cjas Cuban Journal of Agricultural Science Cuban J. Agric. Sci. 2079-3480 Ediciones ICA 00004 ANIMAL SCIENCE Fungal contaminants in Sicilian livestock feeds and first studies on the enzymatic activity of Aspergillus isolates Mirabile G. 1 Bella P. 1 Conigliaro G. 1 Giambra S. 1 Alberto Vazquez M. 2 Davino S. 1 Torta L. 1 * Dipartmento di Scienze Agrarie, Alimentari e Forestali (SAAF), Università degli Studi di Palermo, Viale Delle Scienze, Ed. 5, 90128 Palermo, Italy Università degli Studi di Palermo Dipartmento di Scienze Agrarie, Alimentari e Forestali (SAAF) Università degli Studi di Palermo Palermo Italy Instituto de Ciencia Animal (ICA), San José de Las Lajas, Mayabeque, Cuba Instituto de Ciencia Animal Instituto de Ciencia Animal (ICA) San José de Las Lajas Mayabeque Cuba Email: livio.torta@unipa.it 05 12 2019 12 2019 53 4 373 386 25 03 2019 01 07 2019 This is an open-access article distributed under the terms of the Creative Commons Attribution License ABSTRACT

The purposes of this study were i) to determine the total fungal contamination in Sicilian raw materials and livestock, ii) to evaluate the occurrence of Aspergillus spp., Penicillium spp. and Fusarium spp, iii) to identify fungi belonging to the genus Aspergillus and iiii) to determine their ability to produce cellulolytic enzymes. Fourteen feed samples were collected in a feed mill near to Palermo (Sicily, Italy). Analysis of the total mycobiota was performed on Sabourad Dextros Agar (SAB) and Potato Dextrose Agar (PDA) and total fungal counts were expressed as CFU/g. Aspergillus spp. isolates were selected on the basis of the frequency of isolation and identified using micro and macro-morphological characteristics and ITS sequence analysis. The ability of the Aspergillus isolates to produce cellulolytic enzymes was tested qualitatively by in vitro assay at two temperature, 25 and 30 °C, and in static and shaking condition. Total fungal population ranged from 1.11x106 to 1.31x108 and from 1.11x103 to 1.58x106 CFU/g on PDA and SAB, respectively. All feed samples showed the recurrent presence of colonies belonging mostly to the ubiquitous genera Aspergillus, Fusarium and Penicillium. Eight isolates of Aspergillus spp. were obtained and identified as A. amstelodami, A. awamori, A. flavus, A. niger, A. oryzae and A. tubingensis. Between them, A. awamori, A. niger and A. tubingensis showed the highest enzymatic activity. The presence of potential mycotoxigenic isolates of Aspergillus spp. in the analysed feeds represents a risk for animal health; moreover their ability to produce cellulolytic enzymes can seriously affect feed quality.

 Key words: toxygenic moulds raw materials animal foods cellulolytic activity INTRODUCTION

Fungal contamination in food and feed cause significant economic losses in primary agricultural yields, in the transforming industries and in livestock farms (Godfray et al. 2016). The main contaminant fungi (molds), belonging to several ubiquitous genera, are characterized by the production of large masses of conidia easily spread by air to the soil, water, plants and animals (Adhikari et al. 2004). Fungal contamination of the raw materials also occurs during pre-harvest (field-produced fungi) and post-harvest periods, such as storage and transformation processes (storage-produced fungi) (Krnjaja et al. 2008 and Whitlow et al. 2010). Contaminating fungi prejudice the preservability and the nutritional value of food and feed by the production of lithic enzymes and pose a potential risk for consumer health by mycotoxigenic activity (McNeil et al. 1984).

The cellulolytic activity is widely distributed in the contaminating fungi belonging to Phylum Ascomycota as in the genera Bulgaria, Chaetomium, Helotium, Neurospora, Aspergillus, Cladosporium, Fusarium, Geotrichum, Myrothecium, Paecilomyces, Penicillium and Trichoderma (Lynd et al. 2002 and Tian et al. 2009). They dominate, both in abundance and in activity, the microbial community responsible for the decomposition of cellulose residues (Wilson, 2011).

Among these, species belonging to the genus Aspergillus are able to produce a large variety of glucanases that allow the complete degradation of cellulose.

Moreover, species belonging to genera Aspergillus, Fusarium and Penicillium, able to produce dangerous mycotoxins, can cause metabolic disorders resulting in biological effects on animals as liver and kidney toxicity, central nervous system effects and estrogenic effects (Greco et al. 2014). Some contaminating mycotoxin due secondary contamination in humans via eggs, meat or milk (carry-over effect), with acute and chronic toxic effects (Volkel et al. 2011).

In particular, the contamination of feeds with fungi and their spores is worldwide described. In tropical regions Aspergillus spp. predominate in several feeds and Penicillium, Fusarium and Alternaria species are recurrent contaminants of kernels and other grains (Prasad et al. 2016). In Sicily, recent studies on local row materials and feeds shown low mycotoxins contamination levels, if compared to imported products, while very few are the data on the level of fungal colonization (Finoli and Vecchio, 2003, Gallo et al. 2008 and Russo, 2015).

The aim of this study was to evaluate, in Sicilian raw materials and livestock feeds, the total fungal contamination and to detect the percentage of the three potential mycotoxigenic genera (Aspergillus, Fusarium and Penicillium). Moreover, the most recurrent Aspergillus isolates, identified at level of specie by morphological and molecular methodologies, was tested to evaluate their cellulolytic activity.

MATERIALS AND METHODS

Sampling. A total of fourteen feed samples (table 1) were collected in a feed mill located in the province of Palermo (Sicily, Italy) following the standard methodologies (Reg. CE 1441/2007; Reg. CE 401/2006; DM 20/04/1978). Aliquots of 600 g were randomLy taken from different parts of the bag or container of each feed both from packaging and storage areas. The samples were placed in sterile plastic bags, transported to the laboratory and stored at 4 °C until further analysis.

Feed samples collected in a feed mill in the province of Palermo
Code Sample Composition Sampling area
1 Flaked broad bean Raw material Packaging area
2 Oat Raw material
3 Poultry feed Soy flour, cornmeal, maize, wheat bran
4 Flaked maize Raw material
5 Swine feed Maize, wheat bran, barley, sunflower flour, carob, citrus peel
6 Horses feed (West Performance) Wheat bran, broad bean, flaked barley, carobs, maize, oat flour, molasses
7 Cattle feed Maize, barley, carobs, broad bean
8 Poultry feed Soy, corn, wheat bran, maize Finished product storage area
9 Horses feed (West Performance) Wheat bran, broad bean, flaked barley, carobs, maize, oat flour, molasses
10 Flacked broad bean Raw material
11 Cattle feed Maize, barley, carobs, broad bean
12 Flaked maize Raw material
13 Horses feed (Superior House) Flaked broad bean, flaked maize, flaked barley, flaked oat, flaked corn, sunflower flour
14 Ruminants feed Broad bean, barley flour, maize, wheat bran

Fungal contamination. Isolation and enumeration of fungal colonies were carried out using serial dilution and spread plate technique (Maina et al. 2016). All samples were ground with a mill up to 0.25 mm and 1 g of each sample was homogenized in 10 mL of distilled sterilized water. Samples were analyzed in triplicate. Aliquots (0.1 mL) of ten-fold serial dilutions were inoculated in Sabouraud Dextrose Agar (SAB) and Potato Dextrose Agar (PDA) in order to evaluate the medium efficiency (the highest number of colonies). All plates were incubated at 22 °C for 9 days and the total fungal count (CFU/g) and relative percentage of Aspergillus, Fusarium and Penicillium colonies were evaluated under stereomicroscope every 3 days. Single colonies of Aspergillus spp. were sub-cultured into PDA and monoconidial pure cultures of each strain were obtained and used for further characterization. All the isolates were maintained into PDA agar slants and cryopreserved in 15% glycerol at -80 °C.

Morphological identification of Aspergillus species. Macro-morphological features of the Aspergillus isolates were determined in PDA, SAB and Czapek dox Agar (CZ) according to Lin and Dianese (1976) and Thathana et al. (2017). For each Aspergillus isolate, plugs from the edge of 7-day-old pure colonies were taken by a sterile 3 mm cork borer and placed singly at the center of 10 cm diameter plates with the three different media, in three replicates. The plates were incubated at 25°C for 9 days and diameter, growth rate, color (conidia and reverse), shape and texture of each colony were recorded every 3 days. Microscopic observations were performed at the microscope Axioskop (Zeiss, Oberkochen, Germany) coupled to an AxioCam MRc5 (Zeiss, Oberkochen, Germany) digital camera. Images were captured using the software AxioVision 4.6 (Zeiss, Oberkochen, Germany). The microscopic features were conidial heads, vesicle shape and diameter, presence of metulae and size and shape of phialides and conidia (30-50 measurements) (table 3). The identification was carried out using taxonomic keys (Barnett and Hunter 1972, von Arx 1981, Cole and Kendrick 1989, Pitt and Hocking 1999 and Klich, 2002).

DNA extraction, PCR and sequencing. Genomic DNA was extracted from pure colonies of the most recurrent Aspergillus isolates following the CTAB-based method (Torta et al., 2015). The DNA was suspended in 100 µL of TE 1x (0,121g of Tris 10 mM and 0,037g of EDTA 1mM in 100 mL of distilled water) quantified by using NanoDrop ND-1000 and stored at -20°C. The primers ITS1F (Gardes and Bruns 1993) and ITS4 (White et al. 1990) were used for the amplification of the ITS1-5.8S-ITS2 region. The PCR assay was performed in a total reaction volume of 25 µL consisting of PCR buffer 10X (Thermo Scientific), 0,2 mM of each dNTPs, 0,3 µM of each primer, 0,5 U of Taq DNA polymerase (Dream Taq, Thermo Scientific) and 1 µL of target DNA. The amplification was carried out in a MultiGene OptiMax thermocycler (Labnet International Inc.) with an initial denaturation cycle at 94 °C for 3 min followed by 35 cycles at 94 °C for 30s, annealing at 55 °C for 30s, elongation at 72 °C for 45 s, with a final extension at 72 °C for 10 min.

PCR products were separated by electrophoresis in 1% agarose gel and amplicons were detected under UV transilluminator (330 nm).

PCR products were purified using Exo I-SAP protocol according to the manufacturer’s instructions (Applied Biosystems, Foster City, CA). Primer ITS1F was used in the sequencing reaction. Sequencing reactions were performed with BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) followed by Ethanol/EDTA/Sodium Acetate precipitation (according to manufacturer’s instruction). Finally, capillary electrophoresis was performed in the 3500 Genetic Analyzer (Applied Biosystems, Foster City, CA). The sequences were aligned against those already deposited in the GenBank databases using BLASTn tool (Altschul et al., 1990). New sequences were deposited in GenBank.

Qualitative determination of the cellulolytic activity. The cellulolytic activity of the Aspergillus isolates was performed according to Mandels et al. (1976) and Ghorbani et al. (2015). The strains were grown in 10 mL of Mandels liquid medium (Mandels et al., 1976) in tubes containing a 1x6 cm Whatman No 1 paper strips and incubated at temperatures of 25 and 30 °C under static condition, in order to evaluate the effect of temperature on the cellulolytic activity. The control was tubes not inoculated. Cellulolytic activity was also evaluated in shaking condition (90 rpm) at 30 °C. After 5, 10, 15 and 21 days, fungal growth and paper maceration were evaluated by using a rating scale from 0 to 5 (0 = no fungal growth and no maceration; 5 = complete paper colonization and maceration).

Statistical analysis. Data on total fungal contamination were subjected to analysis of variance (ANOVA) through the Statgraphics Plus 5.1 program. The averages were compared by the Fisher LSD multiple comparison test (P ≤ 0.05).

 RESULTS

Fungal contamination of feed samples. All feed samples showed fungal contamination, and total fungal count varied within the samples and also in relation to culture medium (figure 1). At the third day of incubation, on PDA, fungal colonies were observed only on sample 1 (flaked broad bean), while on SAB they were present in 12 out of 14 feed samples. At 6 days number of fungal colonies increased in all the samples both on SAB and PDA. These values remained fairly constant until the ninth day but in PDA total fungal population was higher than in SAB. Total population ranged from 1.11x106 to 1.31x108 and from 1.11x103 to 1.58x106 CFU/g on PDA and SAB respectively. Statistically significant differences were observed at nine days of incubation within the feed samples on both media, although total fungal contamination was similar in the two sampling areas (table 2). Oat (sample 2) and poultry feed (sample 8) showed the highest level of total fungal contamination on PDA and on SAB respectively (table 2). Regarding the potential mycotoxigenic fungal genera, colonies belonging to Aspergillus, Fusarium and Penicillium were obtained on 3 (samples 1, 3, 12, on PDA) and on 4 (1, 3, 11, 12 on SAB) out of 14 samples. The frequency of the three isolated genera is shown in figure 2. In general, Fusarium sp. was the prevalent species isolated (from 10 and 7 samples, respectively on PDA and SAB). Penicillia, isolated from 5 and 4 samples on PDA and SAB respectively shown the lower frequency. Nevertheless, the percentage of the three genera varied depending on the culture medium. Aspergillus spp. colonies were more frequently isolated on SAB, while Fusarium spp. and Penicillium on PDA. With regard to total fungal population, the frequency of isolation of Aspergillus colonies was about 10% on both media (data not shown), whereas in each sample the percentage ranged from 14 to 36% and from 1.33 to 70% on PDA and SAB respectively.

Total fungal contamination detected in the 14 feed samples at 3, 6 and 9 days of incubation, on PDA (above) and SAB (below)

Total fungal contamination in the analyzed feed samples at 9 days of incubation on PDA and SAB, expressed in Log of UFC/g and relative percentage of Aspergillus colonies.
SAMPLE PDA %Aspergillus SAB %Aspergillus
1= Flaked broad bean 6.37 + 2.28 a 0 5.05 + 1.84 ab 0
2= Oat 8.12 + 0.39 c 25 5.20 + 0,30 ab 59
3= Poultry feed 7.88 + 0,40 abc 0 5.48 + 0.22 ab 0
4= Flaked maize 7.37 + 0.46 ab 36 5.09 + 1.85 ab 0
5= Swine feed 7.46 + 0.41 ab 31 5.94 + 0.08 bc 3
6= Horses feed 8.01 + 0.88 bc 0 5.05 + 1.74ab 0
(West Performance)
7= Cattle feed 7.91 + 0.17 bc 18 4.43 + 0.09 a 8
8= Poultry feed 7.85 + 0.14 abc 14 6.20 + 0.26 c 1.33
9= Horses feed 7.65 + 0.09 ab 20 5.12 + 0.52 ab 70
(West Performance)
10= Flacked broad bean 7.05 + 2.50 a 0 5.05 + 1.63ab 66
11= Cattle feed 6.05 + 2.17 a 0 3.05 + 1.17a 0
12= Flaked maize 7.15 + 0.59 a 0 3.52 + 1.22a 0
13= Horses feed 7.35 + 0.66 ab 0 3.74 + 1.30 a 60
(Superior House)
14= Ruminants feed 7.35 + 2.50 ab 0 4.60 + 1.54a 0

abcdIn each column, values followed by same letters are not statistically different according to Fisher LSD Test (P≤0.05).

Frequency of Aspergillus, Fusarium and Penicillium isolated at 9 days of incubation on PDA (above) and SAB (below) from 14 feed samples

Identification of Aspergillus spp. Aspergillus spp. were isolated from 8 out of 14 feed samples and eight isolates belonging to Section Nigri, Section Flavi and Section Nidulantes, were selected on the basis of the frequency of isolation and of the macro-morphological features. The strains belonging to the Section Nigri presented colonies with dark-brown to black colour and reverse pale or light yellow color; the conidiophores bearing spherical vesicles were uniseriate or biseriate producing globular or subglobular conidia (smooth, finely rough or rough). Isolates belonging to the Section Flavi were characterized by typical yellow-green mature conidia, reverse light yellow to yellow colour; mainly uniseriate conidiophores with globular vesicle producing globular conidia (smooth or finely rough). Isolates of Section Nidulantes presented colonies with pale yellow or yellow colour and reverse yellow or pale orange color; microscopically they presented uniseriate conidiophores, subglobular vescicles, subglobular rough spores and characteristic Hulle’s cells. Based on morphological characteristics, the isolates were identified as Aspergillus amstelodami (L. Mangin) Thom & Church (figure 3), Aspergillus awamori Nakaz., Aspergillus flavus Link., Aspergillus niger Tiegh., Aspergillus oryzae (Ahlb.) Cohn. and Aspergillus tubingensis Mosseray (table 3).

An amplicon from about 500 to 600 bp of the ribosomal region including the two non-coding ITS1 and ITS2, and the 5.8S rDNA gene was amplified from 8 Aspergillusspp. isolates. Apergillus spp. isolates had high match with published sequences in GenBank showing maximum identities of 99-100% with Apergillus sections Nigri (A. awamori, A. niger, A. tubingensis), Flavi (A. flavus, A. oryzae) and Nidulantes (A. amstelodami) (table 3).

Morphological features of the 8 <italic>Aspergillus spp</italic>. isolated from feed samples. T= teleomorph (sexual reproductive stage); A= anamorph (asexual reproductive stage)
Isolates Feed samples Colour on PDA Colony growth at 25°C (mm) Stage Conidiophores Conidia shape Conidia size (µm) Vesicle size (µm) Hulle’s cell Species GenBank Accession number
PDA SAB CZ ITS
SAAF 7 5 Black 55x57 60x65 66x67 A Biseriate Rough 4.6-5.7 55-70 - A. niger MK503962
SAAF 12 8 Black 83x83 85x85 85x85 A Biseriate Rough 4.5-6 25-40 - A. niger MK503964
SAAF 15 9 Black 79x81 80x82 83x84 A Biseriate Rough 4.6-5.8 30-45 - A. niger MK503966
SAAF 14 7 Dark-brown 35x36 32x33 45x47 A Biseriate Smooth 3.5-4.1 20-40 - A. tubingensis MK503965
SAAF 10 12 Black 80x80 85x85 85x85 A Biseriate Smooth 4-6 40-50 - A. awamori MK503963
SAAF 4 7 Green 67x70 75x75 83x83 A Biseriate Smooth -finely rough 3.2-5.8 18-36 - A. flavus MK503960
SAAF 17 10 Yellow-green 75x75 80x80 75x75 A Biseriate Smooth 5.3-7.2 25-40 - A. oryzae MK503967
SAAF 6 2 Yellow 25x25 25x25 30x30 T Uniseriate Rough 4.3-6.5 20-25 + A. amstelodami MK503961

Macroscopic and microscopic features of <italic>A. amstelodami</italic> SAAF 6. 10 days- old colonies grown on PDA (a = F; b = R), SAB (c = F; d = R) and CZ (e = F; f = R); g, h ) Conidiophores, vesicles and conidial head; i) ascus and ascospores. F = Front, R = Reverse. Bar: g, h, = 25 µm; i = 10 µm

Qualitative determination of the cellulolytic activity. All Aspergillus strains showed cellulolytic activity, growing on the filter paper (table 4). In all the control tubes the filter paper was not altered. Strains A. niger SAAF 7, A. awamori SAAF 10 and A. tubingensis SAAF 14 shown the highest cellulolytic activity, completely macerating the paper at the end of the test. On the other hand, the temperature seems affects the cellulolytic capacity. At 25 ºC the filter paper was generally less macerated, whereas A. amstelodami SAAF 6 showed cellulolytic activity only at this temperature. Agitation induced greater or lesser maceration of the filter paper depending on the Aspergillus strain.

Cellulolytic activity of eight Aspergillus strains, detected up to 21 days after inoculation at 25 and 30 °C in static (S) and shaking (Sh) condition.
Strains 25 ºC S 30 ºC S 30 ºC Sh
5 10 15 21 5 10 15 21 5 10 15 21
A. amstelodami SAAF 6 1 1 2 3 0 0 0 0 0 0 0 0
A. awamori SAAF 10 1 1 2 2 1 1 1 2 1 2 3 5
A. flavus SAAF 4 1 1 1 1 1 1 1 1 1 1 1 1
A. tubingensis SAAF 14 1 1 2 2 0 1 3 5 1 1 3 5
A. niger SAAF 7 1 1 1 1 1 3 4 5 1 1 3 5
A. niger SAAF 12 1 2 3 4 1 1 2 2 0 1 2 4
A. niger SAAF 15 1 1 1 1 1 2 2 3 1 1 1 1
A. oryzae SAAF 17 1 1 1 1 1 2 2 2 1 1 2 4

DISCUSSION

In this study, for the first time, fourteen feed samples collected in two sampling area in a mill in Sicily were monitored to detect the presence of contaminating fungi. The level of fungal contaminations in all feed samples ranged from about 3 to 8 Log CFU/g with the recurrent presence of colonies belonging mostly to the ubiquitarious genera Aspergillus, Fusarium and Penicillium, the main toxigenic molds. Between them Fusarium was the predominant genus isolated from samples, followed by Aspergillus and Penicillium, according with the results of other studies conducted in Italy and Europe (Chadd 2004, Covarelli et al. 2011 and Gregori et al. 2013). Moreover, the culture medium efficiency was also tested revealing differences in the estimation of fungal populations on the two utilized agar media. The universal medium PDA, ensuring the isolation of the highest number of fungal colonies, allow to assess the total fungal population, while to evaluate the Aspergillus population SAB should be preferred (Krnjaja et al. 2008). Regarding the two sampled area ours data showed no significantly differences, but the levels of total fungal contamination and above all the presence of toxigenic fungi revealed inappropriate techniques during the feed producing chain, indicating a low quality and bad treatment of the materials.

Usually, the population of these fungal contaminants in feed and food is strictly related to the relative concentrations in mycotoxins, in particular in bad-stored or long-exported materials (Dalcero et al. 1998, Krysinska-Traczyk et al. 2001, Gonzalez Pereyra et al. 2012; and Greco et al. 2014). On the contrary, in Sicilian raw materials and finished livestock feeds, despite the presence of these fungal contaminants, previous studies indicate that the dangerous metabolites (afla-, ochra- and Fusarium-toxins) resulted to be absent or detected at low level (Finoli and Vecchio 2003, Gallo et al. 2008 and Russo 2015). However, high level of fungal contaminants cause in feed and food the loss of nutritional value, due to their degradation by enzymatic activity (Driehuis and Oude Elferink 2000, Megan et al. 2003, andAmigot et al. 2006).

Among the contaminating fungi, some species within the genus Aspergillus are the most critical for their mycotoxigenic and cellulolytic activity, causing a potential risk for animal and human health and for the degradation of trophic substance (Hanif et al. 2004 and Patyshakuliyeva 2016). The production of cellulolytic enzymes by Aspergillus strains isolated from analysed feed samples was qualitatively evaluated. A. niger, A. tubingensis and A. awamori showed the highest expression level of cellulase enzymatic complex. On these basis, oat and poultry feed, the most contaminated livestock tested feeds, may have lost most of their organoleptic and nutritional quality.

As presented in the results, the highest cellulolytic activity is shown in the Aspergillus species that are part of the "Nigri section", in this case A. niger, A. awamori and A. tubingensis, the most used at the industrial level. These fungi, characterized by a high distribution worldwide, are also considered the most common fungi that occur in the decomposition of food (Raper and Fennell 1965) by the production of a great variety of enzymes, such cellulases, xylanases, proteases and phytases, mainly, and also α-amylases, pectinases, amyloglucosidases and lactases (Krishna 2005 and Aguiar, 2010). As regards the effect of temperature on the cellulolytic activity of microorganisms, a greater degradation rate was observed for most of the cases at 30ºC. It is necessary to point out that temperature is one of the main factors that affect the biomass yield, an aspect that is closely related to the type of microorganism that is being tested. Each type of microorganism has a certain optimum growth temperature where it expresses its highest productivity. Temperatures close to this can also have a similar effect. Several authors propose optimum temperatures for the growth of different species of Aspergillus between 25-35ºC (Passamani et al., 2014). On the other hand, temperature affects not only the growth of the biomass but also the production of different metabolites. Several authors have reported optimal temperatures for the production of cellulases in Aspergillus species between 30-35ºC (Bastawde 1992, Velkovska et al. 1997, Shahriarinour et al., 2011 and Saithi et al. 2016).

On the other hand, once the microorganisms have secreted their enzymes into the media, the enzymatic activity is conditioned by different factors such as pH, ionic strength, temperature, among others (Seager et al. 2016). These factors condition an optimal range of activity that varies depending on the type of enzyme. Although, in relation to temperature, an increase of this brings an increase in the speed of the reaction and consequently in the enzymatic activity (Voet et al. 2016). Regarding to the effect of the agitation, it is observed that at the end of the 21 days of experimentation all the strains with agitation, except in the case of A. niger, had a higher cellulolytic activity respect to those without agitation. This is mainly due to the fact that the agitated systems allow a greater interaction of the microorganism with the substrate, which allows a better colonization and use of the specific substrate surface that results in a greater production of enzymes. (Jeong et al. 2006).

In regard to the mycotoxigenic activity (not evaluated in this study), among the six identified Aspergillus species, A. niger and A. flavus are reported as agents of human and animal mycotoxicosis, the first associated with the production of ochratoxin A, and the second with aflatoxins B1 and B2 (Yu, 2012). However, not all the strains of these species are able to metabolize dangerous mycotoxins. As matter of fact, it is known that in A. flavus species only about 40-50% of strains produce aflatoxins and only about 20% of A. niger strains are ochratoxigenic (Abarca et al. 2001 and Davari et al. 2015). Although the detection of toxigenic fungi in the analyzed samples not necessarily indicate that mycotoxins are naturally occurring in the feed, it alerts to the potential risk of contamination. Other species isolated from feed samples, such as A. awamori and A. oryzae, not associated with the production of toxic metabolites, are largely used in food biotechnologies (Siedenberg et al. and 1998; Takagi 2014).

CONCLUSIONS

This first study on the evaluation of the level of fungal contamination in Sicilian raw materials and livestock showed the presence of Aspergillus spp., Penicillium spp. and Fusarium spp, in the sampled materials. In particular, Apergillus sections Nigri (A. awamori, A. niger, A. tubingensis), Flavi (A. flavus, A. oryzae) and Nidulantes (A. amstelodami) were identified by morphological and molecular methodologies. The production of cellulolytic enzymes was observed in all the Aspergillus strains, but A. niger SAAF 7, A. awamori SAAF 10 and A. tubingensis SAAF 14 shown the highest degradation activity.

On the basis of these results it is assumed that the most contaminated feed by these fungi can be the most degraded from a nutritional point of view.

This study highlights the importance of continued monitoring and control of fungal contamination in feed and food. Strategies aimed to prevent them in field, during storage and in all the feed production chain should be implemented. The control of this fungal contaminants and corrects techniques of animal feed production, in fact, can assure not only high level of animal’s health but also high quality level of feeds both in palatability and nutritional values.

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Toxins. 4: 1024-1057. doi:10.3390/toxins4111024. Yu J. 2012 Current understanding on aflatoxin biosynthesis and future perspective in reducing aflatoxin contamination Toxins 4 1024 1057 10.3390/toxins4111024 CIENCIA ANIMAL Contaminantes fúngicos en piensos para ganado siciliano y primeros estudios sobre la actividad enzimática de aislados de Aspergillus. Mirabile G. 1 Bella P. 1 Conigliaro G. 1 Giambra S. 1 Vazquez M. Alberto 2 Davino S. 1 Torta L. 1 * Dipartmento di Scienze Agrarie, Alimentari e Forestali (SAAF), Università degli Studi di Palermo, Viale Delle Scienze, Ed. 5, 90128 Palermo, Italy Instituto de Ciencia Animal (ICA), San José de Las Lajas, Mayabeque, Cuba Email: livio.torta@unipa.it RESUMEN

Los objetivos de este estudio fueron: 1) determinar la contaminación fúngica total en materias primas y ganado sicilianos, 2) evaluar la aparición de Aspergillus spp., Penicillium spp. y Fusarium spp, 3) identificar hongos pertenecientes al género Aspergillus y 4) determinar su capacidad de producir enzimas celulolíticas. Se recolectaron catorce muestras de alimento en una fábrica de piensos cerca de Palermo (Sicilia, Italia). El análisis de la micobiota total se realizó en Sabourad Dextrose Agar (SAB) y Potato Dextrose Agar (PDA) y los recuentos totales de hongos se expresaron como UFC/g. Los aislados de Aspergillus spp. se seleccionaron en función de la frecuencia de aislamiento y se identificaron utilizando características micro y macro morfológicas y análisis de secuencia ITS. La capacidad de los aislados de Aspergillus para producir enzimas celulolíticas se probó cualitativamente mediante un ensayo in vitro a dos temperaturas, 25 y 30 °C, y en condiciones estáticas y de agitación. La población total de hongos varió de 1.11x106 a 1.31x108 y de 1.11x103 a 1.58x106 UFC/g en PDA y SAB, respectivamente. Todas las muestras de pienso mostraron la presencia recurrente de colonias pertenecientes, principalmente, a los géneros ubicuos Aspergillus, Fusarium y Penicillium. Ocho aislados de Aspergillus spp. se obtuvieron e identificaron como A. amstelodami, A. awamori, A. flavus, A. niger, A. oryzae y A. tubingensis. Entre ellos, A. awamori, A. niger y A. tubingensis mostraron la mayor actividad enzimática. La presencia de potenciales aislados micotoxigénicos de Aspergillus spp. en los piensos analizados representan un riesgo para la salud animal; además, su capacidad para producir enzimas celulolíticas puede afectar seriamente la calidad del pienso.

 Palabras claves: mohos toxigénicos materias primas alimentos de origen animal actividad celulolítica INTRODUCCIÓN

La contaminación fúngica en alimentos y piensos causa importantes pérdidas económicas en los rendimientos agrícolas primarios, en las industrias transformadoras y en las explotaciones ganaderas (Godfray et al. 2016). Los principales hongos contaminantes (mohos), que pertenecen a varios géneros ubicuos, se caracterizan por la producción de grandes masas de conidias que se propagan fácilmente por aire al suelo, al agua, a las plantas y a los animales (Adhikari et al. 2004). La contaminación fúngica de las materias primas también se produce durante la precosecha (hongos producidos en el campo) y los períodos poscosecha, como los procesos de almacenamiento y transformación (hongos producidos por el almacenamiento) (Krnjaja et al. 2008 y Whitlow et al.2010). Los hongos contaminantes perjudican la preservabilidad y el valor nutricional de los alimentos y piensos mediante la producción de enzimas líticas y representan un riesgo potencial para la salud del consumidor por la actividad micotoxigénica (McNeil et al. 1984).

La actividad celulolítica está ampliamente distribuida en los hongos contaminantes pertenecientes al Tipo Ascomycota, como en los géneros Bulgaria, Chaetomium, Helotium, Neurospora, Aspergillus, Cladosporium, Fusarium, Geotrichum, Myrothecium, Paecilomyces, Penicillium y Trichoderma (Lynd et al. 2002 y Tian et al. 2009). Dominan, tanto en abundancia como en actividad, la comunidad microbiana responsable de la descomposición de los residuos de celulosa (Wilson 2011).

Entre estas, las especies que pertenecen al género Aspergillus son capaces de producir una gran variedad de glucanasas que permiten la degradación completa de la celulosa.

Además, las especies que pertenecen a los géneros Aspergillus, Fusarium y Penicillium, capaces de producir micotoxinas peligrosas, pueden causar trastornos metabólicos que producen efectos biológicos en animales como toxicidad hepática y renal, efectos en el sistema nervioso central y efectos estrogénicos (Greco et al. 2014). Algunas micotoxinas contaminantes se deben a la contaminación secundaria en humanos a través de huevos, carne o leche (efecto de arrastre), con efectos tóxicos agudos y crónicos (Volkel et al. 2011).

En particular, la contaminación de los piensos con hongos y sus esporas se describe en todo el mundo. En las regiones tropicales, Aspergillus spp. predominan en varios piensos y las especies de Penicillium, Fusarium y Alternaria son contaminantes recurrentes de los granos (Prasad et al. 2016). En Sicilia, estudios recientes sobre materias primas y piensos locales mostraron niveles bajos de contaminación por micotoxinas, en comparación con productos importados, mientras que muy pocos son los datos sobre el nivel de colonización de hongos (Finoli y Vecchio 2003, Gallo et al. 2008 y Russo 2015).

El objetivo de este estudio fue evaluar, en las materias primas sicilianas y los piensos para ganado, la contaminación fúngica total y detectar el porcentaje de los tres géneros micotoxigénicos potenciales (Aspergillus, Fusarium y Penicillium). Además, los aislados de Aspergillus más recurrentes, identificados a nivel de especie mediante metodologías morfológicas y moleculares, se analizaron para evaluar su actividad celulolítica.

 MATERIALES Y MÉTODOS

Muestreo. Un total de catorce muestras de piensos (tabla 1) se recogieron en una fábrica de piensos ubicada en la provincia de Palermo (Sicilia, Italia) siguiendo las normas metodológicas (Reg. CE 1441/2007; Reg. CE 401/2006; DM 20/04 / 1978). Se tomaron aleatoriamente partes alícuotas de 600 g de diferentes partes de la bolsa o contenedor de cada pienso, tanto de las áreas de empaque como de almacenamiento. Las muestras se colocaron en bolsas plásticas estériles, se transportaron al laboratorio y se almacenaron a 4°C hasta su posterior análisis.

Feed samples collected in a feed mill in the province of Palermo
Code Sample Composition Sampling area
1 Flaked broad bean Raw material Packaging area
2 Oat Raw material
3 Poultry feed Soy flour, cornmeal, maize, wheat bran
4 Flaked maize Raw material
5 Swine feed Maize, wheat bran, barley, sunflower flour, carob, citrus peel
6 Horses feed (West Performance) Wheat bran, broad bean, flaked barley, carobs, maize, oat flour, molasses
7 Cattle feed Maize, barley, carobs, broad bean
8 Poultry feed Soy, corn, wheat bran, maize Finished product storage area
9 Horses feed (West Performance) Wheat bran, broad bean, flaked barley, carobs, maize, oat flour, molasses
10 Flacked broad bean Raw material
11 Cattle feed Maize, barley, carobs, broad bean
12 Flaked maize Raw material
13 Horses feed (Superior House) Flaked broad bean, flaked maize, flaked barley, flaked oat, flaked corn, sunflower flour
14 Ruminants feed Broad bean, barley flour, maize, wheat bran

Contaminación fúngica. El aislamiento y la enumeración de colonias fúngicas realizaron utilizando una técnica de dilución en serie y placa de extensión (Maina et al. 2016). Todas las muestras se molieron en un molino hasta 0.25 mm y 1 g de cada muestra se homogeneizó en 10 mL de agua destilada esterilizada. Las muestras se analizaron por triplicado. Se inocularon partes alícuotas (0.1 mL) de diluciones en serie de diez veces en Sabouraud Dextrose Agar (SAB) y Potato Dextrose Agar (PDA) para evaluar la eficacia del medio (el mayor número de colonias). Todas las placas se incubaron a 22 ° C durante 9 días y el recuento total de hongos (UFC/g) y el porcentaje relativo de colonias de Aspergillus, Fusarium y Penicillium se evaluaron con estereomicroscopio cada 3 días. Colonias individuales de Aspergillus spp. se subcultivaron en PDA y se obtuvieron cultivos puros monoconidiales de cada cepa y se utilizaron para una caracterización adicional. Todos los aislados se mantuvieron en agar PDA en plano inclinado y se crioconservaron en glicerol al 15 % a -80°C.

Identificación morfológica de especies de Aspergillus. Las características macro-morfológicas de los aislados de Aspergillus se determinaron en PDA, SAB y Agar Czapek dox (CZ) de acuerdo con Lin y Dianese (1976) y Thathana et al. (2017). Para cada aislado de Aspergillus, los tapones del borde de las colonias puras de 7 días se tomaron con un perforador de corcho estéril de 3 mm y se colocaron individualmente en el centro de placas de 10 cm de diámetro con los tres medios diferentes, en tres repeticiones. Las placas se incubaron a 25°C durante 9 días y el diámetro, la tasa de crecimiento, el color (conidios y reverso), la forma y la textura de cada colonia se registraron cada 3 días. Se realizaron observaciones microscópicas en el microscopio Axioskop (Zeiss, Oberkochen, Alemania) acoplado a una cámara digital AxioCam MRc5 (Zeiss, Oberkochen, Alemania). Las imágenes se capturaron utilizando el software AxioVision 4.6 (Zeiss, Oberkochen, Alemania). Las características microscópicas fueron las cabezas conidiales, la forma y el diámetro de las vesículas, la presencia de metulas y el tamaño y la forma de los fialidos y las conidias (30-50 mediciones) (tabla 3). La identificación se realizó mediante claves taxonómicas (Barnett y Hunter 1972, von Arx 1981, Cole y Kendrick 1989, Pitt y Hocking 1999 y Klich, 2002).

Extracción de ADN, PCR y secuenciación. El ADN genómico se extrajo de colonias puras de los aislados de Aspergillus más recurrentes siguiendo el método basado en CTAB (Torta et al. 2015). El ADN se suspendió en 100 µL de TE 1x (0,121 g de Tris 10 mM y 0,037 g de EDTA 1 mM en 100 mL de agua destilada) cuantificado usando NanoDrop ND-1000 y almacenado a -20°C. Los cebadores ITS1F (Gardes y Bruns 1993) e ITS4 (White et al. 1990) se usaron para la amplificación de la región ITS1-5.8S-ITS2. El ensayo de PCR se realizó en un volumen de reacción total de 25 µL que consta de tampón de PCR 10X (Thermo Scientific), 0,2 mM de cada dNTPs, 0,3 µM de cada cebador, 0,5 U de ADN polimerasa Taq (Dream Taq , Thermo Scientific) y 1 µL de ADN objetivo. La amplificación se llevó a cabo en un termociclador MultiGene OptiMax (Labnet International Inc.) con un ciclo de desnaturalización inicial a 94°C durante 3 minutos seguido de 35 ciclos a 94°C durante 30 segundos, alineamiento a 55°C durante 30 segundos, elongación a 72°C durante 45 s, con una extensión final a 72°C durante 10 min.

Los productos de PCR se separaron por electroforesis en gel de agarosa al 1 % y se detectaron amplicones bajo transiluminador UV (330 nm).

Los productos de PCR se purificaron utilizando el protocolo Exo I-SAP de acuerdo con las instrucciones del fabricante (Applied Biosystems, Foster City, CA). El cebador ITS1F se usó en la reacción de secuenciación. Las reacciones de secuenciación se realizaron con BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) seguido de precipitación con etanol/EDTA/acetato de sodio (según las instrucciones del fabricante). Finalmente, la electroforesis capilar se realizó en el analizador genético 3500 (Applied Biosystems, Foster City, CA). Las secuencias se alinearon contra las ya depositadas en las bases de datos GenBank utilizando la herramienta BLASTn (Altschul et al. 1990). Se depositaron nuevas secuencias en GenBank (tabla 3).

Determinación cualitativa de la actividad celulolítica. La actividad celulolítica de los aislados de Aspergillus se realizó de acuerdo con Mandels et al. (1976) y Ghorbani et al. (2015). Las cepas se cultivaron en 10 mL de medio líquido Mandels (Mandels et al. 1976) en tubos que contenían tiras de papel Whatman No 1 de 1x6 cm y se incubaron a temperaturas de 25 y 30°C en condiciones estáticas, para evaluar el efecto de temperatura en la actividad celulolítica. El control fueron tubos no inoculados. La actividad celulolítica también se evaluó en condiciones de agitación (90 rpm) a 30°C. Después de 5, 10, 15 y 21 días, se evaluó el crecimiento de hongos y la maceración de papel utilizando una escala de calificación de 0 a 5 (0 = sin crecimiento de hongos y sin maceración; 5 = colonización completa del papel y maceración).

Análisis estadístico. Los datos sobre contaminación fúngica total se sometieron a análisis de varianza (ANOVA) a través del programa Statgraphics Plus 5.1. Los promedios se compararon mediante la prueba de comparación múltiple Fisher LSD (P ≤ 0,05).

 RESULTADOS

Contaminación fúngica de muestras de pienso. Todas las muestras de pienso mostraron contaminación fúngica, y el recuento total de hongos varió dentro de las muestras y también en relación con el medio de cultivo (figura 1). Al tercer día de incubación, en PDA, se observaron colonias de hongos solo en la muestra 1 (habas en hojuelas), mientras que en SAB estuvieron presentes en 12 de las 14 muestras de pienso. A los 6 días, el número de colonias de hongos aumentó en todas las muestras tanto en SAB como en PDA. Estos valores permanecieron bastante constantes hasta el noveno día, pero en PDA la población total de hongos fue mayor que en SAB. La población total varió de 1.11x106 a 1.31x108 y de 1.11x103 a 1.58x106 UFC/g en PDA y SAB respectivamente. Se observaron diferencias estadísticamente significativas a los nueve días de incubación dentro de las muestras de pienso en ambos medios, aunque la contaminación fúngica total fue similar en las dos áreas de muestreo (tabla 2). La avena (muestra 2) y el pienso para aves (muestra 8) mostraron el nivel más alto de contaminación fúngica total en PDA y en SAB respectivamente (tabla 2). Con respecto a los posibles géneros micotoxigénicos de hongos, se obtuvieron colonias pertenecientes a Aspergillus, Fusarium y Penicillium en 3 (muestras 1, 3, 12, en PDA) y en 4 (1, 3, 11, 12 en SAB) de 14 muestras. La frecuencia de los tres géneros aislados se muestra en la figura 2. En general, Fusarium sp. fue la especie aislada prevalente (de 10 y 7 muestras, respectivamente en PDA y SAB). La Penicillia, aislada de 5 y 4 muestras en PDA y SAB respectivamente, mostró la frecuencia más baja. Sin embargo, el porcentaje de los tres géneros varió según el medio de cultivo. Las colonias de Aspergillus spp. se aislaron con mayor frecuencia en SAB, mientras que Fusarium spp. y Penicillium en PDA. Con respecto a la población total de hongos, la frecuencia de aislamiento de las colonias de Aspergillus fue de aproximadamente 10 % en ambos medios (datos no mostrados), mientras que en cada muestra el porcentaje varió de 14 a 36 % y de 1.33 a 70 % en PDA y SAB respectivamente.

Total fungal contamination detected in the 14 feed samples at 3, 6 and 9 days of incubation, on PDA (above) and SAB (below)

Total fungal contamination in the analyzed feed samples at 9 days of incubation on PDA and SAB, expressed in Log of UFC/g and relative percentage of Aspergillus colonies.
SAMPLE PDA %Aspergillus SAB %Aspergillus
1= Flaked broad bean 6.37 + 2.28 a 0 5.05 + 1.84 ab 0
2= Oat 8.12 + 0.39 c 25 5.20 + 0,30 ab 59
3= Poultry feed 7.88 + 0,40 abc 0 5.48 + 0.22 ab 0
4= Flaked maize 7.37 + 0.46 ab 36 5.09 + 1.85 ab 0
5= Swine feed 7.46 + 0.41 ab 31 5.94 + 0.08 bc 3
6= Horses feed 8.01 + 0.88 bc 0 5.05 + 1.74ab 0
(West Performance)
7= Cattle feed 7.91 + 0.17 bc 18 4.43 + 0.09 a 8
8= Poultry feed 7.85 + 0.14 abc 14 6.20 + 0.26 c 1.33
9= Horses feed 7.65 + 0.09 ab 20 5.12 + 0.52 ab 70
(West Performance)
10= Flacked broad bean 7.05 + 2.50 a 0 5.05 + 1.63ab 66
11= Cattle feed 6.05 + 2.17 a 0 3.05 + 1.17a 0
12= Flaked maize 7.15 + 0.59 a 0 3.52 + 1.22a 0
13= Horses feed 7.35 + 0.66 ab 0 3.74 + 1.30 a 60
(Superior House)
14= Ruminants feed 7.35 + 2.50 ab 0 4.60 + 1.54a 0

abcdIn each column, values followed by same letters are not statistically different according to Fisher LSD Test (P≤0.05).

Frequency of Aspergillus, Fusarium and Penicillium isolated at 9 days of incubation on PDA (above) and SAB (below) from 14 feed samples

Identificación de Aspergillus spp. Aspergillus spp. se aislaron 8 de las 14 muestras de pienso y se seleccionaron ocho aislamientos pertenecientes a la Sección Nigri, Sección Flavi y Sección Nidulantes, en función de la frecuencia de aislamiento y de las características macro-morfológicas. Las cepas pertenecientes a la Sección Nigri presentaron colonias de color marrón oscuro a negro e inversa de color amarillo pálido o amarillo claro; los conidióforos que portaban vesículas esféricas fueron uniseriados o biseriados produciendo conidios globulares o subglobulares (lisos, finamente rugosos o rugosos). Los aislados pertenecientes a la Sección Flavi se caracterizaron por conidios maduros amarillo verdosos típicos, inverso de color amarillo claro a amarillo; principalmente conidióforos uniseriados con vesículas globulares que producen conidios globulares (lisos o finamente rugosos). Los aislados de la sección Nidulantes presentaron colonias de color amarillo pálido o amarillo e inverso, color amarillo o naranja pálido; microscópicamente presentaron conidióforos uniseriados, vesículas subglobulares, esporas rugosas subglobulares y células características de Hulle. Basado en las características morfológicas, los aislados se identificaron como Aspergillus amstelodami (L. Mangin) Thom & Church (figura 3), Aspergillus awamori Nakaz., Aspergillus flavus Link., Aspergillus niger Tiegh., Aspergillus oryzae (Ahlb.) Cohn. y Aspergillus tubingensis Mosseray (tabla 3).

Un amplicón de aproximadamente 500 a 600 pb de la región ribosómica, que incluye los dos ITS1 e ITS2 no codificantes, y el gen de ADNr 5.8S se amplificó a partir de 8 aislados de Aspergillus spp. Los aislados Apergillus spp. tuvieron una alta coincidencia con las secuencias publicadas en el GenBank mostrando identidades máximas de 99 a 100 % con secciones Nigri de Apergillus (A. awamori, A. niger, A. tubingensis), Flavi (A. flavus, A. oryzae) y Nidulantes (A. amstelodami) (tabla 3).

Morphological features of the 8 <italic>Aspergillus spp</italic>. isolated from feed samples. T= teleomorph (sexual reproductive stage); A= anamorph (asexual reproductive stage)
Isolates Feed samples Colour on PDA Colony growth at 25°C (mm) Stage Conidiophores Conidia shape Conidia size (µm) Vesicle size (µm) Hulle’s cell Species GenBank Accession number
PDA SAB CZ ITS
SAAF 7 5 Black 55x57 60x65 66x67 A Biseriate Rough 4.6-5.7 55-70 - A. niger MK503962
SAAF 12 8 Black 83x83 85x85 85x85 A Biseriate Rough 4.5-6 25-40 - A. niger MK503964
SAAF 15 9 Black 79x81 80x82 83x84 A Biseriate Rough 4.6-5.8 30-45 - A. niger MK503966
SAAF 14 7 Dark-brown 35x36 32x33 45x47 A Biseriate Smooth 3.5-4.1 20-40 - A. tubingensis MK503965
SAAF 10 12 Black 80x80 85x85 85x85 A Biseriate Smooth 4-6 40-50 - A. awamori MK503963
SAAF 4 7 Green 67x70 75x75 83x83 A Biseriate Smooth -finely rough 3.2-5.8 18-36 - A. flavus MK503960
SAAF 17 10 Yellow-green 75x75 80x80 75x75 A Biseriate Smooth 5.3-7.2 25-40 - A. oryzae MK503967
SAAF 6 2 Yellow 25x25 25x25 30x30 T Uniseriate Rough 4.3-6.5 20-25 + A. amstelodami MK503961

Macroscopic and microscopic features of <italic>A. amstelodami</italic> SAAF 6. 10 days- old colonies grown on PDA (a = F; b = R), SAB (c = F; d = R) and CZ (e = F; f = R); g, h ) Conidiophores, vesicles and conidial head; i) ascus and ascospores. F = Front, R = Reverse. Bar: g, h, = 25 µm; i = 10 µm

Determinación cualitativa de la actividad celulolítica. Todas las cepas de Aspergillus mostraron actividad celulolítica, creciendo en el papel de filtro (tabla 4). En todos los tubos de control no se alteró el papel de filtro. Las cepas A. niger SAAF 7, A. awamori SAAF 10 y A. tubingensis SAAF 14 mostraron la mayor actividad celulolítica, macerando completamente el papel al final de la prueba. Por otro lado, la temperatura pareció afectar la capacidad celulolítica. A 25 ºC, el papel de filtro generalmente estaba menos macerado, mientras que A. amstelodami SAAF 6 mostró actividad celulolítica solo a esta temperatura. La agitación indujo una maceración mayor o menor del papel de filtro dependiendo de la cepa de Aspergillus.

Cellulolytic activity of eight Aspergillus strains, detected up to 21 days after inoculation at 25 and 30 °C in static (S) and shaking (Sh) condition.
Strains 25 ºC S 30 ºC S 30 ºC Sh
5 10 15 21 5 10 15 21 5 10 15 21
A. amstelodami SAAF 6 1 1 2 3 0 0 0 0 0 0 0 0
A. awamori SAAF 10 1 1 2 2 1 1 1 2 1 2 3 5
A. flavus SAAF 4 1 1 1 1 1 1 1 1 1 1 1 1
A. tubingensis SAAF 14 1 1 2 2 0 1 3 5 1 1 3 5
A. niger SAAF 7 1 1 1 1 1 3 4 5 1 1 3 5
A. niger SAAF 12 1 2 3 4 1 1 2 2 0 1 2 4
A. niger SAAF 15 1 1 1 1 1 2 2 3 1 1 1 1
A. oryzae SAAF 17 1 1 1 1 1 2 2 2 1 1 2 4

DISCUSIÓN

En este estudio, por primera vez, se monitorearon catorce muestras de pienso recolectadas en dos áreas de muestreo en un molino en Sicilia para detectar la presencia de hongos contaminantes. El nivel de contaminaciones fúngicas en todas las muestras de pienso varió de aproximadamente de 3 a 8 Log CFU/g con la presencia recurrente de colonias que pertenecen principalmente a los géneros ubicuos Aspergillus, Fusarium y Penicillium, los principales mohos toxigénicos. Entre ellos, Fusarium fue el género predominante aislado de las muestras, seguido de Aspergillus y Penicillium, de acuerdo con los resultados de otros estudios realizados en Italia y Europa (Chadd 2004, Covarelli et al. 2011 y Gregori et al. 2013). Además, la eficacia del medio de cultivo también se probó revelando diferencias en la estimación de las poblaciones de hongos en los dos medios de agar utilizados. El PDA medio universal, que garantiza el aislamiento del mayor número de colonias de hongos, permite evaluar la población total de hongos, mientras que para evaluar la población de Aspergillus se debe preferir SAB (Krnjaja et al. 2008). Con respecto a las dos áreas muestreadas, nuestros datos no mostraron diferencias significativas, pero los niveles de contaminación fúngica total y, sobre todo, la presencia de hongos toxigénicos revelaron técnicas inapropiadas durante la cadena de producción de pienso, lo que indica baja calidad y mal tratamiento de los materiales.

Por lo general, la población de estos contaminantes fúngicos en piensos y alimentos está estrictamente relacionada con las concentraciones relativas en micotoxinas, en particular en materiales mal almacenados o exportados durante mucho tiempo (Dalcero et al. 1998, Krysinska-Traczyk et al. 2001, Gonzalez Pereyra et al. 2012 y Greco et al. 2014). Por el contrario, en materias primas sicilianas y piensos terminados para ganado, a pesar de la presencia de estos contaminantes fúngicos, estudios previos indican que los metabolitos peligrosos (toxinas afla, ochra y Fusarium) resultaron estar ausentes o detectados a bajo nivel (Finoli y Vecchio, 2003, Gallo et al. 2008 y Russo 2015). Sin embargo, el alto nivel de contaminantes fúngicos causa en los piensos y alimentos la pérdida de valor nutricional, debido a su degradación por la actividad enzimática (Driehuis and Oude Elferink. 2000, Megan et al. 2003 y Amigot et al. 2006).

Entre los hongos contaminantes, algunas especies dentro del género Aspergillus son las más críticas por su actividad micotoxigénica y celulolítica, causando un riesgo potencial para la salud animal y humana y para la degradación de la sustancia trófica (Hanif et al. 2004 y Patyshakuliyeva 2016). Se evaluó cualitativamente la producción de enzimas celulolíticas por cepas de Aspergillus aisladas de muestras de pienso analizadas. A. niger, A. tubingensis y A. awamori mostraron el nivel de expresión más alto del complejo enzimático de celulasa. Sobre esta base, los piensos de avena y para aves, los piensos para animales más contaminados, pueden haber perdido la mayor parte de su calidad organoléptica y nutricional.

Como se presenta en los resultados, la mayor actividad celulolítica se muestra en las especies de Aspergillus que forman parte de la "sección Nigri", en este caso A. niger, A. awamori y A. tubingensis, las más utilizadas a nivel industrial. Estos hongos, caracterizados por alta distribución en todo el mundo, también se consideran los hongos más comunes que ocurren en la descomposición de los alimentos (Raper y Fennell, 1965) por la producción de gran variedad de enzimas, tales como celulasas, xilanasas, proteasas y fitasas, principalmente, y también α-amilasas, pectinasas, amiloglucosidasas y lactasas (Krishna 2005 y Aguiar 2010). En cuanto al efecto de la temperatura en la actividad celulolítica de los microorganismos, se observó mayor tasa de degradación en la mayoría de los casos a 30ºC. Es necesario señalar que la temperatura es uno de los principales factores que afectan el rendimiento de la biomasa, un aspecto que está estrechamente relacionado con el tipo de microorganismo que se está probando. Cada tipo de microorganismo tiene una cierta temperatura óptima de crecimiento donde expresa su mayor productividad. Las temperaturas cercanas a esta también pueden tener un efecto similar. Varios autores proponen temperaturas óptimas para el crecimiento de diferentes especies de Aspergillus entre 25-35ºC (Passamani et al. 2014). Por otro lado, la temperatura afecta no solo el crecimiento de la biomasa sino también la producción de diferentes metabolitos. Varios autores han informado temperaturas óptimas para la producción de celulasas en especies de Aspergillus entre 30-35ºC (Bastawde 1992, Velkovska et al. 1997, Shahriarinour et al., 2011 y Saithi et al. 2016).

Por otra parte, una vez que los microorganismos han secretado sus enzimas en los medios, la actividad enzimática está condicionada por diferentes factores como el pH, la fuerza iónica, la temperatura, entre otros (Seager et al. 2016). Estos factores condicionan un rango óptimo de actividad que varía según el tipo de enzima. Aunque, en relación con la temperatura, un aumento de esta trae un aumento en la velocidad de la reacción y, en consecuencia, en la actividad enzimática (Voet et al. 2016).

Con respecto al efecto de la agitación, se observa que al final de los 21 días de experimentación, todas las cepas con agitación, excepto en el caso de A. niger, tenían mayor actividad celulolítica con respecto a aquellas sin agitación. Esto se debe, principalmente, al hecho de que los sistemas agitados permiten una mayor interacción del microorganismo con el sustrato, lo que permite mejor colonización y uso de la superficie específica del sustrato que resulta en mayor producción de enzimas. (Jeong et al. 2006).

Con respecto a la actividad micotoxigénica (no evaluada en este estudio), entre las seis especies de Aspergillus identificadas, A. niger y A. flavus se informan como agentes de micotoxicosis humana y animal, la primera asociada con la producción de ocratoxina A, y la segunda con las aflatoxinas B1 y B2 (Yu, 2012). Sin embargo, no todas las cepas de estas especies pueden metabolizar micotoxinas peligrosas. De hecho, se sabe que en las especies de A. flavus solo alrededor del 40-50 % de las cepas producen aflatoxinas y solo alrededor del 20 % de las cepas de A. niger son ocratoxigénicas (Abarca et al. 2001 y Davari et al. 2015).

Aunque la detección de hongos toxigénicos en las muestras analizadas no necesariamente indica que las micotoxinas se producen naturalmente en el pienso, estas alertan sobre el riesgo potencial de contaminación. Otras especies aisladas de muestras de piensos, como A. awamori y A. oryzae, no asociadas con la producción de metabolitos tóxicos, se utilizan en gran medida en biotecnologías alimentarias (Siedenberg et al.1998 y Takagi 2014).

 CONCLUSIONES

Este primer estudio sobre la evaluación del nivel de contaminación por hongos en las materias primas y ganado sicilianos mostró la presencia de Aspergillus spp., Penicillium spp. y Fusarium spp, en los materiales muestreados. En particular, las secciones de Apergillus Nigri (A. awamori, A. niger, A. tubingensis), Flavi (A. flavus, A. oryzae) y Nidulantes (A. amstelodami) se identificaron mediante metodologías morfológicas y moleculares. Se observó la producción de enzimas celulolíticas en todas las cepas de Aspergillus, pero A. niger SAAF 7, A. awamori SAAF 10 y A. tubingensis SAAF 14 mostraron la mayor actividad de degradación.

Sobre la base de estos resultados, se supone que el pienso más contaminado por estos hongos puede ser el más degradado desde el punto de vista nutricional.

Esta investigación resalta la importancia de un monitoreo y control continuo de la contaminación por hongos en los alimentos y piensos. Deben implementarse estrategias destinadas a prevenirlos en el campo, durante el almacenamiento y en toda la cadena de producción de piensos. El control de estos contaminantes fúngicos y corregir las técnicas de producción de piensos para animales, de hecho, puede asegurar no solo alto nivel de salud del animal sino también alto nivel de calidad de los piensos, tanto en palatabilidad como en valores nutricionales.