Exposure of chitosan to UV / ozone : Structural information and antibacterial activity

Article history: Received on: 01/10/2016 Revised on: 14/11/2016 Accepted on: 26/11/2016 Available online: 28/12/2016 In the present work, variation in the group coordination of chitosan due to the exposure to UV/ozone was considered by the FTIR reflectance. Pronounced variations in the intensities of reflectance bands and shifts in their positions were detected. This could be attributed to the change in the molecular configuration of chitosan. On the other hand, the well diffusion method was used to investigate the anti-bactericidal activities against two common bacterial strains [Bacillus subtitles (B. subtilis) and Escherichia coli (E. coli)] by detecting the mean inhibition zone diameters (IZD) against the microorganisms. The achieved data revealed that the chitosan exposed to UV/ozone confirmed a high potential of antibacterial activity.


INTRODUCTION
Chitin is a natural polysaccharide synthesized by a great number of living organisms and functions as a structural polysaccharide.Chitosan is a modified natural carbohydrate polymer of glucosamine and N-acetyl glucosamine derived from chitin by deacetylation (Tolaimate et al., 2000).Chitosan possesses positive ionic charges, which give it the facility to chemically bind with negatively charged fats, lipids, cholesterol, metal ions, proteins, and macromolecules (Li et al., 1992).Chitosan has some advantageous properties, such as biocompatibility, biodegradable polymer of high molecular weight non toxic and antimicrobial activity, that encourage its applications in many fields including agricultural (Rodriguez . et al., 2007, Nguyen Anh Dzung et al., 2011), paper industry, food and textile industries, pharmaceutics (Ouattara et al., 2000, Muzzarelli et al., 2005), biochemistry, biotechnology, cosmetic, and biomedical applications (Li et al., 2005, Alves and Mano 2008, Dzung 2014, Ngo and Kim 2014).Chitosan shows good performance in drug delivery and analgesia (Wang et al., 2002).Despite the enlarged knowledge of microbial pathogenesis and application of modern therapeutics, the morbidity and mortality associated with the microbial infections remain high (Dutta et al., 2010).Bacterial resistance to antibiotics has been addressed by looking for new antibiotics and modified antibiotics.In recent years considerable attention has established the antimicrobial activity of chitin, chitosan and their derivatives against different groups of microorganisms (Khanafari et al., 2008, Dutta et al., 2010, Yang et al., 2010), such as bacteria, yeast, and fungi.The antimicrobial activities of chitosan are greatly reliant on its physical characteristics, molecular weight (MW) and degree of deacetylation (% DDA).
Chitosan with a higher degree of deacetylation inclines to have a higher antimicrobial activity (Liu et al., 2001).In addition, higher antimicrobial effect against Escherichia coli is presented by water insoluble chitosan (Acharya et al., 2005).Furthermore, improvement of the antibacterial activity of chitosan by adding Jojoba liquid wax against Staphylococcus aureus and Bacillis subtilis, by using the agar disc diffusion method is previously reported (Osiris et al., 2013).
Many researchers (Chien and Chou 2006), from their obtained results, have declared that the molecular weight is one of the main factors affecting the antibacterial activity of chitosan.Also, chitosan antimicrobial activity of chitosan is dependent on both the chitosan and the microorganism used (Park and Kim 2010).
Ultraviolet (UV) radiation represents a part of a broad spectrum of wavelengths emitted from the sun.The Ozone layer absorbed most UV radiation while some of it gets through to reach the surface of the Earth.UV-radiation has positive and also negative applications.Although the wide spectrum of the advantages of UV is also harmful; any polymers (plastics, nylon, polystyrene, …etc) used in consumer items are broken down and/or lose strength due to exposure to UV-radiation.In addition, fabrics, furnishings and paintings need protection from UV to prevent color change or loss.Products made of natural polymer such as chitosan for biomedical applications need to be sterilized.For the sterilization of materials made of polymers, both UVradiation and high temperature can be used (Struszcyk 2002, Sionkowska et al., 2014).
Irradiation by ultraviolet, which possesses the energy sufficient to cleavage covalent bonds, results in formation of free radicals which they can initiate further reaction such as degradation and/or recombination reactions (Ramani and Rangananthaiah 2000).On other hand, the photolysis of polymer leads mainly to a random scission of chain backbone.Random homolitic scission of main-chain carbon-carbon bonds and photolysis or photodissociation of side groups are two major degradation reactions can occur.After irradiation, oligomer is formed which is mainly due to chain depolymerization after photolytic scission (Wasikiewicz et al., 2005).
UV-radiation as an accessory factor has been found to be an effective and efficient means for the accelerated degradation of chitosan treated with ozone.Therefore, the behavior of the UVradiation and ozone radiation is important on the development of chitosan in several fields, such as biomedical, cosmetic and industry ones.The structure of the degraded chitosan obtained by ozone treatment combined with ultraviolet radiation is characterized by FTIR (Mao et al., 2004, Wu et al., 2009).
The aim of the present work was to study the variation in the group coordination of chitosan in the FTIR reflectance in the range of 4000-450 cm -1 due to exposure to UV/ozone for three diverse intervals.The well diffusion method was processed in order to investigate the anti-bactericidal activities of chitosan exposed to UV/ozone against two bacterial strains Bacillus subtitles (Gram positive) and Escherichia coli (Gram-negative).

Material
Off-white Shrimp chitosan powder (CAS No: 9012-76-4) with average molecular weight of 900,000 to 1,000,000 Dalton and deacetylation above 90% and free of E. coli and Salmonella was supplied from Oxford Lab Chem.Mumbai, India.

UV/Ozone Treatment
Chitosan powder samples were exposed to UV/ozone for three episodes of 55, 110 and 220 minutes.High intensity, lowpressure mercury lamp without outer envelope -LRF 02971, 200 watt, 220 volt, 184.9 nm (Poland) was used as the UV/ozone source placed in a cubic box of dimensions 60 x 60 x 60 cm at National Institute for Standards, Giza, Egypt (Robert andFrancis 1996, Michael et al., 2004).Atomic oxygen is generated both when molecular oxygen is subjected to the184.9 nm radiation and when ozone is irradiated at 253.7 nm.The 253.7 nm radiation is absorbed by most hydrocarbons and also by ozone.The samples are placed around the source at a distance 20 cm.

Fourier Transform Infrared (FTIR)
The FTIR spectra of both the unexposed and the exposed chitosan samples were achieved using potassium bromide (KBr) pellet technique (Sankar et al., 2004, Kassai 2009, Smith 2011).Pellets are prepared by mixing 1 mg of the powdered sample with 100 mg of dried KBr powder.Mixing is carried out using a pestle and agate mortar.The mixture is then pressed using a special die at a pressure of 10,000 pounds per square inch to produce a pellet.The FTIR reflectance spectra were performed over the range of 4000-450 cm -1 at room temperature using a Bruker Vector 22 Spectrophotometer (Germany) with accuracy better than ±1% and a resolution of 4 cm −1 .

Antimicrobial Activity
The antibacterial activity of the unexposed and exposed chitosan to UV/ozone against two common bacterial strains: Bacillus subtilis ATCC6633 as Gram-positive and Escherichia coli ATCC25922 as Gram-negative, was carried out using the agar well diffusion method (Magaldi et al., 2004, Valgas et al., 2007).The medium is sterilized by autoclaving at 120 o C (15 lb/in 2 ).About 30 mL of the agar medium with the respective strains of bacteria is reassigned aseptically into each sterilized Petri plate.The agar plate surface was inoculated with 1 x 10 7 colony forming unit (CFU) for bacteria.The plates were left at room temperature for solidification.Then, a well with a diameter of 0.6 to 0.8 cm was punched aseptically using a sterile cork borer (wells are 2.0 cm apart from one another) and a volume (20-100 mL) of the antimicrobial agent at desired concentration is introduced into the well.Then, agar plates were incubated under suitable conditions at 37±2 o C for 24 h depending upon the test microorganism, under aerobic conditions.After incubation, confluent bacterial growth is detected.The standard disc 0.6 cm diameter with ciprofloxacin (50 μg/disc) is used as control for antibacterial activity.The diameter of the inhibition zone was measured around the well (in cm) using Vernier caliper.All tests are accomplished under sterile conditions in duplicates and repeated three times for each sample and the average diameter zone of inhibition is calculated.

Fourier Transform infrared (FTIR) Spectral Analyses
FTIR spectroscopy is one of the most common characterization methods for chitin and chitosan due its simplicity, relative instrument availability and independence of sample solubility (Ng et al., 2006).The FTIR reflectance spectra of chitosan before and after exposure to UV/ozone with frequency range of 4000-450 cm -1 were shown (Fig. 1).The chemical assignments for the unexposed and exposed chitosan were considered (Table 1).
It was noticed that the FTIR spectrum of unexposed chitosan designated the details of functional groups present in correlation with that of earlier reports (Kweon et al., 2001, Zheng et al., 2001, Pawlak and Mucha 2003, Qi et al., 2004, Pranoto et al., 2005, Zvezdova 2010, Hawary et al., 2011, Puvvada et al., 2012, Long 2013, Vimal et al., 2013, Kumari and Kumar-Rath 2014, Arafat et al., 2015) and was comparable to that of the exposed chitosan.The absorption bands of unexposed chitosan were matching to those of standard chitins.Different stretching vibration bands were observed in the range of 3439-2852cm -1 related to ν (N-H) in ν (NH 2 ) associated with primary amines.The band at 3439 cm -1 could be assigned to ν (N-H), ν (O-H) and ν (NH 2 ) which was present in chitosan in diverse amounts among which NH 2 groups being the least.The presence of methyl group in NHCOCH 3 , methylene group in CH 2 OH and methylene group in pyranose ring was evidenced by the corresponding stretching vibrations of these groups in the range of 2921-2852 cm -1 .The band at 1593 cm -1 had a higher intensity than that of 1659 cm -1 , which proposes effective degree of deacetylation.When chitin deacetylation happened, the band observed at 1659 cm -1 decreased, while a growth of another band at 1593 cm -1 occurred, designating the prevalence of NH 2 groups.When the same spectrum of chitosan after exposure to UV/ozone was observed, the band from 1500-1700 cm -1 was stressed, designating an intensification of the peak around 1593 cm -1 while, a reduction around 1659 cm -1 .These observations indicated the degree of deacetylation (DDA) (Zvezdova 2010, Kumari andKumar-Rath 2014).
The FTIR results showed that, there was a noticeable variation in the intensities of the reflectance bands and shifts in the band positions.This could be attributed to the change in the molecular configuration of chitosan that occurred due to the exposure to UV/ozone.In addition, the observed change in the spectral position of some bands after exposure to UV/ozone could be accredited to some of the sensitive monomer units of chitosan.
Using the FTIR spectrum attained, accurate DDA could be calculated using the Baxter, Dillon et al. formula (Baxter et al., 1992): A 1655 and A 3450 were the absorptions bands at a wavelength of 1655 and 3450 cm -1 , respectively.The induced change in the degree of deacetylation (%DDA) of the unexposed and exposed chitosan to UV/ozone for various exposure times were determined by FTIR and their percentage changes were revealed in Table 2.

Antibacterial Activity
The antibacterial activities of unexposed and exposed chitosan were experienced against two common bacterial strains: Bacillus subtilis (ATCC6633) as Gram-positive and Escherichia coli (ATCC25922) as Gram-negative.The antibacterial activity was articulated as inhibition zone diameters (IZD) measured in cm of samples against the pathological strains based on the well diffusion assay.The effect of exposure to UV/ozone for 55 minutes (as an example) on: B. subtilus (a) and E. coli (b) bacteria were shown (Fig. 2).The average, standard deviation and their percentage changes in inhibition zone diameters for unexposed and exposed chitosan were interpreted in Table 3.The results revealed that the inhibition zones diameters upsurge upon exposing to UV/ozone in both Gram positive and Gram negative bacteria.In addition, the results testified that the highest inhibition zone was achieved with chitosan exposed to UV/ozone for 55 minutes with very high significant inhibitions against B. subtilis and E. coli.Furthermore, the data emphasized that the exposure of chitosan to UV/ozone validated a high potential for enlightening the antibacterial activity.This coincides with the findings of Pranoto et al. (2005) who concluded that the antibacterial effect of chitosan occurred without migration of active agents.On other hand, several studies approved that an increase in the positive charge of chitosan binds it into the bacterial cell walls more intensely (Liu et al., 2006).Therefore, chitosan exposed to UV/ozone improves its antibacterial efficacy.Moreover, diffused antibacterial activity of chitosan, motivated the clearing zone of the bacterial growth.In addition, the cause of the inhibition of microbial cells by chitosan exposed to UV/ozone upsurges the interaction with anionic groups on the cell surface, due to its poly-cationic nature.This may motivate the formation of an impermeable layer around the cell, which prevents the conveyance of necessary solutes (Kamala et al., 2013).
From the obtained data, better efficacy by 55 minutes exposure to UV/ozone instead of the other two longer exposures (110 and 220 minutes) was observed.This may arise from the variation in the total number available states caused by UV/ozone according to the compromise between the degradation and crosslinking processes due to UV/ozone exposure (Chikwenze andNnabuchi 2010, Abd El-Kader et al., 2010).Also, this better efficacy may be attributed to the change of the molecular configuration by exposure with UV/ozone which leads to rupture of the bonds and formation of free radicals as a result of the degradation process.On other hand, by increasing the time of UV/ozone exposure up to 220 minutes, the cross-linking process was more pronounced and then, the obtained change may be due to the structural changes in the chitosan matrix.This means that both degradation and cross-linking processes were existed due to UV/ozone exposure and the degradation process was more pronounced at shorter exposure while cross-linking process was more significant at longer exposure time.

CONCLUSIONS
The present study delineated that the FTIR results pronounced variations in the intensities of the reflectance bands and shifts in their positions were detected.This could be attributed to changes in the molecular configuration of chitosan due to the exposure to UV/ozone.
In addition, the achieved data highlighted that the chitosan exposed to UV/ozone gained a prospective antibacterial activity.Furthermore, chitosan degradation by ozone treatment combined with ultraviolet radiation is a very promising technique that can be applied successfully.

Fig. 1 :
Fig. 1: Variations in FTIR spectra of unexposed and exposed chitosan to UV/ozone.

Table 1 :
Positions and assignments of unexposed and exposed chitosan to UV/ozone.

Table 2 :
Variation in the % degree of deacetylation (% DDA) of unexposed and exposed chitosan to UV/ozone by FTIR and their percentage exchange %Δ (%DDA).

Table 3 :
Average, standard deviations and their percentage variations of the inhibition zone diameters of chitosan against the microorganisms Bacillus subtilis and Escherichia coli.Cibrofloxacin (50 µg) used as standard for antibacterial activity.