Preparation of Water-Soluble Colloidal Chitin (WSCC) from Prawn Waste and Its Characterization

Abstract

Chitin, the shell material of prawn, is a biodegradable polymer and environmentally biocompatible with low toxicity. Chitosan is the deacetylated form of chitin, which consists of poly-D-glucosamine units with no or few N-acetyl-D-glucosamine units. Commercial applications of these natural polymers are increasing in various sectors. Therefore, in addition to the environmental benefit, it may be economical to recover chitin from prawn waste. Chitosan is soluble in various organic acids, solvents and water. The poor solubility of chitin is the major limiting factor in its use in industrial applications. Number of studies have investigated to overcome the solubility problem of chitin. This research focuses on a new way of developing water-soluble colloidal chitin (WSCC) from prawn waste and investigates its fundamental rheological and antibacterial properties. WSCC films studied during this research may be used in food packaging or in medical applications. The use of WSCC biodegradable films will protect the environment in the future and will be an effective alternative to plastics that threatens the environment. The antibacterial study may be applied in pharmaceutical, medical and food packaging and coating applications. This research was conducted at the University of New South Wales, Australia in 2008.

1. Introduction

Pollution of soil, air and water contributed to environmental deterioration; its control is necessary. Plastics have become part of our lives; the treatment of waste plastics has become a serious problem because of the difficulty of land reclamation and disposal by incineration [1]. Recent interests have focused mainly on biodegradable plastics that are biocompatible to the environment [2]. Chitin and chitosan are examples of biodegradable, biorenewable and biofunctional polymers derived from seafood processing waste [3, 4, 5, 6, 7, 8]. Therefore, in addition to the environmental benefit, it may be economical to recover chitin from prawn waste. The poor solubility of chitin is the major limiting factor in its use in industrial applications [9]. But chitosan is soluble in various organic acids, solvents and water [10]. Several studies have investigated the solubility problem of chitin. This research focuses on a new way of developing water-soluble colloidal chitin (WSCC) from prawn waste and investigates its fundamental rheological and antibacterial properties.

2. Literature review

2.1 Chitin and chitosan

Chitin is derived from the Greek word chiton, which means a coat of nail. Chitin is the major component of the exoskeleton of invertebrates and the cell wall of fungi and yeast [11, 12], from mushrooms [13]. Chitosan is usually obtained by alkaline or enzymatic deacetylation of chitin. The importance of chitin and chitosan has grown partly because they represent a renewable and biodegradable source of materials, and partly because of the recent increased understanding of their functionality in various applications [2, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25]. Usually, chitin is prepared from crustacean waste through deproteinization using alkali or enzyme, demineralization or decalcification using acid followed by decolourization using decolouring agents in order to remove the proteins, calcium and colour, respectively. The chitin thus obtained can then be deacetylated either by alkali or enzyme to produce chitosan [26]. The properties of chitin and chitosan depend on the processing conditions. Chitosan prepared from chemical and enzymatic deacetylation of chitin differs in their degree of deacetylation (%), distribution of acetyl groups, chain length and conformational structure of chitin and chitosan molecules. These factors affect the characteristics of chitin and chitosan [27].

2.2 Solubility of chitin and its derivatives in water

The most remarkable difference between chitin and chitosan is their solubility. Chitin is insoluble in almost all solvents; chitosan dissolves in almost all aqueous acids. The insolubility of chitin is the major disadvantage to its use. Solvents of chitosan are generally safe to consume, thus allowing its use in various industries including the food industry. Most solvents used for the dissolution of chitin are toxic, hence, they cannot be used in food processing applications [28]. This research investigates the preparation of a water-soluble chitin derivative rather than chitosan because there are many studies on the preparation of chitosan water-soluble derivatives.

The solubility of chitin is achieved by the destruction of the strong hydrogen bonds in chitin molecules and their reorganization form a chitin gel [29]. Various procedures to make chitin water-soluble and its characterization are reported: [17, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53]. The water-solubility of chitin can be obtained by structural modification and by controlling the degree of deacetylation. Such modifications affect the properties of water-soluble chitin derivatives.

The degree of deacetylation of chitin/ chitosan plays a major role in their solubility in water. Modified chitins having 50% degree of deacetylation become soluble in water [54, 55, 56, 57]. Deacetylated chitin has 50% degree of deacetylation with tosyl, iodo, trimethylsilyl and glucosyl groups soluble in water as well as in organic solvents [58]. This research mainly investigates on preparing water-soluble chitin derivatives having the degree of deacetylation similar to that of natural chitin.

2.3 Characterization of chitin and its derivatives

Characterization of chitin derivatives is by solubility, crystallinity, viscosity, degree of deacetylation, molecular weight, mechanical, thermal and moisture retention properties and antimicrobial properties and is helpful to determine their suitability in specific applications.

2.3.1 Solubility and crystallinity

Solubility is an important parameter for the use of chitin and its derivatives in a wide range of industrial applications. The sorption ability of chitin increases as the number of amino groups grows as the degree of deacetylation increases chitin is being converted into chitosan. The solubility characteristics of chitin/ chitosan are governed mostly by the extent of degree of deacetylation, the distribution of acetyl groups, degree of dissociation, processing methods, pH and the ionic strength [28, 59, 60]. The solubility of chitin and its derivatives can be demonstrated using chitosan in a dilute acidic medium. In this system, chitosan tends to be at equilibrium Eq. (1).

$$\text{Chitosan}-{\mathrm{NH}}_2+\mathrm{HOH}\rightleftharpoons \text{Chitosan}-{{\mathrm{NH}}_3}^{+}+{\mathrm{OH}}^{–}$$E1

Chitosan is soluble when pH is lower than 6 or 5.5. At lower pH, the amino groups in chitosan are fully protonated and the positively charged polymer chains will repel each other and fall apart in solution thus resulting in its dissolution. At pH above 6.5, chitosan will precipitate [54].

Polymer swelling reduces the crystallinity of chitin derivatives in a solution [61]. The crystallinity of chitin derivatives varies with the substitution of other functional groups onto the polymer chains [54]. The crystallinity index of chitin (85%) is higher than that of the water-soluble chitin derivative (48 to 57%) [42]. Because the acid or alkali treatments depolymerize the polymer chains during processing [62, 63]. Chitin shows a crystalline structure whereas water-soluble chitin derivatives show an amorphous structure due to structural modification [32, 54, 64].

2.3.2 Viscosity

Viscosity refers to the resistance to flow in liquids while elasticity refers to energy recovery in solids. Polymeric material may be time-dependent, acting more like a solid during short processing time (rapid movement) or acting more like a fluid during long processing time (slow movement). When a polymeric material has both fluid as well as solid behaviours, it is called viscoelastic. Like solubility, viscosity of chitin derivatives is also an important property in processing these polymers. The viscosity data also provides the information on the structure and properties of chitin-derived polymers [47]. This data will be helpful while designing new and innovative chitin-based films for various industrial applications. The dibutyryl chitin with an intrinsic viscosity of greater than 1 dl/g had a good spinnability and film-forming ability [65]. Apparent viscosity [η] or just viscosity, commonly used in place of dynamic viscosity, is defined as the ratio of the imposed shear stress [τ] to the shear rate [$\dot{\gamma }$]. See Eq. (2).

$$\mathrm{\eta }=\mathrm{\tau }/\dot{\gamma }$$E2

In a Newtonian fluid, the shear stress is proportional to shear rate, viscosity is therefore constant. If the viscosity of a fluid varies with respect to shear rate or shear stress, then it is termed a non-Newtonian fluid. A liquid such as water, alcohol etc, which is composed of a single substance is usually a Newtonian fluid. On the other hand, a polymer solution or a colloidal solution containing high molecular weight compounds and/or suspended solids is generally a non-Newtonian fluid.

Mathematical equations have been derived by many researchers to form flow models for describing the rheological behaviour of a material in terms of shear rate and shear stress. The simplest model that is used to describe the flow behaviour of a Newtonian fluid is Eq. (3). In case of non-Newtonian fluid, a power law model is usually used to describe the flow characteristics Eqs. (3) and (4) [66].

$$\mathrm{\tau }=\mathrm{\eta }\dot{\gamma }$$E3

$$\mathrm{\eta }=\mathrm{K}{\left[\dot{\gamma }\right]}^{\mathrm{n}–1}$$E4

$$\mathrm{\tau }=\mathrm{K}{\left[\dot{\gamma }\right]}^{\mathrm{n}}$$E5

where,

τ—Shear stress (Pa)

$\dot{\gamma }$—Shear rate (s⁻¹)

η—Apparent viscosity (Pa. s)

K—Consistency coefficient (Pa. sⁿ)

n—Flow behaviour index, dimensionless number (n = 1 for Newtonian fluids; n <1 for shear thinning or pseudoplastic fluids and n >1 for shear thickening fluids).

Rheological study of both chitin, as well as water-soluble O-carboxymethylated chitin derivatives in N,N-dimethyl acetamide/ lithium chloride solvent system, exhibits non-Newtonian shear-thinning behaviour. The power law indices of these solutions increase along with the temperature [67]. Therefore, a solution containing chitin and/or its derivatives is a non-Newtonian fluid.

2.3.3 Degree of deacetylation (%)

The degree of deacetylation has an influence on all physiochemical properties such as molecular weight, viscosity and solubility. The presence of 50% amine groups defines the boundary between chitin and chitosan; chitin has less than 50% deacetylation and chitosan has more [68]. The degree of deacetylation is affected by the concentration of alkali, processing temperature, reaction time, previous treatment of chitin, particle size and chitin concentration. The degree of deacetylation and the distribution of the acetyl groups influence the solubility [69]. This research used the colloidal titration method to determine the degree of deacetylation of chitin and its derivatives [27, 32, 70].

2.3.4 Thermal analysis

A ‘glass’ can be defined as a solid, brittle material that has an amorphous liquid-like structure with very little flexibility or any obvious fluidity. Glass can be achieved by melting the ordered form of the material and then by rapid cooling or supercooling. A perfectly crystalline polymer will melt at a well-defined temperature; this melting transition is defined as a first-order transition. Melting causes discontinuous changes in volume, enthalpy and primary thermodynamic variables [71]. In an amorphous polymer, the molecular motion of the polymer chains is immobile at low temperatures. The state of the polymer is glassy. When the polymer is heated, the molecules obtain sufficient energy to slide over one another. The polymer becomes viscous, flexible or rubbery at the glass transition [72]. The glass transition temperature is highly specific to each anhydrous amorphous material and depends on experimental conditions, moisture content and molecular weight [73, 74].

The final glass transition temperature has significant impact on the final texture, diffusivity and the rate of deterioration. Stiffness or brittleness of the polymers is lost by the reduction in the glass transition temperature owing to the plasticizing effect of water. This will make the polymer unsuitable for making films [74, 75, 76]. Thermal behaviour of chitin derivatives is conducted by thermomechanical analysis (TMA), thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). DSC is an effective technique to evaluate the thermal behaviour as well as to determine the degree of deacetylation of chitin derivatives [64, 77].

Figure 1 shows the typical chromatogram of a chitin derivative during DSC analysis. In the DSC chromatogram, the first endothermic peak indicates the loss of absorbed moisture by the films. The second exothermic peak(s) indicates the degradation of chitin derivatives. Finally, the phase transition of the chitin polymer occurs. Various studies have been performed to analyze chitin and its derivatives using DSC [58, 77, 78, 79, 80]. Both chitin and water-soluble carboxymethyl-chitin exhibit the endothermic peak that relates to the loss of water during DSC [64]. The decomposition of chitin during DSC is obtained in two stages: one peak at a temperature range of 200 to 260°C and the second peak at 300°C to 360°C [79]. In contrast, a single-stage decomposition of chitin is obtained at around 400°C [77]. Chitin shows better thermal stability than chitosan it contains fewer amine groups [77]. The increase in molecular weight causes a proportional increase in glass transition temperature. DSC analysis of high molecular weight chitin and chitosan shows no glass transition even up to a temperature of 550°C [64, 77, 79].

Figure 1.

DSC chromatogram of a chitin derivative.

2.3.5 Moisture absorption

Food packaging technology requires the use of low oxygen and carbon dioxide permeable materials. The presence of water in the polymer influences the way in which these gases are sorbed and diffused [81]. Biomedical or pharmaceutical activity depends on how the water molecules are associated with the polymer. Moreover, the swelling characteristics of polymer gels are dominated by the nature of the polymer and the state of water [82]. Moisture absorption in polymeric films is important for a variety of industries ranging from microelectronics to adhesives and coatings. In many applications, water absorption leads to reliability problems such as the degradation of dielectric properties, corrosion or delamination. A significant number of studies covering many polymer systems have focused on characterizing the absorption and diffusion properties of water in polymer films [83]. It is very interesting to obtain a better understanding of the water sorption phenomena and the mechanical strength of chitin-based films prior to their use in food, medical and pharmaceutical applications. The solubility and strong swelling of the finished films in water decrease the stiffness of the films [47].

Better understanding of moisture absorption mechanisms and controlling steps may help not only in optimizing the use of chitin films but also in designing new chitin-based polymers. Equilibrium moisture absorption properties are frequently controlled by diffusion properties [especially intra-particle diffusion], degree of deacetylation, chemical structure and physical modification of the polymer; when the size of sorbent particles increases, moisture absorption performance may drastically decrease, the time required to reach equilibrium exponentially increases and sometimes the sorption capacity at equilibrium diminishes [64, 81, 84]. The moisture absorption ability of water-soluble chitin derivatives depends on their chain conformation in solution and their molecular weight [39].

2.3.6 Antimicrobial properties of chitin

Although there are many studies about the antimicrobial activity of chitosan, few studies have been performed to analyze the antimicrobial activity of natural chitin or water-soluble chitin. This is because chitosan has higher degree of deacetylation. An increase in the degree of deacetylation of chitin and hence the number of amino groups increases the antimicrobial activity [26, 85, 86, 87]. Therefore, there is a relationship between the antimicrobial activity and the degree of deacetylation of chitin and its derivatives. This research mainly focuses on the antibacterial property of water-soluble chitin derivatives against Bacillus cereus.

3. Materials and methods

3.1 Materials used

Due to the difficulty in sourcing adequate commercial prawn waste, raw eastern school prawns (Metapenaeus macleayi, approximately 9 cm body length) were obtained from Department of Primary Industries, Fisheries Conservation Technology Unit, NSW, Australia and hand-peeled to obtain prawn waste to conduct this research (moisture content 74%; Ash 23% and 11% chitin dry basis). This prawn waste was stored at –22°C until used for research. All solvents used were HPLC grade supplied by Lab Scan Analytical Sciences. All chemicals were AR grade.

3.2 Preparation of water-soluble colloidal chitin (WSCC)

Step 1: Natural chitin was recovered from prawn waste before it was converted into WSCC. First, prawn pigment, astaxanthin complex was extracted from prawn waste followed by deproteinization using 10% sodium hydroxide (1:2, w/v) at 100°C for 6 hours and then demineralization using 2M hydrochloric acid (1:3, w/v) for 48 hours at ambient temperature. The residue was natural chitin [88]. The moisture content chitin and ash content were measured using Equation 2.8 [89]. Care was taken that the ash content of chitin was less than 1%.

Step 2: This natural chitin was freeze-dried at ambient temperature for 24 hours at 0.4 mbar and stored in the desiccator. Finely ground freeze-dried natural chitin (100 to 125 mesh) (5%, w/v) was digested in sodium hydroxide (50%, w/v) for 6 hours at 87°C to prepare chitosan. The chitosan was thoroughly washed to neutral pH and freeze-dried [88].

Step 3: Chitosan hydrochloride was prepared by dissolving 2.5 g of freeze-dried chitosan in 100 mL of 10% acetic acid followed by precipitation using concentrated hydrochloric acid. The precipitate was thoroughly washed using methanol several times to get chitosan hydrochloride free of chloride. The presence of Clˉ was tested by adding 1% silver nitrate to the filtrate; a white precipitate indicated presence of Clˉ. The chloride-free precipitate called chitosan hydrochloride was dried in the oven at 50°C and moisture content was determined.

Step 4: Two methods were studied to prepare WSCC.

1. Method 1: Oven-dried chitosan hydrochloride (0.5 g) was dissolved in 2 mL of distilled water and then re-acetylated by adding a mixture of acetic anhydride (5 mL) and pyridine (2.5 mL). The reaction mixture was stirred in a magnetic stirrer overnight at ambient temperature to evaporate the solvent. This WSCC was dried in the oven at 50°C and the moisture content was measured.

2. Method 2: Oven-dried chitosan hydrochloride (0.5 g) was re-acetylated by mixing with an equal volume of acetone (5 mL) and acetic anhydride (5 mL). The mixture was stirred in a magnetic stirrer overnight at ambient temperature to evaporate the solvent. It was then dried in the oven at 50°C. The moisture content of the oven-dried WSCC was measured.

3.3 Preparation of WSCC film for antibacterial study

WSCC film of 0.22 mm thickness was prepared by pouring 15 mL of WSCC solution in water (5%, w/v) on a glass petri dish underlined with a layer of microwave-safe all-purpose food packaging film without imperfections. WSCC film of 0.52 mm thickness was prepared by pouring 100 mL of WSCC solution in water (5%, w/v) on a glass plate (16 cm × 24 cm) underlined with a layer of GLAD wrap. These two samples were then oven-dried at 40°C. Careful attention was given that the prepared film was free from air bubbles and physical imperfections. The thickness of the dry WSCC films was measured using a vernier calliper at five random positions and averaged.

3.4 Characterization of WSCC

3.4.1 Viscosity of WSCC using different viscometers

In this study, the viscosity of WSCC dissolved in distilled water was evaluated using two viscometers. They are rotational Haake viscotester® VT550 and SV-10 AND Vibro viscometer (SV series 300). The results obtained from these two viscometers were then compared. Rotational Haake viscometer measures the viscosity by measuring the running torque of the cylindrical rotors immersed in a sample because viscosity is directly proportional to a running torque required to develop steady rotating motion. Its temperature is controlled by a re-circulating water bath and water jacket. SV-10 AND Vibro viscometer measures the viscosity by controlling the amplitude of the sensor plates immersed in a sample and measuring the electric current to drive the sensor plates. SV-10 AND viscometer vibrates with sine-wave of frequency about 30 Hz and amplitude of approximately 0.2 mm. The temperature at the geometric centre is measured, not controlled. These two viscometers have different shear rates. The shear rate of the Haake viscometer is controlled. The nominal shear rate values for SV-10 AND Vibro viscometer are shown in Table 1.

Viscosity coefficient (mPa.s)	Maximum shearing rate (s^−1)	Effective shearing rate (s^−1)
1	590	420
10	130	92
100	42	30
1000	17	12
10000	10	7

Table 1.

Nominal shear rate values of a viscosity standard Newtonian fluid using SV-10 AND Vibro viscometer.

3.4.2 Viscosity testing using Haake viscometer

In the Haake viscometer, a sample solution (9 mL) of WSCC (5%, w/v) in distilled water was taken in a concentric cylinder (NV type, system no: 8, radius: 20.5 mm) together with an NV rotor (system no: 8, radius: 17.85 mm, height: 60 mm). Prior to the analysis, the sample solution and sensor system were placed in the water bath at the respective temperature for 15 minutes for equilibration. The flow curves were observed at shear rates 0.13 to 300 s⁻¹ and at temperatures 20°C, 30°C and 40°C. The parameters for power-law models were noted. The operating parameters used during viscosity analysis are given below. Ramp1 eliminated start-up effects and was not taken into account while analyzing the results.

1. Ramp1: CR 0.13 s⁻¹; t = 10 s; #5

2. Ramp2: CR lin, 0.13–300 s⁻¹; t = 120 s; #100

3. Ramp3: CR lin, 300–0.13 s⁻¹; t = 120 s; #100

Power-law models of flow curves were calculated for each temperature Eq. (4). The apparent viscosity at shear rates 5, 10, 100 s⁻¹ was calculated from each model Eq. (5) [66]. The temperature dependence of viscosity follows the Arrhenius exponential relationship Eq. (6).

$$\mathrm{\eta }={\mathrm{Ae}}^{\mathrm{E}/\mathrm{RT}}$$E6

where,

E—Activation energy of the sample

A—Empirical constant

T—Temperature of reaction mixture

R—Universal gas constant

η—Apparent viscosity

This exponential relationship of Arrhenius applies to a polymer system of low molecular weight and low viscosity [90]. From the results obtained, WSCC was a low molecular weight polymer with low viscosity. Therefore, Arrhenius models were constructed for WSCC solutions using Eq. (7). Arrhenius models were obtained by plotting the natural logarithmic of apparent viscosity (η) on Y-axis and the inverse of temperature (K⁻¹) on X-axis for each shear rate. Arrhenius constants were determined from the slope and the intercept of the Arrhenius model Eq. (7). Activation energy values (E) at each shear rate were calculated by multiplying the respective slope of the Arrhenius equation with the gas constant value (R). The intercept was the natural logarithmic of the empirical constant, A.

3.4.3 Viscosity testing using SV-10 AND viscometer

Sample solution (10 mL) of WSCC (5%, w/v) in distilled water was analyzed using SV-10 AND Vibro viscometer, which delivers a single shear rate, and the temperature and viscosity profile were recorded over time. An Arrhenius plot was constructed and Arrhenius constants were determined.

Apparent viscosity values (η_S) at 20°C, 30°C and 40°C were interpolated using the Arrhenius model, and the corresponding shear rates ($\dot{\gamma }$) were calculated from the effective shear rate vs viscosity co-efficient standard curve. The calculated shear rates were then substituted in the power-law equations of Haake viscotester Eqs. (4) and (5). The apparent viscosity values (η_H) corresponding to the SV-10 AND vibratory viscometer shear rates at 20°C, 30°C and 40°C were calculated. Finally, the apparent viscosities of WSCC in distilled water were obtained using both the viscometers and were compared.

3.4.4 Degree of deacetylation determination

Degree of deacetylation of chitin samples was measured by the colloidal titration method. Oven-dried chitosan hydrochloride (1 g) samples prepared from methods 1 and 2 were titrated against 0.1M sodium hydroxide using phenolphthalein indicator. The degree of deacetylation of the sample was calculated Eq. (7) [91]. Standard deviation and significant differences were calculated.

$$\%\mathit{DD}=\frac{N1V1}{1000}x\frac{V0}{V2}x\left(\mathit{\text{MWCTS}}-\mathit{Cl}\right)x\left\{\frac{100}{W4x\left(1-\%\mathit{MC}/100\right)}\right\}$$E7

where,

%DD—Degree of deacetylation of chitin sample (%)

N₁—Concentration of sodium hydroxide (M)

V₁—Volume of sodium hydroxide used (mL)

V₀—Total volume of chitosan chloride solution (mL)

V₂—Volume of chitosan chloride solution used for titration (mL)

MW_CTS-Cl—Monomer weight of chitosan chloride

W₄—Weight of chitosan chloride taken for titration (g)

%MC—Moisture content of chitosan chloride (%)

3.4.5 Differential Scanning Calorimetric [DSC] analysis of WSCC film

Differential scanning calorimetric measurements were performed using Universal V4.3A TA Instruments. WSCC films [sample weight 3 mg, 0.22 mm thickness] were equilibrated in a desiccator or in the relative humidity chamber (92.5%) for a week and the DSC analysis was performed under a dynamic nitrogen atmosphere (50 mL/minute) at a heating rate of 5°C/minute. Samples equilibrated to a range of relative vapour pressures (36.1%) between these two were also tested. Intermediate relative humidity (36.1%) of WSCC film was obtained by equilibrating the film at ambient conditions. Accurately weighed sample (±0.1 mg) was placed into a covered aluminium sample holder. An empty sample holder was used as reference and two runs were performed for each sample by heating the sample from 25°C up to 450°C.

In another study, two samples treated at the ambient relative humidity were tested individually using DSC. The DSC curves were performed under a dynamic nitrogen atmosphere (50 mL/minute) at a heating rate of 5°C/minute. Accurately weighed samples (±0.1 mg) were placed into an aluminium sample holder and sealed. An empty sample holder was used as reference and the runs were performed by heating the samples from 25°C up to 110°C with an isothermal for 15 minutes to remove the moisture present in the samples. The samples were then reweighed and reheated from 25°C up to 480°C [77]. The results are then compared with the phase behaviour of the moist WSCC films.

3.4.6 Moisture absorption isotherm of WSCC film

In this study, moisture absorption behaviour of WSCC was investigated at different relative humidities. Moisture absorption isotherm of WSCC film of 0.22 mm thickness was prepared using saturated salt solutions of different relative humidities (Table 2). WSCC films (2.5 × 2.0 cm) were placed in each of the relative humidity chambers and the samples were kept at a controlled temperature of 25°C for equilibration. The moisture gained by each of the samples was measured after a week and the moisture absorption isotherm was prepared by plotting the relative humidity on X-axis and the moisture content of the film on Y-axis.

No.	Saturated Solutions	Equilibrium relative humidity (%) at 25°C
1	Potassium acetate [CH3COOK]	22.5
2	Magnesium chloride [MgCl2. 6H2O]	32.7
3	Potassium carbonate [K2CO3. 2H2O]	43
4	Sodium nitrite [NaNO2]	64
5	Sodium chloride [NaCl]	75.1
6	Potassium chloride [KCl]	84.2
7	Potassium nitrate [KNO3]	92.5

Table 2.

Relative humidity standards used for the experiment [83, 92].

3.4.7 Antibacterial activity of WSCC film

Bacillus cereus 043800 was supplied by the culture collection of Department of Biological Sciences, UNSW and stored at –80°C freezer. The inoculum suspension (10 μL) was spread on WSCC film (2 cm × 1.5 cm). A control WSCC film was made without inoculum. Each treatment was carried out in duplicate. The films were then placed in the aseptic plastic Petri plates and autoclaved glass Petri plates separately. Petri dishes containing the films were then sealed using adhesive tape and incubated at 30°C for a day. Then inoculated films were taken out and the films were placed into a ‘stomacher’ bag with 10 mL of peptone/water (0.1%, w/v). The stomacher bags were initially massaged by hand to loosen the adhesion of cells to the film followed by ‘stomaching’ using stomacher for 5 minutes and then allowed to sit for 5 to 10 minutes. The bag was again massaged by hand before plating to ensure homogeneous distribution of the suspension. 50 μL of each of these samples was spread-plated onto a brain heart infusion agar plates. The plates were incubated at 30°C, and colonies were counted 1 day later [93].

4. Results and discussion

4.1 Solubility of WSCC

The WSCC prepared by Method 1 was soluble in water and the oven-dried material was not readily soluble in water. Moreover, this method was not reproducible, and the strong odour of pyridine was other major concern. When the processing conditions of chitin to chitosan were changed in Method 2, the degree of deacetylation of the prepared chitosan was less and the oven-dried WSCC readily formed a colloidal suspension in water. Method 2 was reproducible in preparing WSCC and this method did not use pyridine. Therefore, Method 2 was chosen to produce WSCC from prawn waste.

4.2 Characterization of WSCC

4.2.1 Viscosity of WSCC using different viscometers

4.2.1.1 Viscosity testing using Haake viscometer

Flow curves obtained for WSCC dissolved in distilled water (5%, w/v) using Haake viscometer at different temperatures and different shear rates were modelled by power-law equations. The value of the power law exponent, n was less than 1 (ranging between 0.8 and 0.9) at all temperatures Thus, WSCC dissolved in distilled water was a shear-thinning, non-Newtonian fluid. As would be expected, the apparent viscosities generally decreased with an increase in temperature and shear rate during the experiment. A flow curve obtained for WSCC dissolved in distilled water (5%, w/v) at 30°C is shown in Figure 2.

Figure 2.

Flow curve obtained for WSCC dissolved in distilled water (5%, w/v) at 30°C using Haake viscometer.

The Arrhenius model for WSCC in distilled water using Haake viscometer at different shear rates and at different temperatures is shown in Figure 3. Arrhenius models at different shear rates were straight lines with negative slope, hence fit into Arrhenius exponential relationship. The R² values were between 0.94 and 1.00. An increase in temperature decreased the apparent viscosities at different shear rates (5 s⁻¹, 10 s⁻¹ and 100 s⁻¹). Therefore, temperature dependence of apparent viscosity of WSCC in distilled water obeyed the Arrhenius exponential relationship. The Arrhenius model constants at different shear rates for this particular sample system are shown in Table 3. In this sample system, the activation energy values increased with an increase in temperature (positive activation energy values) and shear rate.

Figure 3.

Arrhenius models of WSCC in distilled water (5%, w/v) at different shear rates and at different temperatures: Haake viscometer.

Shear rate (s^−1)	Activation energy, E (KJ/mol.K)	Empirical constant, ln(A)
5	0.0005	−4.6568
10	0.0006	−4.7567
100	0.001	−5.0889

Table 3.

Arrhenius model constants of WSCC in distilled water (5%, w/v) at different shear rates: Haake viscometer.

4.2.1.2 Viscosity testing using SV-10 AND vibratory viscometer

The relationship between time, temperature and apparent viscosity profile of WSCC in distilled water (5%, w/v) using SV-10 AND vibratory viscometer is shown in Figure 4. As expected, the apparent viscosity decreased with increase in temperature.

Figure 4.

Temperature and apparent viscosity profile of WSCC in distilled water (5%, w/v) using SV-10 AND vibratory viscometer.

The Arrhenius model of natural logarithmic values of apparent viscosity against the reciprocal of absolute temperature for WSCC in distilled water (5%, w/v) using SV-10 AND viscometer is shown in Figure 5. The relationship was linear with negative slope (R² = 0.99), thus followed the Arrhenius exponential relationship. The Arrhenius model for this system obtained using SV-10 AND viscometer was y = −8252.4x + 32.297. The intercept, which is the natural logarithmic value of the empirical constant, was –32.297. The activation energy for this sample system calculated from the slope of the Arrhenius model was 68.61 KJ/mol.K, which is a positive value. Positive activation energy for the same sample system was obtained in the Haake viscometer as well (Table 3).

Figure 5.

Arrhenius model of WSCC in distilled water (5%, w/v) at a single shear rate: SV-10 AND vibratory viscometer.

The Arrhenius models constructed for WSCC in distilled water (5%, w/v) using both viscometers showed negative slope. The Arrhenius models obtained from both viscometers confirmed that the apparent viscosity decreased with an increase in temperature. As mentioned in 3.4.1, the apparent viscosities (η_S) of WSCC dissolved in distilled water (5%, w/v) at 20°C, 30°C and 40°C were calculated from the Arrhenius model of the SV-10 AND vibratory viscometer, which are shown in Table 4. The corresponding shear rate values for the calculated apparent viscosities at 20°C, 30°C and 40°C were obtained from the effective shear rate vs viscosity standard curve of the SV-10 AND vibratory viscometer. These values are listed in Table 4. The apparent viscosities were not constant with varying shear rates. Thus, WSCC in distilled water (5%, w/v) behaved like a non-Newtonian fluid. A similar result was obtained when analysed using the Haake viscometer.

Temperature (°C)	Apparent viscosity (mPa.s)	Shear rate (s^−1)	Apparent viscosity ηH (mPa.s)
20	15.82	48.55	9.43
30	6.25	32.14	8.75
40	2.62	21.85	8.32

Table 4.

Apparent viscosities and shear rates of WSCC in distilled water (5%, w/v) at different temperatures: SV-10 AND vibratory viscometer.

The obtained shear rate values from the SV-10 AND vibratory viscometer values at 20°C, 30°C and 40°C were then substituted into the power-law model of Haake viscometer. The apparent viscosities (η_H) for those shear rates were then worked out using Haake viscometer power-law equations. The obtained apparent viscosities (η_H) are given in Table 4.

The apparent viscosities for the respective shear rates obtained using both viscometers were different (Table 4). The differences in the calculated apparent viscosities by these two viscometers are due to the different working principles. In Haake viscometer, shear rate can be controlled whereas the shear rate cannot be controlled in SV-10 AND Vibro viscometer. Both these viscometers showed that the system containing WSCC in distilled water (5%, w/v) was a non-Newtonian fluid with shear thinning behaviour and an increase in temperature reduced the apparent viscosities. Similar results are obtained for chitin and water-soluble o-carboxymethylated chitin dissolved in N,N-dimethyl acetamide/ lithium chloride solvent system [67]. Thus, the apparent viscosity of WSCC dissolved in distilled water is dependent on the temperature and the shear rate.

4.2.2 Degree of deacetylation of WSCC

There was no significant difference between the degree of deacetylation of chitin and WSCC (Table 5). This is because acetylation with a mixture of acetic anhydride-acetone gives rise to the complete acetylation of amino groups [94, 95]. Thereby the original degree of deacetylation of chitin was restored by WSCC. The yield of WSCC was 82 g/kg of prawn waste (dry basis).

Sample	Degree of deacetylation (%)
Chitin	27.64 ± 0.61
Chitosan (Method 1)	88.22 ± 0.46
Chitosan (Method 2)	54.08 ± 0.69
WSCC	28.46 ± 1.08

Table 5.

Degree of deacetylation of chitin and its derivatives.

4.2.3 Differential Scanning Calorimetric (DSC) analysis of WSCC film

The DSC curves obtained for WSCC films equilibrated in high relative humidity (92.5%) (moisture: 16.4%) and in a desiccator (moisture: 6.52%) are shown in Figures 6 and 7 with exothermic peaks facing up. A generic DSC curve obtained for the WSCC film equilibrated in high relative humidity is also shown in Figure 1. Samples equilibrated to a range of relative vapour pressures between these two were also tested. The DSC thermogram for both these samples showed an endothermic peak (Peak 1) after 70°C followed by an exothermic peak around 180°C (Peak 2).

Figure 6.

DSC curve for high-moisture WSCC film.

Figure 7.

DSC curve for dry WSCC film.

Various studies indicate that the endothermic peak at 100°C is attributed to the evaporation of absorbed water and the first exothermic peak is probably due to the degradation of chitosan studied during DSC [64, 82, 96, 97, 98]. In another study, the endothermic peak corresponds to the loss of moisture seen at a lower temperature (70°C), which is similar to this experiment [77]. The onset of the endothermic peak is related to pressure build-up because of water evaporation inside the sealed sample cups during DSC. The pressure at which the seam of the cups started to leak corresponds to approximate vapour pressure of water. The leaking of sealed sample pans during DSC also relates to the sample weight loss after the run [64].

Peak 1 in this study is due to loss of absorbed moisture by the WSCC film. As expected, a smaller peak of water loss (Peak 1) for a low moist sample was obtained compared to a high moist sample. It was also expected that the sample equilibrated in the desiccator would be truly anhydrous. However, sample treated in desiccator showed endothermic peak for water loss. Some water in the film was not completely removed during sample drying in the desiccator. Therefore, it is suggested that desiccators be evacuated to accomplish faster and complete removal of water. Moreover, totally anhydrous chitin samples are difficult to obtain because of the high water affinity presented by these polymers [77].

In this study, Peak 2 is due to the degradation of chitin derivatives. The first exothermic peak for degradation of chitin derivatives is obtained at around 180°C in this study. The exothermic peak for the decomposition of chitin derivatives depends on the molecular weight and the presence of hydrophilic groups [64]. Viscometric analysis of WSCC shows that the molecular weight of WSCC is lower than chitin. Because of this reason, low molecular weight WSCC derivatives degrade faster than the high molecular weight chitin. The degradation of WSCC film occurred at relatively lower temperature than the published data of chitin and water-soluble carboxymethyl-chitin run [64]. The thermal stability of WSCC is poorer compared to chitin and water-soluble carboxymethyl-chitin.

The degradation temperature (T_d) of high-moist WSCC film was low compared to low-moist WSCC film. However, noticeable differences in the degradation temperature of both low-moisture and high-moisture samples were hard to see (Table 6). WSCC film did not show a considerable increase in the absorption of moisture over a wide range of relative humidities (Figure 8). Therefore, moisture content of WSCC film slightly influences the degradation of WSCC derivatives in this study.

Samples	Tm (°C)		Td (°C)		Tg (°C)
	Run 1	Run 2	Run 1	Run 2	Run 1	Run 2
Low-moisture WSCC film	72.82	70.32	185.97	186.91	364.05	364.45
Standard Deviation	1.41		0.67		0.28
High-moisture WSCC film	76.96	77.69	181.23	185.10	360.07	363.48
Standard Deviation	0.52		2.74		2.41

Table 6.

Glass transition temperature (T_g) of WSCC films determined using DSC.

Figure 8.

Moisture absorption isotherm of WSCC film.

The glass transition temperature (T_g) for WSCC film was observed at around 360°C. The glass transition temperature (T_g) of high-moisture WSCC film was lower than that of low-moisture WSCC film (Table 6). Higher molecular mobility accelerates reactions limited by diffusion and decreases stiffness [84]. The thermal degradation of the material is sensitive to moisture content confirms that degradation reactions were diffusion-limited. Therefore, moisture content did influence the phase behaviour of WSCC film. There was, however, sample weight loss in both high-moisture (57%) and low-moisture (50%) WSCC films after DSC run. The difference in the sample weight loss is due to the difference in the moisture content. The sample weight-loss (15%) of water-soluble carboxymethyl-chitin film after DSC run is also reported by Ref. [64]. It was beyond the scope of this project to exhaustively test the rheological and thermo-physical properties of chitin and chitosan films against a range of other polymers. Such tests would be more profitably carried out at a larger scale of production, which would enable rolled or extruded films to be made. Nevertheless, the tests done show that the recovery of prawn chitin and its processing can lead to films of high and reproducible quality.

4.2.4 Moisture absorption isotherm of WSCC film

The specific moisture absorption isotherm of WSCC film (0.22 mm thickness) appeared to follow the expected sigmoid curve usually obtained over the whole range (0 to 1) of relative vapour pressure (Figure 8). This kind of isotherm is also called a type II isotherm in reference to the Brunauer–Emmett–Teller (BET) model [99]. A similar result was obtained for chitosan films by Ref. [99].

The specific moisture absorption of WSCC film increased with increasing relative humidity. The absorbed moisture by WSCC films varied from 40% to 55% between low (22%) and high relative humidity (92.5%). In this case, there was not much difference obtained in terms of the absorption of specific moisture over a wide range of relative humidities. The other study shows that the absorbed moisture content by chitosan films varies from 5 to 45% over relative humidities from 20% to 80% [99]. This indicated the moisture absorption of WSCC films was less compared to chitosan films over a wide range of relative humidities. The solubility and strong swelling of the finished film in water decreases the stiffness of the films which in turn decreases the suitability of the film in various applications [47]. The specific moisture absorption behaviour of WSCC film may not vary considerably over a range of relative humidities. This property will increase the selectivity of the finished films when they are used as membranes, packaging films or coating materials. In-depth study on the moisture absorption mechanism of WSCC films will be carried out during the scale-up of this process. Moisture absorption isotherm of WSCC films will be useful to determine the suitability of the film for maintaining proper moisture content of a particular product.

4.2.5 Antibacterial activity of WSCC film

The antimicrobial activity of WSCC film against the gram-positive bacterium Bacillus cereus 043800 was investigated. The average values of the number of colonies of B. cereus during the antibacterial study are presented in Table 7. The empty glass and plastic Petri plates incubated alone for sterility check did not show the presence of B. cereus on brain heart infusion agar. WSCC film inhibited around 90% of the growth of B. cereus 043800. This shows that films made by WSCC have excellent antibacterial activity against B. cereus. The interesting finding in this study is that although the degree of deacetylation of WSCC was less (28.5%), WSCC film inhibited almost 90% of the growth of Bacillus cereus 043800. The extension of this work would test the overall antimicrobial activity of WSCC film against different microorganisms. Recently, water-soluble chitin derivation was investigated for the film coating of Ricotta cheese, and it proved to be efficient in prolonging the shelf life of Ricotta cheese [17].

Antibacterial study details	Plastic Petri plate	Glass Petri plate
(A) Cell concentration in stock suspension (cfu/mL)	8.7 × 10^7	8.7 × 10^7
(B) Cell concentration in inoculum suspension (cfu/mL)	2.98 × 10^6	2.63 × 10^6
(C) Average number of bacteria in the control (WSCC film without bacterial inoculum) (cfu)	1.98 × 10^4	2.13 × 10^4
(D) Average no. of bacteria inoculated onto film (cfu)	2.98 × 10^4	2.63 × 10^4
(E) Average no. of bacteria present in the film after incubation (cfu)	3 × 10^3	3.1 × 10^3
Bacterial colonies inhibited (100−(E/D × 100)) (%)	89.2	88.2

Table 7.

Concentration of Bacillus cereus 043800: An antibacterial study of WSCC film.

5. Conclusions

In conclusion, this study has clearly demonstrated the fundamental characteristics of WSCC. The study of WSCC will be helpful in evaluating its suitability in various industrial sectors including food and pharmaceuticals. To effectively use WSCC as a functional ingredient, relationships between the functional properties and characteristics of WSCC must be constantly monitored for proper quality control. In the current study, limited relevant information on aspects of such relationships was obtained. More extensive investigations are needed for a better understanding of the relationships reported in the present research, especially in view of current worldwide interest in commercial use of water-soluble chitin derivatives. This will be best done during the scale-up of this current project.

The biodegradable water-soluble colloidal chitin recovered from prawn waste offers exciting possibilities because of its water solubility. These chitin derivatives can be obtained from the prawn waste changing the waste stream into a valuable resource that is commercially viable. The antibacterial property of water-soluble colloidal chitin may be applied in pharmaceutical, medical and food packaging and coating applications. Biodegradable films made from these natural polymers obtained from renewable sources will protect the environment in the future and will be an effective alternative to plastics that threatens the environment. However, vegetarians may object to the use of animal polymers.

6. Recommendations

The study of the preparation of water-soluble colloidal chitin from prawn waste recommends scale-up to pilot scale and reexamination of the stability of water-soluble colloidal chitin. Further microbial investigation is required to explore the water-soluble colloidal chitin film’s antimicrobial activity on a wide range of microorganisms. An appropriate method needs to be developed to determine the molecular weight and physio–chemistry of the water-soluble colloidal chitin following scaled-up production. Detailed study about the toxicity of water-soluble colloidal chitin is necessary prior to its use in industrial applications, especially in food applications because these compounds may contain chemical/solvent residues. Water-soluble colloidal chitin can be tested as an antimicrobial coating in fruits and vegetables. The application of water-soluble colloidal chitin in food packaging applications can be studied in the future.

Further, the use of sodium hydroxide and hydrochloric acid during preparation of water-soluble colloidal chitin generates chemical waste. World over efforts is on to find out an alternative to chemical method of preparation of chitin/ chitosan. Therefore, an alternative way of preparing water-soluble chitin derivatives using enzymes or simply by adding or substituting functional group to chitin molecule is recommended. Such material can be used for preparing films and the related studies.

Acknowledgments

I acknowledge Prof. Wilem F. Stevens, Mahidol University, Thailand, who guided me during this research, when required. I also acknowledge Katherine Zerdin, CSIRO, Sydney, Australia for generously sharing the resources to perform the antibacterial work.

Notes/thanks/other declarations

Many thanks to the Department of Primary Industries, Fisheries Conservation Technology Unit, NSW, Australia for supplying raw Eastern School prawns for the research. I also thank the Australian Government for the financial support by providing the International Postgraduate Research Scholarship to undertake this research.