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, 21000 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) seeming-ly whole, undamaged kernels, without change of colour, and b) seemingseeming-ly damaged ker-nels, 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. ni-ger, A. ochraceus, A. versicolor, A. wentii, Cladosporium cladosporioides, Eurotium her-bariorum, Penicillium aurantiogriseum, Rhizopus stolonifer and Trichoderma harzia-num). Alternaria alternata, Aspergillus flavus, A.ochraceus, A. versicolor and Eurotium herbariorum were isolated on both media. Aspergillus candidus, A. versicolor, C. Cla-dosporioides, 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
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 star-ted during the World War I, but more intensive production dates from 1930. 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 con-tamination depends on diverse factors, such as: species, sort, hybrid, cultural practices during the growing and maturation phases, conditions during harvest, transport and sto-rage, 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), sunflo-wer 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 Hypho-mycetes (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.
MATERIALS AND METHODS
The sunflower samples used for mycological investigation were protein hybrids, ran-domly 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 frac-tions: a) seemingly whole undamaged kernels without discoloration and b) seemingly da-maged kernels, broken or with present discoloration.
Determination of the total mould count per kernel/g, their isolation and identifi-cation. 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 shakenon 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 an-tibiotics (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 du-ring 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
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 1.6 1.3
Massive seed fraction before the peeling 2.1 1.2
Kernels after the peeling 4.8 3.6
Hull 1.5 1.6
Seemingly whole undamaged kernels 4.1 2.2
Seemingly damaged broken kernels 5.6 7.5
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 harzia-num. From Table 2 it could be seen that the genus Aspergillus was presented with 6 dif-ferent 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).
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 Toxins
Alternaria alternata Alternariol, alterotoxin, tenuazonic acid
Aspergillus candidus Candidulin, terphenyllin, xanthoascin
A.flavus 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 Sterigmatocystin
Penicillium aurantiogriseum
Ochratoxin A, penicillic acid, xanthomegnin, viomellein, viridicatin (terrestric acid, penitrem A)
Rhizopus stolonifer Toxic cyclic peptide
CONCLUSION
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 pro-duce different toxic metabolites.
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