Journal of Chemical, Biological and Physical ...

JCBPS; Section B; Nov . 2014 – Jan. 2015, Vol. 5, No. 1; 545-564

E- ISSN: 2249 –1929

Journal of Chemical, Biological and Physical Sciences An International Peer Review E-3 Journal of Sciences Available online atwww.jcbsc.org

Section B: Biological Sciences CODEN ( USA): JCBPAT

Research Notes

Growth and antimicrobial activity of different mushroom strains grown in submerged culture Mohamed Osman, Hassan, F.R.H.1 , W.A. Ahmed2 , Soad Nady3 , Heba El-sayed. Department of Botany and Microbiology, Faculty of Science, Helwan University, Egypt. *Food Technology Research Institute, Agriculture Research Center, Giza, Egypt 2 Department of Cancer Biology, National Cancer Institute, Cairo University, Egypt. 3 Department of Entomology and Zoology, Faculty of Science, Helwan University, Egypt. Received: 21 November 2014; Revised: 10 December 2014; Accepted: 21 December 2014

Abstract: The present study was carried out to evaluate the antibacterial and antifungal activity of aqueous extract of mycelia from five mushroom strains on selected bacterial pathogens such as Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Staphylococcus aureus and Streptococcus pneumoniae and five fungal strains Penicillium sp., Aspergillus flavus, Aspergillus niger, Fusarium oxysporium and Rhizocotonia sp. As well as the activity against Candida spp.Mycelial extracts from submerged cultures of mushrooms showed potential antimicrobial activities against the selected bacterial and fungal strains.Flammulina velutipes 6 showed a broad effect against most of tested bacteria and inhibited the growth of Aspergilus niger and Fusarium oxysporum. Lentinula edodes LC2141, Lentinula edodes LC202 extract showed antifungal activity against all tested fungal strains. Candida albicans and Candida tropicalis were inhibited by Lentinula edodes LC2141 and Flammulina velutipes 13.Tested mushrooms showed higher levels of endopolysaccharides (IPS) than exopolysaccharides(EPS). Key words: Antimicrobial activities, Crude hot water extracts, mushroom, growth, submerged culture.

INTRODUCTION Mushrooms belong to a special group of macroscopic fungi. Macromycetes arranged in the phylum Basidiomycota and some of them in the Ascomycota are known as the higher fungi 1, 2 . It is estimated the existence of about 140.000 different species of mushrooms in the planet, however, only about 10% is 545

J. Chem. Bio. Phy. Sci. Sec. B, Nov. 2014 – Jan. 2015; Vol.5, No.1; 545-564.

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known. Half of them present nutritious properties. 2.000 species of mushrooms are safe and, approximately, 70 are known for presenting some pharmacological properties. Edible mushrooms are attractive because of their flavor, taste, and delicacy 3 . Although many species of edible mushrooms exist in the nature, less than 20 species are used as food and only 8–10 species are regularly cultivated in significant extent. Flammulina velutipes, one of the most popular edible mushrooms, has attracted considerable attention of biochemistry and pharmacology due to its biological activities. The Flammulina fungus is also known as winter mushroom or velvet stems with worldwide distribution4 . Different groups of bioactive compounds such as polysaccharides, protein glucans complex, sterols, lectins, peroxidases, laccases, cellulases and proteases, with medicinal and pharmaceutical properties (immunomodulating, antitumor, anti-oxidant, thrombolytic, fibrinolytic, antibacterial, antifungal, antiviral, mitogenic were isolated from F. velutipes5 . Polysaccharides and a low-weight protein-bound polysaccharide with high antitumor activity were also isolated from thismushroom6 . Flammulin, a basic simple protein from F. velutipes is able to markedly inhibit tumorcells7 . Shiitake (Lentinula edodes) is the second largest cultivated and most popular mushroom in world 7 . Shiitake have several functional properties such as antitumor and hypocholesterolemic effects, and antimicrobial and antioxidant features that have been intensively investigated8, 9 .Shiitake mushrooms are the main sources of some antioxidant minerals. Polyphenolic compounds have also been detected in shiitake, and may contribute to the antioxidant potential of this kind of mushroom 10 . The purpose of the present study was to cultivate different mushroom strains in submerged culture by using standard liquid medium and comparison between the production yield of exopolysaccharides and endopolysaccharides. Also, to investigate the antimicrobial activity of the crude hot water extracts from the mycelia of the tested strains. MATERIALS AND METHODS Fungal Strains: Five edible fungal strains; Lentinula edodes LC2141, Lentinula edodes LC202, Flammulina velutipes 5, Flammulina velutipes 6 and Flammulina velutipes 13 were kindly obtained from Fujian Agriculture Univ., China. Strains were maintained on Potato Dextrose Agar (PDA) slants and incubated at 25°C for 7days then stored in refrigerator at 4°C for routine culture and storage purposes11 . Test microorganisms: Antimicrobial activity of extracts from the investigated mushrooms was tested against bacteria and fungi. The following strains of bacteria were used: Pseudomonas aeruginosa, Salmonella typhi, streptococcus pneumoniae, Staphylococcus aureus, Staphylococcus haemophilus, Bacillus subtilis, Micrococcus sp., Klebsiella sp., Proteus sp. and Escherichia coli, while Aspergillus niger, Aspergillus flavus, Rhizocotonia sp., Fusarium oxysporum, Penicillium sp., Candida albicans and Candida tropicalis were the tested fungi and yeasts in this study. All tested fungi were obtained from Mycology lab of Faculty of Science, Helwan University. While the tested bacteria were kindly provided from Pharmaceutical Control Authority, Giza, Egypt. Table 1: List of microorganisms used in this study

Gram-positive bacteria Gram-negative bacteria Plant pathogenic fungi Pathogenic yeast

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Microorganisms Bacillus subtilis, Staphylococcus aureus, Micrococcus luteus,Staphylococcus haemophilus and streptococcus pneumoniae Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi, Klebsiella sp.and Proteus sp. Aspergillus niger, Aspergillus flavus, Rhizocotonia sp., Fusarium oxysporum, Penicillium sp. Candida albicans and Candida tropicalis


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Culture Media and conditions: The fungal strains were initially grown on PDA medium in Petri dishes for 7–8 days .submerged fermentations were performed in a 250 ml flask containing 50 ml of mushroom fermentation medium (MFM). Each flask was inoculated with 10 agar plugs 0.7 cm of the agar plate culture with sterilized cork porer. Cultures were incubated under Static and shaking conditions. MFM medium consisted of the following components (g/liter): glucose (35), peptone (5), yeast extract (5), KH2 PO4 ·H2 O (1), MgSO4 ·7H2 O (0.5) and vitamin B1 (0.05)12 . Determination of dry weight: The fungal mats were separated from culture media by filtration and washed several times with distilled water, then dried at 60°C to a constant weight. Preparation of crude endopolysaccharide (IPS) extract: The dried mycelia were pulverized and 5.0g of the powdered sample were extracted with 80% (v/v) ethanol for 24 h, and then filtered. The residues were dried and extracted with distilled water at 100 °C three times. The whole extract was filtered and centrifuged. After centrifugation (6000 rpm, 10min), the supernatant was concentrated by evaporation under vacuum and treated with three volumes of ethanol for precipitation at 4 °C overnight. The precipitate was obtained by centrifugation, and dried to give a crude extract13 . Exopolysacchride (EPS) production: EPS was extracted from the culture medium. The culture supernatant was mixed with three volumes of 95% ethanol, stirred vigorously, and then left overnight at 4°C. The precipitated EPS was collected by centrifugation at 4000 rpm for 10 min, and the supernatant was discarded. Then, the precipitates were resolved in water and the EPS content was measured by the same procedures used for the extraction of IPS. Extraction yield (%) = (Weight of extract/weight of dried sample) ×100 Properties of crude polysaccharides: Total carbohydrate content of the polysaccharide was determined by phenol- sulfuric colorimetric method using glucose as the standard14 . The reducing sugar was determined by Dinitrosalicylic acid (DNS) developed by Miller reducing sugar was calculated using D-glucose as a standard.

15

. The total

Total polysaccharide was the subtraction of reducing sugar from total carbohydrates. Determination of protein content: The total protein content was measured by Bradford method16 . Antimicrobial activity: The percent inhibition of mycelial growth (PIMG) was used to determine the antifungal effect of the crude extracts. The crude extracts of mycelia of the tested mushrooms were added to PDA at concentrations 20 mg/ml. After autoclaving at 121o C for 15 minutes, these media were poured in 5 cm sterilized Petri dishes. Agar discs (5 mm) were taken from 7 days old cultures of 5 pathogenic fungi (Aspergillus niger, Aspergillus flavus, Rhizocotonia sp., Fusarium oxysporum and Penicillium sp.) and placed separately on the center of the Petri plates. For control, same size agar discs of the tested fungi were placed on a fresh PDA plate. All pairings of cultures were carried out in replicates and incubated at 25○C for 6 days. Inhibitory activity was assessed by measuring the radial growth of mycelium on the treated media (R2) and the radial growth on fresh PDA as control (R1). The two measurements were transformed into PIMG using the formula18 , where PIMG = {(R1 − R2)/R1} × 100. Candida albicans and Candida tropicalis were incubated at 28°C for 48 hrs and tested by well diffusion method. Antibacterial activity: Antibacterial activity of the mushroom extracts was tested using well diffusion method 19 . Petri dishes with nutrient agar were inoculated with a 100 μl suspension of each bacterial culture. Wells were made on the agar surface with 6mm cork borer. The extracts (100 µl) were poured into the well using sterile syringe. The bacteria seeded plate placed in the refrigerator for 8 hours at 4o C. 547


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Then, plates were incubated at 37±2o C for 24 hours. The plates were observed for the inhibition zone formation around the wells. The zone of inhibition was calculated by measuring the diameter of the inhibition zone around the well (in mm) including the well diameter. The measurements were taken in three different fixed directions and the average values were tabulated. The concentration of the extract used was 20 mg/ml and sterile distilled water was used in the dilution of the extract. Statistical Analysis: Statistical analysis of data was carried out by using one way analysis of variance (ANOVA) followed by homogenous subsets (Duncan) at confidence level of 5% (0.05) using the Statistical Package for the Social Science (SPSS) version17. Duncan’s multiple range tests were used to compare between means of treatments20 . RESULTS AND DISCUSSION Cultivation of mushrooms using submerged liquid Culture: Mushroom strains were grown on mushroom fermentation medium (MFM) under static and shaking (150 rpm) conditions. The data given in fig.1 showed that the obtained biomass of Lentinula edodes LC2141was 4.2 g d. wt/L with endopolysaccharide production 0.76g/L. Also, Lentinula edodes LC202 showed biomass of 2.6 g d. wt/L and endopolysaccharide production (0.62g/L) under shaking. Flammulina velutipes 6, Flammulina velutipes 5 and Flammulina velutipes 13 showed the highest growth (9.1, 6.2 and 14.7 g d. wt/l respectively) with crude extract production (4.3, 2.1 and 2.5 g/l respectively) under static incubation. Production of exopolysaccharides as compared with endopolysaccharides of the tested mushrooms showed that all tested strains produced endopolysaccharides yield more than the produced exopolysaccharides as shown in fig. 2. Similar results were reported that IPS production was a main contributor to the total polysaccharide level in several mushroom strains21 .

Figure 1: Cultivation of tested mushroom strains on standard liquid medium under shaking and static incubation.

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Figure 2: Production of exopolysaccharides and endopolysaccharides of the tested mushroom strains. Comparison of Extraction Yield and extract composition from the tested mushroom strains: Polysaccharides were extracted from the mycelia of the tested mushroom strains by hot water and precipitation by three volumes of ethyl alcohol. These results showed that the yield of polysaccharides (10%) in Flammulina Velutipes 6 extract was the highest than that of other strains. Total carbohydrate contents in hot water extract of Lentinula edodes LC2141, Lentinula edodes LC202, Flammulina velutipes 6, Flammulina velutipes 5 and Flammulina velutipes 13 were estimated (446, 497, 700, 520 and 420 mg/g d. wt respectively) using the phenol–sulfuric acid method. Table 2: The yield of crude polysaccharides, total carbohydrates, reducing sugar and protein content of crude hot water extract of five mushroom strains. Crude hot water extract

Appearance

% IPS Yield (w/w)

% EPS Yield (w/w)

Total carbohydrate (mg/g d.wt)

Total polysaccharide (mg/g d.wt)

Reducing sugars (mg/g d.wt)

Protein (mg/g d. wt)

Lentinula edodes LC2141

Brown viscous extract

5.0

1.9

446.0

240.0

206.0

11.2


Brown viscous extract

4.2

1.3

497.0

250.0

247.0

18.0

Flammulina velutipes 6

White powder

10.0

3.8

700.0

570.0

130.0

25.8


White powder

2.1

0.8

520.0

410.0

110.0

28.8


White powder

2.5

1.2

420.0

330.0

90.0

4.2

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The total yield of the crude extracts obtained from each of the mushroom species was relatively low and this could probably be due to the employed method of extraction. The gelling of some of these mushrooms components in hot water into thin slime may reduce the total yield as it made filtration through the filter paper somewhat slower and difficult22 . Generally, the higher yield of hot water extracts compared to ethanol extracts may be explained by higher proportion of water-soluble constituents in mushrooms 23 . This result is in contrast with Obi and Onuoha 24 who reported that ethanol extraction of plant ingredients were better than water extract. Antimicrobial activity: The aqueous extracts of the tested mushroom strains were screened against five pathogenic fungi to check their antifungal activities. The percent inhibition of mycelial growth (PIMG) was used to determine the antifungal effect of the crude extracts against pathogenic fungi as shown in tables4 and 5. Lentinula edodes LC202showed the highest PIMG (66.7%) against Aspergillus niger followed by Flammulina velutipes 6 (29%) then Lentinula edodes LC2141 (18%). Poor reproductive structures formation was observed in the treated plates. The highest and lowest PIMG of Rhizocotonia was 17.0% (Lentinula edodes LC202) and 6.3% (Lentinula edodes LC2141) respectively. The inhibitory effect of polysaccharide extracts of Flammulina velutipes 6 (42.0%) was better against Fusarium oxysporum than Lentinula edodes LC202 (22.0%) and Lentinula edodes LC2141 (17.0%). All plates of Fusarium oxysporum treated with these extracts showed low pigmentation as compared with the control plates. Penicillium was inhibited by extracts of Flammulina velutipes 5 (39.4%), Lentinula edodes LC2141 (27%), Flammulina velutipes 13 (18.2%) and Lentinula edodes LC202 (10%). The highest and lowest PIMG of Aspergillus flavus was 17.0% (Lentinula edodes LC202) and 10.4% (Lentinula edodes LC2141) respectively and failure in the formation of reproductive structure. The extract of Flammulina velutipes 13 and Lentinula edodes LC2141 also inhibited Candida albicans and Candida tropicalis growth while Lentinula edodes LC202extract inhibited Candida tropicalis.

a.

Plate seeded with Lentinula edodes LC4121extract.

b.

Control plate of Rhizocotonia sp.

a.

Plate seeded with Lentinula edodes LC202 extract.

b.

Control plate of Aspergillus niger.

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a.

Plate seeded with Lentinula edodes LC202 extract.

b.

Control plate of Rhizocotonia sp.

a.

Plate seeded with Lentinula edodes LC4121extract.

b.

Control plate oxysporum.


of

Fusarium

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a. Plate seeded with Flammulina Plate seeded with Flammulina velutipes 6 extract velutipes 6 extract b. Control plate of Aspergillus niger b. Control plate of Fusarium oxysporum Figure 3: Inhibitory effects of endopolysaccharide extracts on the mycelial growth of different pathogenic fungi. a.

Antibacterial activity: Antibacterial activity of hot water extracts of the tested mushrooms was tested using well diffusion method. The tested mushroom strains used in this study were found to exhibit various degrees of antimicrobial activities against the tested microorganisms. This was evidenced by the clear zone of inhibition formed around the wells of the tested mushroom extracts. Lentinula edodes LC2141 showed antimicrobial activity against Bacillus subtilis and Pseudomonas aeruginosa. While Lentinula edodes LC202 had the minimum inhibitory activity in comparison of all other tested strains. It showed antimicrobial activity against Proteus sp. and Candida tropicalis only. Flammulina velutipes 6 was the most effective mushroom extract against all tested bacterial strains except Micrococcus luteus, Salmonella typhi and Proteus sp. but it had no inhibitory activity against yeast strains. Flammulina velutipes 5 inhibited the growth of Staphylococcus aureus, Staphylococcus haemophilus, and streptococcus pneumonia and only the Gram negative bacteria, proteus. The extract of Flammulina velutipes 13 also inhibited Bacillus subtilis and Pseudomonas aeruginosa. Table 3: Antimicrobial activity of the hot water extracts from different mushroom strains by well diffusion method.

Bacterial strains Gram- positive bacteria Gram- negative bacteria

Bacillus subtilis Staphylococcus aureus Staphylococcus haemophilus Micrococcus luteus streptococcus pneumoniae Escherichia coli Pseudomonas aeruginosa Salmonella typhi Klebsiella sp. Proteus sp.

Lentinula edodes LC2141* 10a

Diameter of inhibition zone (mm) Lentinula Flammulina Flammulina edodes velutipes 6* velutipes 5* LC202* -ve 13d -ve

-ve

-ve

18b

20b

-ve

-ve

-ve

15c

10c

-ve

-ve

-ve

-ve

-ve

-ve

-ve

-ve

20a

25a

-ve

-ve

-ve

8e

-ve

-ve

10a

-ve

18b

-ve

13b

-ve

-ve

-ve

-ve

-ve

-ve -ve

-ve 18a

13d -ve

-ve 20b

-ve -ve

*: Means in the same column with different letters have significant differences between each other. -ve: Negative antibacterial activity.

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Flammulina velutipes 13* 15a

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Researchers have reported antimicrobial activity of several mushrooms. Few of medicinal mushrooms Ganoderma, Lentinula, Pleurotus have been found with effective antimicrobial activity25, 26 .It is interesting to note that the pathogenic microorganism, Pseudomonas aeruginosa, which is resistant to conventional synthetic antibiotics like gentamycin and tetracycline 27, 28 was found to show susceptibility to hot water extracts of mushroom. Mushrooms produce various antiviral, antifungal compounds to survive in the wild against competing or pathogenic agents 29, 30 . Also observed in this study is that there were variations in the degree of antimicrobial activities of mushrooms. This result is in agreement with the reports of Jaggadish 31 and Udu-Ibiam32 . The broad spectrum activity of mushrooms was also brought to light as the extracts of mushrooms showed inhibitory effects on clinical isolates used for this investigation. This suggests that the bioactive products of the investigated mushrooms showed varying degrees of antimicrobial activity. Further studies will be required to reveal the active components in the mycelial extracts as well as investigating their cytotoxicity. Table 4: Antifungal activity of the hot water extracts from different mushroom strains

Candida albicans

Diameter of inhibition zone (mm) Lentinula Lentinula Flammulina edodes edodes velutipes 6* LC2141* LC202* 15b -ve -ve

Candida tropicalis

20a

Yeast

15a

-ve

Flammulina velutipes 5*

Flammulina velutipes 13*

-ve

20a

-ve

15b

*: Means in the same column with different letters have significant differences between each other. -ve: Negative antifungal activity.

Table 5: Antifungal activity of the hot water extracts from different mushroom strains Percent of inhibition of mycelial growth (PIMG) % Tested fungi Mushroom species Lentinula edodes LC2141



Flammulina velutipes 5 Flammulina velutipes 13

Aspergillus* niger

Penicillium* sp.

Rhizocotonia*

Fusarium oxysporum*

Aspergillus flavus*

18.0c Poor reproductive structures 66.7a Poor reproductive structures 29.0b Poor reproductive structures -ve

27.0b

6.3b

17.0c Low pigmentation

10.0d

17.0a

22.0b Low pigmentation

-ve

-ve

42.0a Low pigmentation

10.4b Poor reproductive structures 17.0a Poor reproductive structures -ve

39.4a

-ve

-ve

-ve

-ve

18.2c

-ve

-ve

-ve

*: Means in the same column with different letters have significant differences between each other. -ve: Negative antifungal activity.

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Corresponding author: Heba El-Sayed Department of Botany and Microbiology, Faculty of Science, Helwan University, Egypt E. mail: [email protected]

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