Antimicrobial and enzymatic activity of anemophilous fungi of a public university in Brazil

To the fungal microbiota the UFPE and biotechnological potential enzymatic and antimicrobial production. Air conditioned environments were sampled using a passive sedimentation technique, the air I ratio and the presence of aflatoxigenic strains evaluated for ANVISA. Icelles were to determine the enzymatic activity of lipase, amylase and protease metabolic liquids to determine antimicrobial activity. Diversity was observed in all CAV environments, CFU/m ranged from 14 to 290 and I/E ratio from 0.1 to 1.5. The of the fungal genera were: Aspergillus (50%), Penicillium (21%), Talaromyces (14%), Curvularia and Paecilomyces (7% each). Aspergillus sydowii (Bainier & Sartory) Thom & Church presented enzymatic activity and the Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenb., Seifert, Peterson, Varga & Frisvad presented antibacterial activity against all bacteria that all environments present fungal species biodiversity no toxigenic or pathogenic fungi were found, according to ANVISA legislation for conditioned environments and airborne filamentous fungi present potential for enzymatic and antimicrobial activity.


INTRODUCTION
Fungi air, water, soil, animals and food.They colonize substrates, have an extensive geographic distribution and are able to grow under environmental conditions (Oliveira and Borges-Paluch 2015).
Fungal contamination is considered due to the production.Of the filamentous fungi under adequate conditions of oxygen, temperature and humidity, secondary metabolites (mycotoxins) both and animals, fungi of the genus Aspergillus and Penicillium.A. flavus Link and A. parasiticus Speare produce aflatoxins (Peluque 2014).
Therefore, monitoring air in artificially airconditioned is according to Resolution RE No. 9 of January 16, 2003, the National Agency of Sanitary Surveillance (ANVISA), which defines reference standards cleaning and maintenance of the health and safety of individuals who attend LAUREANA V. SOBRAL et al. public and collective places with conditioned environments.The recommended maximum acceptable contamination of ≤750 CFU/m 3 and ≤1.5 for the relation between internal and external air the presence of pathogenic and toxigenic fungi (Brazil 2003).
Biotechnically, microbial enzymes are by industries reduced production time, increased scale and purification, specificity and stabilization, as well as easy genetic manipulation (Nagarajan 2012) and high biochemical diversity (Silva et al. 2015) is related to the minimization production (Reinehr et al. 2014) and waste and electric consumption (Silva et al. 2015).
Dispersion and appearance of commercially available antimicrobial-resistant microbes, thus the search for sources of effective antimicrobial agents (Santos et al. 2014) researches with the purpose of intervening, preventing or intensifying the conventional bacterial resistance (Catão et al. 2014).
The antimicrobial using mycelia and liquid metabolites of anemophilous filamentous fungi this may be the first activity.The present study aimed to evaluate the microbiological quality of air, the presence of aflatoxigenic fungi in the climatic environments of the Federal University of Pernambuco/Vitória Academic Center and enzymatic production and antimicrobial activity.

SAMPLING AND COLLECTION OF AIR
The study was conducted at the Federal University of Pernambuco/Vitória Academic Center (UFPE/ CAV) and 82 artificially air-conditioned 3 board of directors, 9 library, 1 auditorium, 15 classrooms, 34 laboratories, 13 administrative rooms, 1 room of the Internal Accident Prevention Commission (CIPA), 5 student assistance centers, 1 teaching staff and an external area located in the courtyard of the main entrance of the CAV for fungi quantification.Non-air-conditioned environments were excluded from the study.
The samples were collected from the central area of the environments through the passive sedimentation technique (Lacaz et al. 1998) on solid culture medium using Petri dishes containing the culture (MEA) and Aspergillus flavus and parasiticus agar (AFPA) for total and aflatoxigenic fungi cultures, respectively.The plates were placed 1m the ground and opened for 15 min bioaerosols, containing microorganisms present in the atmospheric air.The plates were incubated at room temperature (28 ± 2°C) for up to days.

MICROBIOLOGICAL ANALYSIS OF AIR
The colonies present in the respective culture media were quantified and the relation between internal and external air (I/E) was calculated to determine the internal air quality.ANVISA the recommended m a x i m u m a c c e p t a b l e m i c r o b i o l o g i c a l contamination value of ≤750 CFU/m 3 and ≤1.5 for the relationship between internal and external air (Brazil 2003).Sedimentation sampling does not directly determine the number of microorganisms present in a given volume of airit is possible to transform the results of this method (number of CFU/unit area) into CFU/volume unit number (Morais et al. 2010).Air sampling by sedimentation was using the following equation described by Friberg et al. (1999): The area of the petri dish 90 x 15 mm (our study 0.006m 2 ).SAR is the ratio between the in the air and on the surface of the culture medium.For environments with spontaneous sedimentation and without apparatus that forces the sedimentation of air, this ratio is 1:23 (Morais et al. 2010).
AIR FUNGI: ANTIMICROBIAL AND ENZYMATIC ACTIVITY 2329

IDENTIFICATION OF AFLATOXIGENIC FUNGI
The presence of aflatoxigenic strains revealed by the orange reverse of the AFPA culture medium was evaluated.ANVISA the presence of pathogenic and toxigenic fungi in artificially climatized environments (Brazil 2003).

ISOLATION, IDENTIFICATION AND PRESERVATION OF FUNGI
After in the MEA solid culture medium, the fungal colonies were purified in the same culture medium.After the fungal species, the macroscopic (coloration, appearance and diameter of the colonies) and microscopic (somatic and reproductive microstructures) (Samson and Frisvad 2004) were observed through culture under coveralls (Riddell 1950) and preserved to the Castelani method distilled water and immersion of pure cultures mineral oil.The lipase reaction was by visualizing the presence of calcium salt crystals of lauric acid and/ or the formation of a clear area around the fungal colony dis.To the production of amylase, 0.1N iodine solution was used, allowing the identification of the enzymatic reaction through a translucent halo around the colony.For protease, due to the chemical reaction it was possible to visualize the formation of translucent halo around the colony.After the development of the enzymatic reactions, a halo of enzymatic activity was measured to determine the production potential of the colonies, which is proportional to the halo diameter.

DETERMINATION OF ANTIBACTERIAL ACTIVITY
Of the species identified, 5 metabolic liquids from the species Paecilomyces variotti Bainier (04), Aspergillus japonicas Saito (24), Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenb., Seifert,Peterson,Varga & Frisvad (28), Curvularia lunata (Wakker) Boedijn (49), Aspergillus parasiticus Speare (67) were randomly selected to perform antimicrobial activity and determine and purified fungal mycelia dis were cultured in 10 mL of liquid culture medium Sabouraud brothand incubated at room temperature for 20 days.The liquid metabolite was separated from the mycelium by filtration on sterile filter paper.The metabolic fluid was stored at 4°C to perform the antibacterial activity tests and the mycelium was frozen for subsequent extraction of metabolites.
positive: Staphylococcus aureus (ATCC 29213) and S. aureus ORSA.The bacterial suspensions were prepared in 0.85% saline and a concentration of 1.5 x 10 5 CFU/mL was obtained, from the dilution of the corresponding suspension to the 0.5 tube of the McFarland scale.Five metabolic liquids produced by filamentous fungi from atmospheric air were tested.All experiments were performed in triplicate.The antibacterial activity test was performed the broth dilution method of the ANVISAM7-A6 technical standard.Serial dilutions of metabolic liquids were performed.Positive control of the test was using chloramphenicol antibiotic and negative control of the test, in the absence of antibiotic, to confirm absences of contaminants in the metabolic liquid used in the test was added a column with the pure metabolic liquid.After distribution of the bacterial suspensions, the plates were incubated at 37°C for 24h.20 μL 2,3,5-triphenyltetrazolium chloride (TTC) (2% v/v) diluted in water was added to all wells after 2h of incubation room temperature, bacterial growth.The wells stained in red indicate bacterial growth (Rahman et al. 2004).
The red coloration results from the TTC reaction with hydrogen ions constituted due to cellular respiration, generated by an insoluble reddish solution called formazan, indicating viable bacterial cells in the medium (Rahman et al. 2004).
The reading was performed through visual observation of the lowest concentration of metabolic liquids that inhibit bacterial growth.The test was performed, where was for Petri dishes containing Mueller-Hinton Agar, the plate incubated at 37°C for 24h.Bacterial growth, indicating bactericidal or bacteriostatic action of the fungal metabolic the wells not presentantimicrobial stained red.
The liquid metabolites used in the study were tested in their crude state after filtration.Therefore, the antimicrobial activity the inhibitory concentrations defined for the method in μL/mL: 500.00;250.00; 125.00; 62.50; 31.25; 15.63; 7.81; 3.91; 1.95 and 0.97μL/mL.

RESULTS AND DISCUSSION
Growth was observed in all environments the lowest amount of CFU/m 3 in the classroom (14 CFU/m 3 ) and with the amount and diversity of the living pharmacy space, the CIPA room and the teaching office (Table I), all the evaluated environments of the CAV were within the ANVISA for microbiological air quality (≤750 CFU/m 3 ).Fungal colonies from environments observed in Figures 1 and 2 the colonies before and after purification of the fungal isolates, respectively.The evaluated environments possibly the access flow of ventilation and passers-by in the environments, atmosphere is influenced by the climate, the interior atmosphere is influenced by building maintenance.Both environments influence human activities, humidity and little temperature, since many indoor fungi grow in a wide temperature range (Araújo and Cabral 2010).
The ratio of fungi quantification of I/E (internal/ external) of the environments surveyed in the CAV (Table I) met the current ANVISA legislation for air quality in conditioned environments (≤1.5), this ratio 0.1 to 1.5, the minimum and maximum, respectively, since the external air quantification was 188 CFU/m 3 .
The environments that presented an I/E statement in the maximum acceptable limit were: library entrance hall, living pharmacy space, CIPA room and laboratory 1 and teaching room.Fact that the entrance hall of the library by visitors, with human activity showing a direct influence on the live pharmacy laboratory handles plant and soil samples CIPA room is an environment used for meetings the room is relatively frequented by students.In these environments, the values of the air fungi quantification in CFU/m 3 were allowed by ANVISA ( 750), but the I/E ratio was the limit.
No environment presented aflatoxigenic fungi (Figure 3) in compliance with the aforementioned     pathogenic prevent epidemics and protect public detect microorganisms (Zhoug et al. 2000).
Biologically, fungi are considered a promising source for new biomolecules with diverse biological activities for enzyme production and antimicrobial activity, due to chemical and structural diversity (Silva et al. 2015).
The majority of studies air quality were performed in hospital settings.Azimi et al. (2013) analyzed the quality of hospital air and found 70% of the samples fungi of Penicillium sp., 14% Aspergillus sp., 12% Cladosporium sp., 25% Alternaria sp., indicating a risk factor for patients and professional these environments.Our study, since in addition to the analyzed environment being different, the most frequent was Aspergillus.
Library are similar with the study by Nascimento (2011) that when analyzing the internal The concern to find the genus Aspergillus and Penicillium in indoor and air-conditioned environments is species these genera produce secondary metabolites with a toxic effect on health (Peluque 2014).Exposure to mycotoxins may lead to reactions including hemorrhages and necrosis, and in abundance such toxins have affinity for specific organs or tissues such as the liver, kidneys and nervous system (IARC 2002).
Kalwasinska et al. ( 2012) evaluated functional and nonfunctional environments of a university library in Torun, Poland.According to the authors, the internal air was according to the legislation, both in the functional (classroom, reading room of periodicals, collection conservation salt), in nonfunctional (bathroom, cafeteria) in which all sectors of the CAV library analyzed were below the limit established by ANVISA (Table I).Boff (2011) environments with fungal agglomerates influenced by environmental factors such as temperature, humidity, airflow, available organic substrates, climatic conditions and seasonal variation, physical factors the shape, size and density of the particles, among other situations that corroborate the increase of conidia in the environment.
Ejdys et al. ( 2013) studied the fungal composition of air from school classrooms and monitored or years, classifying according to biosafety level.The authors isolated 151 species of fungi, of which 22 and 61 species of fungi were classified as class 1 of biological risk.
Hayleeyesus and Manaye (2014) studied university from Ethiopia and observed that the according to the legislation, with concentrations of fungi ranging from 367 to 2.595 CFU/m 3 .
Aspergillus sp., Penicillium sp., Alternaria sp., Cladosporium sp. and Fusarium sp.most frequently found found for the library and other environments of the CAV.Many fungal species, including those present in the air, may be responsible for the production of metabolites with the most varied industrial and biological applications, enzymatic and antimicrobial activities (Viriato 2014).
The sources of extraction to study biological activities are soil, water, plants and more recently marine environments, exploring the secondary metabolism in order to perform tests using liquids and metabolic extracts or isolated metabolites by chromatography.The existing fungi in atmospheric air containing a range of pollutants still unexplored for the isolation of fungi some biological activity.
From the air, 67 specimens were initially isolated and then tested with 14 isolates after morphological identification to determine the enzymatic activity for the production of amylase, lipase and protease.The species of filamentous fungi identified in the air of the CAV were Paecilomyces variotti Bainier (04), P. fellutanum Biourge ( 06 II).
Not all isolates of a fungal species necessarily have the same enzyme the same intensity.Therefore our study that although the isolates 14, 27 and 67 belong to the same species Aspergillus parasiticus Speare, the same enzymatic reaction LAUREANA V. SOBRAL et al.
was not observed among them.For, the isolate 27 only produced lipase and the isolates 14 and 67 produced all three enzymes, results the isolates of Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenburg, Seifert, Peterson, Varga & Frisvad.The fungi species that presented the best amiolitic activities were Penicillium fellutanum Biourge (7 mm) and P. Oxalicum Currie & Thom (7 mm) and lipase activity were Aspergillus parasiticus Speare (10 mm) and A. sydowii (Bainier & Sartory) Thom & Church (14 mm).The production of protease, although the one of lower intensity for the producing species, had the isolate 18 of A. sydowii (Bainier & Sartory) Thom & Church as the best producer (5 mm) (Table III).The intensity of the in solid substrate can be directly influenced the solubility, diffusibility, enzyme, other factors.
The results presented in this study show that amiolytic activity was the most common among the species, corroborating with the finding of Wenzel et al. ( 2013) evaluated endophytic fungi of soybean and revealed a greater quantitative enzymatic activity for the amylase (83.3% 20).
The amylase reaction halos in our study varied between 4 and 7 mm the results found by Firmino and Furtado (2014) the fungus Ceracystis sp., whereas Tavares et al. (2012) 29 mm halos evaluating Aspergillus isolated from noni (Morinda citrifolia L.).
A study by Soares et al. (2010) for amylase production, suggest that fungi under stress conditions interfere with enzymatic production.Firmino and Furtado (2014) also evaluated lipase activity with isolates of the fungus Ceracystis sp. did not observe any activity for this enzyme.The authors also tested the protease and halos did not exceed 1 mm.
The averages of enzymatic activities of our study differ from the results presented by Griebeler et al. (2015) who found higher values halos above 7.08 mm for amylase, lipase and protease.These authors worked with isolated fungi from different sources, such as soil, olive and soybean oil, cheeses, tomato extract, cream of milk, meat, soybean meal and contaminated culture media.
A study evaluated fungi isolated Baru seeds, bark and pulp (Dipteryx alata Vog.) and observed that none of the fungi had proteolytic activity, but all of them were able to present some amiolytic and lipolytic activity (Molina et al. 2012).The evaluation performed by Bezerra et al. (2012) with isolates of a Caatinga plant (Opuntia ficus-Mil) revealed that a strain of A. japonicas Saito did not produce proteolytic activity, corroborating with our study.Sunitha et al. (2013) enzymatic production differs among fungi and is related to the habitat where it is found.
Aboul-Nasr et al. ( 2013) tested 110 fungal isolates from intensive care unit air and hospital operating room in India and found that among the 110 isolates tested, 73% produced protease, lipase and urease, and this was the only the literature testing the enzymatic and mycotoxin production potential of anemophilous fungi.Among the lipase and protease isolates were Aspergillus sp., Cladosporium sp., Myrothecium sp. and Fusarium sp.According to the authors, these results are important since proteases and lipases play a role In relation to the tests of antibacterial activity of the fungi isolated from the air of the CAV, three metabolic liquids presented activity against all bacteria tested, represented by the species Paecilomyces variotii Bainier (04), Talaromyces purpureogenus Samson,Yilmaz,Houbraken,Spierenb.,Seifert,Peterson,Varga & Frisvad (28) and Aspergillus parasiticus Speare (67).The metabolic liquid of Curvularia lunata (Wakker) Boedijn ( 49) no antibacterial activity against the bacteria tested (Table IV).
The metabolic liquid of the species Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenburg, Seifert, Peterson, Varga & Frisvad (isolate 28) presented the best antibacterial activity against all the bacteria tested, reaching the of 125 μL/mL, none of the metabolic liquids tested presented bactericidal action, only bacteriostatic.
Duarte ( 2006) reports that no consensus what is acceptable the level of inhibition for antimicrobial activity related to natural products versus standard antibioticssome authors consider only antibioticlike results, although others understand that levels inhibition good potential.
The study by Paraginskia et al. ( 2014) analyzed all bacterial strains evaluated in our study, but used different concentrations of triazenes compounds for antimicrobial activity and obtained bacteriostatic action in at least one of the six evaluated compounds.

CONCLUSIONS
It was concluded that the environments analyzed in the UFPE/CAV presented great diversity of fungi species they met the requirements of the ANVISA resolution.It was also verified that not present fungi potentially harmful to human health in the environments.Our study demonstrated that some species of anemophilous filamentous fungi are capable of producing the amylase, lipase and during microbial infection and suggest these results are important since the extracellular lipases play an important role during microbial infection, since they digest lipids to obtain nutrients by pathogenic microbes, allowing the growth of microbes where lipids are the only source of carbon and proteolytic degradation of lung tissues has been identified as a key phenomenon in the pathophysiology of Aspergillus fumigatus, several species of Aspergillus secrete protease.
AIR FUNGI: ANTIMICROBIAL AND ENZYMATIC ACTIVITY 2331 was these 79 isolates.Several mycotoxins are detected by TLC, the aflatoxins being zearalenone, gliotoxin, fumigiline, Mycotoxin production is a virulence factor.The frequency of the fungal genera found in the analyzed environments, whose were: Aspergillus (50%), Penicillium (21%), Talaromyces (14%), Curvularia and Paecilomyces (7% each) as shown in Figure 4.Anemophilous fungal infections in the medical literature and inhaled airborne spores have been implicated responsible for various allergic(Furtado and Ferraroni 1982).In addition to allergy, many opportunistic fungi such as those shown in Figure4, are responsible for diseases otitis, mycotoxicosis, urinary infections, onychomycosis, eye infections fungemia.Since are dispersed abundantly in the environment(Grumach 2001).Therefore, monitoring the environment detect potential

Figure 1 -
Figure 1 -Diversity of colonies of air bioaerosols of different environments of the Academic Center of Vitória -UFPE/CAV.Internal Commission for Accident Prevention -CIPA (a), Laboratory of Effort Physiology (b), Library -consultation collection room (c), Laboratory of Maternal and Child Nursing -LABEMI (d), Laboratory of Dietetic Technique (e), Laboratory of Semiology 2 and Clinical Evaluation (f), Nutrition Center (g), Library -study hall of the ground floor (h).

Figure 2 -
Figure 2 -Diversity of colonies after purification of fungi isolates from different environments of the Vitória Academic Center -UFPE/CAV.

Figure 3 -
Figure 3 -Negative detection of aflatoxigenic fungi in CAV air in AFPA medium (absence of orange color on reverse of fungal colonies).

Figure 4 -
Figure 4 -Frequency of fungal genera found in the air of the analyzed environments of the CAV.

TABLE III Identification of the species and metabolic product of fungi isolated in the air of the CAV/UFPE and halo of enzymatic activity (in mm).
Identification code of fungal isolate, (mm) Millimeter, (A) Amylase, (L) Lipase, (P) Protease, (+) Produced enzymatic activity, (-) It did not produce enzymatic activity.LAUREANA V. SOBRAL et al. protease enzymes the fungi Aspergillus parasiticus Speare and A. sydowii (Bainier & Sartory) Thom & Church better amiolytic potential among the other species and enzymes under the conditions of this experiment.The antimicrobial activity of the species Talaromyces purpureogenus Samson, Yilmaz, Houbraken, Spierenburg, Seifert, Peterson, Varga & Frisvad was the one that presented the best result however, showed low bactericidal potential.