Comparative Study of Antibacterial Activity of Chitin and Chemically Treated Chitosan Prepared from Shrimp ( Macrobrachium Rosenbergii ) Shell Waste

The antibacterial activity of chemically deacetylated chitin i.e. chitosan produced from shrimp shell. The antibacterial activity was tested against gram negative (Escherichia coli) bacteria, and gram positive (staphylococcus aureus) bacteria. To test the antibacterial activity institute of clinical laboratory standards (ICLS) approved dilution methods was used. The study was carried out by broth dilution method and used turbidity estimations to determine the growth characteristics of a bacterial culture with or without chitin or chitosan. Chitosan possess higher antibacterial activity against both types of bacteria, while chitin possess very lower activity. For staphylococcus aureus the average absorbance in every O.D value measured, staphylococcus aureus with chitin showed far higher absorbance than chitosan, indicates lower level of activity of chitin. After 32 hours of incubation s.aureus with medium absorbance was 1.15, while chitosan with s.aureus absorbance recorded 0.32, chitin with s.aureus recorded 0.733. Lower absorbance for chitosan containing E.Coli medium. At the peak point Medium and E.coli absorbance was 1.07, while chitosan with E.coli recorded 0.282 and chitin with E.coli absorbance was found 0.831. The ratio between medium E.coli and chitosan: medium and E.coli=0.26:1, and the ratio of medium S.aureus and chitosan : medium and S.aureus = 0.28:1, indicates G (+Ve) bacteria gives 0.02 times higher absorbance than G (-Ve) bacteria. The between chitin and chitosan, chitosan was found 2.2 times more active than chitin against S.aureus and 3.0 times more active than chitin against E.coli.


Introduction
Chitin is a polysaccharide of animal origin found abundantly in nature and characterized by a fibrous structure.It forms the basis of the main constituent of the outer skeleton of insects and crustaceans like shrimp, crabs and lobster (Kumar et al., 2005).According to Chen (1998) the chitin structure can be modified by removing the acetyl groups, which are bond to amine radicals in the C2 position on the glucan ring, by means of a chemical hydrolysis in concentrated alkaline solution at elevated temperature to produce a deacetylated form known as chitosan.No, H.K. et al., (2002) stated that antibacterial activity of chitosan is effective in inhibiting growth of bacteria.The antimicrobial properties of chitosan depend on its molecular weight and the type of bacterium.For gram-positive bacteria, chitosan with 470 KDa was the most effective, except for Lactbacillus sp., whereas for gramnegative bacteria, chitosan with 1,106 KDa was effective.Chitosan generally showed stronger bactericidal effects for gram positive bacteria (Listeria monocytogenes, Bacillus megaterium, B. cereus, Staphylococcus aureus, Lactobacillus plantarum, L. brevis, and L. bulgaris) than for gram-negative bacteria (E.coli, Pseudomonas fluorescens, Salmonella typhymurium, and Vibrio parahaemolyticus) in the presence of 0.1% chitosan.The antimicrobial activity of chitosan is described to be associated with molecular weight, degree of acetylation, concentration of chitosan and bacterial inoculum size was showed by Chen, M.C et al., (1996) and Fernandes, J.C et al., (2008).It is reported by Peter E. J. C. et al. (2008) that lower molecular weight chitosan is more effective against Gram-negative bacteria, whereas high molecular weight chitosan is effective against Gram-positive bacteria in atomic force microscopy of cell wall structure and nano-indentation study.In this research work we tried to show the activity of chitin and chitosan against two pathogenic bacteria, one gram positive strain staphylococcus aureus and one gram negative strain Escherichia coli.These pathogenic bacteria are all around us contaminating our food, water readily, causing spoilage of perishable food items.The bacterial effectiveness on gram-positive or gram-negative bacteria is however, somewhat controversial.Some authors have stated that chitosan generally showed stronger effects for gram-positive bacteria (e.g.Listeria monocytogenes, Bacillus megaterium, B. cereus, Staphylococcus aureus, Lactobacillus plantarum, L. brevis, L. bulgaris, etc.) than for gram-negative bacteria (e.g.E. coli, Pseudomonas fluorescens, Salmonella typhymurium, Vibrio parahaemolyticus, etc.) Coma et al, Jeon et al. andNo et al. (2001-03), andDutta (2009).Conversely, it has been demonstrated that hydrophilicity in gram-negative bacteria is significantly higher than in gram-positive bacteria, making them most sensitive to chitosan stated by Chung et al., (2004).The charge density on the cell surface is a determinant factor to establish the amount of adsorbed chitosan.More adsorbed chitosan would evidently result in greater changes in the structure and in the permeability of the cell membrane.This would suggest that the antibacterial mode of action is dependent upon the host microorganism Másson, M. et al. (2008).
The antimicrobial activity of chitosan is described to be associated with molecular weight, degree of acetylation, concentration of chitosan and bacterial inoculum size was described by Chen M. C. et al 1996C. et al , Fernades J.C et al. (2008)).It is reported that lower molecular weight chitosan is more effective against Gram-negative bacteria, whereas high molecular weight chitosan is effective against Gram-positive bacteria in atomic force microscopy of cell wall structure and nanoindentation study by Peter E.J. C et al. (2008).
In this research work we tried to show the activity of chitin and chitosan against two pathogenic bacteria, one gram positive strain staphylococcus aureus and one gram negative strain Escherichia coli.
The result obtained from the research work will contribute to evolve a more active natural antibacterial agent which may be applied to preserve food items, may be used as antimicrobial agent in pharmaceuticals, and may also find uses in agriculture as a more active Seed coating, fertilizer, controlled agrochemical release agent.In drug industry it may be used to formulate weight loosing supplement, which is already produced in many countries.As Bangladesh is an agro based country we think production of chitin and chitosan will confined into variety of uses.Chitosan production from shrimp waste certainly save us from serious environment pollution, we should use this biopolymer as a safe preservative in food item, also can be apply as seed coatings, fertilizer, we are with serious water problem in municipal area, chitosan may be used as water purification agent.
Based on the above investigation the following objectives were undertaken: 1.The study investigates chitosan (produced by chemical deacylation) and chitin produced exclusively from shrimp of our country (as region or source may affect activity) whether possess antibacterial activity and 2. To compare whether chitin is more active than chitosan and vice-versa against E. coli and Staphylococci.

Methodology
Chitin is extracted from crustacean shell waste such as crab, shrimp, lobster, and crawfish.We prepared chitin from shrimp shell (Macrobrachium rosenbergii) locally known as Galda chinri.Isolation of chitosan from shirmp shell wastes involves four traditional steps demineralization (DM), deproteinization (DP), decolorization (DC), and deacetylation (DA).the method was modified from No and Meyers, (1995).Two bacterial strains were tested for the antimicrobial activity of chitosan and chitin include gram negative bacteria Escherichia coli gram positive Staphylococcus aureus (pure culture was obtained from Sono Diagonostic & hospital, Kushtia) pure Staphylococci culture was procured from Food microbiology research lab-2, Islamic university, kushtia).Antibacterial susceptibility test (AST) was used to determine the efficiency of potential antimicrobials and initial inoculums bacterial count was made.AST methods are used to screen antimicrobial activity but are largely used to determine usefulness of an antimicrobial to combat against infections.The current study was carried out by broth dilution method, since it is most economical method for AST.In this exercise we used turbidity estimations to determine the growth characteristics of a bacterial culture.For this we first ensure same characteristics of the inoculation medium.The sequence of procedure to accomplish this is as follows-Growth Media: The sterile growth media was poured into 18 sterile test tubes in aseptic condition using laminar air flow cabinet.In every test tube 9ml of nutrient broth media was taken.Few test tubes will be used as standard in spectrophotometer measurement.
Addition of Chitin and Chitosan: 3% (v/v) 100 ml acetic acid preparation: 3ml concentrated (99%) acetic acid was taken into a conical flask and made up to 100ml volume mark by distill water.
1.5gm chitin and chitosan was taken into two test tube (sterile) and 10ml 3% acetic acid was poured into it gradually.To increase the solubility the solution stirred and heat was also applied in water bath at 40º C. The solution of chitin and chitosan was then added into the test tubes.The upper soluble portion of each sample was added with medium, we did not take the supernatant from the test tube.In 6 test tubes 0.5 ml chitosan (in each test tube) solution was taken by micropipette.Similarly another test tube was prepared with chitin solution (0.5ml in each test tube).Both for chitin and chitosan, from these 6 test tubes one was used as standard (media+ chitin / media+ chitosan), one for negative control (media+ chitin / media+ chitosan), two for gram negative bacteria inoculation (media+ chitin+ gram negative / media+ chitosan+ gram negative), and two for gram positive bacteria inoculation( media+ chitin+ gram positive / media+ chitosan+ gram positive).Of the remaining 6 test tubes one was used standard (only media), one for negative control (only media), two for gram negative inoculation (media+ gram negative), and two for gram positive inoculation (media+ gram negative).
pH Control: We adjusted the pH of the medium.The pH of only medium was 6.5.The pH of chitin with medium, chitosan with medium was also adjusted between 6.2-6.5 range, using NaOH solution.All these were done in laminar air flow cabinet.

Inoculation of Bacteria:
After adjustment of the pH we inoculated the test tubes, with gram positive staphylococcus aureus and gram negative Escherichia coli.The following table shows the number of test tubes inoculated, with G (+Ve) and G (-Ve) bacteria respectively.The experiment was conducted in a view to compare the growth of bacterial strain in only media, and in media with chitin and chitosan.

Turbidimetric Measurement of Bacterial
Growth: Growth is commonly measured by determining the turbidity of a cell suspension with an instrument known as a spectrophotometer or colorimeter.In this exercise we used turbidity estimations to determine the growth characteristics of a bacterial culture.In liquid culture, the medium appears more and cloudier as the bacteria increase in number by division.A tube of bacteria will tend to reflect light so that less light is transmitted through the tube.A spectrophotometer can measure the amount of light passing through the tube, or conversely the amount of light absorbed.These measurements of turbidity or optical density (OD) are not direct measurements of bacterial numbers, but an indirect measurement of cell biomass that includes both living and dead cells.As the bacterial cell population increases, the amount of transmitted light decreases, increasing the absorbance reading on the spectrophotometer.If one takes readings of the same culture over time, the absorbance readings will increase as the cell number increases.Comparative Study:

Results and Discussions
Chitosan possess strong antibacterial activity against both G (+Ve) and G (-Ve) bacteria, while chitin possess lower level of activity against these two types of bacteria.The result was numerically presented to determine which types of bacteria is mostly affected by chitosan and chitin:

Comparative Study of Antibacterial Activity against G (+Ve) and G (-Ve) of Chitosan
The absorbance for medium and S.aureus at the peak was 1.15.While the absorbance for medium and S.aureus +chitosan at the peak was 32.The ratio between these two absorbance is 0.28:1.So the ratio of medium and S.aureus +chitosan: medium + S.aureus = 0.28:1.
The absorbance for medium and E.coli at the peak was 1.07, while the absorbance for medium and E.coli +chitosan at the peak was 0.282.The ratio between these two absorbance 0. Koide S.S., (1998) stated that chitin and chitosan in vitro showed antibacterial and anti-yeast activities.One of chitosan derivatives, i.e., Ncarboxybutyl chitosan, was tested against 298 cultures of different pathogenic microorganisms that showed bacteriostatic and bactericidal 16 activities, and there were marked morphological alterations in treated microorganisms when examined by electron microscopy according to Muzzarelli R, et al., (1990).There was a contradict among researcher, Chen YM, Chung YC, Wang LW, Chen KT, Li SY cited that chitosan or its derivatives is more effective for gram-negative bacteria than gram-positive bacteria , other authors Jonathan Rhoades and Bob Rastall cited the activity of chitosan is followed by Grampositive bacteria and finally Gram-negative bacteria.The result reveals that chitosan produced by chemical treatment from shrimp (Macrobrachium rosenbergii) waste was more active antibacterial agent, inhibits both gram negative and gram positive bacteria.So it is a broad spectrum antibacterial agent.
The major constituent of their cell wall is peptidoglycan and a little amount of protein.The cell wall of Gram negative bacteria on the other hand is thinner but more complex and contains various polysaccharides, proteins and lipids beside the peptidoglycan.Also, the cell wall of Gram-negative bacteria has an outer membrane which constitutes the outer surface of the wall (Black, 1996) the complex structure of gram positive bacteria is a possible cause of the less effective than gram negative strains.

Table 5 . Determination of O. D Value for E.Coli (28h-52h)
The O.D value for E.coli clearly demonstrate antibacterial activity against gram negative E.coli.After 36hours of inoculation the maximum growth for the inoculum and the Absorbance was 1.07, while chitosan with E.coli ABS was 0.282, and chitin with E.coli Absorbance was .831.Chitosan shows strong activity against E.coli, while chitin has much lower activity.The data graphically below:

Shows the Ratio between Medium S.aureus and Chitin or Chitosan with Medium and S.aureus.
Dutta J. et al., (2012)unit absorbance for medium and S. aureus at the same time or same hour of incubation S. aureus with chitosan found 0.28 and chitin with S. aureus recorded 0.64.So chitosan is about 2.2 times more active against S.aureus than chitin.The chitin and chitosan was found to be bacteriostatic against S. aureus and E. coli.Dutta J. et al., (2012)