Gamma irradiation can control the number of psychotrophic bacteria in Agaricus bisporus during storage

1 Universidade Estadual Paulista, UNESP, Faculdade de Ciências Agronômicas, FCA, Departamento de Produção Vegetal/Defesa Fitossanitária, Módulo de Cogumelos. Rua José Barbosa de Barros, 1780 Fazenda Lageado. Caixa Postal 237, 18610-307. Botucatu, SP, Brasil. 2 Universidade de São Paulo, USP, Escola Superior de Agricultura, ESALQ, Departamento de Agroindústria, Alimentos e Nutrição. Av. Pádua Dias, 11, Caixa Postal 9, 13418-900. Piracicaba, SP, Brasil.


INTRODUCTION
Edible mushrooms are a food of high nutritional quality.However, the useful life of the mushrooms as well as their nutritional value vary depending on the species, strain, processing postharvest, developmental stage of *Corresponding author.3811-7206.mushroom, and the type of substrate used (Andrade et al., 2008;Bononi et al., 1995;Minhoni et al., 2005).As mushrooms are highly perishable, they tend to lose the quality immediately after harvest.The short shelf life (1 to 3 days at room temperature) is a disadvantage for the distribution and marketing of fresh product.In this period, changes occur in the mushroom, such as darkening, opening of the pileus, stem elongation, increase in the diameter of the hat, weight loss, and change in texture due to high respiration rate and lack of physical protection to prevent water loss or microbial attack (Akram and Kwon, 2010;Sommer et al., 2010;Singh et al., 2010).
Food irradiation is one of the best and most satisfactory techniques for food preservation.However, the irradiation dose required to control microorganisms on food depends on various factors such as the strength of each particular kind and degree of contamination of the food.For edible mushrooms, irradiation commonly is performed in many countries with low doses (1 to 3 kGy), aiming to reduce the number of spoilage microorganisms and prolonging its life and sensory qualities (Fernandes et al., 2012;Farkas, 2006).
In Brazil, it is still common practice to irradiate mushrooms as a method for postharvest storage, and for Agaricus bisporus, the production is largely marketed in the preservative solution, for instance, calcium chloride and glucanalactone (Kuyper et al., 1993;Rodrigo et al., 1999).However, it is known that such solutions change the original taste of fresh mushrooms and their physicochemical and sensory properties.Recently, Moda (2008) evaluated the shelf life of Pleurotus sajor-caju irradiated with 125, 250, 500, and 750 Gy and found that a dose of 750 Gy was the most suitable and its estimated shelf life of five days, being this time superior for nonirradiated mushrooms.However, in Brazil, no scientific reports were published of the use of gamma radiation to A. bisporus, being necessary to check the feasibility of applying this technique in growing conditions prevailing in the country.Thus, we evaluated the effect of irradiation doses (0, 125, 250, and 500 Gy) in control of psychrotrophic bacteria in strains of A. bisporus (ABI-07/06, ABI-05/03, and PB-1) grown in two kinds of composts (brachiaria and oats) during 14 days of storage at 4 ± 1°C and 90% RH.

MATERIALS AND METHODS
The production of mushrooms was developed on the premises Mushroom Module, Department of Plant Protection, Faculty of Agronomic Sciences (FCA/UNESP), Botucatu/SP, Brazil.
Data were subjected to analysis of variance, and means were compared by Tukey test, using the SISVAR 4.2 statistical program, developed by Department of Mathematical Sciences from UFLA (Federal University of Lavras), MG, Brazil.

Strains of Agaricus bisporus
We used pure culture (primary matrix) strains ABI-07/06, ABI-05/03, and PB-1; the strain ABI-07/06 originated from Piedade/SP, the strain ABI-05/03 originated from Cabreúva/SP, and the strain PB-1 from the company Brasmicel (Suzano-SP), and that, according to the same log files, was acquired by Paul Stamets (Washington, USA) in 2000.These strains are maintained in culture stock, the base culture medium (CA compost agar) submersed in mineral oil sterilized and maintained in biological oxygen demand (BOD) adjusted to 8°C, the Bank Matrix Module mushrooms, located at the Department of Production Plant of the Faculty of Agricultural Sciences, UNESP, Botucatu/SP, Brazil.

Composting, growing, and harvesting
We formulated two types of composts based on brachiaria (Brachiaria spp.) and oat (Avena sativa) straws, with an initial C/N ratio of about 25/1, for the cultivation of A. bisporus strains.All stages of composting, growing, and harvesting followed procedures used by Andrade et al. (2008).

Postharvest
The mushrooms collected from all culture conditions proposed were packaged in plastic polypropylene boxes each containing 200 g.Transport of samples was done in cool boxes to the Center for Nuclear Energy in Agriculture (CENA), University of São Paulo (USP), Brazil, where they were irradiated with 125, 250, and 500 Gy in Cobalt-60 irradiator, type Gammacell with 220 kGy dose rate 0.740 h -1 .The control (nonirradiated) and other treatments were maintained at 4 ± 1°C and 90% of moisture in an incubator for the realization of physicochemical analyzes on the 1st and 14th day of storage.

Sample preparation and dilutions
Analyses were performed on samples of 25 g of fresh mushrooms, weighed aseptically and placed in sterile elemeyers with 225 ml of peptone water (0.1%) sterile, constituting 10 -1 dilution after stirring for 2 min in a peristaltic homogenizer.One milliter of 10 -1 dilution was pipetted in 9 ml of sterile peptone water (0.1%) and from this dilution 10 -2 , 10 -3 to 10 -6 dilutions were tested.

Psychrotrophic count
We used for the enumeration of psychrotrophic bacteria the dilutions 10 -3 to 10 -6 in duplicates.We added 0.1 ml of dilution in Petri dishes containing culture medium plate count agar (PCA) previously prepared and sterilized, these being subsequently inverted and incubated at 7°C for 10 days.After this period, we counted the number of colonies by MPN g -1 food (most probable number).This same methodology procedure and evaluation was repeated after 14 days of storage of the mushroom samples.Thus, microbiological evaluations were made for two periods (on the 1st and 14th day of storage), totaling 384 Petri dishes, with 192 in each period.
Table 1.F values from analysis of variance of the number of psychrotrophic microorganisms colonies present in mushrooms of Agaricus bisporus strains ABI-05/03, ABI-07/06, and PB-1, newly irradiated (initial), and stored at 4 ± 1°C for 14 days (final), grown on two kinds of composts based on oat straw and Brachiaria and submitted to irradiation doses 0, 125, 250, and 500 Gy.

RESULTS AND DISCUSSION
F values from analysis of variance of the number of psychrotrophic bacterial colonies present in A. bisporus are shown in Table 1.For all strains of A. bisporus tested at doses of 250 and 500 Gy, there was no significant difference in the number of psychrotrophic bacterial colonies from mushrooms grown on composts based on oats and Brachiaria grass, both in newly irradiated and in stored mushrooms (Figure 1).Moreover, in nonirradiated (control) mushrooms, the number of psychrotrophic bacteria was influenced by the compost type.These results indicate that irradiation at doses of 250 and 500 Gy was effective in eliminating the effect of the compost on the number of psychrotrophic bacteria and indicated a significant reduction of microorganisms compared to control in both periods.
The highest average number of psychrotrophic colonies, after 14 days of storage, was observed in the ABI-07/06 strain (1.30 × 10 8 MPN g -1 ) in nonirradiated mushrooms, coming from Brachiaria compost (Figure 1).This same strain under the same storage conditions, coming from the same compost type but after a dose of 500 Gy, showed a significant reduction in mean number of psychrotrophic bacterial colonies (2.25 × 10 4 MPN g -1 ).These results differ from those obtained by Moda (2008), which evaluated the increased shelf life mushroom P. sajor-caju with application of gamma radiation and also found that the average psychrotrophic bacteria at a dose of 500 Gy (4.5 × 10 8 MPN g -1 ) in mushrooms P. sajorcaju stored for 10 days was not lower than the average obtained on samples of nonirradiated mushrooms (2.5 × 10 7 MPN g -1 ).
It is known that the type of mushroom and irradiation dose applied directly influence the results of increasing the mushrooms' shelf life.About this, Rivera et al. (2011) evaluated the effect of gamma irradiation on the microbial population of Tuber melanosporum; during 35 days of storage at 4°C, they found that the dose of 1500 Gy did not increase the useful life of mushrooms and maintenance of the quality of truffles.Moreover, Jiang et al. (2010), evaluating the effect of integrated application of gamma irradiation (1000, 1500 and 2000 Gy) and modified atmosphere on the microbiological properties of shiitake mushroom (Lentinula edodes), found that the dose of 1000 Gy was the most efficient in maintaining the firmness level of mushrooms.
Comparing the mean number of psychrotrophic bacterial colonies of strains of A. bisporus, it was found that there were significant differences in the nonirradiated treatments in two periods (Figure 2).Furthermore, in samples of mushrooms subjected to doses of 250 and 500 Gy, the values for the strains of A. bisporus were similar to each other as was the reduction in the number of psychrotrophic bacteria (compared to control) in both irradiated and fresh mushrooms stored, resulting in an increase in the mushrooms' shelf life.Beaulieu et al. (2002) also reported the benefit of preserving irradiation on the mushroom A. bisporus reporting that the useful life was extended to 4 days with a dose of 4500 Gy of irradiation.Also, Lescano (1994) reports that the dose of 3000 Gy, combined with Poly-Vinyl Chloride (PVC) film packaging and storage of 10 ± 2°C, increased the shelf life A. bisporus, providing a white color retention, and growth and the opening of the pileus, acceptable for commercialization for 11 days and for consumption up to 16 days of storage.
Regarding the mean colonies of psychrotrophic bacterial colonies on mushrooms when submitted to irradiation, it was found that all doses tested favored reducing these colonies compared to control (Figure 3).For ABI-05/03 strain grown on oat-based compost, the average psychrotrophic colony count in the nonirradiated treatment was 1.85 × 10 5 MPN g -1 .Already at a dose of 500 Gy, this number was reduced to 1.00 × 10 3 MPN g -1 .Similar results were obtained by Moda (2008) who also found a reduction of the number of psychrotrophic bacteria in P. sajor-caju between 1 day before and 1 day after irradiation (500 Gy) of 6.2 × 10 6 MPN g -1 for 1.3 × 10 4 MPN g -1 , respectively.

Conclusion
The irradiation doses tested favored the reducing number of psychrotrophic bacteria colonies, regardless of composts and A. bisporus strains.

ACKNOWLEDGMENT
To the coordination of improvement of personnel of superior level, by the conception of the master scholarship to the first author.