How gamma-rays and electron-beam irradiation would affect the antimicrobial activity of differently processed wild mushroom extracts?

Aims: The effects of irradiation (gamma-rays and electron-beams), up to 10 kGy, in the antimicrobial activity of mushroom species ( Boletus edulis , Hydnum repandum , Macrolepiota procera and Russula delica ) differently processed (fresh, dried, freeze) were evaluated. Methods and Results: Clinical isolates with different resistance proﬁles from hospitalized patients in Local Health Unit of Mirandela, Northeast of Portugal, were used as target micro-organisms. The mushrooms antimicrobial activity did not suffer signiﬁcant changes that might compromise applying irradiation as a possible mushroom conservation technology. Conclusions: Two kGy dose (independently of using gamma-rays or electron-beams) seemed to be the most suitable choice to irradiate mushrooms. Signiﬁcance and Impact of the Study: This study provides important results in antimicrobial activity of extracts prepared from irradiated mushroom species.


Introduction
The interest of the scientific community for mushrooms (extracts and derived compounds) has been increasing due to their potential in prevention or treatment of different diseases of the modern world. Different mushroom species were previously reported for their anticancer, anti-inflammatory, immunosuppressive and antibacterial properties (Asfors and Ley 1993;Wieczorek et al. 1993;Ferreira et al. 2010;Alves et al. 2014).
In particular, several authors reported the antimicrobial activity of extracts prepared from different mushroom species including Boletus edulis Bull. (Kosani c et al. 2012), Hydnum repandum L. Fr., (Ozen et al. 2011), Macrolepiota procera (Scop.) Singer and Russula delica Fr. (T€ urko glu et al. 2007;Yaltirak et al. 2009;Alves et al. 2012b). In this sense, mushrooms have been recognized as functional foods and as a source for the development of drugs and nutraceuticals (Lindequist et al. 2005;Poucheret et al. 2006).
Nevertheless, and in spite of these undeniable qualities, mushrooms are one of the most perishable products and tend to loose quality immediately after harvest. A short shelf life (1-3 days at room temperature) is one of the disadvantages for their distribution and marketing as a fresh product. Their shelf life is limited due to postharvest changes, such as browning, cap opening, stipe elongation, cap diameter increase, weight loss and texture changes, but also due to a high respiration rate and lack of physical protection to avoid water loss or microbial attack (Akram and Kwon 2010;Fernandes et al. 2012).
Irradiation is recognized as a safe and effective preservation method, being used worldwide to increase the shelf life of foods (e.g. fruits and legumes, spices, grains, meat or seafood) (Andrews et al. 1998;Fernandes et al. 2012). Gamma-irradiation (Beaulieu et al. 2002) and electron-beam (Koorapati et al. 2004) are considered potential tools in extending the shelf life of fresh mushrooms. Furthermore, different regulatory agencies ensure that food irradiation is a safe process in relation to the processing of food for humans (US-FDA 1991;WHO 1994).
Our research group has been investigating the effects of mushrooms (including the above mentioned species B. edulis, H. repandum, M. procera and R. delica) irradiation by evaluating their nutritional, physical and chemical parameters, concluding that these parameters are not affected in high extension with this preservation technology (Fernandes et al. 2013a(Fernandes et al. ,b, 2014a. Nevertheless, to the authors' knowledge, there are no available reports about the effects of irradiation on mushrooms antimicrobial activity. Therefore, in this study, the antibacterial properties of extracts prepared from irradiated (gamma-radiation and electron-beam) wild mushrooms were assessed against clinical isolates with different resistance profiles from hospitalized patients in Local Health Unit of Mirandela, Northeast of Portugal.

Standards and reagents
To estimate the dose and dose rate of gamma-irradiation, it was used a chemical solution sensitive to ionizing radiation, Fricke dosimeter, prepared in the lab following the standards (ASTM 1992) and Amber Perspex dosimeters (batch V, from Harwell Co., Oxfordshire, UK). To prepare the acid aqueous Fricke dosimeter solution, the following reagents were used as follows: ferrous ammonium sulphate(II) hexahydrate, sodium chloride and sulphuric acid, all purchased from Panreac S.A. (Barcelona, Spain) with purity PA (proanalysis), and water treated in a Milli-Q water purification system (Millipore, model A10, Bilerica, MA).
Methanol was of analytical grade purity from Lab-Scan (Lisbon, Portugal). The culture media Muller Hinton broth (MHB), Wilkins-Chalgren Broth (WCB) and Columbia agar (CA) with 5% horse blood were obtained from Biomerieux (Marcy l 0 Etoile, France), respectively. The dye p-iodonitrotetrazolium chloride (INT) was purchased from Sigma-Aldrich (Spruce Street; St. Louis, MO) to be used as microbial growth indicator.

Samples and samples irradiation
Macrolepiota procera, B. edulis, H. repandum and R. delica ( Fig. 1) were collected in Tr as-os-Montes, in the Northeast of Portugal; the first mushroom species were collected in November 2011, and the three other species were collected in November 2012 and further irradiated.
Boletus edulis and H. repandum fresh fruiting bodies were divided in three groups (each species) with three mushrooms per group and submitted to gamma-irradiation according to the procedure described by Fernandes et al. (2013a): control (nonirradiated, 0 kGy), sample 1 (irradiated with 1 kGy) and sample 2 (irradiated with 2 kGy). Macrolepiota procera fruiting bodies were divided in two groups with nine mushrooms per group, and further submitted to different processing technologies according to the procedure described by Fernandes et al. (2014a): freezing (at À20°C in a freezer) and drying (at 30°C in an oven). Each group was further subdivided in three subgroups and submitted to gamma-irradiation: control (nonirradiated, 0 kGy); sample 1 (irradiated with 0Á5 kGy) and sample 2 (irradiated with 1 kGy). Russula delica fruiting bodies were divided in four groups with six mushrooms per group, dried at 30°C in an oven and then submitted to electron-beam irradiation according to the procedure described by Fernandes et al. (2014b): control (nonirradiated, 0 kGy), sample 1 (irradiated with 2 kGy), sample 2 (irradiated with 6 kGy) and sample 3 (irradiated with 10 kGy).

Extracts preparation
Each mushroom lyophilized sample (approx. 1Á5 g) was extracted using a methanol : water (80 : 20; 30 ml) mix- Figure 1 Representative samples of the control population of Macrolepiota procera, Boletus edulis, Hydnum repandum and Russula delica (from the left to the right). Mushrooms of the other groups (irradiated) are similar to the control. ture at À20°C for 1Á5 h. After 15 min in an ultrasonic bath, filtered through Whatman no 4 paper. The residue was then re-extracted with additional 30 ml of methanol: water mixture at À20 C for 1Á5 h, and the previous steps were repeated. The combined extracts were evaporated at 40°C under reduced pressure to remove methanol (rotary evaporator B€ uchi R-210, Flawil, Switzerland), lyophilized, redissolved in water, at a concentration of 200 mg ml À1 and stored at À20°C for further use.

Micro-organisms and culture media
The micro-organisms used were clinical isolates from patients hospitalized in various departments of the Local Health Unit of Mirandela, Northeast of Portugal.
Two Gram-negative bacteria (Escherichia coli and Proteus mirabilis, isolated from urine) and two Gram-positive bacteria (MSSA-Methicillin-sensitive Staphylococcus aureus, isolated from Wound exudate and MRSA-methicillin-resistant Staph. aureus, isolated from expectoration) were used to screen the antimicrobial activity of the mushroom extracts.

Characterization of antibiotic susceptibility of target strains
The characterization of antibiotic susceptibility and the identification of the target strains were performed using VITEK â 2 Compact card (Biom erieux, Lyon, France). These cards allow the simultaneous determination of susceptibility to antimicrobial agents and the strain identification, including aerobic and facultative anaerobic Gramnegative bacilli (VITEK â 2 Compact AST-N192), and Gram-positive cocci such as some fastidious aerobic Gram positive (VITEK â 2 Compact AST-P619). The sensibility of the micro-organisms to each antibiotic is identified by the MIC.
Determination of the antimicrobial activity of the extracts MIC determinations were performed by the microdilution method and the rapid p-iodonitrotetrazolium chloride (INT) colorimetric assay following the methodology suggested by Kuete et al. (2011a,b) with some modifications.
Initially, 50 ll of each mushroom extract (200 mg ml À1 ) was diluted in 450 ll of MHB (final concentration of 20 mg l À1 ), and then, 200 ll of this extract solution was added in each well (96-well microplate). Dilutions were carried out over the wells containing 100 ll of MHB and afterwards, 10 ll of inoculum (1 9 10 8 CFU ml À1 ) was added to all the wells. Two negative (one with MHB and the other with the mushroom extract) and one positive (with MHB and the inoculum) controls were prepared. The plates were incubated at 37°C, for 24 h, in an oven (Jouan, Berlin, Germany).
The MIC of the samples was detected after adding INT (0Á2 mg ml À1 , 40 ll) and incubating at 37°C for 30 min. Viable micro-organisms reduced the yellow dye to a pink colour. MIC was defined as the lowest mushroom extract concentration that prevented this change and exhibited complete inhibition of bacterial growth. All the assays were carried out in duplicate.

Results
Before analysing the potential antimicrobial activity of the studied mushrooms, the resistance profiles of the bacterial isolates are described in Table 1. From its interpretation, it is possible to conclude that E. coli and MSSA constitute wild strains with small resistance to the tested antibiotic groups, excluding the result obtained with MSSA against benzylpenicillin. On the other hand, P. mirabilis and MRSA showed resistance against the quinolone group (ciprofloxacin and levofloxacin) and b-lactamic antibiotics (amoxicillin/clavulanic acid). In addition, P. mirabilis was also resistant to trimethoprim/ sulfasoxazole (antimetabolic) and nitrofurantoin. Overall, the obtained MIC values are in the same range as those obtained in a previous study (Alves et al. 2012b). However, the magnitude of the MIC values obtained using antibiotics is nearly 1000-fold lower when compared with the same values obtained using mushroom extracts (Table 2). Nevertheless, the values depicted in each table should not be directly compared, as results in Table 1 were achieved using pure compounds, while those in Table 2 derive from using crude extracts with several possible interferences.
Centering the discussion in the antimicrobial activity of mushroom extracts, a primary observation allows concluding that the Gram-positive bacteria presented more susceptibility, in agreement with other reports (Barros et al. 2007(Barros et al. , 2008aVenturini et al. 2008;Alves et al. 2012b), despite the results obtained by Ozen et al. (2011), which indicated that H. repandum extracts had a maximal antibacterial activity against a Gram-negative bacteria (Pseudomonas aeruginosa).
Among all tested extracts, those obtained from fresh B. edulis presented the highest activity against all tested bacterial strains, followed by dried samples of M. procera. Similar resistance levels were previously reported for B. edulis samples assayed against E. coli and MSSA (Kosani c et al. 2012). The results reported herein for dried R. delica extracts indicate slightly higher antibacterial activity, namely against P. mirabilis and MRSA, than those obtained in our laboratory with the same species (Alves et al. 2012b). Nevertheless, the results from both studies might be considered as belonging to the same range. As the studied species have no phenolic compounds (Vaz et al. 2011), the antimicrobial activity could be due to the presence of steroids, sesquiterpenes, organic acids, or peptides, which represent antimicrobial compounds commonly found in mushrooms (Alves et al. 2012a).
The mushroom species studied herein, among several others, might benefit from an increased shelf life when submitted to irradiation, either using gamma-rays (Beaulieu et al. 2002;Fernandes et al. 2013aFernandes et al. ,b, 2014a or electron-beams (Koorapati et al. 2004;Fernandes et al. 2014b,c) as energy sources. In all studied cases, the chemical composition and antioxidant activity of each mushroom species were not negatively affected by irradiation treatment. Considering the ability to maintain the antioxidant activity, we hypothesized that irradiated mushrooms might likewise present the same antimicrobial activity. The results obtained for differentially processed mushrooms (fresh, freeze or dried) treated with gamma-irradiations or electron-beam irradiations with varying doses are presented in Table 2. As a first remark, it should be clarified that the used heterogeneous samples were defined to prove the feasibility of irradiation treatment in the maintenance of antimicrobial activity, independently of mushroom species, mushroom processing, irradiation source and irradiation dose. As it can be concluded from Table 2, the antimicrobial activity was not adversely affected in most of the cases, except gamma-irradiated B. edulis against P. mirabilis. In fact,  this activity was sometimes potentiated, as it was verified for the frozen samples of M. procera against E. coli and MSSA, or the dried sample of R. delica against E. coli. Some matrix effects must indeed be considered. For instance, the 1 kGy dose does not seem to be the preferable choice to treat B. edulis and H. repandum (which have a better response to the 2 kGy dose), but the same dose had advantageous effects when used to irradiate the M. procera samples. This indicates that the chemical composition of a determined mushroom might impart different outcomes as a result of the same irradiation dose. Nevertheless, the results obtained for irradiated samples and controls are very similar, indicating that there are no significant changes in the antimicrobial activity, which might compromise the feasibility of this technology (under the limitations of the used sampling). As a general conclusion, the results indicate that the 2 kGy should be considered as the best choice in terms of antimicrobial activity maintenance.

Discussion
The development of antibacterial agents' resistance and the decrease of effective antifungal agents are leading the researchers towards investigating novel chemical structures and considering new natural sources of compounds. Usually, compounds found in natural matrices exert their antimicrobial activity by interacting with the microorganism's cell membrane or cell wall, altering its permeability and causing cell destruction. The antimicrobial activity might also be explained by infiltration into bacterial cells, followed by promoting the coagulation of the cell content (Taguri et al. 2006;Tian et al. 2009).
The antimicrobial activity of different mushroom species was previously reported, particularly in what regards their effectiveness against Gram-positive and Gram-negative bacteria (T€ urko glu et al. 2007;Ozen et al. 2011;Venturini et al. 2008;Yaltirak et al. 2009;Alves et al. 2012a, b;Kosani c et al. 2012;Alves et al. 2014;Smolskait_ e et al. 2015). In this study, instead of using commercial bacterial cultures, the antibacterial activity was evaluated using clinical isolates from different body fluids. The obtained bacteria had the common feature of showing some resistance against the typically used antibiotics.
After several studies have been conducted proving that neither the chemical composition nor the antioxidant activity of different mushroom extracts were adversely changed by irradiation treatment, the feasibility of this technology was tested herein by verifying its effects in the antimicrobial activity of different mushroom species. Besides using different mushrooms, samples were processed differently (fresh, dried, freeze) according to the most common practices applied to mushrooms. Likewise, different doses (0Á0, 0Á5, 1, 2, 6 and 10 kGy) and two irradiation sources (gamma-rays and electron-beams) were evaluated. Under all the assayed conditions, the antimicrobial activity did not suffer significant changes that might compromise applying irradiation as a possible mushroom conservation technology. As a final remark, the 2 kGy dose (independently of using gamma-rays or electron-beams) seemed to be the most suitable choice. Nevertheless, future assays with additional mushrooms and/or bacterial cultures will certainly be needed before guaranteeing the complete adequacy of this treatment.