1.  Introduction

Recent studies have shown that biodegradable films and films made from renewable and natural polymers such as starch, cellulose derivatives, gelatin, protein and lipids are receiving attention [1]. Biodegradable films are not meant to completely replace synthetic packaging films but they possess the ability to replace conventional packaging in some cases. Biodegradable films have the advantage of being completely degraded by microorganisms without emission of toxic gases. [1].

Starch is one of the most studied natural polymers for the development of biodegradable films because of its low-cost carbohydrate polymer, easy to obtain and good ability to form films [2]. Thus, it is a potential packaging material in agriculture, medicine, and packaging industries [3].

Tacca leontopetaloides also known as Polynesian arrowroot starch is a wild perennial herb belonging to the Dioscoreaceae species [4]. This plant is naturally distributed from Western Africa through Southern Asia to Northern Australia [5]. This starch serves as an important food source for many Pacific Island cultures and is additionally used to stiffen fabrics in some of the islands. Application of this starch was reported as a treatment of stomach ailments. It is also believed to treat diarrhea and dysentery [4].

The process of biodegradable film production involves the use of native and slightly modified starches whose advantages are that they are cheap, abundant and renewable. However, the use of starch itself in bio-plastic manufacturing is unsuitable because of various disadvantages [6]. These include the brittleness of the material in the absence of suitable plasticizers and the hydrophilic nature of starch. As a result, the mechanical properties deteriorate upon exposure to environmental conditions like humidity. Thus, starch needs to be blended with other synthetic polymers or plasticizers to eliminate these disadvantages.

Varieties of plasticizers to improve the processing properties and product performance of biodegradable films have been evaluated. Studies show that plasticizers used in promoting the plasticization of starch are glycerol, glycol, xylitol, sorbitol, sugars, and amides, such as urea, formamide and ethylenebisformamide [7].

Despite the considerable contribution of petroleum-based plastics to the global economy exceeding billions of dollars, their non-degradability represents a huge challenge for the ecosystem, leading to many environmental crises [8]. Concentrated efforts toward the development of biodegradable materials with properties that ensure food safety and security while minimizing the environmental impact of their uses have been the focus of different studies. Most of these biodegradable polymers exhibit excellent properties comparable to those of petroleum-based plastics but their applications in food packaging are limited by certain poor barrier and mechanical properties [9].

Studies have been carried out using common starches such as corn, potato, cassava and wheat [10]. However, these starches have important roles in the human food hierarchy system and large industrial production of starch has been focused on these crops. Therefore, there is a need to find alternative sources of starch among the underutilized crops and to apply novel technologies in extending their applications in more special or severe circumstances.

The advantages of starch for bio-plastic production include its renewability, good oxygen barrier in the dry state, abundance, low cost and biodegradability. Tacca starch in which the percentage of amylose to amylopectin is quite similar to corn and cassava starch among other properties, promises to provide elastic properties and become an alternative to common starch in the production of biodegradable film. Adversely, native starch-based materials are reported in many investigations to be very brittle with many surface cracks and are difficult to handle. However, these drawbacks can be resolved by the addition of plasticizers to pure starch to improve its workability and suppress film brittleness [11]. Thus, the blending of different plasticizers with tacca starch seems attractive for the optimization of the film in different aspects [12]. This study investigated the use of tacca starch for use in the production of biodegradable films which can be utilized for food packaging; starch was extracted from tacca crop and biodegradable films were developed in combination with different blends of plasticizers (Glycerol and Sorbitol) using different process temperatures. The physical (thickness, density and moisture content) and mechanical properties (tensile strength and elongation) of the film were determined. The individual and interactive effects of the process parameters on the films were determined; the plasticizers and process temperatures were also optimized.

2.  Materials and methods

2.1 Materials

The material used for starch production was Tacca tubers obtained from a local market in Shendam Local Government Area of Plateau State, Nigeria. Other materials for the starch extraction were knife, bowl, grinder, sieves, filter clothes and distilled water. Sorbitol and glycerol were purchased from SIM BEST Scientific and Chemicals Minna, Niger State.

2.2 Methods

2.2.1 Starch preparation

The Tacca tubers were peeled and washed with potable water. This was followed by mechanical grating of the tuber. The grated Tacca was mixed with water (3 times the volume of the grated tacca). The mixture was sieved and filtered using coarse sieve and filter clothes respectively. Thereafter, the filtrate was allowed to settle for twelve hours. This process is known as starch washing. At the end of the twelfth hour, it was decanted. The wet starch was dewatered manually and oven-dried using Mqeco-4t4030 oven. It was dried at a temperature of 105^oC for twelve hours. This was to make sure that the starch was in the barest minimum moisture content of 12% [13]. The flowchart for the starch preparation is shown in Fig. 1.

Figure 1. Flow Chart for Tacca starch preparation [13]

2.2.2  Film preparation and experimental design

The method described by [14] was adopted with slight modifications. The Tacca starch films were produced by conventional solution-casting techniques at the Chemical Engineering Laboratory facility of the Ahmadu Bello University (ABU), Zaria. The formulations were done based on the experimental design (Table 1).

Table 1. Box-Behnken’s experimental design for film formulation

The experiments were conducted using Box Behnken experimental design for investigating the individual and interactive effects of ingredient proportions (tacca starch, glycerol and sorbitol concentration) and process temperature on the film thickness, density, moisture content, tensile strength and elongation of Tacca starch-based film in which 29 different trials were conducted. Box-Behnken is a spherical, revolving response surface methodology design that provides an efficient solution compared with a three-level full-factorial design and reduces the number of required experiments, which becomes more significant as the number of factors increases [15].

5-15g (w/w) of Tacca starch was dissolved in 100ml distilled water. This was followed by heating the film forming solution at a temperature range of 75^oC to 95^oC (75^oC, 85^oC and 95^oC) for 15 minutes under constant stirring in a water bath. Temperature control was achieved by putting off the heat when the thermometer indicated the required temperature measurement. This step helped to provide homogeneous dispersion by disintegrating the starch granules. Thereafter, the different plasticizers were added into the dispersions at 0 - 4.5g (w/v) of each plasticizer (Glycerol and Sorbitol) representing 0-30% (w/w starch basis). The heating process for each was continued for an additional 15 minutes for each temperature for proper gelatinization. The gelatinized film solutions were cooled down, prior to their casting in glass petri-dishes. The glass petri-dishes served as casting surfaces, enabling the film to have a smooth and flat surface. The fresh casted films were placed in an oven (45°C) to allow evaporation. After 36 hours of drying and a constant weight, films were peeled from the casting surfaces and stored in desiccators with 53% relative humidity (RH). The selection of the above levels of the filmogenic components were based on preliminary studies and reviewed literature [16, 17]. The film preparation process is shown in Fig. 2. The formulation, heating and casting of the films are shown in Plate I.

Figure 2. Flow chart for film preparation [14]

Plate I: (a) Film formulation and heating (b) Casting and oven dried film samples

2.3 Determination of film properties

The following properties of the Tacca starch film were investigated: Physical (thickness, density, and moisture content) and Mechanical (tensile strength and elongation).  Film thickness, density and moisture content were determined according to the method described by [18] with slight modifications. Tensile strength and elongation were determined using a standard method of [19].

2.4 Statistical analysis

Statistical Analysis of variance (ANOVA) was done to study the effects of the different independent parameters on all dependent variables by Response Surface Methodology (RSM) using statistical design software (Design Expert 11 version 27. 3D).

2.5. Optimization and verification of models

Numerical optimization based on the desirability function using Design expert software was used to analyze the responses for obtaining optimum process parameters. Model equations were developed for each response to calculate the optimum condition for both dependent and independent variables.

3.  Results

The mean results of the film thickness, density, moisture content, tensile strength and elongation properties of Tacca starch-based film are presented in Table 2. These parameters were chosen among other quality indices due to their important roles in determining the handling, workability as well as the shelf-life of biodegradable films.

4.  Discussion

The influence of the concentration of Tacca Starch (A), Sorbitol (B), Glycerol (C) and Temperature (D) over the physical (film thickness, density and moisture content) and mechanical (tensile strength and elongation) properties of Tacca Starch biodegradable films were analyzed through the Response Surface Methodology (RSM) using the Box-Behnken Design (BBD). The different formulations of the Box-Behnken’s experimental design and the response variables are obtained for each combination as shown in Table 2. The developed models were investigated for adequacy and fitness between process variables and response variables for the Tacca starch biodegradable film as shown in Tables 3 to 8.

Table 2. Physical and mechanical properties of the Tacca starch-based films

4.1 Thickness (mm)

Table 3 showed that only the mass concentration of tacca starch (A) significantly (P < 0.05) affected the film thickness. Plasticizer (Sorbitol and Glycerol) blends and process temperature had no significant (p > 0.05) effects on the film thickness. The kind and concentration of plasticisers play a significant part towards attaining successful plasticization [20].

Table 3. Summary Statistics of the Thickness of TSF

Fig. 3a shows that film thickness is well augmented with the higher level of starch and this effect is attributed to the formation of intermolecular hydrogen bonds between starch and dry matter content as well as the interaction between polysaccharides. This inference is drawn on the basis that plasticizer blends did not influence the film thickness since there was negligible significant difference in the film thickness variation in plasticizer concentration and process temperature. This real effect on the film thickness might be due to the large amount of the mass of starch with a larger surface area, which improves when interacted with plasticizers [21].  A linear model was obtained for the film thickness as follows:

Thickness (T) = 0.356787 + 0.150400A + 0.038889B + 0.054741C + 0.005100D                                                  (1)

From the above model equation, the regression coefficient depicts a rise in the film thickness as the starch level (A) increased by 0.1504 (codded value). From Table 4, the coefficient of determination (R²) of 0.7200 designates a moderate correlation between the experimental and predicted values. Fig 1b also attests to the correlation between the predicted and actual values. The predicted R² value of 0.6867 is in reasonable agreement with the adjusted R² value of 0.6077. This implies that the model can be used to navigate the design space.

Table 4. Summary Statistics of the Density of TSF

Furthermore, the model F-value of 6.00 and a corresponding p-value of <0.0017 implies the model is significant indicating its suitability for packaging. According to the Indian Government Packaging Standard, packaging films must be at least 0.05 mm thick [22]. The film thickness is an important parameter which influences both the mechanical properties and WVP of the biodegradable film [23]. Fig 3 is the 3-D response plot for film thickness and the comparison between predicted and experimental values for TSF for the quality index.

Figure 3. (a) Response surface plot (3-D) for film thickness and

(b) comparison between predicted and experimental value for film thickness

4.2 Density g/cm³

Table 4 reveals that the concentration of plasticizer (B and C) blends and process Temperature (D) showed no significant (p > 0.05) effects on the film density.

Only the mass concentration of tacca starch (A) had significant effect on the film density at 5% level of significance. This is similar to a report by [24] that no significant effect was observed in density by adding glycerol plasticizer in corn starch film. This can be ascribed to change in film formulation that caused a simultaneous rise in volume, which increased the thickness of the film, hence, no significant difference in film’s density. The empirical relationship between the observed experimental results and input effects was expressed by a linear equation fitted according to the experimental design used in this study as follows:

Density = 0.974052 + 0.015500A - 0.002222B - 0.000370C - 0.000667D                  (2)      

The regression coefficients as depicted by the above model equation showed a rise in density as starch level (A) increased at constant plasticizer blends and process temperature. On the other hand, a decrease in density results from increase in plasticizer concentration and process temperature at a constant mass concentration of tacca starch.

From Table 4, the R² of 0.7451 and the plots in Fig. 4 showed a good correlation between the experimental and predicted values, revealing the adequacy and fitness of the model. Also, there was a reasonable agreement between the adjusted R² (0.7027) and the predicted R² (0.6157) for the film density. The model F-value of 17.54 and a corresponding p-value of <0.0001 implies that the model is significant and can be used to navigate the design space.

Figure 4. (a) Response surface plot (3-D) for film density and (b) comparison between predicted and experimental value for film density

4.3 Moisture content (%)

The influence of mixture components and process temperature as indicated by ANOVA (Table 5) showed that the mass concentration of Tacca starch (A) and plasticizer (B and C) blends had significant (p<0.05) effects on the film moisture contents while process temperature (D) had no significant (p>0.05) effect on the film moisture content. This implies that both starch and plasticizers blends have a real or true effect on the film moisture content in the research study while the effects exerted by the process temperature and solely by chance or some random factors.

Table 5. Summary Statistics of the Moisture content of TSF

Similar results were reported by [25] and [26] for glycerol and Sorbitol as plasticizers. Also, a high concentration of plasticizers favors the adsorption of water molecules due to their hydrophilic nature. This may be due to the hydrophilic nature of both starch and the plasticizers which assists in the formation of hydrogen bonding with free OH groups [27]. Water acts as a solvent and causes textural degradation, chemical and enzymatic reactions. Also, the water activity of food is an important parameter in relation to the shelf life of the food. Therefore, in low moisture food, a low level of water activity must be maintained to minimize the deterioration, chemical and enzymatic reactions and prevent textural degradation.

The empirical relationship between the observed experimental results and input effects  was expressed by a linear equation fitted according to the experimental design used in this study as follows:

Moisture Content = +1.01293 + 0.500000A - 0.185185B - 1.99244C + 0.041667D                                                    (3)

The model coefficients reveal a decrease in moisture content as a result of increase in sorbitol (B) and glycerol (C) as depicted by -0.18518 and -1.99244 (coded) respectively when other variables are kept constant. Conversely, the model coefficients of 0.500 and 0.041 for starch level (A) and process temperature (D) reveal an increase in moisture content due to their increase, when other variables remain constant. The model R² value of 0.6159 and the plot in Fig 5 showed the correlation between the experimental and predicted values. Also, the adjusted R² and predicted R² of 0.5352 and 0.5124 respectively, implies a good fit for the model. The model F-value of 6.39 and a corresponding p-value of 0.012 implies the model is significant.

Figure 5. (a) Response surface plots (3-D) for film moisture content and

(b) comparison between predicted and experimental value for film moisture content.

4.4 Tensile strength (MPa)

Table 6 revealed that plasticizer (B and C) blends, and their interaction (BC) exerted a significant (p < 0.05) effect on the tensile strength of the biodegradable film while starch level (A) and process temperature (D) had no significant effect (p > 0.05) on the tensile strength of the Tacca starch biodegradable film at 5% level of significance. On the other hand, the quadratic terms of glycerol (C²) and temperature (D²) had significant effects (P < 0.05) on the tensile strength of the biodegradable film. The possible reason for the real effect of plasticizer concentration on the tensile strength of the biodegradable film is the domination of strong hydrogen bonds produced by starch–starch intermolecular interaction over starch–plasticizer attraction. This phenomenon can be explained through the role of plasticizers in diminishing the strong intra-molecular attraction between the starch chains and promoting the formation of hydrogen bonds between plasticizers and starch molecules. [28] reported similar observation that glycerol plasticizer induces great effect on the tensile strength starch based biodegradable films.

Table 6. Summary Statistics of the Tensile Strength of TSF

Tensile strength was found to have a quadratic relationship with the process variables as per the following equation:

Tensile Strength = + 168.62540 + 0.832190A – 1.25251B – 2.38020C – 3.91480D – 0.088178AB – 0.043289AC – 0.007710AD – 0.763160BC – 0.012444BD – 0.030789CD + 0.018510A² + 0.137430B² + 0.601208C² + 0.024136D^2.          (4)

From the empirical model, the quantitative effects of the individual mixture components and process parameter (temperature) as obtained from the above regression coefficients infer that an increase in either plasticizer (B and C) blends, process temperature (D), the interaction between starch level and sorbitol (AB), the interaction between starch level and glycerol (AC), the interaction between starch level and temperature (AD), the interaction between the plasticizers (BC), interaction between sorbitol and temperature (BD) or the interaction between glycerol and temperature (CD) would lead to a decrease in the tensile strength of the biodegradable film. On the other hand, the model coefficients also imply an increase in the tensile strength for any increase in starch level (A), and the quadratic terms of starch (A²), sorbitol (B²), glycerol (C²) or temperature (D²) when other variables were kept constant.

The model F-value of 3.62 and a corresponding p-value of 0.0110 implies the model is significant. Also, the coefficient of determination R² value of 0.7836 implies a good correlation between predicted and experimental values as depicted by the plot in Fig. 6. The adjusted R² of 0.6671 and the predicted R² of 0.6073 showed an agreement within the values and good fit for the model.