypt, Arab Universities. J. Agric. Sci. 8 (2000) Afzal R., Mughal, S.M., Munir, M., Sultana, K., Qureshi, R., Arshad, M. and Laghari, M.K.: Mycoflora associated with seeds of different sunflower cultivars and its management. Pak. J. Bot. 42, 1 (2010) Vaidehi, B.K.: Seed mycoflora of sunflower a perspective. Frontiers in Micro Biotec. Plant pathol. 1 (2002) Morar M.V., Dancea, Z., Bele, C., Salegean, D., Beke, A. and Baonca, I.: An approach upon the qualities of the raw material and raw oil from sunflower seeds resulting in process of low capacities. Buletinul-Universitatii-de-Stiinte-Agricole-si-Medicina- Veterinara-Cluj-Napoca-Seria-Agricultura 60 (2004) Pitt, J.I. and Hocking, A.D.: Fungi and food spoilage, CSIRO Division of Food Research, Sydney (1985)

7 APTEFF, 43, (2012) UDK: ]: Ellis, M.B.: Dematiaceous Hyphomycetes, Commonwealth Mycological Institute, Kew, Surrey, England (1971) Samson, R.A., Hoekstra, E.S. and Frisvad, J.C.: Introduction to food- and airborne fungi. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands (2004) СЕМЕ КОНЗУМНОГ СУНЦОКРЕТА КАО СУПСТРАТ ЗА РАСТ МИКОПОПУЛАЦИЈА Марија М. Шкрињар, Жарко M. Петровић, Невена Т. Благојев и Владислава М. Шошо Универзитет у Новом Саду, Технолошки факултет, Нови Сад, Србија Миколошка испитивања у овом раду баве се узорцима сунцокрета из редовног гајења на подручју Војводине. Узорци су испитивани у различитим фазама производње: полазно семе из силоса, крупнија фракција одвојена на љуштилици и која иде на љуштење, језгро које излази након љуштења са љуштилице, љуска, финални производ, тј. језгра пречишћена од видљивих нечистоћа на конвејерској траци и та зрна су на основу визуелног прегледа ручно одвојена у две фракције: а) наизглед цела, неоштећена зрна без промене боје, и б) наизглед оштећена зрна, поломљена или са променом боје. За испитивање укупног броја плесни и њихово изоловање, коришћене су паралелно две подлоге: Sabouraud-малтозни агар (SMA) и сладни/квасни екстракт са 50% глукозе (MYG50). Сви тестирани узорци су били контаминирани плеснима. Укупан број плесни по зрну је варирао од 1.6 (SMA) и 1.3 (MYG50) на пријему, до 5.6 (SMA) и 7.5 (MYG50) cfu/зрно у узорцима видљиво оштећеног језгра (финални производ). Из зрна, језгра и љуске изолован је велики број плесни, које су сврстане у 8 родова и 13 врста (Alternaria alternata, Arthrinium phaeospermum, Aspergillus candidus, A. Flavus, A. niger, A. ochraceus, A. versicolor, A. wentii, Cladosporium cladosporioides, Eurotium herbariorum, Penicillium aurantiogriseum, Rhizopus stolonifer and Trichoderma harzianum). Врсте Alternaria alternata, Aspergillus flavus, A.ochraceus, A. versicolor и Eurotium herbariorum су изоловане са обе подлоге. Aspergillus candidus, A. Versicolor, C. cladosporioides, P. aurantiogriseum и T. harzianum су изоловане само на SMA подлози, док су А. niger, A. Wentii и R. stolonifer изоловане само са MY50G подлоге. Најраспрострањенија врста је била Alternaria alternata, која је изолована из свих испитиваних узорака, док су A. candidus, C. Cladosporioides и T. Harzianum изоловане само из зрна сунцокрета на пријему у силос, и то само на SMA подлози. Кључне речи: сунцокрет, микопопулације, ксерофилне плесни, SMA, MY50G Received: 17 August 2012 Accepted: 27 September