Antibacterial activity of Chito-oligosaccharides derived from Fish Scales

 

Gul-e-Saba Chaudhry1*, Thirukanthan CS1, Nor Atikah Mohamed Zin1,

Yeong Yik Sung1, Tengku Sifzizul Tengku Muhammad1, Effendy AWM1,2

1Institute of Marine Biotechnology, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia.

2Faculty of Fisheries and Food Science, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Malaysia.

*Corresponding Author E-mail: gul.saba@umt.edu.my/sababiochem@gmail.com, effendy@umt.edu.my

 

ABSTRACT:

This study aimed to evaluate the antibacterial activity of chito-oligosaccharides (COS) from Tilapia fish scales. There is a massive potential in sustainably prospecting from these resources: algae, vertebrate, crustaceans, molluscs, residues from fish farms, and fisheries such as bones, skin, and fins, internal organs and non-bio-degradable substances such as scales—the production COS achieved by chemical hydrolysis involving demineralization, deproteinization and deacetylation. The antibacterial activities performed against Staphylococcus aureus, Bacillus cereus, Streptococcus agalactiea, Escherichia coli, Pseudomonas aeruginosa, Salmonella typhi. The bacteriostatic and bactericidal effect measured by taking Minimum Inhibitory Concentrations (MIC), and the Minimum Bactericidal Concentrations (MBC). Chitin yielded 32.74% from 100 grams of dried fish scales in this study, whereas Chito-oligosaccharides yielded 24.12% from 100 grams of chitin. The ratio of MBC/MIC determination reveals the bacteriostatic and bactericidal effect of a COS. The bactericidal properties were visible in COS. The potential antibacterial effect of COS obtained by Tilapia fish scales could be utilized for biomedical purposes such as wound healing.

 

KEYWORDS: Chito-oligosaccharides, Antibacterial, Molecular weight, MIC, MBC.

 

 


INTRODUCTION:

In the past three decades, ever since the boom in bio-based materials, bio-prospecting from aquatic-derived substances have discussed among researchers since more than 70% of the planet's surface is covered by ocean and other aquatic environments. There is a vast potential in sustainably prospecting from these resources, namely algae and vertebrate, including crustaceans, molluscs, residues from fish farms, and fisheries.1-4

 

The antibacterial activity of various potential compounds derived from plants has shown remarkable effects.5-14 However, the need to utilized waste products of natural origin is there.  The annual fish waste that is discarded amounts to approximately 20 million tonnes or constitutes about 25% of the total production in Malaysia.15

 

Previous study reported that most of this waste produced from fish processing industries in Malaysia,16 where this waste is often discarded directly or indirectly into the environment without being treated appropriately. The waste classified into readily biodegradable elements, namely bones, skin, fins, internal organs and non-bio-degradable substances such as scales. Fish scales are composed of a surface layer consisting of hydroxyapatite, calcium carbonate, and collagen type I, making them a very stable substance degraded by scavenger microbes. Fish waste load is a globalized problem where numerous studies have been done and carried out to produce value-added products.  Bulk amount of fish scales are inevitably generated from fish fillet factories and mainly disposed of as solid wastes. Marine waste from seafood processing plants contributes to the ever-rising coastal pollution when dumped into the sea. To resolve this issue, scientists have developed a solution to sustainably exploit these resources to convert them into a value-added substance known as chitin and chitosan.17 This study attempted to optimize chito-oligosaccharides (COS) extraction from Tilapia (Oreochromis sp.) fish scales. The phsyico-chemical characterization and antimicrobial activities of the COS evaluated. This will surely aid in development and application of bio-polymers such as chito-oligosaccharides in the vast industrial fields.

 

MATERIALS AND METHOD:

Isolation of Chito-oligosaccharides from fish scales:

COS extracted by chemical hydrolysis following the methods described by Chang et al. (2000).18 Firstly, 10g of chitosan was hydrolyzed in 8M 500 mL of HCl at 72ºC for 3 hours. The solution neutralized with 2M Sodium Hydroxide to a final pH of 7. Ultra-pure methanol was added to this solution to precipitate chito-oligosaccharides. Residues precipitated on the bottom of the flask were filtered through a 0.22-µm nylon membrane.

 

Antibacterial Assay:

The antibacterial activity of gram-positive bacteria (Staphylococcus aureus, Streptococcus agalactiea, Bacillus cereus) and gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella typhii) determined accordingly. The colonies picked using aseptic techniques from 24h old cultures grown on nutrient agar. The colonies then used to make a suspension in a sterile saline solution adjusted to an OD reading of 0.1 at 600nm. Then, 0.1mL of this bacterial suspension was then transferred to 9.9mL of sterile Luria Bertani broth (LB Miller, Merck) at a dilution factor of 1:100 to yield a final concentration of 1× 106 CFU ml-1.

 

MIC and MBC Assay:

For determination of the minimum inhibitory concentration of COS, according to reported method,19 first 100µL the bacteria suspension in the broth is placed inside each well of a sterile 96-well plate. Two-fold serial dilution to a final concentration ranging from 0.039mg/mL – 5 mg/mL. 100µL of the diluted samples were placed based on their respective concentrations into the 96-well plate. Triplicate samples performed for each test concentration. The plates incubated for 24 hours at 37ºC under aerobic conditions. The plates then checked for visible signs of growth using a microplate reader (Shimadzu UV-1800) at 630nm. The lowest concentration of COS that inhibited the tested bacterial strains' growth were considered the minimum inhibitory concentration (MIC). The minimum bactericidal concentration (MBC) determined by gently mixing the contents of each well by flushing them with a sterile pipette19. 100uL was drawn out from these wells, placed onto a petri dish with nutrient agar, and spread evenly using a hockey stick. Plates incubated for 24 hours at 37ºC under aerobic conditions.

 

Mechanisms of Antibiosis Determination (MIC Index:

The mechanism of antibiosis of COS evaluated using the method reported.20 The MBC/MIC values ratio showed the bactericidal or bacteriostatic (MBC/MIC > 4) and MBC/MIC≤ 4 considered as antibacterial effects of COS.

 

Time-Kill Assay:

In this study, we employed the standard protocols of M26-A by CLSI and ASTM-2315. The COS prepared at a concentration of 4 times the MIC value. An inoculum size of 1.0 × 106 CFU/mL of bacterial strains prepared in sterile 100mL conical flasks. Four times, the MIC concentration of COS respectively to each bacterial strain was added and incubated at 37ºC on an orbital shake for 6 hours to these flasks. At an interval period of 30minutes apart, 1.0mL of the incubated medium drawn out and the turbidity of the aliquots examined at an OD of 630nm using a spectrophotometer (Shimadzu UV-1800). A blank control test performed with bacterial broth without the addition of COS. The results extrapolated in a graph (OD against time).

 

Statistical Analysis:

The results expressed as the mean±Standard deviation (SD). The experiment data undergoes to two-way analysis of variance (ANOVA) via using Origin 8 SR4. The significant data were determined at P-value < 0.05.

 

RESULTS:

Yield:

The yield for the production of chitin, chitosan and chito-oligosaccharides is shown in table 1. Chitin yielded 32.74% from 100grams of dried fish scales in this study; Chito-oligosaccharides yielded 24.12% from 100grams of chitin.

 

Table 1: Yield of chitin, and COS derived from fish scales

Chitin

Chito-oligosaccharides

Sample weight (g)

Yield weight (g)

Yield (%)

Sample weight (g)

Yield weight (g)

Yield (%)

100

32.74

32.74

100

24.12

24.12

 

MIC and MBC of COS

Results revealed that the COS significantly exhibited lower MIC and MBC values for all tested bacterial strains, concluding the promising results of COS as a stronger antimicrobial agent. As expected, the MBC values obtained were higher than those obtained for the MIC. The study revealed that MIC and MBC more significant against gram-positive bacteria than gram-negative bacteria in COS (Table 2).

 

Table 2: The Minimal Inhibitory Concentration (MIC), and Minimum Bactericidal Concentration (MBC) of COS against bacterial stains.

Test bacteria

COS

M.I.C. (mg/mL)

M.B.C.(mg/mL)

Staphylococcus aureus

0.156

0.313

Bacillus cereus

0.156

0.313

Streptococcus agalactiea

0.313

0.625

Escherichia coli

0.313

0.625

Pseudomonas aeruginosa

1.25

2.5

Salmonella typhii

0.313

0.625

 

Mechanism of Antibiosis:

The horizontal axis represents the concentration of COS, and the vertical axis represents the viability of the test strains presented through the optical density of the aliquot (Figure 1). Results revealed that as the concentration of COS increased from 0mg/mL to 5mg/mL, so did the inhibition ability of COS against all tested bacteria. The COS shows enhance inhibition in gram (+) bacteria compared to gram (-) bacteria. However, the antimicrobial effect can considered significant where 100% inhibitions observed at the 2.5 mg/mL COS concentration.

 

Figure 1: Effect of different concentration of COS against the growth of S. aureus, S.agalactiea, B. cereus, S. typhii, P.aeruginosa and E.coli

 

Table 3 shows the mechanism of antibiosis or MICindex of COS. The ratio of MBC/MIC determination reveals the bacteriostatic and bactericidal effect COS. Results revealed that COS act as a bactericidal agent against all tested bacterial strains.

 

Table 3: Comparative MBC/MIC ratios of COS

Bacteria

COS

Antibacterial effects

Staphylococcus aureus

2.00

Bactericidal

Bacillus cereus

2.00

Bactericidal

Streptococcus agalactiea

1.99

Bactericidal

Escherichia coli

1.99

Bactericidal

Pseudomonas aeruginosa

2.00

Bactericidal

Salmonella typhii

1.99

Bactericidal

 

Time Kill Assay:

The growth curve of ascending concentrations of COS (0mg/mL – 5mg/mL) against the viability of 6 tested bacterial strains are shown in Figure 2 and 3, respectively. The horizontal axis represents the concentration of COS, and the vertical axis represents the viability of the test strains presented through the optical density of the aliquot. Results revealed that as the concentration of chitosan increased from 0 mg/mL to 5mg/mL, so did the inhibition ability of chitosan against all tested bacteria. The antimicrobial effect of COS can be considered as much significant where 100% inhibitions observed at the 2.5mg/mL COS concentration.

 

DISCUSSION:

Chitosan derivatives have reported of demonstrating antimicrobial activities by many studies despite the actual mode of action yet to be fully understood. This study revealed that the COS exhibited remarkable antibacterial activity as COS showed effective inhibitory effects towards gram-positive bacteria compared to gram-negative bacteria stains. The previous studies supported our studies, which shows that stronger antibacterial activity was apparent against Gram-positive bacteria vs Gram-negative bacteria.21-22 Whereas, few studies supports the Gram-positive bacteria to be more susceptible.23

 

The enhanced antibacterial activity of COS might be due to the solubility of COS, where it releases all of the free amines that could freely interact with the negatively charged outer membrane of bacterial cells. Once the interaction established, it readily adsorbed, congregates and precipitates the cells that lead to cell death.24 Another study conducted revealed that COS could be absorbed by bacterial cells causing inhibition to DNA transcription and RNA and protein synthesis.25 The study shown the physicochemical and biological properties of COS where they have depicted the mechanisms involved in the event leading to the microbial death by COS and its conjugates26. It has shown that the COS lower molecular weight and a higher degree of deacetylation had an easier bacteria cellular, therefore increasing the antimicrobial activities of COS. This penetration correlates with our study where the COS derived from fish scales had a molecular weight of 4.6 kDa and degree of deacetylation of 94.65%, respectively. Well, our previous study also utilized the waste to get the potential anti-bacterial activity but originated from plant base such as lychee peel.27 The reported showed that the mode of action of chitosan derivatives exhibited a dose-dependent growth-inhibitory effect.28 Similar effects visible in our study, where the growth curve of ascending concentrations of COS against the bacterial test strains was dose-dependent. The higher the concentrations, the more eminent the inhibitory effects observed.


 

Figure 2: Time kills assay of COS at 4x the MIC value against gram-positive bacteria; (A) Staphylococcus aureus; (B) Bacillus cereus; (C) Streptococcus agalactiea.

 

 

Figure 3: Time kill assay of COS at 4x the MIC value against gram-negative bacteria; (A) Salmonella typhii, (B) Escherichia coli, (C) Pseudomonas aeruginosa

 


CONCLUSION:

In conclusion, the antibacterial activities of COS derived from fish scales demonstrated in this study exhibited good antibacterial activity when examined against both gram-positive and negative bacteria. The molecular weight, degree of deacetylation and solubility of chitosan and COS were the most decisive factors affecting the antibacterial activity. The antibacterial activities of COS more pronounced against gram-positive bacteria.  Further investigation is warranted; particularly needed are those studies designed to extend the findings into the mechanism of actions of these antibacterial activities for novel antimicrobial solutions.

 

CONFLICT OF INTEREST:

The authors have no conflict of interest.

 

ETHICAL STATEMENT:

This article does not contain any animal or human study.

 

REFERENCES:

1.      Kerton FM, Liu Y, Omari KW, Hawboldt K. Green chemistry and the ocean-based biorefinery. Green Chem 2013;15: 860–871.

2.      Lopes C, Antelo LT, Franco-Uria A, Alonso AA, Perez-Martin R. Valorization of fish by-products against waste management treatments—Comparison of environmental impacts. Waste Manag 2015;46: 103–112. doi: 10.1016/j.wasman.2015.08.017

3.      Chew KW, Yap JY, Show PL, Suan NH, Juan JC, Ling, et al. Microalgae biorefinery: High value products perspectives. Bioresour Technol 2017;229: 53–62. doi.org/10.1016/j.biortech.2017.01.0.06

4.      Ferraro V, Cruz IB, Jorge RF, Malcata FX, Pintado ME, et al. Valorization of natural extracts from marine source focused on marine by-products: A review Food Res Int 2010;43: 2221–2233. doi.org/10.1016/j.foodres.2010.07.034.

5.      Mona AEl-S, Mohamed MK, Mohamed AEl-R, Samir M. Osman, et al. Study of Antibacterial Activity of Some Plant Extracts Against Enterohemorrhagic Escherichia coli O157:H7. Research J Pharm and Tech 2013;6(8): 916-919. doi: 10.5958/0974-360X.

6.      Pooja K, Umakant, Gyanendra S. Synthesis and Antibacterial activity of some newer Benzimidazole derivatives. Research J Pharm and Tech 2020;13(6): 2597-2600. doi: 10.5958/0974-360X.

7.      Poojashree, Anitha R. In-vitro Antibacterial activity of Ethyl Acetate extract of Sesbania grandiflora leaf against E. faecalis – A root Canal threat. Research J. Pharm. and Tech 2016;9(12): 2147-2149. DOI: 10.5958/0974-360X.

8.      Disha MD, Shahare HV, Gedam SS, Bhoyar PK, Ganjiwale RO. Aging of Honey Enhances Its Antibacterial Activity. Research J Pharm and Tech 2009; 2 (4): 872-873. DOI: 10.5958/0974-360X.

9.      Reena K, Surjit S, Neha P, Shreta C, Loganathan K, et al. RSM Optimized Media to Increase the Antibacterial Activity of Wild and Mutated Strain of Nocardiopsis VITSRTB. Research J Pharm and Tech 2014;7(2): 213-220. DOI: 10.5958/0974-360X.

10.   Shruthi C, Geetha RV. Antibacterial Activity of the Three Essential Oils on Oral Pathogens- An In-vitro Study. Research J Pharm and Tech 2014 7(10):1128-1129. DOI: 10.5958/0974-360X.

11.   Aravind KS, Lakshmi T, Arun AV. Invitro Antibacterial Activity of Acacia catechu ethanolic leaf extract against selected acidogenic oral bacteria. Research J Pharm and Tech 2012;5(3): 333-336. DOI: 10.5958/0974-360X.

12.   Kanagavalli U, Mohamed SA, Sathishkumar, Rajeshkumar S. Plant Assisted Synthesis of Silver Nanoparticles Using Boerhaavia diffusa Leaves Extract and Evolution of Antibacterial Activity. Research J Pharm and Tech 2016;9(8): 1064-1068. DOI: 10.5958/0974-360X.

13.   Jobin J, Dhidhin R, Prashanth N. Microspheres - Novel Drug Delivery Carrier for Plant Extracts for Antibacterial Activity. Research J Pharm and Tech 2018;11(4): 1681-1684. DOI: 10.5958/0974-360X.

14.   Saravana K, Avijit M, Vanitha J, Ganesh M, Venkateshwaran K, et al. Antibacterial Activity of Methanolic Extract of Sesbania Grandiflora (Fabaceae). Research J Pharm and Tech 2008;1(1): 59-60. DOI: 10.5958/0974-360X.

15.   FAO The state of world fisheries and aquaculture. Food and Agriculture Organization of the United Nations 2012.

16.   Nurdiyana H, SitiMazlina MK, SitiNor FM. Optimization of protein extraction from freeze dried fish waste using response surface methodology (RSM). Int J Eng Technol 2008;1: 48–56.

17.   Dhillon GS, Kaur S, Brar SK, Verma M. Green synthesis approach: extraction of chitosan from fungus mycelium. Crit Rev Biotechnol 2013;33: 379–403. doi: 10.3109/07388551.2012.717217.

18.   Chang KLB, Lee J, Fu WR. HPLC analysis of N-acetyl-chito-oligosaccharides during the acid hydrolysis of chitin. Journal of Food and Drug Analysis 2000;8(2): 75-83. doi.org/10.38212/2224-6614.2837.

19.   Qi L, Xu Z, Jiang X, Hu C, and Zou X. Preparation and antibacterial activity of chitosan nanoparticles. Carbohydrate Research 2014;339(16):2693–2700. doi.org/10.1016/j.carres.2004.09.007.

20.   Gnanamani A, Priya KS, Radhakrishnan N, Babu M. Antibacterial activity of two plant extracts on eight burn pathogens. J Ethnopharmacol 2003;86: 59-61. doi: 10.1016/s0378-8741(03)00044-8.

21.   Chung et al , Chung STL, Legge GE, Cheung SH. Letter recognition and reading speed in peripheral vision benefit from perceptual learning Vision Research 44;2004:695-709. doi: 10.1016/j.visres.2003.09.028.

22.   Raafat D, Sahl HG. Chitosan and its antimicrobial potential--a critical literature survey. Microb Biotechnol 2009;2(2): 186-201.

23.   Tsai, JL, Chentsova-Dutton Y, Friere-Bebeau L, & Przymus DE. Emotional expression and physiology in European Americans and Hmong Americans. Emotion 2002;2: 380-397. doi: 10.1037/1528-3542.2.4.380.

24.   Khan MM, Kalathil S, Lee J, Cho MH. Synthesis of cysteine capped silver nanoparticles by electrochemically active biofilm and their antibacterial activities Bull. Korean Chem Soc 201233(8): 2592-2596.

25.   Rabea EI, Badawy MET, Stevens CV, Smagghe G and Steurbaut W. (2003). Chitosan as antimicrobial agent: Applications and mode of action. Biomacromolecules 4(6): 1457–1465. doi.org/10.1021/bm034130m.

26.   Lucas P, Muhammad N, Imran SM, Li B, Di B. Chitooligosaccharide: An evaluation of physicochemical and biological properties with the proposition for determination of thermal degradation products. Biomedicine & Pharmacotherapy. 2018;102: 438-451. doi: 10.1016/j.biopha.2018.03.108.

27.   Perveen S, Safdar N, Chaudhry GE, Yasmin A. Antibacterial evaluation of silver nanoparticles synthesized from lychee peel: individual versus antibiotic conjugated effects. World J Microbiol Biotechnol 2018 14;34(8): 118. DOI: 10.1007/s11274-018-2500-1.

28.   Raafat D, von , Haas K, Sahl HG. Insights into the mode of action of chitosan as an antibacterial compound. Applied Env Microbiol 2008;74(12): 3764–3773. doi: 10.1128/AEM.00453-08.

 

 

 

 

Received on 16.05.2021           Modified on 14.10.2021

Accepted on 18.01.2022         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(7):3081-3085.

DOI: 10.52711/0974-360X.2022.00515