Antioxidant, antibacterial and shelf-life extension of chitosan extracted from the shells of shrimp (Fenneropenaeus indicus and Litopenaeus vannamei)
Sandip P. Gondake*, Santosh R. Kshirsagar, Sagar I. Shinde, Valmik S. Kapase Department of Chemistry, Dada Patil Mahavidyalaya, Karjat, Ahmednagar, Maharashtra, India
*Corresponding author: Email: [email protected]
Abstract
The present study aimed to show antioxidant, antibacterial and shelf life extension of chitosan of chitosan extracted from the shrimp (Fenneropenaeus indicus and Litopenaeus vannamei) shells. Additionally, Chitosan coating was evaluated in vegetables. It has been found that the antibacterial action of the chitosan coating solutions against gut pathogens is successful. From the results obtained, the operation of chitosan 2, isolated from the shells of Litopenaeus vannamei, was found to be more successful than that of Fenneropenaeus indicus chitosan. It should also be proposed for the pharmaceutical industry.
Introduction
Owing to biochemical, microbiological or physical modifications during post-mortem preparation, shrimp are extremely perishable, resulting in short commodity shelf life [1].
Common shrimp preserving strategies, such as cold drying, freezing and chilling, do not efficiently suppress spoilage. In addition, to increase the shelf life of foods, industrial preservatives such as antioxidants, chelating agents and antimicrobial compounds can be applied [2]. Researchers have been working on different natural products due to consumer appetite for preservative-free foods. Recently, there has been a growing interest in creating film-forming materials with antimicrobial properties that lead to enhancing food safety and shelf life. Edible coatings can increase the consistency of food products by avoiding lipid degradation, loss of protein functionality, odour removal, discoloration and lack of moisture. Chitosan is a polysaccharide and is the key ingredient of the crustacean exoskeletons produced by deacetylation of chitin. Chitosan has many benefits over other polymers, such as biocompatibility, biodegradability and non-toxicity [3].
Chitosan theoretically has a range of functional properties, including antimicrobial and antioxidant behaviours, film-forming capacities, texturizing and binding actions [4]. The major downside of chitosan, however, is its water vapour permeability, which limits its use as a self- standing packaging medium. Crosslinking is a way of changing the water resistance of chitosan by certain chemical therapies, however it has been reported that an improvement in crosslinking reduces the antimicrobial ability of chitosan films. Mixing them with another polysaccharide or protein is one of the successful methods to enhance the physical, mechanical and barrier properties of chitosan films. The film-forming ability of gelatin obtained by partial collagen degradation can be used as an outer surface cover to protect food against light and oxygen [5-8].
In order to increase consistency and prolong shelf life, some workers have researched chitosan- gelatin films and coatings for fish products [9-11].
Materials and methods
Collection and processing of sample
The fresh shrimps (F.indicus, L.vannamei) were collected from the nearby local fish market. The shrimp’s shells were collected by removing the exoskeleton separately including the uropod. The shells were washed with flowing water to remove the soil and edible matter. The shells were sun dried for 2-3 days. The dried samples of both the shrimp shells were grinded into a fine powder and kept in different containers for further analysis.
Extraction of chitosan
The chitosan can be extracted from the shrimp shells by three major steps. 10g of finely grounded samples were taken to carry out the following process.
Deproteinization
The dried shrimp shells powder were treated with 4%(1:20g/ml) NaOH solution for 24 hours at room temperature. After which the alkali solution were drained and the samples were repeatedly washed with distilled water until the pH becomes neutral. The samples were kept overnight at 60°C in the Hot air oven.
Demineralization
The deproteinised shrimp shell powder were demineralized by treating it with 4%(1:15g/ml) HCL at room temperature for 12 hours. The acid solution were drained off and the samples were washed with distilled water until the pH becomes neutral. The samples were kept overnight at 60°c in the hot air oven. The end product is Chitin.
Deacetylation
For deacetylation, the chitin were treated with a strong alkali (i.e) 50% NaOH solution for 3days at 42°c to convert it to chitosan. The alkali solution were drained off and the pH was adjusted to neutral by repeated washing it with distilled water. The chitosan were kept overnight in the Hot air oven at 60°c. The resultant is chitosan.
Verification of chitosan Solubility test
The quality of chitosan produced were checked by solubility test with 1% Acetic acid.
1gram of chitosan were added to 50ml of 1% acetic acid and kept in the BOD shaker for 24 hours.
DMSO test
For DMSO test, 7g of chitosan samples were added to 15ml of Dimethyl sulfoxide. It was kept in the shaker for 30minutes. Chitosan being organic, should completely dissolve in DMSO.
FTIR analysis
The FTIR spectra of chitosan samples were obtained using an infrared spectrometer (PerkinElmer Spectrum version 10.4.4). The chitosan powder were weighed 1mg and individually mixed with vaccum dried potassium bromide (KBr) and pressed into pellets by hydraulic press. The infrared spectra were obtained with the frequency range of 4000 - 400cmˉ¹ The degree of deacetylation of the chitosan samples were calculated using the formula:
DD% = A1655 × 100 A3450 1.33
Where A1655 and A3450 were the absorbance at 1655 cm-1 of the amide-I band as a measure of the N-acetyl group content and 3450 cm-1 of the hydroxyl band as an internal standard to correct for disc thickness. The factor '1.33' denoted the value of the ratio of A1655 / A3450 for fully N-acetylated chitosan.
Antioxidant activity
The antioxidant activity was tested using DPPH( 1,1-diphenyl-2-picrylhydrazyl) free radical scavenging assay. The chitosan samples at different concentrations(2µl,4µl,8µl,16µl,20µl) were mixed with 1ml of methanolic DPPH. They were incubated for 30mins at room temperature. A colour change from violet to pale yellow is noted due to the formation of stable non radical form of DPPH. The absorbance was measured at 517nm using UV spectrophotometer. The experiment were done in triplicates. The percentage of DPPH free radical quenching activity was determined using the following equation:
Radical-scavenging activity(%) = Absorbance of control - Absorbance of sample × 100 Absorbance of control
Chitosan coating
collection of vegetable samples
The vegetables were collected fresh from the nearby local market. The vegetables used for coating were Brassica oleracea (cauliflower), Solanum melongena (brinjal), Capsicum annum (Green chilli), Coccinia grandis (ivy gourd). They were washed and left aside for coating.
Preparation of the coating sample
The deacetylated chitosan samples were weighed for 1gram. Then 1% chitosan solutions were prepared by adding 1g of chitosan to 50ml of water with high speed magnetic stirrer, while
agitation 50ml of 2% acetic acid was added by continuous stirring at 60°C for 2 hours. After stirring, the solution were filtered through Whatmann filter paper to remove undissolved particles and stored at room temperature.
Application of the coating on vegetable samples
The vegetable samples were equally distributed as five per experiment. Each vegetable was dipped on the solutions and left to air dry for 15minutes and then dipped again for second coating. The non coated samples were kept as controls. The vegetables were stored at room temperature.
Determination of weight loss
To calculate the weight loss of the stored vegetables they were weighed on the first day and fifth day of storage and noted. The following equation were used to calculate the weight loss.
Weight loss(%) = DW × 100 FDW
(DW- weight of the fruit at concrete day of storage, FDW - weight of the fruit at first day) Antibacterial activity
The antibacterial activity of chitosan coating solutions were tested against four bacterial species with gram positive and gram negative strains (S.aureus, E.coli, E.faecalis, B.subtilus).
The antibacterial activity were tested by well diffusion method. The Muller Hinton agar was autoclaved, poured into sterile petriplates and allowed to solidify under the laminar air flow hood. The four bacterial strains were swabbed on the petriplates. Then the wells were made and variable concentrations (20µl, 40µl, 60µl, 80µl, 100µl ) of 1% chitosan coating solution were loaded. A well with 10µl of 1% acetic acid was used as a control. The plates were incubated at 37°C for 24 hours and then observed for antibacterial activity.
Results and Discussion Extraction of chitosan
The chitosan were extracted from the shells of Fenneropenaeus indicus and Litopenaeus vannamei by chemical method and the total yield were calculated (Figure 1). The chitosan extracted from the shells of F. indicus is named as Chitosan 1. The yield (%) were calculated to be 18.03 ± 0.51. Chitosan 2 were prepared from the shells of L . vannamei .The total yield(%) were calculated to be 39.05 ± 0.47.
Figure 1.Chitosan of F.indicus, L.vannamei and Commercial chitosan Verification of chitosan
The solubility test with 1% acetic acid chitosan 1 showed complete solubility, whereas the Chitosan 2 showed partially solubility. In DMSO test, both chitosan 1 & 2 were found to be completely dissolved as it is organic in nature. This confirms that the extracted product is chitosan. The yield percentage of chitosan 1 was found to be 18.03% whereas yield percentage of Chitosan 2 was recorded as 39.05 %. Which was found to be higher than the previous studies.
The percent yield was associated with the effectiveness in the removal of minerals and proteins present in them. The variation could be due to the different types of shrimp waste used and also the difference in the age of shrimps from which the sample was taken. The present findings showed that, the chitosan 1 was soluble in acetic acid whereas chitosan 2 was partially soluble. The solubility of chitosan in organic acids like acetic acid, lactic acid and formic acid is primarily due to the ease of protonation of the amino group in chitosan at low pH. Chitosan solubility is primarily associated with its molecular weight, DDA, chitosan source, pattern of substitution of its monomers i.e. N-acetyl-D-glucosamine and D-glucosamine, ratio and concentration between acid and chitosan.
Degree of deacetylation
The Fourier Transform Infrared Spectroscopy (FT-IR) is used to find the functional groups of the given sample by analyzing the peaks. The FT-IR graph is plotted for both the chitosan 1 & chitosan 2 and shown in Figure 2 and 3. The major absorption band is observed at 3450/cm in chitosan 1 which denotes the amino group (-NH2) which is the characteristic peak of chitin and chitosan. Further, chitosan 1 showed absorbance band at 1632, 1664, 1562, 2935, 3450, 1078. This shows the confirmation of the chitosan. The chitosan 2 showed absorbance band at 3467/cm and 1634/cm denotes the (-NH2) free amino group at C2 position of glucosamine, a major group of chitosan. The percentage for the degree of deacetylation of synthesized chitosan powder were determined by the peak at 1655/cm as measuring band and the peak at 3450/cm as reference band. Hence, the DD% of chitosan 1 is found to be 80.15%
The degree of deacetylation is an important characteristic of chitosan. The higher deacetylation degree denotes the better quality of chitosan. In this study, the degree of deacetylation was
calculated by FTIR analysis using PerkinElmer spectrum. The major bands on NH2 stretching, CH stretching, Amide C=O stretching were observed (Table 1 and 2).
Figure 2. FT-IR plot for chitosan 1
Figure 3. FT-IR plot for chitosan 2 Table. 1 Wavelength of main bands for chitosan 1
Sl.no Wavenumber cm-1 (exp)
Wavwnumber cm-1 (lit.) Absorption bond
1 3450 3300-3500 NH2 stretching
2 2935 2700-3300 CH stretching
3 1664 1650-1780 C=O stretching
4 1632 1550-1650 C=N stretching
5 1564 1500-1600 Ring stretching
6 1078 1050-1250 C-O stretching
Table 2. Wavelength of main bands for chitosan 2 S.no Wavenumber/cm
(exp)
Wavenumber /cm (lit.) Absorption band
1 3467 3300-3500 NH2 stretching
2 1634 1550-1650 C=N stretching
3 1553 1500-1600 Ring stretching
Antioxidant activity
The antioxidant activity of extracted chitosan was tested by free radical scavenging activity using DPPH. The scavenging activity was increased with increase in concentration of both the chitosan 1 and chitosan 2. The chitosan 2 showed better activity than chitosan 1 as it depends on the factors like degree of deacetylation, molecular weight etc. The results of free radical scavenging activity of chitosan 1 and chitosan 2 were given in Figure 4.
Figure 4. Free radical scavenging activity of chitosan 1 &chitosan 2 Chitosan coating
The vegetables are easily perishable hence the shelf extension property was tested using chitosan 1 & 2. On the 5th day of storage the control samples of ivy guard, brinjal, green chilli and cauliflower were highly spoiled, whereas both the chitosan 1 & 2 coated samples showed good results than the control samples. The control samples showed the signs of shrinking, ripening, decaying but the chitosan coated samples didn’t show such signs. For observation, the storage period of the chitosan coated samples was extended three days. On the 8th the observed samples showed partial spoilage but there is no signs of microbial and fungal growths were noted. As a parameter the samples were tested using weight loss. The weight loss in the
2µl 4µl 8µl 16µl 20µl
F. indicus 6.25 6.37 7.46 8.002 8.965
L. vannamei 3.18 4.271 10.1 10.95 17.26
0 5 10 15 20
% Activity
Concentration
DPPH
corresponding vegetable samples were measured, calculated and plotted in the Figure 5 and 6.
The chitosan 2 coating solution showed better results than chitosan1 coating solution. The present findings revealed that the scavenging activity of chitosan increased with increase in concentration for both chitosan 1 and 2. Chitosan eliminates various free radicals by the action of nitrogen on the C-2 position of the chitosan. The scavenging activity of chitosan may be due to the reaction between the free radicals and the residual free amino group to form stable macromolecule radicals by absorbing hydrogen ions from the solution.
Figure 5. Chitosan coatings on vegetable samples
Before storage After storage
Figure 6. Ivy Guard Antibacterial activity
The zones of inhibition test were performed in order to check the antibacterial activity of chitosan.. The zones of inhibitions were clearly observed on the plates against four gut pathogenic bacterial (Bacillus subtilus, Staphylococcus aureus, lEnterococcus faecalis, Escherichia coli) after 24 hours of incubation. Both the chitosan coating solution of chitosan 1 &
2 showed the antimicrobial activity with increase in concentration. The observed zone of inhibition were measured and mentioned in the Table 3. The present finding on the zone of inhibitions revealed that the Chitosan coating solution 1 extracted from the shells of F. indicus
Cauliflower Brinjal Ivy Guard Green Chilli
Control 31% 67% 72% 53%
Chitosan 1 44% 72% 79% 59%
Chitosan 2 61% 74% 83% 67%
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
Weight loss
Vegetable sample
showed highest activity on Bacillus subtilus and least activity on Enterococcus faecalis.
Whereas, the chitosan coating solution 2 extracted from the shells of L. vannamei showed highest activity on Escherichia coli and lowest on Bacillus subtilus. The inhibition activity of chitosan depends on the type of shells, concentration of alkali used in the extraction, molecular weight, degree of deacetylation and the concentration of the chitosan coating solutions.
Table 3. Inhibitory effect of chitosan 1 & 2
S.No Bacterial strains
Zones of inhibitions(mm) Chitosan (1) Chitosan (2) 1. Bacillus subtilus 14 ± 0.2 11.2 ± 0.3 2. Staphylococcus aureus 12.7± 0.3 14. 4 ± 0.2 3. Escherichia coli 13.2 ± 0.2 14.6 ± 0.2 4. Enterococcus faecalis 9.1 ± 0.4 11.7 ± 0.3 Conclusion
In the present study, the extracts of chitosan from the shrimp shell waste was analysed for antioxidant activity, antimicrobial activity and shelf life extension activity of vegetables.
Chitosan are biodegradable and biocompatible hence, it is suitable for pharmacological industry and the food industry. The degree of deacetylation is considered as an important characteristic of chitosan. It was observed that the higher the deacetylation degree, the higher the activity of chitosan. The applications of chitosan were dependent upon the deacetylation degree. The radical scavenging activity of the chitosan will be great helpfull in the replacement of synthetic additives. Chitosan act as a preservative material which will delay the ripening process by inhibiting the respiration rate in fruits. Chitosan was found to be an effective preservative against the spoiling of vegetables. Hence chitosan coatings can be suggested for storing and maintaining the quality of organic food materials. The antibacterial activity of the chitosan coating solutions against gut pathogens was found to be effective. From the obtained results, the activity of chitosan 2, extracted from the shells of Litopenaeus vannamei found to be more effective when compared to chitosan from Fenneropenaeus indicus. Hence, it can be suggested for pharmaceutical industry.
Therefore, the findings of the In vitro study revealed that the chitosan, a natural polymer extracted from the shrimp bio waste has immense industrial applications. The results also prove that recycling the bio waste to chitosan will reduce environmental pollution and will be helpful in the pharmaceutical industry and Food industry. Further, the study of chitosan can be employed in the field of agriculture as a biopesticide and as a preservative.
Acknowledgments
The authors are gratefully acknowledge the Central Instrumental Facility, Savitribai Phule, Pune University, Pune, India and DST - FIST sponsored Central Instrumentation Laboratory, Dada Patil Mahavidyalaya, Karjat Dist- Ahmednagar, India.
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