Lactic acid demineralization of shrimp shell and chitosan synthesis

 

Alewo Opuada AMEH*, Muhammed Tijani ISA, Mariam AYINLA, Samuel Okon BASSEY, and Blessing Mayowa OLATUNJI

 

Department of Chemical Engineering, Ahmadu Bello University, Zaria, Nigeria.

E-mails: aoameh@abu.edu.ng; mtisaz@yahoo.com; mariam2hot@gmail.com; okonbassey56@gmail.com; olatunji.blessing@yahoo.com

* Corresponding author, phone: +2348035600744

 

 

Abstract

The use of lactic acid was compared to hydrochloric acid for shrimp shell demineralization in chitosan synthesis. Five different acid concentrations were considered for the study: 1.5M, 3.0M, 4.5M, 6.0M and 7.5M. After demineralization, the shrimp shell were deproteinized and subsequently deacetylated to produce chitosan using sodium hydroxide solution. The synthesized chitosan samples were characterized using solubility, FTIR, SEM, XRD and viscosity. The SEM, FTIR and XRD analysis indicated that chitosan was synthesized with a high degree of deacetylation (83.182.11 when lactic acid was used and 84.25.00 when HCl was used). The degree of deacetylation and the molecular weight of the chitosan samples were also estimated. ANOVA analysis (at 95% confidence interval) indicated that acid type and concentration did not significantly affect the solubility, degree of deacetylation, viscosity and molecular weight of the chitosan within the range considered.

Keywords

Chitosan; Demineralization; Lactic acid; Shrimp

 

 

Introduction

 

Chitin is a natural polysaccharide composed of N-acetyl-D-glucosamine units and is the second most abundant biopolymer after cellulose [1]. It occurs as a principal structural polymer in the covering of insects and crustaceans and in the cell walls of many fungi. Chitin and chitosan have also been prepared from fish scale [2]. Chitin composition may range from trace quantities up to about 40% of the body weight of the organism [3]. In shrimp processing, between 40 and 50% of the total mass ends up as waste. Crustacean shell waste consists mainly of 30-40% protein, 30-50% calcium carbonate, and 2030% chitin [4], though these may vary with species and location [5].

Chitosan is a unique natural biopolymer which is commercially derived from chitin and has found numerous applications in food, agriculture, cosmetic, biomedicine, textile, water treatment and pharmaceuticals [6].

In the preparation of chitin from natural sources, chemical treatment step to remove calcium carbonate (demineralization) and protein (deproteinization) was usually required. Hydrochloric acid seemed to be the preferred reagent for demineralization [7]. However, the harsh nature of the chemical treatments could have resulted in an anomerization and hydrolytic effects on the chitin structure [8]. The molecular weight decrease of chitin and chitosan was associated with the use of chemicals for pretreatment [9]. Waste treatment was also a fundamental drawback of the process. As such, substitution with milder and environment-friendly chemicals continues to gain attention [10, 11]. Furthermore, the use of organic acids may have had other benefits such as the fact that they might have been produced from low cost biomass and the resulted organic salts from the demineralization process could have found other applications [12]. The objective of this manuscript was to synthesize chitosan using similar concentrations of lactic acid and hydrochloric acid and investigate the effect of the acid treatments on chitosan property.

 

 

Material and method

 

Shrimp was obtained from Samaru market and taken to the Department of Biological Science, Ahmadu Bello University for identification. The exoskeleton of the shrimp was manually removed, washed, dried, and ground to pass through a 250m sieve. Lactic acid (AnalaR, BDH was procured and prepared into five concentrations (1.5, 3.0, 4.5, 6.0, and 7.5M).

8.57ml of 1.5M lactic acid solution was introduced into a conical flask (250ml) with stirring on a magnetic stirrer at room temperature (≈ 27C). 10g of the prepared shrimp shells were quickly introduced and stirred for 6 hours after which the content of the flask was quickly filtered and washed with deionized water until neutrality as determined using a pH meter (Kent EIL 7055). The demineralized samples were dried and weighed in an oven.

The entire procedure was then repeated for the other lactic acid concentrations of 3.0, 4.5, 6.0 and 7.5M after which similar concentrations and quantities of hydrochloric acid were used to demineralize the shrimp shells. The resulting samples after demineralization were weighed and treated in 0.62M Sodium hydroxide solution at a ratio of solute to solvent of 1:10 (w/v) for 16 hours in a 250ml conical flask at ambient temperature (27C) for 16 hours to dissolve the proteins and sugars thus isolating the crude chitin.

The samples were then washed thoroughly with deionized water until neutrality then filtered and dried to constant weight using an oven. The extracted chitin obtained from the process of deproteination was weighed and treated in 25M NaOH solution (1:10w/v) for 20 hours at a temperature of 115C in an autoclave in order to dissolve the remaining proteins and remove acetyl groups from the chitin.

The extracted chitosan was then washed properly with deionized water until neutrality then filtered and dried to constant weight using an oven.

0.2g of dried chitosan samples obtained from Acetic acid demineralization and HCl demineralization was weighed and dissolved in 1% acetic acid followed by mild stirring and then filtered. Solubility was computed as the fraction that dissolved.

The viscosity of the solution of the various samples obtained by dissolving 0.2 g of the dried chitosan in 1% acetic acid was measured using a viscometer.

The molecular weights of the various samples were determined using the Mark-Houwink equation:

[η] = KM

(1)

where M is the viscosity average molecular weight; K and a are constants, whose values depend on the polymer type and the chosen solvent. As shown in [13] for chitosan and the solvent 0.1M acetic acid these constants are 3.5104 and 0.76, respectively, and they do not depend on the deacetylation degree. In addition, [η] is intrinsic viscosity.

The degree of deacetylation of the various chitosan samples obtained was determined using relationship [14]:

DD = 118.883- [40.1647(A1655/A3450)]

(2)

where A1655 and A3450 are absorbance bands at 1655 and 3450 from FTIR spectra.

The structural differences in the synthesized chitosan was taken for Fourier transform infrared spectroscopy analysis (8400S spectrophotometer, Shimadzu), Scanning Electron Microscopy (JEOL JSM 630 J) and X-Ray Diffraction (X'Pert PRO MRD XL) analysis.

 

 

Results and Discussion

 

Table 1 shows the yield of Chitosan obtained from 10g of dried shrimp shell powder using the various acid demineralization.

 

Table 1. Effect of demineralizing acid type and concentration on the yield of chitosan

Acid concentration (M)

Yield for Lactic acid demineralization, %

Yield for HCl demineralization, %

1.5

21

14

3.0

15

19

4.5

12

11

6.0

11

11

7.5

14

13

 

The results of chitosan yield of Table 1 were within reported values of 15-20% for crabs (chinoecetes opilio) and shrimps (pandalus borealis) chitosan [15]. Statistical analysis (ANOVA at a confidence interval of 0.05) gave P-values greater than 0.05 indicating that acid type and concentration did not significantly affect the yield of chitosan.

Tables 2 and 3 present the properties of extracted chitosan. From Tables 2 and 3, the solubility of the extracted chitosan from the demineralization of both lactic acid and Hydrochloric Acid at the five different concentrations was within the range of 35-45% in 1% acetic acid solution. ANOVA gave P-values of 0.161458 and 0.771715 which indicates that the solubility of the extracted chitosan is independent of the type acid used, and the concentration of the acid used in the demineralization of extracted chitosan respectively.

 

Table 2. Properties of chitosan synthesized after lactic acid demineralization

Concentration (M)

Solubility (%)

Viscosity (cps)

Molecular weight (g/mol)

1.5

25

2.5

1.18 x 105

3.0

30

3.0

1.50 x 105

4.5

15

2.7

1.30 x 105

6.0

15

1.9

0.82 x 105

7.5

20

2.0

0.88 x 105

 

Table 3. Properties of chitosan synthesized after Hydrochloric acid demineralization

Concentration (M)

Solubility (%)

Viscosity(cps)

Molecular weight (g/mol)

1.5

20

2.0

0.88 x 105

3.0

25

4.5

2.55 x 105

4.5

35

3.0

1.50 x 105

6.0

30

2.5

1.18 x 105

7.5

45

3.0

1.50 x 105

 

The various samples showed low viscosity using the viscometer within the range of 3.0 - 3.5 cps, this low viscosity is attributed to the poor solubility of the extracted chitosan in 1% solution of acetic acid. However, the viscosity of the extracted chitosan was not significantly affected by the acid used in the demineralization process and the concentration of acid.

Using the Mark-Houwink equation the various molecular weight of the samples was calculated within the range of 1.5 105 1.83 105g/mol though within the values reported by [15]. However, several factors during production, including high temperature, concentration of alkali, reaction time, previous treatment of the chitin, particle size, chitin concentration, dissolved oxygen concentration and shear stress may influence the Molecular weight of chitosan.

Figures 1 and 2 present typical FTIR spectra obtained. The sample of FTIR spectra of synthesized chitosan presented in Figure 1 and Figure 2 shows the bands typical of all the chitosan samples produced using both lactic acid and HCl demineralization. The spectra indicate the presence of absorption band observed between 1220 and 1020 cm-1, which represents the free amino group (-NH2) at C2 position of glucosamine, a major group present in chitosan.

Figure 1. FTIR spectra of extracted chitosan after demineralization using 3.0M Lactic Acid

Figure 2. FTIR spectra of extracted chitosan after demineralization using 4.5M HCl

 

Furthermore, the sample showed the absorption bands for the free amino group between 1026 and 1259 cm-1 while the peak at 1374 cm-1 represents the C-O stretching of primary alcoholic group (-CH2 - OH). The absorbance bands of 3268, 2930,2878,1563, and 1418 cm-1 indicated the N-H stretching, symmetric CH3 stretching and asymmetric CH2 stretching, CH stretching, C=O stretching in secondary amide (amide I) and CN stretching in secondary amide (amide II) respectively. Absorbance bands were observed at 3283, 2921, 2865,1643,1552,1421, 1022, 893and 752 cm-1 which confirms the structure of chitosan. Table 4 shows the various degree of deacetylation of the chitosan derived after Lactic acid and HCl demineralization using various acid concentrations.

Having confirmed that the synthesized samples contained chitosan using FTIR, the degree of deacetylation (DD) was calculated using Equation (2).The values obtained were within 83.182.11 when lactic acid was used and 84.25.00 when HCl was used. Demineralization using 3.0M and 4.5M gave the highest DDs for lactic acid demineralization (86.2%) and HCl demineralization (89.2%) respectively. However, statistical analysis using ANOVA indicated that the effects of acid type and concentration did not significantly affect the DD as they recorded P-values of 0.97 and 0.32 respectively (at a = 0.05).

The DD is an important parameter affecting solubility, chemical reactivity, and biodegradability. Depending on the source and preparation procedure, DD may range from 30% to 95 % [16]. It is rare however to have the production of chitosan with 100% degree of deacetylation. Therefore, commercial chitosan with various degree of deacetylation in the range of 7585% is commonly found.

Table 4 compares the degree of deacetylation for the treatment methods used. XRD patterns of extracted chitosan are illustrated in Figure 3.

 

Table 4. Chitosan Degree of Deacetylation (DD)

Concentration (M)

Lactic Acid demineralization

HCl demineralization

1.5

80.5

84.4

3.0

86.2

79.2

4.5

82.1

89.4

6.0

83.7

79.1

7.5

83.4

88.9

 

Figure 3. XRD Diffractogram of prepared chitosan

 

XRD patterns of extracted chitosan are illustrated in Figure 3, indicating a striking similarity between the lactic acid and the hydrochloric treated samples. The XRD pattern of chitosan exhibits broad diffraction peaks at 2θ = 10 and 21 which are typical fingerprints of semi-crystalline chitosan [17]. Yen and Mau [18], found that fungal chitosan showed two crystalline reflections at 9.7 and 19.9. It has been reported that the XRD patterns of shrimp chitosan showed two major characteristic peaks at 2θ = 9.9-10.7 and 19.8-20.7 [19]. It is also reported that the two characteristic crystalline peaks with slightly fluctuated diffraction angles found in the XRD patterns indicated that two types of α- and γ-chitosan exhibited comparable degree of crystallinity and had two consistent peaks of 9-10 and 19-20.

Figure 4 and 5 presents the SEM micrographs for the treatment methods used.

C:\Users\BASSEY\Desktop\research project\Dr Ameh 2\LACTIC0001.jpg

Figure 4. The SEM Morphology of extracted chitosan after 3.0M lactic acid demineralization

C:\Users\BASSEY\Desktop\research project\Dr Ameh 2\1.0M HCL0001.jpg

Figure 5. The SEM Morphology of extracted chitosan after 4.5M Hydrochloric acid demineralization

 

Figure 4 and 5 presents the SEM micrographs illustrating the morphology of chitosan prepared after lactic acid and hydrochloric acid demineralization respectively. Under the electron microscopic examination, chitosan showed non homogenous and non-smooth surface with straps and shrinkage which corresponds to the SEM morphology of extracted chitosan [20].

 

 

Conclusions

 

The following conclusion may be drawn from the results of this work: chitosan was synthesized from shrimp exoskeleton after demineralization using lactic acid and hydrochloric acid. The properties of the synthesized chitosan where found to be independent of acid type and concentration as indicated by ANOVA analysis. Lactic acid which is friendlier to the environment can replace hydrochloric acid in chitosan synthesis.

 

 

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