COMPARISON OF THE PHYSICOCHEMICAL PROPERTIES
OF CHITOSAN EXTRACTED FROM SHRIMP SHELL
WASTE WITH DIFFERENT DEACETYLATION DEGREES
Aygul Kucukgulmez1,*, A Eslem Kadak2, Ladine Celik3, Afsin Farivar3, Mehmet Celik1
1Cukurova University, Faculty of Fisheries Department of Seafood Processing Technology, Adana, Turkey 2 Kastamonu University, Faculty of Fisheries, Kastamonu, Turkey
3Cukurova University, Faculty of Agriculture, Department of Animal Science, Adana, Turkey
ABSTRACT
In this study chitosan with different degrees of deacetylation have been extracted from deep sea pink shrimp (Parapenaeus longirostris) shells not evaluated and discarded as waste products to inves-tigate the effect of deacetylation on physicochemical properties of chitosan. In order to determine physi-cochemical characterization of the chitosans; mois-ture and ash contents, degree of deacetylation, mo-lecular weight, apparent viscosity, water and fat binding capacities, Fourier Transform Infrared Spec-troscopy, Scanning Electron Microscopy and X-ray diffraction analyses were applied. In addition, the physicochemical characteristics of the chitosan ex-tracted from P. longirostris shells were compared to the commercial chitosan. Low degree deacetylated chitosan exhibited a higher molecular weight, higher viscosity and higher water and fat binding capacities compared to the high degree deacetylated chitosan. Study findings are considered to be useful for the economic use of shrimp wastes in Turkey and light the way for future studies on other crustacean spe-cies.
KEYWORDS:
Chitosan, Deacetylation degree, Parapenaeus longirostris Physicochemical properties
INTRODUCTION
Chitin (poly- β-(1,4)-2-asetamide-2-deoxi-β-D-glucopyranose) is known as the most available bi-opolymer after cellulose in the world. Firstly pro-duced from fungi, in our day chitin is mostly ex-tracted from shellfish (crab, shrimp, lobster) and in-sect skeletons [1]. Chitosan (poly-[β-(1,4)-2-amino-2-deoxi-β-D- glucopyranose) is the deacetylated form of chitin and because of its polycationic struc-ture, it can be more easily dissolved compared to the chitin; and it has more superior features [2].
Chitosan has different reactive functional groups and the chemical modification of these groups enables chitosan to be a useful material in
several different fields[3]. It is commonly used in different sectors including especially food, chemis-try, biotechnology, agriculture, veterinary, cosmetic, medicine, dentistry, environmental protection, tex-tile, paper making and packaging [4]. Physicochem-ical properties of chitosan significantly affect the use of chitosan in all these sectors. These physicochem-ical properties can be ranged as deacetylation degree, molecular weight, viscosity, solubility, and colour. The most important one among these is “Deacetyla-tion Degree.”
Deacetylation degree is the removal of acetyl group from aminoacetyl groups which are found in the structure of chitin. Thereby, only amine group re-mains behind. Deacetylation degree affects chi-tosan’s physical, chemical, and biological qualities such as base and electrostatic characteristics, biodeg-radability, self-aggregation, and sorption properties [5]. Deacetylation degree varies in accordance with the source of chitin, extraction method, the duration of reaction, NaOH concentration, and the tempera-ture of reaction [6, 7]. Determination of the qualities of chitosan acquired in different deacetylation de-grees is significantly important in different usage ar-eas of chitosan. For this rar-eason, in the present study, chitosan with different degrees of deacetylation were prepared to investigate the effect of deacetylation on physicochemical properties of chitosan. In the char-acterization of the chitosan, deacetylation degree, moisture and ash content, molecular weight, appar-ent viscosity, water and fat binding capacity, fourier transform infrared spectroscopy, scanning electron microscopy and X-ray diffraction were measured. In addition, chitosan extracted from P. longirostris was compared to commercial chitosan.
MATERIALS AND METHODS
Materials. Fresh samples of deep sea pink
shrimp (Parapenaeus longirostris) wastes were col-lected from a local shrimp processing factory. Shells were completely separated from these wastes in la-boratory, washed in pure water and dried at 60ºC.
Two commercial chitosan (low and high deacetylation degree) of Sigma Chemical was used
as a control to compare with the extracted chitosan in this study.
Extraction of Chitosan. Chitosan was
ex-tracted by the method of Chang et al. [8]. Deprotein-ization and demineralDeprotein-ization steps were carried out with 2.5 N NaOH at 65 ºC for 6 hours and 1.7 N HCl at 25 ºC for 6 h, respectively. The chitin residue was treated with 9 vol. of hydrogen peroxide and dried at 90 ºC for 2 h. Two different deacetylated chitosan (low and high degree) was prepared by alkali treat-ment of chitin using % 50 (w/v) NaOH in distilled water at 120 ºC (50 and 240 minutes). The reactants were filtered, washed with deionized water to neutral pH and dried at 90 ºC for 2 h. LDEC: Low degree deacetylated extracted chitosan, HDEC: High degree deacetylated extracted chitosan, LDCC: Low degree deacetylated commercial chitosan, HDCC: High de-gree deacetylated commercial chitosan.
Characterization of Chitosan. (1) Deacetyla-tion Degree. DeacetylaDeacetyla-tion degree of chitosan was
determined by a potentiometric titration method. 250.0 mg portion of chitosan were dissolved in 10.0 ml of 0.30 M HCl and after being diluted to 50.0 ml with ultrapure water, it was titrated with 0.10 M NaOH [9].
(2) The moisture and ash contents. The
mois-ture was determined after drying samples for 24 h at 105ºC, and ash content was determined by heating at 530ºC for 20 h.
(3) Molecular Weight Determination. The
viscosity average molecular weight of chitosan was calculated from experimental intrinsic viscosity data by using the Mark-Houwink equation [10].
(4) Apparent Viscosity. Chitosan solution was
prepared in 1% (v/v) acetic acid at 1% (w/v) concen-tration on a moisture-free basis. Viscosity of chi-tosan was determined with an AMVn Automated Micro Viscometer (Anton Paar, Graz, Austria). Re-sults were averages of seven determinations and s were reported as centipoises units (cP).
(5) Water and fat binding capacity. Water
binding capacity (WBC) and fat binding capacities (FBC) of chitosan were measured using a modified method of Wang and Kinsella [11]. Water and fat absorption were carried out by weighing a centrifuge tube containing 0.5 g of sample, adding 10 ml of wa-ter and mixing for 1 min to disperse the sample and centrifuged at 3500 rpm for 25 min. After the super-natant was decanted, the tube was weighed again. The same process was applied for fat binding capac-ity.
(6) Fourier Transform Infrared Spectros-copy (FTIR). The infrared spectrum (IR) was
per-formed in a Perkin Elmer spectrometer (model 550 FT-IR/FIR/NIR Spectrometer Frontier, ATR), in the region 500-4000 cm-1. The spectrum was obtained using sample of chitosan powder, previously dried in oven for 24 hours at temperature of 90°C.
(7) Scanning Electron Microscopy (SEM).
SEM characterization was carried out using a Zeiss-Supra 55 type instruments. Prior to analysis, the dried chitosan powder was coated with platinum with automatic coating machine. Images of the sam-ples surfaces were recorded at different areas and magnifications.
(8) X-Ray Diffraction (XRD). X-ray
diffrac-tion data were collected on a Rigaku-SmartLab dif-fractometer the voltage and the current used were 40 kV and 30mA, and 2θ with a scan angle from 4 to 45, respectively. Chitosan was prepared by com-pressing it in the cassette sample holder without any adhesive substances.
RESULTS AND DISCUSSION
The results of the physicochemical parameters (deacetylation degree, moisture, crude ash, molecu-lar weight, apparent viscosity, water and fat binding capacities) of chitosan extracted from P. longirostris shells are shown in Table 1.
Deacetylation degree shows the amount of the number of deacetylated N-acetyl-D-glucosamine units in proportion to total unit number. Physico-chemical properties of chitosan change in accord-ance with the deacetylation degree and deacetylation degree can be regulated with regard to the place of use in industry. In the present study, the deacetyla-tion degree determined through the potentiometric titration method was measured as 72.86% in the low-degree group while it was determined to be 93.70% in the high deacetylation degree group. There are various methods available to increase or decrease the degree of deacetylation. In the deacetylation phase, NaOH concentration, and duration and temperature of the reaction are important factors on the deacety-lation degree.
No statistical difference was observed in the moisture (1.52-1.80%) and raw ash (0.12-0.18%) contents of the chitosan with low and high deacety-lation degree. High quality chitosan’s raw ash con-tent should be lower than 1% [12]. Hence, the mois-ture content of the both feamois-tured chitosan produced in this study is under this value. Similarly, Tajik et al. [12] determined the raw ash content of the chi-tosan acquired from Artemia to be between 0.19-0.51%.
TABLE 1
Physicochemical properties of chitosan extracted from P. longirostris with different deacetylation degrees
Deacetylation Degree
Parameters Low High
Deacetylation Degree (%)* 72.86±3.21 93.70±2.20
Moisture (%) 1.80±0.65 1.52±0.04
Crude ash (%) 0.12±0.01 0.18±0.07
Molecular weight (kDa)* 14.08±1.34 2.15±0.04
Apparent viscosity (cP)* 1450±141.42 150.00±43.58
Water binding capacity (%)* 808.17±10.44 654.16±23.32
Fat binding capacity (%)* 910.23±27.67 692.30±30.12
* indicates a significant difference between two groups (p<0.01)
FIGURE 1
FTIR Spectra of Chitosan (a) LDEC (b) HDEC (c) LDCC (d) HDCC
TABLE 2
FTIR bands of chitosan with different deacetylation degrees
Functional Group/Wavenumber a b c d
C=O Amide I band 1655 1647,53 1647,48 1647,99
C=O-C-N 1587,70 1577,19 1550,70 1587,80 CH2 vibration 1421 1420,20 1418,60 1416,30 CH-CH3 vibration 1375,49 1376,38 1375,02 1375,22 Amide bands 1319 1320,21 1309,70 1314,30 NH 1261 1263,30 1263,30 1263,30 C-O-C 1150,42 1149,99 1151,68 1150,80 C=O 1062,97 1059,73 1065,51 1062,80 C=O 1026,37 1026,55 1027,22 1027,17
Fingerprint region, SH, PO4 vibration 894,31 892,38 897,23 893,27 (a) LDEC (b) HDEC (c) LDCC (d) HDCC
In this study, it was detected that with the in-creasing deacetylation degree of the chitosan, there was a statistically important decrease in the viscosity value (p<0.01). Jeon et. al. [13] reported that the vis-cosity of the chitosan was closely related to the deacetylation duration; and in parallel with the pre-sent study’s results, chitosan had the highest viscos-ity in the shortest deacetylation duration. In parallel
with the viscosity values, molecular weight results also showed that chitosan with low deacetylation de-gree had higher values compared to the chitosan with high deacetylation degree. During chitosan produc-tion, several factors such as temperature rating, al-kali concentration, duration of reaction, duration of
chitin acquirement, particle size, chitin concentra-tion, and dissolved oxygen concentration may affect molecular weight [12].
Water and fat binding capacities of the chitosan group with high deacetylation degree were found to be lower (Table 1). Water and fat binding capacities of the both groups have shown similarities with the results of different studies carried out with different chitosan samples [12, 14, 15]. Similar to this study, Trung et al. [16] examined the functional qualities of chitosan with three different deacetylation degrees (75, 87, 96 %) in the same molecular weight (ap-proximately 810 kDa) and reported that the chitosan with the lowest deacetylation degree had the higher water binding capacity. All these physicochemical properties of chitosan vary depending upon the source of chitin, isolation method, deacetylation de-gree, the duration of being processed with sodium hydroxide, concentration, and the temperature dur-ing the process.
FT-IR spectrums of the chitosan with low and high deacetylation degree, which were acquired from P. longirostris, and the commercial chitosan are demonstrated in Figure 1; and the details of spec-trums are demonstrated in Table 2. As the deacety-lation degree of the chitosan acquired from shrimp shell increased, 1655 peak in the group with low
deacetylation feature was replaced by 1647 peak. In commercial chitosan groups, this value remained stable at 1647 peak. For both natural and commercial chitosan, it was seen in spectrum results that with the increase in the deacetylation degree, some functional groups moved away from the structure between 800-450 bands. As the deacetylation degree increased, 610 peak in the natural chitosan with low deacetyla-tion degree was observed on 617 band. While this peak was not observed in commercial chitosan with low deacetylation degree, it was observed on 610.97 band in the high commercial. It was determined that with the increase in deacetylation degree, this peak became evident. In the general sense, it was observed that there were changes in the intensity of peaks be-tween C=O Amide I band and Fingerprint region groups.
SEM pictures of the chitosan with different deacetylation degrees are presented in Figure 2. Chi-tosan’s fibrillary structures are seen in the images, and it can be said that the acquired chitosan has a lamellar structure. Although generally a spherical structure is observed, particles comprising uneven shapes are also seen. It was determined that the group with high deacetylation had a more fibrillary structure compared to the group with low deacetyla-tion.
FIGURE 2
Scanning Electron Microscopy (SEM) of Chitosan at 15.00 KX (a) LDEC (b) HDEC (c) LDCC (d) HDCC
FIGURE 3
X-Ray Diffraction Patterns of Chitosan (a) LDEC (b) HDEC (c) LDCC (d) HDCC
XRD results of chitosan are shown in Figure 3. XRD results of chitosan extracted shrimp shell show two strong peaks 10.8 and 20.37° (low deacetylated), 10.01 and 20.04° (high deacetylated), respectively. XRD results of commercial chitosan show two strong peaks 9.56 and 19.82° (low deacetylated), 10.56 and 19.89° (high deacetylated), respectively. While teta-10 was under 200 in chitosan samples with low deacetylation quality, teta-20 was meas-ured around 800. In the group with high deacetyla-tion, the value of teta 10 was determined to be around 400 and the value of teta 20 was determined to be higher than 1000. With the increase in deacety-lation, an increase was observed in both peaks.
In conclusion, with the development of shell-fish processing technology, the recycling of wastes has become an important issue. Shellfish wastes, which are not utilized in factories in our country, have constituted a great potential. The utilization of wastes is an important topic both for aquaculture in-dustry and public health. With the expansion in the usage possibilities of chitosan’s different forms in different industries, the use of productions with no side effects particularly for human health will be en-abled; and, it will be possible both to generate an
in-come economically and to prevent the environmen-tal pollution created by the wastes of processed shell-fish.
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Faculty of Fisheries Department of Seafood Processing Technology Adana – Turkey
