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Antimicrobial Properties of Chitosan Extracted from Freshwater Shrimp (Astacus Leptodactylus) Caught from Aras Lake M

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Antimicrobial Properties of Chitosan Extracted from Freshwater Shrimp (Astacus Leptodactylus) Caught from Aras Lake M Irani an Journ al of A quati c Ani m al Heal th 6(1) 105-117 2020 Antimicrobial properties of chitosan extracted from freshwater shrimp (Astacus leptodactylus) caught from Aras Lake M. Gholampoor1, G. Mahmoodi2, A. Moshfegh*3, A. Tehranifard3 1 Department of Marine Biology, Lahijan Branch, Islamic Azad University, Lahijan, Iran 2 Department of Biology, Kermanshah Branch, Islamic Azad University, Kermanshah, Iran 3 Department of Biology, Lahijan Branch, Islamic Azad University, Lahijan, Iran Received: June 2020 Accepted: August 2020 Abstract With the emerging of resistant Extracted chitosan showed a stronger microorganisms to conventional antibiotics, inhibitory activity against S. aureus as well as consumers' concerns about the side compared to E. coli. It was also observed that effects of chemical drugs, the tendency for with increasing chitosan concentration, the natural bioactive compounds is increasing. inhibitory zone diameter increased against Astacus leptodactylus is considered for both S. aureus (p = 0.001) and E. coli (p = export and consumption in Iran, but during its 0.122). It was also observed that the acidic processing a large amount of waste material solution of chitosan had a stronger could be produced. The aim of the present antibacterial activity than aqueous solution of study was to investigate the antimicrobial chitosan. The methanolic extract did not properties of chitosan extracted from A. show significant effects on the studied leptodactylus caught from Aras Lake. The microorganisms. Chitosan had a weak shrimp was caught from Lake Aras and antifungal activity, although it showed a transferred to the Microbiology Laboratory greater effect on A. niger than C. albicans. of the Islamic Azad University of Lahijan close to ice. Chitosan was extracted from Keywords: Astacus leptodactylus, Chitosan, shrimp shells, and methanol extract of shrimp Antibacterial activity, Escherichia coli, tissue was prepared. Antimicrobial activity of Staphylococcus aureus chitosan and extracts were evaluated against Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ] Introduction Gram-positive bacteria Staphylococcus According to significant advances in medical aureus and Gram-negative bacteria science and access to a variety of drugs, the Escherichia coli as well as two species of increasing prevalence of infectious and Aspergillus niger and Candida albicans. inflammatory diseases has caused medical *Correspondence A. Moshfegh, researchers to face the problem of pathogen Department of Biology, Lahijan Branch, resistance and inefficiency of existing drugs Islamic Azad University, Lahijan, Iran. and increase the associated costs (Tanha, (Email: [email protected]). Karimzadeh & Zahmatkesh, 2017). There are 105 Azam Moshfegh, Antimicrobial properties of chitosan extracted from Astacus leptodactylus concerns about the complications and side & Aksu, 2011). During the processes of the effects of common chemical drugs, including Astacus leptodactylus, about 15% of the the carcinogenic effects of these drugs, which harvesting is converted to fillet and the also increase the tendency to replace them remaining will be as waste (Moody & Martin, with natural origin drugs (Chen et al., 2014). 2000). Among these, water resources due to the Extraction of beneficial compounds from large area, diversity of biological conditions these wastes and using them in animal diets and consequently the diversity and evolution as an additive or supplement has been of existing organisms make it possible to reported in previous studies (Kumar & Pal, extract a variety of compounds for this 2016; Flick, 1994). It has also been reported purpose (Bhatia & Chugh, 2015). that king shrimp’s shell has significant Many bioactive compounds have been amounts of Astaxanthin pigment, flavoring obtained from marine organisms such as compounds, chitin, etc. (Escobar, Wells & sponges, corals, sea hares, sea sludge, and Waikar, 1991; Meyers & Lee, 1989; Kumar alga. Also, wastes of production and & Pal, 2016). Chitin is a Mucopolysaccharid processing plants of fish cause environmental and has a structural role, which is found in problems and increase the costs of abundance in the external skeleton of maintenance and disposal of these wastes. arthropods such as shrimp, crab and in insects Surely, high value-added compounds and as well as Cryptogamae. Some applications products can be extracted from these wastes of chitin in fish rearing and medical sciences if they were properly processed. (Kumar & include disinfection, increase of seed growth, Pal, 2016). protective coating of seeds against pests, A. leptodactylus is widely consumed in antifungus, treatment of skin burns, many countries as a commercial and special orthopedics, enrichment with a variety of aquatic food due to its high content of omega- drugs, antibiotics and vitamins. Also, one of 3 fatty acids such as Eicosapentaenoic acid the most important derivatives of chitin is a and Docosahexaenoic acid (Martin, Carter & compound called chitosan, which unlike Davis, 2000; Harlioğlu, 2004). A. chemical polymer compounds, is non-toxic leptodactylus is the only genus of Astacus and biodegradable in nature. Chitosan is used habitats in Iran caught from internal in a wide range of industries including freshwater sources. Among the water pharmaceuticals, agriculture, food industries, Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ] resources, the reservoir lake behind Aras water treatment, biotechnology, medicine, Dam located in Poldasht city of West cosmetics, textiles, papermaking, etc. Azerbaijan province is one of the main Chitosan can be used in the food industry as habitats and the only commercial fishing antibacterial, antifungal, antioxidant, food place of this species in Iran, which more than coatings, food concentrates, strengthening, 200 tons of this species is exported annually water absorption, transparency in products to European countries (Karimpour, Harlioğlu such as juices, to prevent food color change, 106 Iranian Journal of Aquatic Animal Health gel production and reduce fat absorption in waters, the necessary measures have been fried products (Kumar, 2000). taken in terms of productivity of these vast Fortunately, in the southern parts of the resources and the necessary planning to country, there are good reserves of shrimp extract these valuable compounds, also and crabs for chitin, but unfortunately, their taking into account the high potential of the skin is removed and discarded without use country, while meeting domestic needs and after fishing. Due to the scarcity and eliminating the need to import this product, it dispersion of aquaculture processing plants, provided the necessary grounds for its export the total amount of catch is divided and to other parts of the world and provided scattered in different areas after reaching the currency for the country. shore, therefore there is no clear plan for the Therefore, the aim of the present study optimal use of these wastes in the aquaculture was to extract Chitosan from A. leptodactylus sector. Also, high perishability, seasonal that was harvested from Lake Aras and to fishing of these fish and high temperature of investigate its antimicrobial properties. fishing areas are other important reasons Materials and Methods which make it impossible for the productivity Equipment and Ingredients of these wastes in the South. Therefore, due to the existence of vast water resources in the The materials, culture media and instruments, North and South of the country and also the which were used in this study, were listed in significant reserves of crustaceans in these Table 1. Table 1. Applied equipment and used ingredients Row Apparatus and material brand 1 Autoclave Hyrayama,Japan 2 Bain-marie Memmert,Germany 3 Incubator Memmet,Germany 4 microbial hood Behdad,Iran 5 Centrifuge Pars Azma C, Iran 6 Oven Memmert,Germany 7 DMSO QueLAB Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ] 8 Muller Hinton Agar growth medium (MHA) Merck 9 Muller Hinton Broth growth medium Merck 10 Tryptic Soy Broth growth medium Merck 11 Antibiotic disc Padtan TEB 107 Aza m Mo shf e gh, A nti mic ro b ial p ro pe rtie s o f c hi to san e xt rac te d f ro m Asta cus lep toda ct ylus Providing samples To conduct the research in summer, A. The PH of the superior phase, which contains leptodactylus were caught from Aras Dam chitosan in acetic acid solution, was reduced Lake located in West Azerbaijan Province to about 9 with Sodium 4 molar. At this point, and transferred to the Microbiology the chitosan was suspended in solution and Laboratory of Islamic Azad University, then precipitated. B o t h resulting residues, Lahijan Branch for identification and crude chitin and chitosan, were washed with preparation of extract processes. A. distilled water, ethanol and acetone, and were leptodactylus samples were identified by dried at 20 °C. Th e resulting precipitate was using the available identification keys. placed in an oven at 105 °C for 24 hours and Extraction of chitosan the dry sample weight was determined After washing, the waste and shell of A. (Chang & Tsai, 1997). leptodactylus were manually removed from Preparation of methanolic extract the flesh and transferred to an oven at 37 °C Each muscle tissue was cut into small and allowed to dry for 24 hours. The waste pieces. This tissue was poured into a sterile and shell were then ground and stored container containing 96% methanol in a ratio separately in a dark glass in a freezer at -20 of 1 ml distilled water with 4 g of tissue and °C until use. 2 M profit was added to 5 g of placed in laboratory temperature for one dried A. leptodactylus shell powder, and i t week, after which the solution was passed was incubated at 25 ° C for 15 minutes to de- through filter paper. About 200 ml of crude proteinize the sample. Then the supernatant methanolic extract was evaporated at room was centrifuged at 2000 rpm for 10 minutes.
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