Comparison of Antibacterial Activity of the Spent Substrate of Pleurotus ostreatus and Lentinula edodes

Nowadays, the uncontrolled use of antibiotics has created the problem of bacterial resistance to them, what has motivated the search for new alternatives of drug for the treatment of bacterial diseases. Here, we compare antimicrobial activity of spent substrate of mushroom Pleurotus ostreatus and Lentinula edodes, against Escherichia coli, Salmonella tiphymorium, Staphylococcus aureus and Micrococcus luteus. We designed two mixtures, barley straw to be used as a substrate of cultivation of mushroom Pleurotus ostreatus and oats or cedar for the cultivation of mushroom Lentinula edodes; and were obtained aqueous extracts from spent substrates; extracts were tested for antibacterial activity. The protocol was a completely randomized assay with a factorial arrangement design. The data were analyzed with PROC GLM, SAS. The results showed that in the case of Escherichia coli the greatest inhibition zone was of 12.66 mm at a concentration of 6 mg mL, with treatment of Lentinula edodes/Cedar; Salmonella tiphymorium showed a greatest inhibition zone of 31.10 mm to a concentration of 5.12 mg mL, with treatment of Pleurotus ostreatus/Barley straw; Staphylococcus aureus showed a greatest inhibition zone of 9.33 mm to a concentration of 100 mg mL, with the treatment of Lentinula edodes/Cedar and finaly, Micrococcus luteus showed a greatest inhibition zone of 15.00 mm to a concentration of 50 mg mL, with the treatment Lentinula edodes/Oats. In conclusion, the results suggest that it is possible to use indistinctly the spent substrate of Pleurotus ostreatus and Lentinula edodes as source of extracts with antibacterial activity.


Introduction
The uncontrolled use of antibiotics has caused serious problems in human and animal health, causing that bacterias develop resistance to them, so World Health Organization considered to infections caused by bacteria resistant to drugs as a public health problem; therefore, it is necessary to find new pharmacological strategies, among which we can find natural products such as plants and fungi (Roca et al., 2015).
Spent mushroom substrate containing carbohydrates as cellulose and hemicellulose, lignin, remnant of edible fungi, is a byproduct of mushroom production industry.Substrate used as growth media to produce mushroom is composed of maize cobs, wheat straw, grass straw, sugarcane bagasse, field hay, corn cobs, cotton seed hulls and some other.After several mushroom harvesting cycles, the productivity of the substrate could decrease so that the substrate is declared as spent (Guoa & Choroverb, 2006;Onyango, Palapala, Arama, Wagai, & Gichimu, 2011).One of the main problems in the production of mushrooms still the treatment and disposal of spent mushroom substrate; many studies have already been carried out for the use of such substrates, among which we mention feeding and/or antimicrobial activity (Zhu, Sheng, Yan, Qiao, & Lv, 2012).
The fungi have an important role in the degradation of organic matter (Chang & Miles, 1984), in addition to being a source of bioactive substances to produce antibiotics or pharmaceutical drugs, such as functional food and additives in feeding stuffs (Santoyo, Ramírez-Anguiano, Reglero, & Soler-Rivas, 2009).Crop of mushrooms (Pleurotus ostreatus) it is a source of products agricultural, their organic waste generated can be used as a source of food with high protein content and as an alternative pharmaceutical treatment; Pleurotus ostreatus is a fungus that has the ability to grow on agricultural wastes, accelerates the biodegradation and recycling them, avoiding its burning and the subsequent environmental pollution (Varnero, Quiroz, & Álvarez, 2010).Lentinula edodes, fungi edible, is the most studied, has been shown that fruiting body and mycelium have antimicrobial properties; likewise, the lentine inhibits mycelium growth of other fungi as Physalospora piricola, Mycosphaerella arachidicola and Botrytis cinerea (Rojas, 2013;Romero-Arenas, Martínez, Damian, Ramírez, & López-Olguín, 2015).
The production of mushroom has been used with a large number of substrates; one of the main is straw, used as a source of carbon to increase the nutritional characteristics and palatability of the fruiting body, getting a better nutritional quality (Sánchez, 2010).The crop of fungi and the quick growth in mushroom production worldwide has resulted in large quantities of spent substrate mushroom (about 13.6 million t year -1 ).The massive amounts of waste can cause environmental problems; this causes, led more research to develop technologies for its treatment or use (Lin et al., 2014).
In recent years, there has been a need study antimicrobial phytochemicals with potential to generate new pharmacological options.Our group previously demonstrated that the use of spent substrate of Pleurotus ostreatus mixed or not with medicinal plants, has antibacterial activity (Ayala et al., 2015).Therefore, the objective of this study was to determine the antibacterial activity of spent substrate of Pleurotus ostreatus and/or Lentinula edodes against Escherichia coli, Salmonella tiphymorium, Staphylococcus aureus and Micrococcus luteus at different concentrations.

Strain, Substrates and Cultivation Mushrooms
The blocks (1 Kg) were obtained from Centro de Investigaciones Biológicas of the Universidad Autónoma del Estado de Hidalgo, México.The blocks were formed by a mixture of barley straw; which were purchased in Central Abastos in Pachuca Hidalgo, Mexico, the taxonomic identification did it the botanist Dr. Miguel Angel Villavicencio Nieto; the specimens were deposited at the Herbarium of the Centro de Investigación de Ciencias Biológicas, of the Universidad Autónoma del Estado de Hidalgo, México.To form the substrate, barley straw was colonized with mycelium of Pleurotus ostreatus UAEH-004 in solid substrate fermentation; mushrooms were harvested at 23 d and was obtained spent substrate; on the other hand, oats or cedar were colonized with mycelium of Lentinula edodes UAEH-015 in solid substrate fermentation; mushrooms were harvested at 90 d and were obtained spent substrate of each one.

Preparation of Organic Extracts
The extracts were obtained by mixing 100 g of spent substrate mushroom of each treatments and 300 mL distilled water in case of Pleurotus ostreatus and 600 mL distilled water in case of Lentinula edodes.Then, the mix was macerated in blender during 1 min.Subsequently, mix macerated was filtered using gauze, to separate solid and liquid parts, was filtered through filter paper Whatman® #41.The extract was placed in a water bath to 70 °C for 48 h, to obtain the sample dry; for each 25 ml of liquid extract were obtained 0.472 g of Pleurotus ostreatus and 0.451 g of Lentinula edodes of dry extract.

Antimicrobial Assay
The antibacterial activity of the spent substrate of Pleurotus ostreatus and/or Lentinula edodes extracts were studied by the method of paper disc diffusion assay with slight modification (Kil et al., 2009).The bacterial pathogens strain were grown in liquid medium for 24 h to yield a final concentration of Escherichia coli 7.7 × 10 6 CFU/200 µL, Salmonella tiphymorium 1.11 × 10 7 CFU/200 µL, Staphylococus aureus 1.0 × 10 7 CFU/200 µL and Micrococcus luteus 1.04 × 10 7 CFU/ 200µL.Next, aliquots of 0.1 ml of the test microorganisms were spread over the surface of agar plates.Sterilised filter paper discs of 6 mm diameter (paper Whatman® #41) were saturated with 50 μl of different concentrations (0, 6, 12.5, 25, 50 and 100 mg mL -1 ) spent substrate of Pleurotus ostreatus and/or Lentinula edodes extracts.The soaked discs were then placed in the middle of the plates and incubated for 24 h at 37 °C (Forma Series II Water Jacket CO 2 , Incubator, Model 3100, Thermo Scientific, USA), after which the diameter (in mm) of each inhibitory zone was measured (scalimeter).Negative control was prepared with distilled water; as positive control was used commercial antibiotic (Penicillin G sodium salt, Sigma-Aldrich, St. Louis, MO, USA) to a concentration 100 mg mL -1 on medium culture Mac Conkey and Estafilococos No. 110.

Statistical Analysis
Data were analyzed using factorial design 3 × 3 and blocked by extract type and extract concentration and bacterial strain as factors.A PROC GLM procedure and LSMEANS option were used (SAS, 2002).

Results
Treatment with spent substrate of Pleurotus ostreatus and Lentinula edodes extracts were effective against four bacterial strains tested; antibacterial activity at different concentrations is showed in Table 1; the tested bacteria were quantitatively assessed by measuring the diameter of inhibition generated for each sample; each result is the mean of three replicates.The results showed that the spent substrate Pleurotus ostreatus/Barley straw extracts presented highest inhibitory effect against Escherichia coli (7.7 × 10 6 CFU/200 µL) at a concentration of 12.5 mg mL -1 with 9.86 mm inhibition halo; Staphylococus aureus (1.0 × 10 7 CFU/200 µL) at a concentration of 25 mg mL -1 with 9 mm inhibition halo and Micrococcus luteus (1.04 × 10 7 CFU/ 200µL) at a concentration of 50 mg mL -1 with 9.66 mm inhibition halo.Salmonella tiphymorium (1.11 × 10 7 CFU/200 µL) at a concentration of 12.5 mg mL -1 with 31.10 mm inhibition halo, showing significant differences with the concentrations 6, 25, 50 and 100 mg mL -1 (P < .05).
When comparing the largest zone of inhibition of the three treatments tested (Pleurotus ostreatus/Barley straw, Lentinula edodes/Oats y Lentinula edodes/Cedar) against each bacteria (Table 2), the results showed in the case of Escherichia coli the largest zone of inhibition was at a concentration of 6 mg mL -1 (12.66 mm) with treatment of Lentinula edodes/Cedar; Salmonella typhimurium showed the largest zone of inhibition at a concentration of 5.12 mg mL -1 (31.10 mm) with treatment of Pleurotus ostreatus/Barley straw; Staphylococcus aureus showed the largest zone of inhibition at a concentration of 100 mg mL -1 (9.33 mm) with the treatment of Lentinula edodes/Cedar and Micrococcus luteus showed the largest zone of inhibition at a concentration of 50 mg mL -1 (15.00 mm) with treatment of Lentinula edodes/Oats.Salmonella typhimurium and Staphylococcus aureus no showed significant differences between each treatments; Escherichia coli treated with Lentinula edodes/Oats is significantly different to the treatment Lentinula edodes/Cedar (P < 0.05), but without showing difference significant with treatment Pleurotus ostreatus/Barley straw, while Micrococcus luteus treated with Lentinula edodes/Oats is significantly different with treatment of Lentinula edodes/Cedar and Pleurotus ostreatus/Barley straw (P < 0.05).

Discussion
In recent years the bacteria have acquired the ability of multi-resistance to antibiotics (Nehra, Meenakshi, & Yadav, 2012) which has generated that recent research are focus in the search for alternative treatments, such as fungi.Edible fungi such as Pleurotus ostreatus, have shown a high nutritional value as food (Patel, Naraian, & Singh, 2012) and anti-inflammatory, antidiabetic, antiviral, antioxidant, anticancer, antitumor, inmunomodulatory and antibacterial activity; however, most of the research has been based on the study of the fruiting body and not in spent substrate (Hearst et al., 2009;Deepalakshmi & Mirunalini, 2014).
A water-soluble polysaccharide named PL was isolated and purified from spent mushroom substrate, the polysaccharide contained two fractions (PL1 and PL2), composed of glucose, rhamnose and mannose; the antibacterial activity of polysaccharide against E. coli was the strongest, while the weakest against Sarcina lutea, the minimal inhibition concentrations of PL2 were 12.5, 25 and 100 μg/mL, respectively (Zhu et al., 2012).We show that aqueous extract of spent substrate of Pleurotus ostreatus/Barley straw has antibacterial activity against Escherichia coli (9.86 mm), Staphylococcus aureus (9.00 mm), Micrococcus luteus (9.66 mm) and Salmonella tiphymorium (31.10 mm), similar to that obtained using extracts of mushroom Pleurotus ostreatus obtained with different organic solvents (24.56 and 14 mm) for Gram positive and Gram negative bacteria (Nehra et al., 2012).
For the cultivation of Lentinula edodes for many years have used various agricultural and industrial wastes, among which we mention sorghum, sugar cane, sawdust, oak, cedar (Grodzínskaya et al., 2002) as carbon source; shiitake mushrooms (Lentinus edodes) is of great importance, due to its attributed not only to its nutritional value, but also potential applications in industrial food and medicine as antibacterial (Hearst et al., 2009) and/or antitumor, among other features; this activity is due to the lentina (one polysaccharide isolated from fruiting body), which acts as an enhancer of host defense; has shown action against Staphylococcus aureus, Bacillus subtilis and Escherichia coli (Hatvani, 2001).Chowdhury, Kubrai, and Ahmed (2015) mentioned antimicrobial activity of 3 edible mushrooms (Pleurotus ostreatus, Lentinula edodes, Hypsizigus tessulatus) methanolic extracts, indicated considerable activity against bacteria and fungi, reveling zone of inhibition ranged from 7 ± 0.2 to 20 ± 0.1 mm; Kazue, Megumi, and Dantas (2001) found that the mycelium of 35 different strains of Lentinus edodes, has antibacterial activity against B. subtilis, with inhibition halos 5-20 mm in diameter similar to our findings.
This work is novel because for the first time is studied the use of spent substrate Lentinula edodes as antibacterial, since only been shown this activity in the fruiting body; they have been used different extraction techniques: high-pressure operations and low-pressure methods.The high-pressure technique was applied to obtain Lentinus edodes extracts using pure CO 2 and CO 2 with co-solvent or organic solvents such as n-hexane, ethyl acetate and dichloromethane (Kitzberger, Smânia Jr., Pedrosa, & Ferreira, 2007); here it is included barley straw as a substrate for the cultivation of Pleurotus ostreatus and oats or cedar for the cultivation of Lentinula edodes, in order to obtain aqueous extracts and determine its antibacterial activity; the findings suggest that it is feasible to use these substrates in the future for obtain antibacterial pharmaceutical compounds and at the same time reduce the pollution by their accumulation.

Table 1 .
Antibacterial activity of spent substrate of Pleurotus ostreatus and Lentinula edodes extracts at different concentrations in vitro

Table 2 .
Comparison of antibacterial activity of spent substrate of Pleurotus ostreatus and Lentinula edodes extracts against Escheriquia coli, Salmonella tiphymorium, Staphylococus aureus and Micrococcus luteus in vitro.Note.ab Literal different ranks indicate significant difference between treatments of each bacteria (P < 0.05) with the Tukey test.