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 Lake M. Gholampoor1, G. Mahmoodi2, A. Moshfegh*3, A. Tehranifard3 1 Department of Marine Biology, Lahijan Branch, Islamic Azad University, Lahijan, 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. 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,

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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

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Providing samples To conduct the research in summer, A. The PH of the superior phase, which contains leptodactylus were caught from chitosan in acetic acid solution, was reduced Lake located in 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. temperature in a rotary apparatus under The surface alkaline liquid was separated and vacuum. This methanolic extract was then discarded. The resulting precipitate was stored at -20 °C. Another 200 ml of crude washed several times with distilled water. 25 methanolic extract with 200 ml of chloroform ml of 0.5 M sulfuric acid was added to the was poured into another container, which precipitate and placed at 50 ° C for 16 hours formed two separate phases after a few to remove minerals. The sample was then minutes. After 3 days, the chloroform section centrifuged at 2000 rpm for 10 minutes. Then was separated and evaporated. The obtained the precipitate was washed with distilled chloroform extract was stored at -20 °C. In water several times. In order to purify the order to use chitosan extracts obtained during

Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ] extracted chitin, dried A. leptodactylus with the mentioned processes, for microbial tests 2% sodium hydroxide solution (1: 30) was and sensitivity determination, was directly deproteinized for 2 hours at 90 ° C. Then the used in aqueous and acidic solvents (1 M) and resulting precipitate and the alkaline mixture along with DMSO applied for anti-fungal were washed. To extract chitosan from the tests) . According to the sources, it is better to above precipitate mixtures, it was placed in mix the extracted chitosan with 45% sodium the presence of 10% acetic acid at a ratio of hydroxide solution in a ratio of 1:10. The 10: 1 (w / v) at 60 ° C for 6 hours and then the above samples were transferred to a bain- crude and pure chitin were separated by marie at 85 ° C at intervals of 20 and 45 centrifugation for 15 minutes at 4000 rpm. minutes.

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The studied microorganisms in the present transferred by sterile forceps to the cultured study, the bacterial strains of S. aureus media. Finally, the plates were incubated at (PTCC: 1113) and E. coli (PTCC: 1763) as 37 °C for 18 hours and after the incubation well as the fungal and yeast strains of time the plates were examined for Aspergillus niger (PTCC: 5154) and Candida observation or non-observation of the growth albicans (PTCC: 5027) were collected and halo diameter and the desired halos were prepared as lyophilized from the Iranian measured. Bacterial Collection Center, Iran was Well diffusion well prepared as lyophilized and were used to Similar to the disk diffusion method, the study the antibacterial effects of chitosan bacteria were first cultured uniformly and extracted from freshwater king prawns. then pasteurized using a sterile pipette, 6 mm Activation of bacterial strains was performed wells with a capacity of 100 μl were punched by transferring the contents of each vial in in agar and cooled molten agar was used to screw tubes containing 10 ml of Tryptic Soy block wells in order to prevent extracts from Broth and incubating for 24 hours at 37 °C. escaping from the bottom of each well. The fungi were also activated in Potato Finally, chitosan extracts extracted from A. Dextrose Agar culture medium and stored for leptodactylus was transferred by means of a 24 hours at 30 °C. To prepare the bacterial sample from specific dilutions into each well suspension equivalent to 0.5 McFarland in embedded in agar. The plates were incubated tubes containing Mueller Hinton broth at 37 ° C for 18 hours and after the incubation medium, 4 to 5 colonies were removed from period the plates were examined for the culture medium after 18 to 24 hours of observation or non-observation of the growth inoculation and inoculated into TSB medium. halo diameter. The tube was shaken vertically to compare Determination of MIC and MBC the resulting uniform solution with the semi- Macro Dilution Broth method was used to McFarland turbidity. The light absorption of determine MIC and MBC. MIC is actually the 0.5 McFarland suspension at 625 nm the minimum concentration of antimicrobial should have an absorption of 0.8-0.13. agent that can inhibit the growth of Finally, if the above conditions were met, the microorganisms, and if this agent is removed microbial suspension would have a from the environment, the microorganism concentration equivalent to 1-2 × 108 will be able to grow again. MBC is the Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ] CFU/ml. minimum concentration of antimicrobial Disk diffusion method agent that can kill 99.9% of bacteria and if First, after 15 minutes of preparing 0.5 secondary culture is carried out, less than McFarland turbidity from the desired strains, 0.1% of primary bacteria can grow. In this culturing was uniformly carried out by a method, different dilutions of chitosan sterile swap on MHA medium and then extract were prepared along with Mueller sterile blank disks were inoculated with 20 μl Hinton broth culture area and each of the of specific doses of chitosan extract. bacteria were added and inoculated with a Extraction of A. leptodactylus was precise sampler in each of the tubes

109 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

containing the culture medium. Each Concentrations of 80 and 100 mg / ml of bacterium equivalent to 0.5 McFarland in the extracts were dissolved in DMSO solvent and amount of 300 μl along with dilution of 10, added in tubes of 0.25 McFarland Nutrient 20, 40, 60, 80, 100 mg / ml of chitosan extract broth along with anti-fungal suspension. It was inoculated to Mueller Hinton broth was then incubated at 35 °C. The growth of culture medium and incubated for 24 h at 37 the fungi was examined for a week and the ° C. For each series of diluted forms, a control MIC was determined. + (including bacteria + culture medium), Results control - (including extracted chitosan + Antibiogram Test culture medium) and a blank tube were The results related to the antimicrobial effect considered to control the culture medium in of chitosan in terms of growth inhibition zone each series. After incubation time, each tube (mm) against S. aureus and E. coli is shown was evaluated for turbidity or non-turbidity in Table 2 and Figure 1. The extracted in which no growth was observed it chitosan showed the maximum inhibitory considered as MIC. In order to determine the activity against Gram-positive bacteria. S. MBC from each concentration, surface aureus showed that at all concentrations culture was performed in a plate containing studied the diameter of the inhibitory aura Nutrient Agar incubated for 24 hours at 37 against S. aureus was more than E. coli ° C . bacteria. It was also observed that with Antifungal Test Method increasing chitosan concentration, The selected fungi for testing were Candida antibacterial effects against both S. aureus (p albicans and Aspergillus niger which were = 0.001) and E. coli (p = 0.012) were collected from the Iranian Collection Center significant and with increasing chitosan of Bacteria and Fungi and used for antifungal concentration, the diameter of the inhibition test. In order to evaluate the antifungal effect halo was significantly higher. In addition, no of extracts Nutrient broth at a temperature of significant difference was observed between 35 degrees was used and compared to the results of the well and disk method (p itraconazole as a reference drug. <0.05). Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ]

Figure 1. Effect of extracted chitosan on S. aureus (A) and E. coli (B)

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The results of the minimum inhibitory had stronger antibacterial activity than the concentration (MIC) and maximum lethal aqueous solution of chitosan, so that the MIC concentration (MBC) of chitosan extracted and MBC of the acidic solution of chitosan against the studied bacteria are shown in against S. aureus were 20 and 40 mg / ml, Table 3. The lowest amount of MIC and respectively, and these values were equal for MBC chitosan extracted against S. aureus the aqueous solution of chitosan extract, 40 was observed while no significant effects and 80 mg / ml, respectively. No inhibitory were observed against E. coli. It was also and inactivating effects were observed in E. observed that the acidic solution of chitosan coli.

Table 2. Antimicrobial effect of chitosan in different concentrations in terms of growth inhibition zone (mm) 20 40 60 80 100 P value

S. aureus 4 11 15 17 19 0.001 PTCC 1113 E. coli 0 0 9 13 14 0.012 PTCC 1763

Table 3. Effects of minimum inhibitory concentration (MIC) and minimum lethal concentration (MBC) of acidic and aqueous solution of chitosan

Aqueous solution of chitosan extracted Acidic solution of chitosan f r om A. leptodactylus extracted from A. leptodactylus MBC mg/ml MIC mg/ml MBC mg/ml MIC mg/ml

S. aureus 80 40 40 20 PTCC1113 E.coli PTCC 1763 No effect No effect No effect No effect

The effect of methanolic extract of A. for total concentration by disk method, which had leptodactylus against the studied microorganisms a growth inhibition zone with a diameter of 9 mm is shown in Figure 2. In the studies obtained from against S. aureus, no significant effects of the effect of total extract to the determination of methanolic extract of A. leptodactylus were susceptibility to disk and welling methods, except observed against the studied microorganisms. Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ]

111 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

Figure 2. Effect of disc containing alcoholic extract of A. leptodactylus against S. aureus (A) and E. coli (B) Assaying the antifungal activity of chitosan a greater effect on A. niger, although it did not extracted against C. albicans and A. niger showed show significant inhibition and did not prevent that acid-soluble chitosan (extracted from acid as hexadecimal growth, but to some extent an aura a chitosan solvent and used in powder form) had of growth was observed (Figure 3). Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ]

Figure 3. Antibiogram tests on Aspergillus niger (A) and Candida albicans (B).

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As shown in Figure 4, the diameter of the non- to standard antibiotics shows that in general S. growth halos of each bacterium relative to the aureus was more sensitive to the studied extracted acidic and aqueous extracts compared antibiotics compared to E. coli.

40

S. aureus PTCC1113 35 32 31 E coli PTCC 1763 29 30 28 27 27 25 25 23 23 22 21 20 20 18 18 18 17 16 16 16

15 14 14 12 12 10 10 9

8 non-growth non-growth halos (mm)

5

0 0 0 0 0 R i f a m p i n Cefazolin Ticarcillin Cefotetan Imipenem Piperacillin Cefotaxime Gentamicin Amoxicillin Ceftazidime Erythromycin Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ] Chloramphenicol Sulfamethoxazole aqueous tntal solution… tntal aqueous Tntal solutixAcidic chitin

Figure 4 . Comparison of growth inhibition zone of antibiotics and total acidic and aqueous extracts on standard strains of E coli and S. aureus

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Discussion Due to the concerns caused by the side effects aureus compared with E. coli, is consistent of common chemical drugs, including the with the results of the present study. It has carcinogenic effects of these drugs and the been reported that one of the main reasons for emergence of antibiotic resistance of higher antimicrobial activities of chitosan microorganisms, the tendency to use against gram-positive compared to gram- compounds of natural origin is increasing day negative bacteria is the difference between by day (Chen, 2014). Chitosan was extracted the cell surfaces as a protective membrane from the leachate of A. leptodactylus and its around the cell wall (Isamu et al. 2003). antibacterial activity was evaluated. The Other reasons for the differences in results show a stronger effect of chitosan antimicrobial activity of chitin and chitosan against S. aureus bacteria compared to E. are relevant to different sources include a) coli. Also, the antifungal activity of the type and age of pathogenic microorganisms, studied chitosan was weaker than its b) physicochemical properties of chitin and antibacterial activity. chitosan, c) source of chitin and chitosan and In most previous studies, it has been d) Extraction conditions (Hongpattarakere & reported that chitosan generally has a Riyaphan, 2008). stronger bactericidal effect on gram-positive In addition, the relatively weak antifungal than gram-negative bacteria (Rout, 2001; activity of chitin and chitosan was attributed Másson et al., 2008). These results are to the effect of antimicrobial compounds on consistent with the results of the present the solid cell wall of fungi, including chitin study, which showed that chitosan has the and glucan. Can be confirmed antibacterial greatest effect of growth inhibition on S. activity (Másson, Holappa & Hjálmarsdóttir, aureus compared to E. coli. On the other 2008). On the other hand, chitosan showed hand, some studies have reported higher significant antifungal properties (Burrows, antimicrobial activity against gram-negative 2007). bacteria (Tareq et al., 2013; Chen, Liau & In the present study, with increasing Tsai, 1998; Chung et al., 2004) and some chitosan concentration, the inhibitory activity have not reported significant differences in against the studied microorganisms this regard (Chen et al., 1996). Therefore, increased, which is consistent with the results more and more comprehensive studies are of the previous studies. Taheri et al. (2014) Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ] necessary. In the study of Taheri et al. (2013) reported that among the concentrations of 5, it was observed that chitosan had a stronger 7 and 10% chitosan, the highest inhibitory antibacterial effect against S. aureus activity was observed for 10% solution compared to E. coli, although in the study of (Taheri, Seyfan & Jalalinezhad, 2013). These these researchers most effects were observed results indicate the fact that chitosan has a against Vibrio ogava (Taheri, Seyfan & certain antimicrobial effect, so increasing the Jalalinezhad, 2013). Results of the study of concentration of chitosan also increases its Taheri et al. (2013) on the stronger effectiveness and lethality. However, some antibacterial effects of chitosan against S. researchers have reported that chitosan’s

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antibacterial activity does not increase by Chitin and chitosan have a wide range of increasing its concentration, which may be microbial activities against gram-positive due to differences in test conditions. Also, bacteria, gram-negative bacteria and fungi, increased chitosan viscosity at higher although the amount of this activity is concentrations and limited number of different for each category. This effective chitosan contacts with the cell antimicrobial activity, which in fact reflects surface of bacterial cells are other reasons for the defense barrier against microorganisms this phenomenon. with chitinous shells, provides significant In this study, the effectiveness of the potential for use in a variety of drugs. extracted chitosan, which was dissolved in Although the mechanism of antimicrobial acid, was more potent than the aqueous activity of chitin and chitosan is not well solution, which can be attributed to both the known, the acceptable mechanism for this type of acidic solution and the effect of the activity is the interaction between the gram-positive wall. Taheri et al. (2013) positive charges of the chitin and chitosan reported that acetic acid soluble chitosan molecules and the negative charges of the cell showed stronger antibacterial activity membrane surface of pathogens. In this compared to water soluble chitosan, which is regard, more positive charges of chitosan consistent with the results of Taheri et al. surface and more antimicrobial activity can (2013) study. Chitosan is in pH less than 6 be expected through this mechanism. polycationic and reacts easily with negatively However, further studies are needed to be charged compounds such as proteins, anionic better understood the mechanism of polysaccharides, fatty acids, and antimicrobial activity of chitin and chitosan. phospholipids, destroying bacterial Overall, the significant effect of extracted membranes. In fact, the antibacterial chitosan extracts, especially against gram- properties of chitosan depend on the positive S. aureus bacteria has been protonation of amino groups, which can react observed, and can be suggested that the waste with a negative charge on the cell surface and of marine organisms such as shrimp skin be cause bacterial cell destruction. It has also used as an available and inexpensive source been reported that because chitosan is for the production of antibacterial and insoluble at pH = 7 and has no amino groups, inhibitory drugs. One of the limitations of the it has low antimicrobial activity (Taheri et al., present study was the lack of chitosan Downloaded from ijaah.ir at 5:20 +0330 on Friday October 1st 2021 [ DOI: 10.29252/ijaah.6.1.105 ] 2013). production to measure its degree of Also, in the present study, it was observed distillation. that the extracted chitosan showed acceptable References inhibitory activity in comparison with the Bhatia, P., & Chugh, A., 2015. Role of studied antibiotics, to which some bacteria marine bioprospecting contracts in had antibiotic resistance, and these results are developing access and benefit sharing consistent with the results of Taheri et al. mechanism for marine traditional knowledge (2013). holders in the pharmaceutical industry.

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