Advances in batch culture fermented Coriolus versicolor medicinal mushroom for the production of antibacterial compounds

Innovative Food Science and Emerging Technologies 34 (2016) 1–8

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Advances in batch culture fermented Coriolus versicolor medicinal mushroom for the production of antibacterial compounds Dunja Duvnjak ⁎, Milena Pantić, Vladimir Pavlović, Viktor Nedović, Steva Lević, Danka Matijašević, Aleksandra Sknepnek, Miomir Nikšić University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080, Belgrade, Serbia

a r t i c l e

i n f o

Article history: Received 17 July 2015 Received in revised form 29 December 2015 Accepted 30 December 2015 Available online 27 January 2016 Keywords: Antibacterial activity Submerged cultivation Coriolus versicolor Exopolysaccharides Mycelium Mushrooms

a b s t r a c t Bioactive compounds obtained from Coriolus versicolor (Trametes versicolor (L.: Fr) Lloyd, 1920.) mushrooms cultivated in a stirred-tank bioreactor were tested to determine their antimicrobial potential. Extracellular polysaccharides were isolated from the fermentation broth by ethanol precipitation. A methanol extract was prepared from mycelium. The cultivation conditions applied during the fermentation process provided for significant biomass 6.63 ± 0.31 g dry weight L−1 and yield of extracellular polysaccharides (EPS) (0.74 ± 0.12 g L−1). Microscopic analyses revealed that the mycelium grew predominately in the form of fluffy pellets. The methanol extract demonstrated very good activity against all the tested Gram-positive bacteria. Bacillus spizizeni and Staphylococcus epidermidis were the most sensitive strains (minimum inhibitory concentration (MIC) b0.3125 mg mL − 1 ). Among the Gram-negative bacteria, Yersinia enterocolitica had the lowest MIC value, 5 mg mL− 1. Microbicidal activity of mycelia methanol extract was established in seven out of ten tested Gram-negative bacteria strains with minimum bactericidal concentration (MBC) values ranged from 20 to 40 mg mL− 1. Enterococcus faecalis and Staphylococcus aureus showed higher sensitivity to the extracellular polysaccharides (MIC values 2.5 mg mL− 1). FTIR analysis revealed a more complex chemical composition of the methanol extract compared to EPS, which might explain the better antibacterial activity of the methanol extract. Our results suggested that the submerged cultivation of Coriolus versicolor followed by ethanol precipitation of EPS and the methanol extraction of mycelia can be a promising process to obtain biological active compounds with significant antimicrobial activity. Industrial Relevance: Mushrooms contain a large number of chemicals with potential use as antimicrobial compounds. One of the biggest challenges for providing biologically active compounds from mushrooms is short-term process standardization with a low risk for contamination. Submerged culture cultivation is the best choice for providing antimicrobial compounds from mushrooms. The submerged culture method represents an effective and energyefficient means to produce novel antibacterial compound from mushrooms. Antibacterial activity testing revealed that methanol extract and isolated exopolysaccharides exhibited strong antibacterial activity, especially against Gram-positive bacteria. © 2016 Elsevier Ltd. All rights reserved.

1. Introduction Medicinal mushrooms have been used in the treatment and prevention of many diseases, since ancient times. Either in the form of a fruiting body, mycelium extract or different chemical compounds, mushrooms are considered as functional food and valuable dietary supplements in the treatment of various disorders. The biological activities of mushrooms are mainly attributed to their polysaccharide and total phenolic contents (Cheung, Cheung, & Ooi, 2003). Polysaccharides could be obtained in

⁎ Corresponding author. Tel./fax: +381 112199 711. E-mail address: [email protected] (D. Duvnjak).

http://dx.doi.org/10.1016/j.ifset.2015.12.028 1466-8564/© 2016 Elsevier Ltd. All rights reserved.

different forms, such as polysaccharides, polysaccharopeptides and proteoglucans, through extraction, separation, and purification (Lin et al., 2008). These compounds are supposed to play a key role in some healthy properties of mushrooms (e.g. enhance macrophage function and host resistance to infections) (Manzi & Pizzoferrato, 2000; Savić et al., 2012; Zaidi, Jain, & Quereshi, 2013). Fungi, both fruiting body and the mycelium, naturally contain metabolites that are antibacterial components that enable them to survive in the natural environment (Alves et al., 2012; Manjunathan & Kaviyarasan, 2010). Researchers have reported antimicrobial and antioxidant activity of basidiocarp and mycelia extracts of more than 2000 fungal species (Alves et al., 2012; Manjunathan & Kaviyarasan, 2010; Turkoglu, Duru, Mercan, Kivrak, & Gezer, 2007).

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Over the years, submerged (SmF) and solid state fermentations (SSF) have emerged as the most suitable techniques for industriallevel production of fungal biomass and its metabolites. The industrial application of mushrooms bioactive compounds in some cases might be difficult. Namely, fruiting body production for commercial purposes is a process that often lasts several months. Likewise, it is a labourintensive procedure with uncertain outcome due to the great possibility of contamination and difficulties in controlling the process parameters. As a result biomass yield is often low and thus the yield of target bioactive compounds. On the other hand, certain types of medicinal mushrooms are host specific, slow growing and rare in nature. In such cases the production of mycelium biomass by submerged culture stands out as the best option and promising alternative for obtaining the bioactive materials. (Borràs, Blánquez, Sarrà, Caminal, & Vicent, 2008; Kim et al., 2005; Tang, Zhu, Li, & Li, 2007). The main advantages of submerged cultivation of higher mushrooms are a constant quality of biomass provided in a defined space and in a shorter time with less chance of contamination. However, growth of Basidiomycetes in liquid is strongly influenced by the fermentation conditions (e.g. temperature, agitation, dissolved oxygen, substrate, metabolite concentration and pH of the medium) and studied by many authors (Borràs et al., 2008; Cui & Chisti, 2003; Ma, Mao, Geng, Wang, & Xu, 2013; Yang, Hsu, Lin, Hsu, & Chen, 2012). Submerged culture cultivation processes for obtaining of the bioactive compounds are always optimized by adjusting the factors mentioned above, all in order to maximize benefit (e.g. profit) and minimize cost (Lübbert, 2003). In submerged culture morphological forms are usually defined as either pelletised or dispersed. Good control of mycelia morphology is important for the performance of the bioreactor and further industrial application. Dispersed filaments exhibit an increase in viscosity and non-Newtonian rheology which leads to reduced transport of oxygen and nutrients and overall productivity. (Cui & Chisti, 2003; Couto & Toca-Herrera, 2007; Borràs et al., 2008;). Bermek, Gülseren, Li, Jung, and Tamerler (2004) reported that growth in dispersed form can cause lower lignolytic enzyme production. By contrast, pellets exhibit low viscosity, approach Newtonian flow behavior and do not adhere to any part of the bioreactor. Additionally, later isolation of fungal biomass is much easier when mycelia grow in form of pellets. Thus, obtaining uniform pellets is desired but not easy, and once formed are the confirmation of a well designed system for submerged fermentation. (Borràs et al., 2008; Couto & Toca-Herrera, 2007; Cui & Chisti, 2003). It has been reported that submerged culture appears to be the preferred option for production of the polymers (e.g. polysaccharopeptides) compared to the fruit body (Cui & Chisti, 2003). There are several limiting factors for industrial exploitation of bioactive compounds from fruit body. The major disadvantages beside long period of growing into mature fruiting bodies are the high purification costs and variable quality. The separation and purification processes of the bioactive compounds, which could be used as pharmaceuticals and functional foods, are much easier and faster from mycelium than from fruiting body, due to the less content of ballast in fungal biomass (Turlo, 2014). It is usually impossible to separate the biomass from the substrate during fungal SSF, because the fungal mycelium binds very tightly to the substrate. Also, mushroom growing on solid substrates originated from industrial areas can accumulate toxic levels of heavy metals (Mitchell & Berovič, 2003). On the other hand, with applying submerged cultivation, polysaccharides can be extracted either from mycelia biomass or cultivation broth (Cui & Chisti, 2003; Sánchez, Montoya, & Vargas, 2015). Secondary metabolites occur in the stationary growth phase and thus, a discontinuous process in a bioreactor is ideal for the production of extracellular polysaccharides. Coriolus versicolor, white-rot fungus is a medicinal mushroom with a broad spectrum of physiological activities that aim to enhance and protect body functions. It can be collected in nature, grown on solid substrate or in submerged fermentation as mycelia biomass. According to traditional Chinese medicine (TCM) classics, it is believed that Coriolus sp. could be useful for removing toxins, strengthening physique,

increasing energy and enhancing the immune function (Chu, Ho, & Chow, 2002). SSF of C. versicolor is mainly used in so-called green biotechnology for bioremediation applications, due to their high production of extracellular lignin mineralizing enzymes (LMEs). (Kadhim, Graham, Barratt, Evans, & Rastall, 1999; Nigam, Armour, Banat, Singh, & Marchant, 2000; Robinson, McMullan, Marchant, & Nigam, 2001; Ullah, Kadhim, Rastall, & Evans, 2000). On the other hand, some of the best established antioxidative and antibacterial compounds are polysaccharopeptides, both extracellular and intracellular, derived from C. versicolor (Chu et al., 2002; Kozarski et al., 2012; Ma et al., 2013). Polysaccharopeptide krestin (PSK) and polysaccharopeptide PSP isolated from C. versicolor mycelia by batch fermentation exhibited antitumor, hepatoprotective and analgesic activities (Chan, Chan, & Sze, 2009; Chu et al., 2002; Cui & Chisti, 2003; Tsang et al., 2003). Also, it was reported that these compounds exhibit significant immunomodulatory activity and have potential antidiabetic properties as α-glucosidase inhibitors (Lin et al., 2008; Yang et al., 2012). Several studies were conducted to improve the production of C. versicolor polysaccharides (Bolla, Gopinath, Shaheen, & Charya, 2010; Lin et al., 2008; Ma et al., 2013; Que, Sun, Xu, Zhang, & Zhu, 2014; Yang et al., 2012). Changes in pH during fermentation, enrichment of culture medium with different plant extracts, changes in carbon and nitrogen sources and optimal recovery conditions were just some of the attempts to upgrade the system. The aim of this research was to test the applied biotechnological process for biomass and EPS production. To the best of our knowledge, the antibacterial activity of C. versicolor EPS isolated by ethanol precipitation has not been the subject of research. To optimize the process, the residual glucose was quantified by HPLC. A scanning electron microscopy was used to analyze the morphology of the formed pellets and the final goal was to examine the antimicrobial activities of the isolated bioactive compounds. 2. Materials and methods 2.1. Microorganism and culture conditions C. versicolor fungus from the collection of the Department of Industrial Microbiology, Faculty of Agriculture, University of Belgrade (Serbia) was used. A culture was grown on malt agar for 7 days. Subsequently, 10-mm diameter discs were cut with a no. 5 cork borer and the discs were transferred into nutrient medium in order to prepare inoculums for the seeding of the bioreactor. The normal nutrient medium for fungal growth contained 4.0% glucose, 0.15% peptone (HiMedia, India), 0.15% KH2PO4, and 0.15% MgSO4·7H2O (Yang et al., 2012). The fungi was cultivated in 500-mL Erlenmeyer flasks, working volume of 100 mL, at 25 °C on a model SI 600R incubated shaker (JEIO TECH, Korea) at 135 rpm (Yang et al., 2012). After five days, the obtained pellets were ground under sterile conditions using a laboratory blender. The biomass was isolated by centrifugation at 5000 g for ten minutes, washed several times with distilled water, and used as inocula for seeding of the bioreactor. 2.2. Bioreactor fermentation Batch fermentation was performed in the same nutrient medium as explained in the previous section. The construction of the 2-L stirredtank is shown in Fig. 1. Air with a pressure at the system input of 67 mmHg was blown through a glass sparger using an air compressor. A cellulose acetate syringe filter, grade 0.22 μm, was placed on the system input in order to provide sterility. The system was placed on Hot Plate Stirrer T-14 (LAB COMPANION, Korea), which provided a constant agitation speed. For the inoculation, a 5% w/v seed culture was used. Fermentation was performed under the following conditions: working volume 1.5 L, temperature 25 ± 2 °C, aeration rate 1 vvm and agitation 150 rpm. After 8 days of cultivation, the biomass was isolated and

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2.6. Methanol extraction Five grams of lyophilized biomass were extracted by stirring on a magnetic stirrer with 150 mL of methanol (25 °C, agitation speed 100 rpm) for 24 h, and then filtrated through Whatman no. 4 filter paper. The residue was re-extracted two more times and the obtained liquid was collected and evaporated under reduced pressure to remove the solvent (Barros, Baptista, Estevinho, & Ferreira, 2007). 2.7. Characterization of the mycelia products 2.7.1. FTIR spectroscopy The FTIR spectra of C. versicolor extract and EPS were recorded using an ATR-FTIR spectrometer IRAffinity-1 (SCHIMADZU, Japan). All FTIR measurements were performed in the spectral range 4000–600 cm−1 with a resolution of 4 cm−1.

Fig. 1. Scheme of constructed bioreactor used for Coriolus versicolor cultivation.

lyophilized for further use. The fermentation broth was used for the isolation of exopolysaccharides.

2.3. Sampling, determination of pH value and dry biomass weight An aliquot of 10 mL from three batches was taken after every 24 h for 8 days under sterile conditions, centrifuged and filtrated through Whatman no. 1 filter paper. The obtained fermentation broth free from mycelia was used to determine the residual glucose by HPLC and for measuring the pH value (pH meter CRISON, Spain). Biomass yield was measured and expressed as mycelia dry weight per litre of fermentation broth (g DW L−1) (Kim et al., 2005). The obtained results are expressed as the mean value ± standard deviation.

2.4. Quantitave analysis of the residual glucose content by HPLC For the quantitative analysis of the samples from the fermentation broth, the Dionex Ultimate 3000 Thermo Scientific (Waltham, USA) HPLC system was used. For glucose determination a carbohydrate column (Hyper REZ XP Carbohydrate Ca2+, 300 mm × 7.7 mm, 8 μm) at 80 °C was employed. Water was used as the sole mobile phase at an elution rate of 0.6 mL min−1 during the analysis. Detection was performed by an RI detector (RefratcoMax 520, ERC, Germany). All data acquisition and processing was realized using Chromeleon Software. The samples were analyzed in triplicate and the results are expressed as the mean value ± standard deviation (Carević et al., 2015).

2.5. Isolation of extracellular polysaccharides (EPS) The fermentation broth obtained after isolation of the biomass was filtered through a Whatman no. 1 filter paper. The samples were evaporated under vacuum in a type R-II rotovapory (BUCHI, Switzerland) to a one-third of their initial volume, and mixed with three volumes of 95% v/v ethanol. Mixture was stirred vigorously and left at 4 °C over night whereby the extracellular polysaccharides separated (Wu et al., 2014). The obtained exopolysaccharides were collected from the surface and washed several times with 95% ethanol (Lee et al., 2004). Lyophilisation was preformed in a 1–4 LSC plus lyophilisator (CHRIST ALPHA, Germany) to preserve the sample until use. For precipitation and estimation of the EPS content, 20 mL of the fermentation broth were used (Lee, Lee, & Lee, 1999). Measurements were conduced in triplicate and the data are expressed as the mean value ± standard deviation.

2.7.2. Microscopic analysis of C. versicolor biomass The microscopic analysis of the mycelia biomass was performed using a Leica DMLS microscope (Leica, Germany) equipped with a DC 300 digital camera (Leica, Germany) and Leica IM 1000 software (Leica, Germany). 2.7.3. Scanning electron microscopy (SEM) The microstructure of the mycelia biomass of the samples was observed using a JEOL JSM-6390LV scanning electron microscope. Prior to the analysis the samples were covered with Au using a Baltec scd 005 sputter coater accessory. 2.8. Antibacterial assay 2.8.1. Bacterial strains and culture preparation For antibacterial activity assay, eight Gram-positive (Enterococcus faecalis ATCC 29212, Bacillus spizizeni ATCC 6633, Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 25923, Staphylococcus epidermidis ATCC 12228, Listeria monocytogenes ATCC 19111, Listeria ivanovii ATCC 19119, Listeria innocua ATCC 33090) and ten Gram-negative strains (Proteus mirabilis ATCC 12453, Proteus hauseri ATCC 13315, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Escherichia coli H7:O157, ATCC 35150, Salmonella ser. Enteritidis ATCC 13076, Salmonella ser. Typhimurium ATCC 14028, Shigella sonnei ATCC 29930, Yersinia enterocolitica ATCC 27729 and Citrobacter freundii ATCC 43864) were used. Bacterial suspensions of E. coli H7:O157 and Listeria strains were prepared in Tryptic Soy Broth (HiMedia, India) while the other bacteria were prepared in Müller Hinton Broth (HiMedia, India). From the Müller Hinton (HiMedia, India) and Tryptic Soy (HiMedia, India) agar plate, a 24-h old colony was sub-cultured to 5 mL of adequate broth and incubated at 37 °C for 18–24 h. The final concentration of the bacteria used for inoculation of the microtitre wells was adjusted to 105 cfu mL−1 (Klačnik, Piskernik, Jeršek, & Možina, 2010). 2.8.2. Broth microdilution method The broth microdilution method was used to determine the minimum inhibitor and minimum bactericidal concentrations of the bioactive compounds of C. versicolor (Klačnik et al., 2010). The samples were dissolved in 5% DMSO/water solution and sterilized by filtration through cellulose acetate syringe filters, grade 0.22 μm, (Sartorius, Germany). Three-fold dilutions were prepared in a 96-well microtitre plate (Sarstedt, Germany). The tested concentrations ranged between 0.3125 mg mL−1 and 40 mg mL−1. As an indicator of cell colour TTC (2,3,5-triphenyl-tetrazolium chloride, Sigma Chemical Co.) was added to the bacteria suspensions in the corresponding broth (Klačnik et al., 2010). For testing Gram-positive strains, resazurin sodium salt was used (Sarker, Nahar, & Kumarasamy, 2007). The prepared bacterial suspensions were added into the wells at different dilutions of the extract or EPS so that the final volume in each well was 100 μL. As a positive

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control, only the bacterial suspension was used, and as a negative control, 5% DMSO was used. After the inoculation, the microtitre plates were incubated at 37 °C for 18–24 h. The minimum inhibitory concentration (MIC) was defined as the lowest concentration of a sample (mg mL−1) for which no visible growth was detected and the indicator of respiratory activity (TTC) remained colourless (Klačnik et al., 2010; Shakeri et al., 2014). When resazurin was used as an indicator, a change in its colour from blue to pink indicated bacteria growth (Shakeri et al., 2014). To determine the minimum bactericidal concentration, the dilutions representing the already established MIC were sub-cultured on the surface of an appropriate agar base. If there were no bacterial growth on the surface, the tested concentration of the sample was considered to be the MBC (Nowacka et al., 2014). 3. Statistical analysis The assays were performed in triplicate and expressed as mean value ± standard deviation. The obtained data were subjected to a one-way analysis of variance (ANOVA). The statistical analysis was performed using the statistical program Origin Pro 9.0. The Tukey HSD test was used at p b 0.05 to identify significant differences among the means. 4. Results and discussion 4.1. HPLC analysis of glucose content, determination of pH value and dry biomass weight The results of the study of glucose consumption by C. versicolor over a period of eight days are shown in Fig. 2. It could be noticed that after 48 h, the glucose concentration started to decrease significantly and had decreased to 20.83 ± 1.27 g L− 1 by the end of the cultivation. According to the data found in the literature, submerged mushrooms cultivations should be conducted for over 30 days in order to achieve maximum glucose consumption (Shih, Tsai, & Hsieh, 2007). Rau et al. (2009) reported that the maximum specific growth rate of C. versicolor was reached after 7.9 days, and that glucose was completely consumed after 11 days. However, in the present study, the glucose consumption slowed down after day seven and in order to achieve higher efficiency (i.e., saving of energy and time), the process was terminated and the products isolated. A relatively high biomass (mycelia) yield of 6.63 ± 0.31 g DW L− 1 was achieved as well as a significant yield of exopolysaccharides, 0.74 ± 0.12 g L−1. Xu, Kim, Hwang, Choi, and Yun (2003) showed that optimisation of submerged mushroom cultivation is critical for higher yields of mycelia and exopolysaccharides. According to the same authors, the optimal glucose concentration, as well as the concentrations of other nutrients, for mycelia and exopolysaccharides

Fig. 2. Glucose consumption (■) and changes in pH (●) value during batch bioreactor fermentation of Coriolus versicolor mushroom.

production was not related. It seems that variation in the process conditions and medium composition could lead to higher productivity in submerged mushroom cultivation. Nevertheless, a relatively high glucose concentration in the medium was tested in the present study and good conversions into mycelia biomass and exopolysaccharides were achieved. The efficiency of submerged mushroom growth is also affected by the conditions and design of the bioreactor system. Mao and Zhong (2004) demonstrated that an optimal oxygen transfer combined with an adequate stirrer system could be critical for maximal production of mushroom metabolites. Based on these findings, gentle stirring of the submerged mycelia was applied with a constant oxygen supply during the whole cultivation period. Constant stirring was necessary since significant changes in the cultivation broth during the submerged growth of C. versicolor were observed. Namely, after the first two days, the individual pellets increased in volume and started to make a compact biomass. Hence, adequate but gentle stirring was required in order to provide a homogeneous distribution of oxygen and nutrients over the whole reactor volume. Zhou, Feng, and Tang (1994) reported that fermentation broths for C. versicolor mushroom are highly viscous due to the presence of extracellular polysaccharides and biomass filaments. Parallel with significant mycelia growth, the pH (Fig. 2) of the cultivation broth started to decrease from ≈ 5.1 (day one) to ≈ 4.3 (day eight). It seems that intensive mycelia growth corresponded with a decreasing of pH. These results are in accordance with those of Que et al. (2014) who reported that the optimum pH for the maximum production of exopolysaccharides for the fungus C. versicolor was slightly lower than for other fungus and ranged between 4.0 and 6.0. In the present experiment, the optimum pH value was achieved without additional adjustments during the process. 4.2. FTIR spectroscopy The chemical properties of the methanol extract from C. versicolor mycelia biomass, as well as of the exopolysaccharides produced during submerged mushroom growth, were studied using FTIR spectroscopy. As it can be seen in Fig. 3, both spectra showed a strong and broad band from OH groups in the spectral region from ≈ 3600 cm− 1 to ≈3000 cm−1. Kozarski et al. (2011) reported that mushrooms extracts exhibit a strong signal in this spectral region due to molecular interaction of the polysaccharide chains. Gonzaga, Menezes, de Souza, Ricardo, and Soares (2013) and Klaus et al. (2015) indicated that mushroom extracts could also show the presence of N–H vibrations in the same spectral region as OH groups. The bands at ≈ 2920 cm− 1

Fig. 3. FTIR spectra of extracellular polysaccharides (a) and mycelium methanol extract (b) of Coriolus versicolor mushroom.

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correspond to CH2 stretching and bending vibrations (Ren et al., 2014). These bands were more pronounced in the case of the samples obtained by methanol extraction, which could be explained by more the complex chemical composition of mycelia extract compared to the composition of extracellular polysaccharides. Moreover, a methanol extract exhibited a more intense peak at ≈ 1730 cm−1 than a sample containing exopolysaccharides. This peak indicates presence of the C = O groups (Xiao et al., 2012). The band at ≈1650 cm−1 is due to the asymmetric stretching vibration of COO− groups (Gonzaga et al., 2013). Two bands could be observed in the spectral region between 1100 cm− 1 and 1000 cm−1; the band at ≈1028 cm−1 is ascribed to C–O stretching vibrations, while that at ≈1079 cm−1 is most probably due to the presence of O-substituted glucose residues. According to Kozarski et al. (2011) a band at ≈1079 cm−1 indicates the presence of β-glucans in mushrooms extracts. However, in the case of exopolysaccharides, analysed in this study this band was shifted to a lower wavenumber suggesting differences in the structure between the samples. This could be expected since β-glucans are located in the structure of mycelia (i.e., the cell wall) and thus, their presence in the extracted exopolysaccharides is limited. The presence of small amounts of proteins in the samples could be noticed by the weak band at ≈ 1411 cm− 1 (Ren et al., 2014). Nevertheless, the results of the FTIR analysis generally showed that both samples from C. versicolor extracts (i.e., mycelia and exopolysaccharides) exhibited bands characteristic for polysaccharides.

4.3. Microscopic analysis of C. versicolor biomass The results of the microscopic analysis of the growth morphology are represented in Fig. 4. As can be seen, C. versicolor grew predominantly in the form of fluffy pellets with presence of hairiness on the surface. It is well known that in submerdged culture, mushrooms can grow in the form of pellets or as free filaments dispersed in the medium, one form usually being predominant (Cui, Van der Lans, & Luyben, 1997). Fungal morphology is the subject of extensive research because it affects the rheology of the fermentation broth and biomass yield, as well as the yield of the target metabolites and biotechnological efficiency of the process (Belmar-Beiny & Thomas, 1991; Daniel, Schonholzer, & Zeyer, 1995; Lee et al., 2004; Packer & Thomas, 2009). Conditions favouring one type of growth are still not fully understood. A number of factors affect growth morphology of mushrooms, for example medium composition, pH, the temperature of fermentation and the aeration rate. Two of the most important factors that affect pellet morphology and size are mechanical forces and the shear stress in the bioreactor (Panda & Rameshaiah, 2009). Another reason for the application of gentle stirring in the present work was, besides ensuring a constant oxygen supply, an attempt to avoid pellet fragmentation. Lee et al. (2004) revealed that

Fig. 4. Light microscope image of Coriolus versicolor pellet.

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during submerge cultivation of Grifola frondosa, compact pellets were formed when the aeration rate was low. They recorded that maximum production of mycelia biomass and polysaccharides was reached when a mushroom pellet was a loose clump with high hairiness. The same authors compared the impact of using air-lift and stirred-tank bioreactors on the yields of mycelia and EPS. They concluded that EPS and mycelia production was better in stirred-tank fermenters due to the better mixing and achievement of pellets with a favourable morphology. Que et al. (2014) reported that the pH value has a significant impact on cellular morphology and that C. versicolor mushroom forms dispersed hyphae at pH 6.0. The same authors concluded that this morphology was suitable for high EPS production from C. versicolor mushroom. 4.4. Scanning electron microscopy (SEM) As can be seen in the Fig. 5, hyphae morphology was typical for the mushrooms. The hyphae were long, very branched and anastomosed forming a pellet form. The density of the hyphae was very high and the presence of septa was not observed. It is known that extracellular fungal material surrounds the C. versicolor hyphae in liquid culture. Their removal from the hyphae surface requires careful washing, while scanning electron microscopy stands out as confirmation (Vesentini, Dickinson, & Murphy, 2005). Since on scanning micrograph EPS were not noticed on the mycelia surface, it could be concluded that the antibacterial activity of the methanol extract originated from bioactive compounds present in the mycelia of the mushroom (Ivarsson & Holmström, 2012). 4.5. Antibacterial assay The results of the antibacterial assay are presented in Table 1. The three Gram-positive strains singled out as being the most sensitive to EPS were E. faecalis, S. aureus and S. epidermidis. The MIC values were 2.5 mg mL− 1, 2.5 mg mL−1 and b 0.3125 mg mL− 1, respectively. For B. cereus and L. ivanovii MIC values were slightly higher (5 mg mL−1 and 10 mg mL− 1, respectively). Other Gram-positive bacteria were equally sensitive with MIC value of 40 mg mL−1. Minimum bactericidal concentrations for EPS were established for only two bacteria, S. epidermidis and B. cereus (MBC values b0.3125 mg mL−1 and 5 mg mL− 1, respectively). Y. enterocolitica and S. sonnei, as Gramnegative bacteria, were also susceptible to EPS but with higher MIC values ranging from 10 to 20 mg mL−1. The E. coli strains were resistant to the tested concentrations of EPS, while for the other Gram-negative bacteria had the same MIC value of 40 mg mL−1. Microbicidal activity was not observed on Gram-negative bacteria. Mahendran, Saravanan, Vijayabaskar, Anandapandian, and Shankar (2013) reported significant antibacterial activity of exopolysaccharides isolated from a liquid culture of Ganoderma lucidum. They showed that G. lucidum EPS possess strong antibacterial activity against B. cereus, B.subtilis, E.coli and Proteus sp. strains, and that this activity depended on the medium composition used for mushroom cultivation. They also reported weak effect of EPS on S. aureus and Ps. aeruginosa. Such effects on S. aureus and E. coli were not in accordance with the present results. On the other hand, similar to the present results, Adebayo, Oloke, Ayandele, and Adegunlola (2012) reported a weak effect of Pleurotus pulmonarius metabolite on E. coli, while Ps. aeruginosa was resistant. The same authors concluded that Pleurotus metabolites exhibited strong antibacterial activity against P. mirabilis. In the present study MIC values for Proteus strains were not determined with the tested EPS concentrations. Suay et al. (2000) tested the ability of C. versicolor fungus to produce secondary metabolites that can be used in the fight against pathogenic microorganisms. The authors concluded that submerged cultivated C. versicolor does not produce secondary metabolites with antibacterial activity, as the sample had no effect on any of the tested strains, including S. aureus, Ps. aeruginosa and B. subtilis. Similarly, Yamac and Bilgili (2006) tested

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Fig. 5. Scanning electron micrographs of Coriolus versicolor mushroom pellet (A) outer pellet surface, with (B) intertwined hyphae with anastomoses.

the antimicrobial activity of organic and aqueous fractions of C. versicolor culture broth. They concluded that both fractions had antimicrobial activity, but the established inhibition zones (b10 mm) pointed to their weak effect. On the contrary, the results obtained in the present study showed that bioactive compounds can be derived from C. versicolor submerged cultivation. Namely, by culturing the mushroom in a stirred-tank bioreactor and using ethanol precipitation of the obtained fermentation broth it was possible to produce EPS, that showed antibacterial activity on 16 of the 18 tested strains. Demır and Yamaç (2008) studied the antimicrobial activity EPS from seven different

Table 1 Antibacterial activity of Coriolus versicolor bioactive compounds expressed as MIC (mg L−1) and MBC (mg L−1) values determined by broth microdilution method. Tested bacteria E. faecalis ATCC 29212 B. spizizeni ATCC 6633 B. cereus ATCC 11778 S. aureus ATCC 25923 S. epidermidis ATCC 12228 L. monocytogenes ATCC 19111 L. ivanovii ATCC 19119 L. innocua ATCC 33090 P. mirabilis ATCC 12453 P. hauseri ATCC 13315 Ps. aeruginosa ATCC 27853 E. coli ATCC 25922 E. coli H7:O157 ATCC 35150 S. Enteritidis ATCC 13076 S. Typhimurium ATCC 14028 S. sonnei ATCC 29930 Y. enterocolitica ATCC 27729 C. freundii ATCC 43864

MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC

Methanol extract

Exopolysaccharides

20.0 ± 0.0a⁎1 nd2 b0.3125 10.0 ± 0.0 5.0 ± 0.0 a nd 10.0 ± 0.0a nd b0.3125 nd 10.0 ± 0.0b nd 40.0 ± 0.0 a 40.0 ± 0.0 nd nd 20.0 ± 0.0a 40.0 ± 0.0 10.0 ± 0.0b 20.0 ± 0.0b 20.0 ± 0.0b nd 40.0 ± 0.0 40.0 ± 0.0 20.0 ± 0.0 nd 20.0 ± 0.0 b 40.0 ± 0.0 40.0 ± 0.0 a nd 20.0 ± 0.0 a 20.0 ± 0.0 5.0 ± 0.0 b 40.0 ± 0.0 10.0 ± 0.0 b 40.0 ± 0.0

2.5 ± 0.0 b nd 40.0 ± 0.0 nd 5.0 ± 0.0 a 5.0 ± 0.0 2.5 ± 0.0 b nd b0.3125 b0.3125 40.0 ± 0.0 a nd 10.0 ± 0.0 b nd 40.0 ± 0.0 b nd 40.0 ± 0.0 a nd 40.0 ± 0.0 a nd 40.0 ± 0.0 a nd nd nd nd nd 40.0 ± 0.0 a nd 40.0 ± 0.0 a nd 20.0 ± 0.0 a nd 10.0 ± 0.0 a nd 40.0 ± 0.0 a nd

*Data are expressed as mean ± standard deviation (n = 3). Means marked by different letters in the same row are significantly different at α = 0.05 (Tukey's HSD). 2 nd, not determined (with the highest tested concentration of samples (40 mg/mL); antibacterial activity was not determined). 1

fungi on bacteria and a yeast culture. They concluded that the mushroom had moderate antimicrobial activity and that the Gram-positive strains were the most sensitive. The present results confirmed previous studies and conclusions that not only the growth conditions and medium composition, but also mushroom species has an influence on antimicrobial activity of the samples. Additionally, it was proved that the isolation methods must be taken into consideration when making conclusions about the potential of some fungi to produce antimicrobial bioactive compounds. Although the sample had the effects on all tested Gram-positive and eight out of ten tested Gram-negative strains, based on obtained MIC and MBC concentration it can be concluded that Gram-positive strains were more susceptible to EPS. It is assumed that variations in the cell wall structure might be the reason for these differences in bacteria resistance. More complex cell wall of Gram-negative bacteria has a role of diffusion barrier providing a higher resistance to the antimicrobial substances (Klačnik et al., 2010). The peptidoglycan content in the cell wall varies between 10% and 60% for Gram-negative and Gram-positive bacteria, respectively. The mechanism of antibacterial activity is mostly related with interferences in the synthesis of the cell wall, modification of plasmatic membrane permeability, interferences in chromosome replication or in protein synthesis (Alves et al., 2012). Researchers reported that polysaccharides can damage the cell wall and cytoplasmic membrane of bacteria. This would cause dissolution of the protein and a leakage of molecules which leads to cell death. Additionally, after entering the cell, the DNA might be decomposed (He, Yang, Yang, & Yu, 2010). The results of testing antibacterial activity of methanol extract showed that the MIC values for Bacillus and Staphylococcus strains, E. faecalis and L. monocytogenes ranged from b 0.3125 to 20 mg mL−1. Methanol extract acted microbicidal on B. spizizeni and L. ivanovii (10 mg mL−1 and 40 mg mL−1, respectively). No visible growth was evidenced for Y. enterocolitica at a concentration of 5 mg mL−1, which was considered as the most sensitive among the Gram-negative bacteria. For the other Gram-negative strains the MIC values were similar and ranged from 10 mg mL−1 to 40 mg mL−1. Also, it was observed that methanol extract exhibited microbicidal effect to seven out of ten tested Gram-negative strains. Concentration of 20 mg mL−1 expressed microbicidal activity against P. hauseri and S. sonnei while concentration of 40 mg mL−1 was lethal to P. mirabilis, E. coli, S. Enteritidis, Y. enterocolitica and C. freundi. Zaidi et al. (2013) tested two type of bioactive compounds isolated from submerged cultivated C. versicolor mushroom. The first was obtained by hot water–ethanol extraction of mushroom pellets and the second by Tris HCl–ethanol extraction. The tested extracts showed no activity on S. aureus bacteria, although high sample concentrations (1000 mg L−1) were used. In case of E. coli and S. Typhimurium very small inhibition zones (5 and 2 mm, respectively) were observed at a concentration of 300 mg mL−1. In the present study, the maximal tested concentration (40 mg mL−1) of methanol extract inhibited E. coli and S. Typhimurium, and had in general a much better antibacterial activity. In the case of S. aureus testing the MIC value was

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even four times lower. Çoban, Isman, and Biyik (2008) extracted polysaccharopeptide (PSP) from a submerged fermentation of C. versicolor and tested its antibacterial activity. Activity was confirmed on S. aureus and B. subtilis but the extract had no effect on E. coli and S. epidermidis. Mehta and Jandaik (2012) reported better antibacterial activity of mycelial compared to fruit body methanol extract of Ganoderma lucidum. Authors showed that both tested extracts contained antibacterial constituents that were able to inhibit the growth of both Gram-positive and Gram-negative bacteria. Higher inhibitory activity of mycelial methanol extract compared to fruit body was possibly due to varying composition of polysaccharide in cell wall. Karaman, Jovin, Malbaša, Matavuly, and Popović (2010) founded that low concentration of C. versicolor fruit body methanol extract (0.05 mg mL− 1) had no effect on Gram-negative strains with the exception of S. Enteritidis. The obtained data revealed that both samples, EPS and methanol extract exhibited equally good effect on Gram-positive bacteria, but that the methanol extract additionally exhibited microbicidal activity on the majority of tested Gram-negative strains. More complex mixture of bioactive compounds in methanol extract might be the reasons for its higher range of antimicrobial activity compared with EPS. Since methanol is higher in polarity than other frequently used solvents, it solubilize more of the fungal material. Research data indicates that in the most cases, methanol extract was the strongest antimicrobial agent (Smolskaitė, Venskutonis, & Talou, 2015). Methanol extracts obtained from fruit bodies of Clitocybe alexandri, Lactarius species, Lepista nuda, Pleurotus saju-caju also indicated inhibitory activity against Gram-negative bacteria. Among all tested strains, E. coli, Klebsiella pneumoniae and P. aeruginosa were the most sensitive (Alves et al., 2012). In this research FTIR analysis showed that the methanol extract most probably contained a greater amount of β-glucans and proteins than the EPS. The presence of these bioactive compounds in greater amounts, most likely, led to the improvement of antibacterial activity. The obtained results showed that submerged cultivated mushrooms beside mycelium, produce bioactive compounds in an extracellular form that could be used against a large number of pathogenic bacteria. 5. Conclusions In this study it was shown that the applied biotechnological process can be successfully used to cultivate C. versicolor mushroom with significant biomass and EPS yields. Applied stirred-tank bioreactor is a simple apparatus which does not require complicate equipment and manipulation. Additionally, it is inexpensive and suitable for the production of bioactive compounds from Basidiomycetes. The microscopic analysis, HPLC analysis of the glucose content and determination of the pH value showed that the fungus grew in a form suitable for the production of bioactive compounds. Furthermore, this biotechnological process enabled the isolation of two promising products, EPS gained from the fermentation broth, and the another one obtained by methanol extraction of the derived mycelia. Additionally, compared to other systems and extraction procedures, the bioactive compounds from C. versicolor with pronounced antimicrobial activity was provided with the special highlight on Gram-negative strains. FTIR spectroscopy revealed differences between the samples in regard to polysaccharides, β-glucans and proteins, which could be a potential explanation for the differences in antimicrobial activity. In general, EPS showed better antimicrobial activity on Gram-positive than on Gram-negative strains while methanol extract exhibited significant activity on both Gram-positive and Gram-negative strains. Acknowledgement This study was financially supported by Ministry of Education, Science and Technological Development of the Republic of Serbia (Projects No. III 46010 and OI 172057) and the EU Commission project AREA, No.

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