Antioxidant Properties and Antimicrobial Activity in the Extracts of Two Edible Mushroom, Pleurotus sajor caju and Schizophyllum commune

Sujjat Al-Azad^1*, Vivian Chong Ai Ping²

¹Borneo Marine Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Malaysia.
²Triniti Farm Enterprise, Kota Belud, Malaysia.

DOI: 10.4236/abb.2022.139023                

Abstract

Extracts of two edible mushrooms, Pleurotus sajor-caju (commercial) and Schizophyllum commune (wild) were used to compare the antioxidant and antimicrobial properties. Aqueous and three types of organic solvents, like 50% of ethanol, methanol and acetone extracts were used in trial. DPPH scavenging activity in P. sajor-caju extract was determined in the range of 53.13% to 85.08%, whereas extracts of S. commune were observed in the range of 54.11% to 97.19% at a concentration of 5 mg/ml. The highest DPPH scavenging activity of 97.19% was observed in ethanol extract of S. commune (97.19%), higher than butyl hydroxytoluene (BTH). Half effective concentration (EC₅₀) in extracts of P. sajor-caju was found in the range of 1.47 to 4.23 mg/ml and that of S. commune in the range of 1.52 to 4.52 mg/ml. The reducing power of P. sajor caju aqueous concentration extract was found to be the closest of 3.353 (700 nm) that of antioxidant activity to BHT (3.445) at 2 mg/ml concentration. The best reducing power EC₅₀ was obtained in P. sajor caju aqueous extract (0.09 mg/ml), but in S. commune with acetone extract (0.22 mg/ml). Minimum inhibition concentration (MIC) was compared in extracts of mushrooms in various Vibrio species. All extracts were able to inhibit V. harveyi growth with MIC of lower than 1.25 mg/ml. In aqueous and methanol extracts of current study showed that bacteria inhibition activity occurred at the concentration of <1.25 mg/ml to 10 mg/ml. Aqueous extract of P. sajor-caju was able to act as reducing agent as functional as the commercial antioxidant agent, BHT. Crude extracts of P. sajor-caju and S. commune were observed to contain antibacterial potential against these mentioned Vibrio bacteria.

Keywords

Mushrooms, Extracts, Antioxidant, Antimicrobial, Vibrio, Bacteria

1. Introduction

Pleurotus sajor caju, a commercially grown and naturally grown Schizophyllum commune are popular edible mushrooms in Sabah (East Malaysia). Taste and smell of these two types of mushrooms are different. Schizophyllum commune is a wild edible mushroom that belongs to the family Schizophyllaceae [1]. Although S. commune growing conditions and substrates are different from commercially grown mushrooms, the nutritional value of this mushroom is as good as commercial mushrooms [2].

Mushroom was proven to be safer, environmentally friendly, pollution free, improve disease resistance, enhance immunity and decrease mortality [3]. Extracts of mushroom are in concentrated fom also gives beneficial effects while being used. There were two common solvents used for mushrooms extractions as which are ethanol and aqueous extract. Pleurotus sajor-caju extract was known for its carbohydrate content, particularly polysaccharide, is soluble in hot water [4]. The mentioned polysaccharide is actively studied for its nutraceutical properties to combat cancer [5], anti-inflammatory [6], anti-obesity and prevent oxidative stress [7]. In addition, this mushroom crude extract revealed antioxidant activity and anti-proliferative activity in aqueous extraction [8]. Crude extract of S. commune was found as good as oxidative stabilizer and free radical scavenging agent to prolong the shelf life of lipid food products [9]. Other traces of antioxidant compounds of S. commune are flavonoid, beta-carotene and lycopene [10]. Schizophyllan has long been identified as medicinal active compound used for cancer research and development in Japan [11].

Methanol extract of P. sajor-caju exhibited anti-bacteria properties that are able to inhibit growth of gram positive and negative bacteria like Escherichia coli, Salmonella typhimurium, Bacillus cereus, Staphylococcus aureus and Micrococcus luteus [12]. In Schizophyllum commune methanol extract, antimicrobial activity was reported significant to clinical importance bacteria namely B. cereus, B. subtilis, S. aureus, Pseudomonas aeuroginosa, Proteus vulgaris, E. coli, Streptococcus mutans, S. sanguis, S. mitis, Shigella sp., S. flexneri, Plesiomonas shigelloides, Salmonella typhi, and Enterobacter faecalis [13]. In methanol extract of S. commune had indicated active antimicrobial inhibitory effect towards gram positive bacteria and fungus [14]. So far, there is limited information on antioxidant and antimicrobial activity in S. commune extract other than methanol. This study will provide information of the chemical selectivity in four extractions from P. sajor-caju and S. commune mushroom. The purpose of this research was to compare the antioxidant and antimicrobial activity of commercially cultured P. sajor-caju and wild variety of S. commune mushrooms extracted with aqueous and three types of organic solvents.

2. Materials and Methods

Sample preparation and extractions used Detail of two types of mushroom collection, drying and making fine powder were mentioned in previous research article. Three types of organic solvents and aqueous extraction were used in the preparation of extracts from two types of mushroom. The detail of extraction techniques and solvents used in the extracts were also stated in other published article [15].

2.1. Antioxidant Activity

2.1.1. Scavenging Effect on 1, 1-Diphenyl-2-Picrylhydrazyl (DPPH) Radical

The scavenging activity of the mushrooms extracts on DPPH radical, a free radical was measured according to the method of [16]. Absorbance of the color was read in 517 nm wavelength. Methanol was used as blank while BHT was used as a standard. Mixture without extract was used as the negative standard and butyl hydroxytoluene (BHT) was used as positive standard. Inhibition of free radical DPPH was calculated with formula:

$\text{Scavengingactivity}\left(\%\right)=\left(A_{\text{1}}-A_{\text{2}}/A_0\right)\times \text{1}00$

where, A₀ is the absorbance of the DPPH without the sample and A₁ is the absorbance of the sample with DPPH, and A₂ is the absorbance of the blank only.

EC₅₀ value (mg/mL) was defined as the half effective concentration. The EC50 value (mg/mL) of DPPH scavenging activity was determined at 50% of various concentration of extract scavenging activity against DPPH from the plotted graph.

2.1.2. Reducing Power Assay

The reducing power was determined according to the method described in research [17]. Absorbance at 700 nm was measured against a blank (distilled water). BHT was used as the positive control. The EC50 value represented the concentration of the reducing power activity at 0.5 (700 nm).

2.1.3. Antimicrobial Activity

The minimum inhibition concentration (MIC) was determined with the technique described by researchers [18] in broth micro-dilution method using 96-well micro-titer plates. Three marine bacteria tested in this study were V. harveyi, V. parahaemolyticus and V. anguillarum. Five two-fold serial dilutions of concentration ranging from 1.25 to 10 mg/mL of extracts were prepared from 100 mg/mL of mushroom extract stock. In each well of a 96-well plate, 90 μL of nutrient broth, 100 μL of mushroom extract solution (1.25 - 10 mg/mL) and 50 μL of bacterial suspension (10⁸ CFU/mL) were added and mixed. Well without mushroom extract was used as blank. After incubation at 37˚C for 24 hours, the absorbance of each incubation was read at 750 nm with a micro-plate reader. The absorbance values of blank and bacteria solution with mushroom extract were compared. The lower absorbance value than blank indicated as positive antimicrobial activity while similar or higher absorbance value than blank indicated as negative. MIC was determined by the lowest concentration of mushroom extract exhibited inhibition activity against bacteria growth.

2.2. Statistical Analysis

Microsoft Excel 2010 was used to calculate the means and standard deviations for all multiple measurements as well as to generate graphs. Antioxidant activities (EC50) results were analysed using analysis of variance to test the mean value and the comparisons of the mean values were done by using turkey test in software program SPSS version 22. The level of significance setting was p < 0.05.

3. Results

3.1. Antioxidant Activity of Varies Mushrooms Extracts

3.1.1. DPPH Radical Scavenging Activity

At concentration of 5 mg/ml, DPPH scavenging activity in P. sajor-caju extract observed in the range of 53.13% to 85.08%, while in S. commune range values (54.11% to 97.19%) observed higher than P. sajor-caju extract (Figure 1). The highest DPPH scavenging activity percentage was observed in ethanol extract of S. commune (97.19%) was higher than BHT (94.24%). The least activity was observed in acetone extracts of both mushrooms (P. sajor-caju = 53.13% and S. commune = 54.11%).

The average half effective concentration (EC₅₀) of P. sajor caju was determined in the range of 1.47 to 4.23 mg/ml and the range of 1.52 to 4.52 mg/ml was observed in S. commune (Table 1). The best EC₅₀ was seen in aqueous extract of P. sajor-caju (1.47 mg/ml) and aqueous extract of S. commune (1.52 mg/ml), which were significant different (p < 0.05) compared to other extracts. Acetone extracts of P. sajor-caju and S. commune were significantly different (p < 0.05) the highest

Figure 1. DPPH activity (%) of Pleurotus sajor-caju and Schizophyllum commune in different extraction solvent and concentration. (P =P. sajor-caju, S =S. commune, W: aqueous extract, E: ethanol extract, M: methanol extract, A: acetone extract and BHT: butylated hydroxytoluene).

EC₅₀ value indicating the weakest DPPH scavenging capability.

3.1.2. Reducing Power

Antioxidant activity (reducing power) of extract was found to increase with 0.5 mg/ml increment of concentration (Figure 2). Among all extracts, P. sajor-caju aqueous concentration at 2 mg/ml had the closest values of 3.353 (700 nm) antioxidant activity to BHT (3.445). The least reducing power reaction was seen in P. sajor-caju ethanol extract.

The value of EC50 in P. sajor-caju extract was obtained in the range of 0.09 mg/ml to 0.81 mg/ml, while in extract of S. commune extract the range values was from 0.22 mg/ml to 0.5 mg/ml. The best reducing power EC₅₀ was determined in P. sajor-caju aqueous extract (0.09 mg/ml) and acetone extract (0.22 mg/ml) of S. commune with significant different of p < 0.05 (Table 2).

Table 1. EC₅₀ (mg/ml) of DPPH scavenging activity from Pleurotus sajor-caju and Schizophyllum commune in various extracts (mean ± sd).

Figure 2. The Absorbance value of Pleurotus sajor-caju and Schizophyllum commune in different extraction solvent and concentration.

Table 2. EC50 (mg/ml) of reducing power activity from Pleurotus sajor-caju and Schizophyllum commune in various extracts (mean ± sd).

3.1.3. Antimicrobial Activity

All extracts were able to inhibit V. harveyi growth with Minimum Inhibition Concentration (MIC) of lower than 1.25 mg/ml. MIC value was determined in V. parahaemolyticus < 1.25 mg/ml and V. anguillarum was observed 10 mg/ml and >10 mg/ml in P. sajor caju extracts. S. commune extracts obtained MIC value of 2.5 mg/ml and 5 mg/ml in V. parahaemolyticus and 5 mg/ml and 10 mg/ml in V. anguilarium. Methanol and acetone extracts of P. sajor-caju were unable to show inhibitory activity against V. anguillarum (Table 3).

4. Discussion

4.1. DPPH Radical Scavenging Activity

Radical scavenging activity is essential to protect tissue damage from deleterious effect of lipid oxidation. S. commune extracted in hot water (autoclaved 121˚C) obtained 79.5% of scavenging activity at 10 mg/mL [19]. Better scavenging activity of 90.27% was obtained at 5 mg/mL of S. commune aqueous extract (room temperature) in current study.

The lower effective concentration (EC₅₀) indicates the better DPPH scavenging capability of the extract. In this study mushroom extraction was conducted under room temperature for three days. Aqueous extract of P. sajor-caju and S. commune was obtained the lowest of EC₅₀ in 1.47 mg/mL and 1.52 mg/mL, respectively. Other P. sajor-caju studies obtained EC₅₀ of 13.38 mg/ml observed in in 24 hours aqueous extraction [20]. The EC₅₀ result of aqueous extract in S. commune reported of 0.8 mg/mL using mushroom over solvent ratio of 1:20 (w/v) [19], comparable to the current aqueous extraction effect. On the other hand, S. commune was not able to obtain any EC₅₀ value within the concentration of 0 to 20 mg/mL in 70% methanol extract with the dry mushroom:solvent ratio of 1:5 (w/v) [21]. Current study using mushroom:solvent ratio of 1:10 (w/v) obtained better EC₅₀ value (3.4 mg/mL) in S. commune 50% methanol extract. Ethanol extract of this study obtained EC₅₀ value of 1.7 mg/mL (P. sajor-caju) and 1.67 mg/mL (S. commune). Ethanol extract of S. commune processed for

Table 3. Minimum Inhibition concentration (mg/ml) of antimicrobial activity from Pleurotus sajor-caju and Schizophyllum commune in various extracts.

two days with twice ethanol extraction obtained 0.883 mg/mL EC₅₀ value [10] better than this study ethanol extract of S. commune.

4.2. Reducing Power

The reducing power of extract exhibited medium antioxidant activity at 0.5 mg/mL and demonstrated the highest activity at the concentration of 2 mg/mL. In this study, at 2 mg/mL (abs = 2.23) of P. sajor-caju aqueous extract was able to act as reducing agent as functional as the commercial antioxidant agent, BHT. Boil aqueous extract of P. sajor-caju was reported 0.37 (700 nm) of reducing power intensity at 0.5 mg/mL extract [22] observed to obtained higher of 0.96 (700 nm) at 0.5 mg/mL of P. sajor-caju aqueous crude extract in the current study. The best EC₅₀ in aqueous extraction of 1.295 mg/mL and 2.237 mg/mL were obtained in P. sajor-caju and S. commune, respectively. EC₅₀ of S. commune was found vast difference in different extractions which were >20 mg/mL in methanol extract [21], 7.6 mg/mL in hot water extract [19] and 0.825 mg/mL in ethanol extract [10]. Antioxidant activity was higher when mushroom was re-extracted twice with ethanol solvent [10]. In addition, aqueous extract of mushroom was expected with high content of phenolic compounds responsible with reductive capabilities [12].

4.3. Antimicrobial Activity

Mushroom P. sajor-caju was reported to be able to inhibit growth of gram positive and negative bacteria using methanol extract [12]. P.sajor-caju extracted in aqueous extract at 4˚C overnight fractionated ribonuclease produced 50% inhibition concentration (IC50) of 51 ± 6 μM, 186 ± 12 μM and 34 ± 4 μM to Pseudomonas aeruginosa, P. fluorescens and S. aureus, respectively [23]. The MIC value obtained from S. commune methanol extract was 500 μg/mL concentration against gram positive bacterias, S. aureus and B. subtilis [14]. The above authors also mentioned that S. commune methanol extract revealed better antimicrobial activity than aqueous extract and ethanol extract. V. harveyi, V. parahaemolyticus and V. anguillarum are clinically important gram negative bacteria lead to vibriosis in marine life [24]. Up to date, V. harveyi has developed resistance to 16 antibiotics, and this alarmed the search for natural alternatives to provide protection against this bacteria [25]. In this work, crude extracts of P. sajor-caju and S. commune were observed to contain antibacterial potential against these species of bacteria. Methanol extract of sea grass (Thalassia hemprichii) was able to obtain MIC value of 1.56 mg/mL against V. harveyi [26]. In this study, Vibrio harveyi was observed sensitive to all types of mushrooms extract at minimum inhibition concentration of <1.25 mg/mL. V. parahaemolyticus was seen vulnerable to P. sajor-caju extracts antimicrobial activity at <1.25 mg/mL concentration. The poorest MIC obtained in V. anguillarum observed no antimicrobial activity at 10 mg/mL with P. sajor-caju methanol and acetone extracts. Methanol extract in betel (Piper betle), a traditional medicinal plant possess strong antimicrobial properties demonstrated inhibitory effect towards V. parahaemolyticus at lower than 0.1 mg/disk but needed a little higher concentration used to inhibit V. anguillarum growth at 0.1 mg/disk [24]. S. commune aqueous extract prepared in boiling water contained no bacteria inhibition activity whereas methanol extract displayed inhibition activity towards gram negative bacteria growth at the concentration of 2 mg/mL [13]. In aqueous and methanol extracts of current study showed that bacteria inhibition activity occurred at the concentration of <1.25 mg/mL to 10 mg/mL.

5. Conclusion

Aqueous extract exhibited the higher antioxidant activities by containing higher reluctant agent and free radical scavenging capability. However different types of extracts have least effect on antimicrobial activity. V. harveyi was susceptible to all extracts while V. parahaemolyticus and V. anguillarum were susceptible to P. sajor-caju and S. commune extracts, respectively.

Acknowledgements

This research was carried out with the help of the grant from Ministry of Education, Government of Malaysia (Grant Number ERGS 0038-STWN-1/2013). Authors also appreciated the supports from hatchery and laboratory staffs of Borneo Marine Research Institute, University Malaysia Sabah, Kota Kinabalu, Sabah, Malaysia.

Conflicts of Interest

The authors declare no conflicts of interest regarding the publication of this paper.

References

[1]	Alexopoulos, C.J., Mims, C.W. and Blackwell, M. (1996) Introductory Mycology. John Wiley & Sons, New York, 869 p.
[2]	Okwulehie, C.I., Nwosu, P.C. and Johnpaul, O.C. (2007) Pharmaceutical and Nutritional Prospects of Two Wild Macro-Fungi Found in Nigeria. Biotechnology, 6, 567-572. https://doi.org/10.3923/biotech.2007.567.572
[3]	Chang, C.-S., Huang, S.-L., Chen, S. and Chen, S.-N. (2013) Innate Immune Responses and Efficacy of Using Mushroom Beta-Glucan Mixture (MBG) on Orange-Spotted Grouper, Epinephelus coioidesm, Aquaculture. Fish & Shellfish Immunology, 35, 115-125. https://doi.org/10.1016/j.fsi.2013.04.004
[4]	Pramanik, M., Mondal, S., Chakraborty, I., Rout, D. and Islam, S.S. (2005) Structural Investigation of a Polysaccharide (Fr. II) Isolated from the Aqueous Extract of an Edible Mushroom, Pleurotus sajor-caju. Carbohydrate Research, 340, 629-636. https://doi.org/10.1016/j.carres.2004.12.032
[5]	Zhuang, C., Mizuno, T., Shimada, A., Ito, H., Suzuki, C., Mayuzumi, Y., Okamoto, H., Ma, Y. and Li, J. (1993) Antitumor Protein Containing Polysaccharides from a Chinese Mushroom Fengweigu or Houbitake, Pleurotus sajor-caju (Fr.) Sings. Bioscience, Biotechnology, and Biochemistry, 57, 901-906. https://doi.org/10.1271/bbb.57.901
[6]	Silveira, M.L.L., Smiderle, F.R., Moraes, C.P., Borato, D.G., Baggio, C.H., Ruthes, A.C., Wisbeck, E., Sassaki, G.L., Cipriani, T.R., Furlan, S.A. and Iacomini, M. (2014) Structural Characterization and Anti-Inflammatory Activity of a Linear β-d-glucan Isolated from Pleurotus sajor-caju. Carbohydrate Polymers, 113, 588-596. https://doi.org/10.1016/j.carbpol.2014.07.057
[7]	Kanagasabapathy, G., Malek, S.N.A., Mahmood, A.A., Chua, K.H., Vikineswary, S. and Kuppusamy, U.R. (2013) Beta-Glucan-Rich Extract from Pleurotus sajor-caju (Fr.) Singer Prevents Obesity and Oxidative Stress in C57BL/6J Mice Fed on a High-Fat Diet. Evidence-Based Complementary and Alternative Medicine, 2013, Article ID: 185259. https://doi.org/10.1155/2013/185259
[8]	Finimundy, T.C., Gambato, G., Fontana, R., Camassola, M., Salvador, M., Moura, S., Hess, J., Henriques, J.A.P., Dillon, A.J.P. and Roesh-Ely, M. (2013) Aqueous Extracts of Lentinula edodes and Pleurotus sajor-caju Exhibit High Antioxidant Capability and Promising in Vitro Antitumor Activity. Nutrition Research, 33, 76-84. https://doi.org/10.1016/j.nutres.2012.11.005
[9]	Yim, H.S., Chye, F.Y., Heng, P.Y. and Ho, C.W. (2011) Oxidative Stability of Sunflower Oil Supplemented with Medicinal Split Gill Mushroom, Schizophyllum commune Fr.: Fr. Extract during Accelerated Storage. International Journal of Medicinal Mushrooms, 13, 357-368. https://doi.org/10.1615/IntJMedMushr.v13.i4.60
[10]	Devi, L.S., Dasgupta, A., Chakraborty, M., Borthakur, S.K. and Singh, N.I. (2014) Chemical Composition and Antioxidant Activity of Schizophyllum commune. International Journal of Pharmaceutical Science Review and Research, 27, 173-177.
[11]	Kojima, T., Tabata, K., Itoh, W. and Yanaki, T. (1986) Molecular Weight Dependence of the Antitumor Activity if Schizophyllan. Agricultural and Biological Chemistry, 50, 231-232. https://doi.org/10.1080/00021369.1986.10867365
[12]	Gogavekar, S.S., Rokade, S.A., Ranveer, R.C., Ghosh, J.S., Kalyani, D.C. and Sahoo, A.K. (2014) Important Nutritional Constituents, Flavour Components, Antioxidant and Antibacterial Properties of Pleurotus sajor-caju. Journal of Food Science and Technology, 51, 1483-1491. https://doi.org/10.1007/s13197-012-0656-5
[13]	Mirfat, A.H.S., Noorlidah, A. and Vikineswary, S. (2014) Antimicrobial Activities of Split Gill Mushroom Schizophyllum commune Fr. American Journal of Research Communication, 2, 113-124.
[14]	Tripathi, A.M. and Tiwary, B.N. (2013) Biochemical Constituents of a Wild Strain of Schizophyllum commune Isolated from Achanakmar-Amarkantak Biosphere Reserve (ABR), India. World Journal of Microbiology and Biotechnology, 29, 1431-1442. https://doi.org/10.1007/s11274-013-1306-4
[15]	Azad, S.A. and Ai Ping, V.C. (2021) Comparison of Protein and Amino Acids in the Extracts of Two Edible Mushroom, Pleurotus sajor-caju and Schizophyllum commune. Advances in Bioscience and Biotechnology, 12, 286-296. https://doi.org/10.4236/abb.2021.129018
[16]	Shimada, K., Fujikawa, K., Yahara, K. and Nakamura, T. (1992) Antioxidative Properties of Xanthan on the Autoxidation of Soybean Oil in Cyclodextrin Emulsion. Journal of Agricultural and Food Chemistry, 40, 945-948. https://doi.org/10.1021/jf00018a005
[17]	Oyaizu, M. (1986) Studies on Products of Browning Reactions: Antioxidative Activities of Products of Browning Reaction Prepared from Glucosamine. Japanese Journal of Nutrition, 44, 307-315. https://doi.org/10.5264/eiyogakuzashi.44.307
[18]	Ren, L., Hemar, Y., Perera, C.O., Lewis, G., Krissansen, G.W. and Buchanan, P.K. (2014) Antibacterial and Antioxidant Activities of Aqueous Extracts of Eight Edible Mushrooms. Bioactive Carbohydrates and Dietary Fibre, 3, 41-51. https://doi.org/10.1016/j.bcdf.2014.01.003
[19]	Klaus, A., Kozarski, M., Niksic, M., Jakovljevic, D., Todorovic, N. and Van Griensven, L.J.L.D. (2011) Antioxidative Activities and Chemical Characterization of Polysaccharides Extracted from the Basidiomycete Schizophyllum commune. LWT—Food Science and Technology, 44, 2005-2011. https://doi.org/10.1016/j.lwt.2011.05.010
[20]	Chirinang, P. and Intarapichet, K.-O. (2009) Amino Acids and Antioxidant Properties of the Oyster Mushrooms, Pleurotus ostreatus and Pleurotus sajor-caju. Science Asia, 35, 326-331. https://doi.org/10.2306/scienceasia1513-1874.2009.35.326
[21]	Wong, J.Y. and Chye, F.Y. (2009) Antioxidant Properties of Selected Tropical Wild Edible Mushrooms. Journal of Food Composition and Analysis, 22, 269-277. https://doi.org/10.1016/j.jfca.2008.11.021
[22]	Boonsong, S., Klaypradit, W. and Wilaipun, P. (2016) Antioxidant Activities of Extracts from Five Edible Mushrooms Using Different Extractants. Aquaculture and Natural Resource, 50, 89-97. https://doi.org/10.1016/j.anres.2015.07.002
[23]	Ngai, P.H.K. and Ng, T.B. (2004) A Ribonuclease with Antimicrobial, Antimitogenic and Antiproliferatve Activities from the Edible Mushroom Pleurotus sajor-caju. Peptides, 25, 11-17. https://doi.org/10.1016/j.peptides.2003.11.012
[24]	Albert, V. and Ransangan, J. (2013) Antibacterial Potential of Plant Crude Extracts against Gram Negative Fish Bacterial Pathogens. International Journal of Research in Pharmaceutical and Biosciences, 3, 21-27.
[25]	Kang, C.-H., Kim, Y.G., Oh, J.S., Mok, J.-S., Cho, M.-H. and So, J.-S. (2014) Antibiotic Resistance of Vibrio harveyi Isolated from Seawater in Korea. Marine Pollution Bulletin, 86, 261-265. https://doi.org/10.1016/j.marpolbul.2014.07.008
[26]	Natrah, F.M.I., Muta Harah, Z., Japar Sidik, B., Izzatul, N.M.S. and Syahidah, A. (2015) Antibacterial Activities of Selected Seaweed and Seagrass from Port Dickson Coastal Water against Different Aquaculture Pathogens. Sains Malaysiana, 44, 1269-1273. https://doi.org/10.17576/jsm-2015-4409-08