Microbiological Characteristics of Wild Edible Mushrooms and Effect of Temperature during Storage of Morchella conica

Background: The continuous worldwide increase of consumption of fresh mushrooms has registered in the recent years. The major goal of this study was to determine the microbiological characteristics of wild edible mushrooms and effect of temperature during storage of Morchella conica. Methods: Wild mushrooms of the species Boletus edulis , Cantharellus cibarius , and Leccinum aurantiacum were collected in a mixed forest of Picea abies , Betula pendula , and Pinus sylvestris located in Tartu territory, Estonia. Faecal indicators, potential patho-gens, spoilage bacteria, and microfungi (yeasts and moulds) were evaluated. M. conica was microbiologically investigated for 24 days under different thermal regimes, including 4, 8, 12, 15, 20, and 28 °C. The statistical analysis was conducted with SAS 9.2 software. Results: The microbial counts of wild mushrooms, ranging from 6.81 to 7.68 log 10 CFU/g for total mesophilic count, were generally higher ( p <0.05) than those registered for marketed samples ranging from 4.60 to 7.39 log 10 CFU/g. The dynamics of total mesophilic microorganisms on M. conica stored at different temperatures indicated that stationary and death phases occurred earlier with increasing temperature and that the highest levels were registered at 28 °C at the 2 nd day of storage. Conclusion: This work highlights consistent differences in terms of microbiological properties among different mushrooms species. The results clearly showed that total mesophilic populations developed at high levels quickly at temperatures more than 15 °C, but also the refrigeration did not stop the microbiological decay of mushrooms.


Introduction
The continuous worldwide increase of consumption of fresh mushrooms registered in the recent years (Venturini et al., 2011) is mainly imputable to their supply in pro-al., 2005). Mushrooms represent an important source of precursors of vitamin D (Mattila et al., 1994). Edible mushrooms also provide bioactive compounds (Valverde et al., 2015) and might exert several positive effects on human health such as hypocholesterolemic, antioxidant, antimicrobial, and anticancer activity (Lindequist et al., 2005;Teoh et al., 2018). Different antimicrobial compounds have been isolated from mushrooms (Nelson et al., 2016;Rathee et al., 2012). Consumption of edible mushrooms showed also positive effects in the prevention of diseases of the gastrointestinal tract due to their generation of short chain fatty acids that influence the intestinal microbial populations, in particular, increasing the abundance of Bifidobacteriales and decreasing that of Fusobacteriales (Zhao et al., 2018). The low content of fat and the high fiber content of mushrooms are particularly appreciated by consumers. Hence, fresh mushrooms are strategic components of a healthy diet (Guillamón et al., 2010).
Fresh mushrooms, like in case of vegetable matrices, are naturally contaminated by eukaryotic (moulds and yeasts) and prokaryotic (bacteria) organisms (Alfonzo et al., 2017;Corsetti et al., 2007). Like in case of vegetable matrices, the environmental contamination of mushrooms might depend on several factors, mainly animal, faeces, soil, and air (Buck et al., 2003). Mushrooms are characterized by a high moisture content, high water activity (≥0.98), and neutral pH; these conditions favor microbial growth after harvesting and during storage of mushroom limiting strongly their shelf life (Venturini et al., 2011).
In the present work, wild mushrooms collected in woods or purchased from markets in Estonia were characterized for the main microbiological parameters. Furthermore, the mushroom Morchella conica collected in Sicily, that has not been object of microbiological characterization in past, was used to evaluate the effect of different thermal regimes applied during storage on the total mesophilic bacterial community.

Sample collection
Wild mushrooms of the species Boletus edulis, Cantharellus cibarius, and Leccinum aurantiacum were collected in a mixed forest of Picea abies, Betula pendula, and Pinus sylvestris located in Tartu territory, Estonia during July 2017. Mushrooms of the same species were purchased in a popular retail market of Tartu, Estonia and were all collected by local mushroom hunters. Ten sporomata of each species were included in the study both for the "wild" and "market" trials. All mushrooms were transported under refrigerated conditions in a portable fridge containing reusable ice packs to the Laboratory of Agricultural Microbiology, SAAF Department-University of Palermo for analyses.

Microbiological analyses
Mushrooms were microbiologically investigated for 10 microbial groups, including Total Mesophilic Count (TMC), Total Psychrotrophic Count (TPC), members of Enterobacteriaceae family, total and faecal coliforms, enterococci, pseudomonads, Coagulase Positive Staphylococci (CPS), Lactic Acid Bacteria (LAB), yeasts and moulds. Ten g of each mushroom were collected (in triplicate, using three sporomata randomly chosen for each species and trial) and subjected to the decimal serial dilution in Ringer's solution (Sigma-Aldrich, Milan, Italy Effect of temperature and time on the microbiological characteristics of mushrooms M. conica was collected in April 2017 in the Ficuzza Forest, Palermo, Sicily, Italy in a mixed forest of Quercus pubescens s.l. and Fraxinus angustifolia. Identification of mushrooms was carried out using fresh ascomata. This species were used as model mushroom to evaluate the effect of storage time and temperature on the microbiological characteristics of wild mushrooms. M. conica samples were stored under different temperatures (4,8,12,15,20, and 28 °C) for 24 days. For this purpose, M. conica harvested in high amounts was divided into 103 samples (in duplicate for a total of 206 Downloaded from jfqhc.ssu.ac.ir at 18:15 IRDT on Wednesday May 22nd 2019 samples) of approximately 15 g and packed into sterile BagLight R 400 Multilayer R bags (Interscience, Saint Nom, France) sealed with a foil welding machine (Laica VT3112, Vicenza, Italy) in order to avoid further contaminations. One aliquot was analyzed soon after packaging and, for each of the six thermal regimes, 17 aliquots were subjected to storage.

Statistical analyses
Microbiological data were subjected to one-way Analysis of Variance (ANOVA). Pair comparison of treatment means was achieved by Duncan's procedure at p<0.05. Differences between wild and market mushrooms were evaluated with the Generalized Linear Model (GLM) procedure. The statistical analysis was conducted with SAS 9.2 software (Statistical Analysis System Institute Inc., Cary, NC, USA).

Results
The results of the microbiological investigation of B. edulis, C. cibarius, and L. aurantiacum are reported in Table 1. Faecal coliforms, enterococci, and CPS were not detected in any sample. All other microbial groups were counted at statistically different levels. The levels of each microbial group were generally significantly different (p<0.05) between wild and market mushrooms with the exception of TPC and pseudomonads for C. cibarius and filamentous fungi for L. aurantiacum. The microbial counts of wild mushrooms, ranging from 6.81 to 7.68 log 10 CFU/g for TMC, were higher than those registered for marketed samples ranging from 4.60 to 7.39 log 10 CFU/g. In particular, the highest viable counts were detected on wild C. cibarius whose levels of TMC and yeasts were 8.18 log 10 CFU/g. Furthermore, for all samples, the levels of TMC were superimposable to those of yeasts, showing that the microbial community of the mushrooms analyzed was dominated by yeasts. Except rod LAB on C. cibarius, for all microbial groups object of the survey, the levels detected on wild mushrooms were higher than those found on the purchased ones belonging to the same species as indicated by p<0.001 resulting from the interaction among the groups of wild and marketed mushrooms for several microbial populations (TMC, members of Enterobacteriaceae family, yeasts, LAB rods, and cocci). The better microbiological quality of the purchased mushrooms in the present investigation was also highlighted by the total absence of total coliforms and the very low levels of members of the Enterobacteriaceae family.  The effect of the temperature and the storage time was thorough evaluated on wild M. conica. To this purpose, TMC were followed by plate count, since they were found to represent the dominant microbial group of wild and purchased mushrooms. The dynamics of TMC is reported in Figure 1; the graphic clearly showed the differences induced by the storage temperature. Except for the stationary phase in the tract 7-11 days for the trials carried out at 12, 15, and 20 °C (with cell densities in the range 10.15-10.19 log 10 CFU/g) and in the tract 14 days-end of observation for the trials carried out at 8 and 12 °C (with cell densities in the range 10.15-10.19 log 10 CFU/g) for which the data were not statistically different (p>0.05), the majority of the differences registered during the experimentation were significant (p<0.05).
Considering the general trend of the effect of temperature on TMC, it was clear that as the temperature increased a more inclined slope of the exponential growth phase, the plateau of living cells was reached earlier and its duration was shorter, while the decline phase occurred faster. As a matter of fact, the death phase began at days 8, 14, 17, and 21 from storage at temperature 28 to 12 °C, respectively. At 4 and 8 °C, the stationary phase started at day 14 and no death phase could be observed. The highest levels of TMC were registered for the trial at 28 °C that reached 10.39 log 10 CFU/g at the 2 nd day of storage.

Discussion
The first part of this work was forwarded to the microbiological analyses of collected wild and also purchased marketed mushrooms in Tartu, Estonia. The results indicated the presence of TMC and yeasts at dominant levels until 8.18 log 10 CFU/g, followed by the presence of psychrotrophic microorganisms, including pseudomonads, moulds, members of Enterobacteriaceae family, and LAB. In a similar work, Venturini et al. (2011) analyzed the microbial properties of fresh cultivated and wild mushrooms sampled from Zaragoza, Spain belonging to several species, including B. edulis and C. cibarius, and commercialized in Spain. B. edulis and C. cibarius were only sampled for the wild trial. The levels of mesophilic aerobes, Pseudomonas genus, Enterobacteriaceae family, LAB, yeasts, and moulds reported by Venturini et al. (2011) were almost superimposable to those showed by our investigation, even though our levels of yeasts for C. cibarius were higher and those of Enterobacteriaceae members were lower both on C. cibarius and B. edulis. Ezekiel et al. (2013) analyzed various mushrooms, including C. cibarius, marketed in Nigeria and reported levels of moulds slightly lower than 3.5 log 10 CFU/g which are comparable to those of C. cibarius analyzed in this study. Low levels of coliforms (below the detection limit) have been reported also for other species of mushrooms (González-Fandos et   , 2000). To our knowledge, L. aurantiacum has been object of study for the content of trace elements (Gorbunov et al., 2013;Orita et al., 2018), but this is the first report on its microbiological characteristics. The differences registered for the microbiological levels of B. edulis, C. cibarius, and L. aurantiacum (p<0.05) might be imputable to the diverse surface/volume ratios of the mushroom species considered in this study, as well as to their different nutrient availability. Regarding the significant differences found among wild and market mushrooms, the reason for the lower counts registered for all marketed mushrooms could be due to the conditions applied during conservation that better preserve their microbiological quality characteristics. A few works had been carried out on the evolution of the microorganisms responsible for the microbiological decay of mushrooms. To this purpose, considering that M. conica has not been object of microbiological characterization in past, our study focused to deepen the knowledge on the microbiological characteristics of this niche edible mushroom. The choice of this species was also due to the fact that, at the season of monitoring, it was easily available in Sicily. Thus, it represented an optimal model to perform a study on the effect of the temperature on the microbiological characteristics of mushrooms minimizing the influence of the transport that might accelerate microbial decay, even if it occurs under refrigeration (La Scalia et al., 2016Scalia et al., , 2017. Our work evidenced a clear effect of the temperature for TMC. With the increasing temperature, they developed until high levels very rapidly. This is due to the fact that mesophilic microorganisms develop at the highest rates between 20 and 30 °C. At lower temperatures, the growth was particularly slow and no death phase was registered for the refrigeration temperatures 4 and 8 °C. A previous investigation on this topic was previously carried out by Parentelli et al. (2007) who focused on Lentinula edodes packaged under several different conditions and stored at 5 °C for 20 days. In that work, the aerobic mesophilic bacteria count decreased soon after the monitoring period and those authors concluded that the shelf-life of L. edodes during storage does not depend on mesophilic bacterial growth. In a study conducted on Agaricus bisporus stored at 17 and 25 °C, the decrease of aerobic bacteria in non-perforated films was observed after 5 days (González-Fandos et al., 2000). Our study confirmed the trend found by Simón et al. (2005) who designed an investigation about the microbiological quality of A. bisporus in fresh sliced form and packaged in modified atmospheres. These researchers stated that aerobic bacteria counts were at very high level (7.5 log 10 CFU/g) since the beginning of the monitoring and increased, depending on the atmosphere composi-tion, until 8-10 log 10 CFU/g at day 12 (Simón et al., 2005).

Conclusion
Although several information are available in literature on the microbiological characteristics of fresh mushrooms marketed or collected in woods/forests, this is one of the first reports on B. edulis, C. cibarius, and L. aurantiacum sampled in Estonia. Furthermore, the edible mushroom M. conica was microbiologically characterized for the first time. This work highlights consistent differences, in terms of microbiological levels and composition of the microbial groups among mushrooms belonging to different species and sampled from different sources such as directly from forests or purchased in retail markets. Since all mushrooms were, indeed, wild and collected by hunters, their microbiological quality, besides environmental factors, could also depend on the post-harvest contamination due to transport and, especially storage conditions. The results clearly showed that total mesophilic populations developed at high levels very quickly at temperatures higher than 15 °C, but also the refrigeration did not stop the microbiological decay for this high perishable product.

Author contributions
L.S. and A.S. designed the project of study; R.G., M.C., P.B., and A.L.R. conducted the experiments; R.G. and G.M. analyzed the data; L.S. wrote the manuscript. All authors read and approved the revised manuscript.

Conflicts of interest
There was no conflict of interest in this study.