IJERD – International Journal of Environmental and Rural Development (2021) 12-2

Research article
erd

Effects of Chemical Extraction Methods on Physicochemical
Properties of Shrimp Chitosan
SOCHEATH TONG*Division of Research and Extension, Royal University of Agriculture, Phnom Penh, Cambodia
Email: tongsocheath@yahoo.com

BORARIN BUNTONGDivision of Research and Extension, Royal University of Agriculture, Phnom Penh, Cambodia

LAIKONG SOPHAL
Division of Research and Extension, Royal University of Agriculture, Phnom Penh, Cambodia

SOKMEAN VANN
Division of Research and Extension, Royal University of Agriculture, Phnom Penh, Cambodia

ANTONIO ACEDO JR.
Institute of Crop Science, University of the Philippines Los Banos, Laguna, Philippines
Received 13 July 2021

Accepted 18 October 2021(*Corresponding Author)

Abstract Chitosan extraction methods have not been applied and optimized in Cambodia
whose seafood industry produced tons of shrimp shell waste yearly. This study adopted thechemical extraction method and optimized the deacetylation step from chitin to chitosan
using different concentrations of sodium hydroxide (NaOH). Shrimp waste (exoskeleton) wassequentially treated with NaOH (3.5%) for deproteination at 80oC; HCl (4%) for
demineralization at 80oC; NaClO (0.315%) for decolorization at ambient temperature, andNaOH (40%, 50%, 60%) at 120oC for deacetylation. Chitosan yield, moisture content, total
ash, lipid, fiber, solubility, nitrogen content, viscosity, and degree of deacetylation weredetermined. The commercial chitosan bought from a local market was included forcomparison. NaOH at 50% was found to be the optimum concentration for deacetylation
based on increased solubility (96.27%), reduced ash content (0.56%) and increased degree ofdeacetylation (83.23) as compared to that of 40% NaOH (93.61% solubility, 1.25% ashcontent, and 74.45 degree of deacetylation) though chitosan yield in the former (20.59%) waslower than that in the latter (25.23%). Increasing the NaOH level to 60% had no significant
advantage. Lipid and fiber contents were not significantly affected. The characteristics ofchitosan extracted with 50% NaOH at deacetylation stage were comparable to that of thecommercial chitosan.
Keywords shrimp shell, chitin, chitosan, deacetylation, NaOH concentration

INTRODUCTION
Cambodia’s seafood industry has long been faced with waste disposal problem, mainly shrimp shells
from factories producing dried shrimps for local and export markets. Habitually, seafood waste isburned, land filled, dumped at sea or left to get spoiled. This has negative impact on the environment,
biodiversity and human health. A sustainable solution is to transform shrimp shell waste into highvalue products, particularly chitin and chitosan which have multiple applications in agriculture, foodand beverage, medical, pharmaceutical. cosmetics and personal care, textile, environment, health,and water sectors. Global demand for chitin and chitosan is rapidly growing and is projected at
281,700 metric tons in 2027 (https://www.strategyr.com/market-report-chitin-and-chitosanderivatives-forecasts-global-industry-analysts-inc.asp). However, global supply may remain short byⒸ ISERD170

IJERD – International Journal of Environmental and Rural Development (2021) 12-2

over 50% of demand as in 2022 projections of global demand of 155,500 MT against supply of only
70,000 MT.
Chitin, a crystalline polysaccharide with high molecular weight, is the second most abundantorganic compound after cellulose (Rinaudo, 2006; Kumari et al., 2015). It is considered as a natural
fiber that is found in shells of marine animals, such as shrimps and lobsters, insect shells and in cell
walls of several fungi and yeasts. In its structure, chitin is often linked to the other major constituentsof the carapace, forming covalent bonds with 30-40% proteins and a complex matrix containing 3050% calcium and phosphate carbonate and 20-30% of chitin. Extraction of chitin from shrimp shells
involves at least two steps: deproteinization which removes proteins and undesired materials such aspigments and is often performed with dilute NaOH solutions, followed by demineralization whichremoves all minerals (e.g. CaCO3) by using dilute HCl solutions. A decolorization step can be added
to remove pigments and produce white product. The chitin produced is further processed by partial
deacetylation to produce chitosan. Chitosan is a cationic semi-crystalline linear aminopolysaccharide of 1-4 linked N-acetyl glucosamine and glucosamine units; a white, hard, inelasticand nitrogenous polysaccharide (Cheba, 2011). It has three reactive functional groups, amine,primary and secondary hydroxyls that offer potential for covalent and ionic modifications allowing
the improvement of its mechanical and biological properties. In addition, chitosan is a biodegradable,
nonallergenic, biocompatible, antimicrobial, renewable, nontoxic product. Both chitosan and chitin
have enormous economic value because of their flexible biological properties as well as their highnitrogen content (6.89%) as compared to synthetically substitute cellulose (1.25%). However, thecrystallinity and insolubility of chitin demote its commercial applications.
The degree of deacetylation of chitosan ranges from 56% to 99% with an average of 80%,depending on the crustacean species and the preparation methods (No and Lee, 1995; No et al., 2000).
Chitin with a degree of deacetylation greater than 50% is generally known as chitosan (Younes et al.
2014). In the deacetylation of chitin, several factors can influence the characteristics of resulting
chitosan product. Rege and Block (1999) found that temperature and processing time were the mostsignificant factors having noteworthy impact on degree of deacetylation and molecular weight. Tsaihand Chen (2003) concluded that molecular weight and deacetylation of chitosan are principally
influenced by the concentration of NaOH, temperature, duration of reaction and recurrence ofalkaline treatment steps.
OBJECTIVE
This study is a first attempt to harness available experience elsewhere on the chemical extraction of
chitosan for shrimp shell waste of Cambodia’s seafood industry and determined the effects ofdifferent NaOH concentrations during the deacetylation step producing chitosan from chitin.
METHODOLOGY
Chitosan Extraction
Figure 1 shows the process flow for chitosan extraction. Shrimp shell waste was collected from alocal dried shrimp factory in Sihanoukville province, Cambodia, and brought to the Faculty of AgroIndustry, Royal University of Agriculture, Phnom Penh, Cambodia. The shells were properly washedwith clean water prior to chitosan extraction which followed the procedures of Divya et al (2014)
and Puvvada and Vankayalapati (2012).
The shrimp shells were deproteinized with 3.5% NaOH at 80oC for 3 hours with constantstirring at a solid to solvent ratio of 1:10 (w/v). Samples were then filtered under vacuum, and thefiltrate was washed with tap water for 30 minutes and oven-dried.
Demineralization was performed by washing the deproteinized shells with 4% HCl for 4 hours
at ambient temperature with a solid to solvent ratio of 1:10 (w/v), then filtered under vacuum. The
filtrate was washed for 30 min with tap water and oven-dried.
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The chitin produced was decolorized with 0.315% sodium hypochlorite for 15 min; with a solid
to solvent ratio of 1:10 (w/v), followed by washing in tap water and drying under vacuum for 2-3hours until the powder was crispy.
Deacetylation of chitin to produce chitosan was done using 40, 50 or 60% NaOH for 3 hours at120oC with a solid to solvent ratio of 1:10 (w/v). The resulting chitosan was washed to neutrality inrunning tap water, rinsed with distilled water, filtered, and dried at 60oC for 24 hr in the oven.
Chitosan yield was calculated by comparing the weight measurements of the raw material to thechitosan obtained after treatment (Kiruba et al., 2013).

Fig. 1 Process flow for the extraction of Chitosan from shrimp shell waste
Proximate Analysis
Moisture, fiber, fat and nitrogen contents were analyzed following the AOAC (2001, 2005) methods.
Total ash content was determined by the method of the Food Safety and Standards Authority of India(2012). Commercial chitosan (Sigma) was included in all relevant analysis as control.
Degree of Deacetylation
The titration method of Czechowska-biskup et al (2012) was followed. Dried chitosan (0.2 g) wasdissolved in 20 ml 0.1 M hydrochloric acid and 25 ml deionized water. After 30 minutes continuous
stirring, next portion of deionized water (25 ml) was added followed by stirring for 30 minutes. Whenchitosan was completely dissolved, solution was titrated with a 0.1N sodium hydroxide (NaOH)
solution using automatic burette (0.01 ml accuracy). Degree of deacetylation (DA) of chitosan was
calculated using formula (1):𝑉 −𝑉

21
𝐷𝐴[%] = 2.03 × 𝑚+0.0042(𝑉 −𝑉 )
2(1)1where m: weight of sample, V1, V2: volumes of 0.1 N NaOH corresponding to the deflection points,
2.03: coefficient resulting from the molecular weight of chitin monomer unit, 0.0042: coefficientresulting from the difference between molecular weights of chitin and chitosan monomer units.
Solubility
Solubility of chitosan was determined by placing chitosan powder (0.1 g in triplicate) into acentrifuge tube (known weight) then dissolving in 10 ml of 1% acetic acid for 30 min using an
incubator shaker operating at 240 rpm and 25oC. The solution was then immersed in a boiling water
bath for 10 minutes, cooled to room temperature and centrifuged at 10,000 rpm for 10 min. TheⒸ ISERD172

IJERD – International Journal of Environmental and Rural Development (2021) 12-2supernatant was decanted. The undissolved particles were washed in distilled water (25 ml) thenagain centrifuged a 10,000 rpm. The supernatant was removed and the undissolved pellets dried at
60oC for 24 hours. The dried pellets were weighed and the percentage solubility was calculated asfollows:[(Initial weight of tube + chitosan) - (Final weight of tube + chitosan)] x 100(Initial weight of tube + chitosan) – (Initial weight of tube)(2)

Viscosity
Viscosity of chitosan was determined with a viscometer. Chitosan solution was prepared in 1% acetic
acid at a 1% concentration on a dry basis. Measurement was made in duplicate using a No. 5 spindle
at 50 rpm on solutions at 25oC with values reported in centipoises (cPs) unit.
Statistical Analysis
A completely randomized design with three replications was used. Data were subjected to analysis
of variance and treatment mean comparison by least significant difference (LSD) test using the SPSS
Statistical Package.
RESULTS AND DISCUSSION
In general, higher deacetylation degree increases the better solubility and viscosity of chitosan, thebetter chitosan quality. Deacetylation with 40% NaOH resulted in highest chitosan yield of about
25.2% (Table 1). Increasing the concentration of NaOH to 50-60% significantly decreased chitosan
yield to about 20.6%. However, the degree of deacetylation was lower with 40% NaOH (74%) thanwith 50-60% NaOH (83%), the latter was comparable to that of commercial chitosan. Similarly,
deacetylation with 50-60% NaOH significantly increased the solubility and viscosity of chitosan
which were comparable to that of the commercial chitosan. Chitosan produced using 40% NaOH had
the lowest solubility and viscosity. Chitosan produced with 50% NaOH had moisture, ash, fiber andnitrogen contents which were statistically comparable to that of the commercial chitosan (Table 2).
With 40% NaOH, the chitosan produced had significantly higher ash and fiber contents and lower
nitrogen content than the commercial control. NaOH at 60% had similar effects as 50% NaOH exceptthat it had the lowest moisture content among treatments. Total fat content did not vary with treatment
and ranged from 1.12-1.49%
Table 1 Physicochemical characteristics of chitosan extracted from shrimp shells
using different NaOCl concentrations for deacetylation
NaOH concentration
Commercial chitosan
40% NaOH
50% NaOH
60% NaOH
Probability
LSD (5%)CV (%)

Yield of chitosan(%)
25.23a20.59b
20.63b
**
0.882
2.00

Degree ofdeacetylation
87.38a74.45b83.23a83.35a
**
5.342
3.00

Solubility (%)

Viscosity (log mPa/s)

95.65a93.61b96.27a96.29a
**
0.287
2.00

3.17b
2.84c
3.20a
3.18ab
**
0.022
0.00

Mean separation within columns by LSD, 5%. a, b, c Duncan's multiple range test

The results indicate that 50% NaOH was the optimum concentration for deacetylation of chitin
to produce chitosan. The chitosan produced had degree of deacetylation, solubility and viscosity of
83.23%, 96.27% and 3.20 log mPa/s, respectively, and proximate composition of 11% moisture
content, 0.56% total ash, 1.33% total fat, 1.47% total fiber and 7.29% nitrogen content which wereⒸ ISERD173

IJERD – International Journal of Environmental and Rural Development (2021) 12-2

statistically similar to that of commercial chitosan. Previous studies have successfully extracted
chitosan from shrimp shell waste (e.g. Kandile et al., 2018; Nacer et al., 2019; Islam et al., 2020;
Rasweefali et al., 2021) with similar results as the present study except for the lower chitosan yieldobtained (Divya et al., 2014; Puvvada and Vankayalapati, 2012). Using lower NaOH concentration
for deacetylation may increase yield of chitosan but other physicochemical properties werecompromised. On the other hand, higher NaOH concentration gave no added benefit and may only
increase the cost of extraction.
Table 2 Proximate analysis of chitosan extracted from shrimp shellsusing different NaOCl concentrations for deacetylation
NaOH concentration
Commercial Chitosan
40% NaOH
50% NaOH
60% NaOH
Probability
LSD (5%)CV (%)

Moisture content(%)
10.53ab11.78a11.00b
10.14c
**
0.208
9.00

Total ash(%)
0.59b
1.25a
0.56b
0.55b
**
0.132
10.00

Total fat
(%)
1.12
1.16
1.33
1.49
ns
0.365
13.00

Fiber (%)
1.33b
1.78a
1.47ab
1.36b
**
1.563
1.00

Nitrogencontent (%)
7.12%a
6.12b
7.29a
7.34a
**
0.287
0.00

Mean separation within columns by LSD, 5%. a, b, c Duncan's multiple range test

CONCLUSION
Deacetylation is the final and critical stage to produce chitosan from chitin, both of which weresuccessfully extracted in this research. NaOH at 50% was optimum for the deacetylation step. The
physicochemical properties of chitosan extracted with 50% NaOH were comparable to that of thecommercial chitosan. Future research could delve into manipulation of other deacetylation conditions(e.g. temperature and duration of deacetylation) that could increase the yield of chitosan without
affecting the physicochemical characteristics.
ACKNOWLEDGEMENTS
This research was funded under the Higher Education Improvement Project (HEIP). International
Development Association (IDA) CREDIT No: 6221-KH, with support provided by the Royal
Government of Cambodia (RGC).
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