Optimization of Lignin–Cellulose Nanofiber-Filled Thermoplastic Starch Composite Film Production for Potential Application in Food Packaging

The optimization of the production of thermoplastic starch (TPS) bionanocomposite films for their potential application in food packaging was carried out, according to the Box–Wilson Central Composite Design (CCD) with one center point, using Response Surface Methodology (RSM) and fillers based on lignin and nanofiber, which were derived from bamboo plant. The effects of the fillers on the moisture absorption (MAB), tensile strength (TS), percent elongation (PE) and Young’s modulus (YM) of the produced films were statistically examined. The obtained results showed that the nanocomposite films were best fitted by a quadratic regression model with a high coefficient of determination (R2) value. The film identified to be optimum has a desirability of 76.80%, which is close to the objective function, and contained 4.81 wt. % lignin and 5.00 wt. % nanofiber. The MAB, TS, YM and PE of the identified film were 17.80%, 21.51 MPa, 25.76 MPa and 48.81%, respectively. The addition of lignin and cellulose nanofiber to starch composite was found to have reduced the moisture-absorption tendency significantly and increased the mechanical properties of the films due to the good filler/matrix interfacial adhesion. Overall, the results suggested that the produced films would be suitable for application as packaging materials for food preservation.


Introduction
Synthetic plastics have dominated every field of human activity, particularly the packaging industries [1]. Despite their many merits, synthetic plastics have been a major environmental concern for some time. Since they are non-biodegradable and also dependent upon a non-renewable petroleum resource, the blooming usage of these plastics has caused grave energy crises, as well as environmental pollution associated with their disposal, including damage to the eco-system, water supplies and sewer systems, as well as rivers and streams. As a result, great attention is being drawn to natural polymers, e.g., starch, due to its potential as a resource for making environmentally friendly useable products to replace those derived from petroleum. The reason for starch being favored for this purpose is because it is inherently biodegradable, renewable, cost effective and available from many plants. In order to make it suitable for industrial application, such as packaging, starch is usually processed in the presence of a plasticizer to obtain a product called thermoplastic (TPS). However, TPSs alone cannot be employed for packaging because they have low mechanical strength and high water sensitivity [2], which falls below the requirements reported in the literature. Moreover, by changing the sources of the lignin and fibers used in TPS, new biocomposites with unique properties can result. In this work, the lignin and nanofibers were obtained from bamboo plant (Bambusa vulgaris). Further to this, work on the optimization of the experimental variables for the development of flexible films from starch for food packaging is limited, hence, the justification for the present study. Therefore, the objective of this research is to produce a lignin-cellulose nanofiber-filled thermoplastic starch composite film, with the optimization of key process variables, for potential application in food packaging.

Film Morphologies
The SEM micrographs of the pure starch (representing the negative control sample) and that of lignin-cellulose nanofiber-filled thermoplastic starch composite film (the target sample) are shown in Figure 1 and Figure 2, respectively. The SEM micrograph of the pure starch, as seen (Figure 1), showed the characteristic near-spherical morphology of the cassava starch granules with varying sizes [14]. The micrograph showed that the starch granules were generally in the order of sizes that ranged between 2 and 7 µm. This morphology is, however, destructured during the starch gelatinization process, subsequent to the incorporation of the fillers, in order to pave the way for good mixing in the composite formation stage. In Figure 2, the SEM micrograph shows that the fillers (lignin and cellulose nanofiber) were homogeneously distributed within the matrix of the starch nanocomposite film, a situation that usually results in improved mechanical properties. The white dots with varying sizes on the composite are considered as the fillers embedded in the starch matrix. The sizes of the cellulose nanofibers and lignin were evaluated using transmission electron microscopy (TEM), and their respective micrographs were as shown in Figure 3a,b. It was observed that the sizes of the cellulose nanofibers varied from 20 to 100 nm, while those of lignin fell between 20 and 200 nm. The determined characteristics of the used cellulose nanofiber are hereby shown in Table 1. mediators for laccase treatment substantially enhanced the water resistance of starch biocomposite, which was detected by a change in the water drop contact angle on the biocomposite surface. So far, studies based on the simultaneous incorporation of lignin and cellulose nanofibers into thermoplastic starch composite film, besides the one referenced above, have not been reported in the literature. Moreover, by changing the sources of the lignin and fibers used in TPS, new biocomposites with unique properties can result. In this work, the lignin and nanofibers were obtained from bamboo plant (Bambusa vulgaris). Further to this, work on the optimization of the experimental variables for the development of flexible films from starch for food packaging is limited, hence, the justification for the present study. Therefore, the objective of this research is to produce a lignin-cellulose nanofiberfilled thermoplastic starch composite film, with the optimization of key process variables, for potential application in food packaging.

Film Morphologies
The SEM micrographs of the pure starch (representing the negative control sample) and that of lignin-cellulose nanofiber-filled thermoplastic starch composite film (the target sample) are shown in Figures 1 and 2, respectively. The SEM micrograph of the pure starch, as seen ( Figure 1), showed the characteristic near-spherical morphology of the cassava starch granules with varying sizes [14]. The micrograph showed that the starch granules were generally in the order of sizes that ranged between 2 and 7 µm. This morphology is, however, destructured during the starch gelatinization process, subsequent to the incorporation of the fillers, in order to pave the way for good mixing in the composite formation stage. In Figure 2, the SEM micrograph shows that the fillers (lignin and cellulose nanofiber) were homogeneously distributed within the matrix of the starch nanocomposite film, a situation that usually results in improved mechanical properties. The white dots with varying sizes on the composite are considered as the fillers embedded in the starch matrix. The sizes of the cellulose nanofibers and lignin were evaluated using transmission electron microscopy (TEM), and their respective micrographs were as shown in Figure 3a,b. It was observed that the sizes of the cellulose nanofibers varied from 20 to 100 nm, while those of lignin fell between 20 and 200 nm. The determined characteristics of the used cellulose nanofiber are hereby shown in Table 1.

Development of Regression Model Equations by Central Composite Design (CCD) for Lignin-Cellulose Nanofiber-Filled Thermoplastic Starch Composite Film
The quadratic models, in terms of the actual factors used in achieving the desired optimum or ideal films, were obtained from an analysis of variance (ANOVA) (Tables 2-5), and are given in Equations (1) to (4). The positive signs in the models indicate synergetic effects, whereas the negative signs, antagonistic effects. The model for predicting the moisture-absorption capacity of the lignin-cellulose nanofiber-filled thermoplastic starch composite film in terms of actual factors is given in Equation (1). where: MAB = moisture-absorption capacity, l = lignin (wt. %), n = nanofiber (wt. %). The model was significant at a p-value of 0.0048, and the coefficient of determination (R 2 ) of 0.9102 was obtained. This indicates that the statistical model could explain 91.02% of the variability, while the remaining 8.98 wt. % could not be accounted for by the independent variables [15]. The predicted of 64.30% reasonably agrees with the adjusted (85.63%), as should normally be the case for model adequacy due to the very small blocking effect of the data used. The precision of the model is high since the adequate precision value obtained was ~11.156 [16].

Development of Regression Model Equations by Central Composite Design (CCD) for Lignin-Cellulose Nanofiber-Filled Thermoplastic Starch Composite Film
The quadratic models, in terms of the actual factors used in achieving the desired optimum or ideal films, were obtained from an analysis of variance (ANOVA) (Tables 2-5), and are given in Equations (1) to (4). The positive signs in the models indicate synergetic effects, whereas the negative signs, antagonistic effects. The model for predicting the moisture-absorption capacity of the lignin-cellulose nanofiber-filled thermoplastic starch composite film in terms of actual factors is given in Equation (1). where: MAB = moisture-absorption capacity, l = lignin (wt. %), n = nanofiber (wt. %). The model was significant at a p-value of 0.0048, and the coefficient of determination (R 2 ) of 0.9102 was obtained. This indicates that the statistical model could explain 91.02% of the variability, while the remaining 8.98 wt. % could not be accounted for by the independent variables [15]. The predicted of 64.30% reasonably agrees with the adjusted (85.63%), as should normally be the case for model adequacy due to the very small blocking effect of the data used. The precision of the model is high since the adequate precision value obtained was ~11.156 [16].

Development of Regression Model Equations by Central Composite Design (CCD) for Lignin-Cellulose Nanofiber-Filled Thermoplastic Starch Composite Film
The quadratic models, in terms of the actual factors used in achieving the desired optimum or ideal films, were obtained from an analysis of variance (ANOVA) ( Tables 2-5), and are given in Equations (1)-(4). The positive signs in the models indicate synergetic effects, whereas the negative signs, antagonistic effects. The model for predicting the moisture-absorption capacity of the lignin-cellulose nanofiber-filled thermoplastic starch composite film in terms of actual factors is given in Equation (1).
where: MAB = moisture-absorption capacity, l = lignin (wt. %), n = nanofiber (wt. %). The model was significant at a p-value of 0.0048, and the coefficient of determination (R 2 ) of 0.9102 was obtained. This indicates that the statistical model could explain 91.02% of the variability, while the remaining 8.98 wt. % could not be accounted for by the independent variables [15]. The predicted R 2 of 64.30% reasonably agrees with the adjusted R 2 (85.63%), as should normally be the case for model adequacy due to the very small blocking effect of the data used. The precision of the model is high since the adequate precision value obtained was~11.156 [16]. The model for predicting the percentage elongation of the lignin-cellulose nanofiberfilled thermoplastic starch composite film in terms of actual factors is given in Equation (2).
where: PE = elongation (wt. %); l and n retained their usual meaning. The model was significant at a p-value of 0.1792, and the coefficient of determination (R 2 ) of 0.8449 was obtained. This indicates that the model could describe 84.49 wt. % variability, while 15.51 wt. % was inexplicable by the independent variables [15]. In addition, the negative predicted R 2 value of −82.39 wt. % implied that the fitted model, though precise (with adequate precision value of 5.37), is not a suitable predictor of the percentage elongation of the starch composite film.
The model for predicting the tensile strength of the lignin-cellulose nanofiber-filled thermoplastic starch composite film with respect to the actual factors is given in Equation (3).
where: TS = tensile strength (MPa); l and n retained their usual meaning. The model was significant at a p-value of 0.0045, and the coefficient of determination (R 2 ) of 0.8346 was obtained. This indicates that the statistical model could explain the 83.46% value of the variability, while a 16.54% value was inexplicable by the independent variables [15]. In addition, the predicted R 2 of 61.72 wt. % agrees with the R 2 (adjusted) of 77.94%, as should normally be the case for adequate model. The model is precise in its capacity to predict the tensile strength of the starch mixture film, since an adequate precision value of 10.91 was recorded [16].
The model for predicting the Young's modulus of the lignin-cellulose nanofiber-filled thermoplastic starch composite film in terms of actual factors is given in Equation (4).
where: YM = Young's modulus (MPa); l and n retained their usual meaning.  The model was found to be significant at a 0.0001 p-value, and the coefficient of determination (R 2 ) of 0.9501 was obtained. This indicates that the model could explain 95.01% of the variability, while 4.99% was inexplicable by the independent variables [15]. The predicted R 2 value of 91.81% reasonably agrees with the R 2 adjusted value of 93.34% for the adequate model, and this was caused by the small block effect of the data used in generating the model. In addition, the model is precise in its capability to forecast the lignin-cellulose nanofiber-filled thermoplastic starch composite film's Young's modulus, since the adequate precision recorded value was 20.51 [16].

Analyses of Response Surfaces
The 3D response surface plots of the joint effects of the independent variables (lignin and cellulose nanofiber) on the MAB, TS, YM and PE of lignin-cellulose nanofiber-filled thermoplastic starch composite film, as performed according to the working conditions stated in Table 6, are presented in Figure 4. The 3D response surfaces were plotted in order to investigate the probable interactions among the variables, and to determine the optimum conditions of each factor for the minimum moisture absorption and maximum strength of the starch nanocomposite films.

Analyses of Response Surfaces
The 3D response surface plots of the joint effects of the independent variables (lignin and cellulose nanofiber) on the MAB, TS, YM and PE of lignin-cellulose nanofiber-filled thermoplastic starch composite film, as performed according to the working conditions stated in Table 6, are presented in Figure 4. The 3D response surfaces were plotted in order to investigate the probable interactions among the variables, and to determine the optimum conditions of each factor for the minimum moisture absorption and maximum strength of the starch nanocomposite films. The response surface plots for the influence of lignin and cellulose nanofiber contents on the MAB, TS, YM and PE of the lignin-cellulose nanofiber-filled thermoplastic starch composite film are presented in Figure 4.   The results revealed that the inclusion of cellulose nanofiber content had the most significant effect on the moisture absorption and mechanical properties of the lignin-cellulose nanofiber-filled thermoplastic starch composite film, followed by lignin inclusion content. The MAB reduced rapidly as the cellulose nanofiber content increased when compared to that of lignin content. The lignin and cellulose nanofiber contents interacted negatively. This shows that the moisture absorption reduced as the lignin and cellulose nanofiber contents increased. The decrease in MAB with the increase in the lignin and cellulose nanofiber contents was because of the strong filler-matrix interaction, which reduced the molecular mobility and diffusivity in the matrix material, thus, limiting the degree of moisture uptake [17]. This result is in accordance with the findings of Khan et al. [18], Mitchelle [19], and Taghizedeh and Sabouri [20], who reported low moisture-absorption rates for cocoyam, tapioca and modified corn starch films, respectively, with an increase in filler loading.
The results also showed that the TS and YM of the starch nanocomposite film increased as the lignin and cellulose nanofiber contents increased, while PE decreased with the increase in lignin and cellulose nanofiber contents. The significant increase in the TS and YM of the lignin-cellulose nanofiber-filled thermoplastic starch composite film with the increase in filler contents, particularly nanofiber, was due to the small sizes (nanorange), smooth surface and large surface area of these fibers, which produced a good fiber/matrix interaction, thus, improving the composite film's strength [21]. It might also be that lignin improved the compatibility between the starch and cellulose nanofiber [22]. Moreover, Suarez et al. [23] also reported the fact that a good interfacial region increases stress transfer efficiency from the matrix to the fillers, thereby increasing the composite's strength.
This result corroborated with that of Wang et al. [22], who confirmed a rise in the TS and YM, but a decrease in the elongation-at-break of a PLA nanocomposite reinforced with lignin-cellulose nanofiber (L-CNF), as the L-CNF content rose from 25 to 35 wt. %. Patpen et al. [24] also reported the effect of cellulose addition on the TS of polylactic acid biocomposite; they observed an increase in this parameter as cellulose loading increased. Tawakkal et al. [25] also reported how kenaf-derived cellulose (KDC)-loaded polylactic affected the material's tensile properties and documented an improvement in both TS and tensile modulus, as KDC loading increased from 30 to 60 wt. %. On the contrary, Sawpan et al. [26] studied the mechanical properties of hemp fiber-reinforced PLA biocomposites The response surface plots for the influence of lignin and cellulose nanofiber contents on the MAB, TS, YM and PE of the lignin-cellulose nanofiber-filled thermoplastic starch composite film are presented in Figure 4.
The results revealed that the inclusion of cellulose nanofiber content had the most significant effect on the moisture absorption and mechanical properties of the lignin-cellulose nanofiber-filled thermoplastic starch composite film, followed by lignin inclusion content. The MAB reduced rapidly as the cellulose nanofiber content increased when compared to that of lignin content. The lignin and cellulose nanofiber contents interacted negatively. This shows that the moisture absorption reduced as the lignin and cellulose nanofiber contents increased. The decrease in MAB with the increase in the lignin and cellulose nanofiber contents was because of the strong filler-matrix interaction, which reduced the molecular mobility and diffusivity in the matrix material, thus, limiting the degree of moisture uptake [17]. This result is in accordance with the findings of Khan et al. [18], Mitchelle [19], and Taghizedeh and Sabouri [20], who reported low moisture-absorption rates for cocoyam, tapioca and modified corn starch films, respectively, with an increase in filler loading.
The results also showed that the TS and YM of the starch nanocomposite film increased as the lignin and cellulose nanofiber contents increased, while PE decreased with the increase in lignin and cellulose nanofiber contents. The significant increase in the TS and YM of the lignin-cellulose nanofiber-filled thermoplastic starch composite film with the increase in filler contents, particularly nanofiber, was due to the small sizes (nano-range), smooth surface and large surface area of these fibers, which produced a good fiber/matrix interaction, thus, improving the composite film's strength [21]. It might also be that lignin improved the compatibility between the starch and cellulose nanofiber [22]. Moreover, Suarez et al. [23] also reported the fact that a good interfacial region increases stress transfer efficiency from the matrix to the fillers, thereby increasing the composite's strength.
This result corroborated with that of Wang et al. [22], who confirmed a rise in the TS and YM, but a decrease in the elongation-at-break of a PLA nanocomposite reinforced with lignin-cellulose nanofiber (L-CNF), as the L-CNF content rose from 25 to 35 wt. %. Patpen et al. [24] also reported the effect of cellulose addition on the TS of polylactic acid biocomposite; they observed an increase in this parameter as cellulose loading increased. Tawakkal et al. [25] also reported how kenaf-derived cellulose (KDC)-loaded polylactic affected the material's tensile properties and documented an improvement in both TS and tensile modulus, as KDC loading increased from 30 to 60 wt. %. On the contrary, Sawpan et al. [26] studied the mechanical properties of hemp fiber-reinforced PLA biocomposites and established a non-linear relationship between TS and fiber content, indicating that the strength of the biocomposite somewhat decreased as the fiber content increased from 0 to 40 wt. %.

Optimization of Lignin-Cellulose Nanofiber-Filled Thermoplastic Starch Composite Film Production
The result of the optimization of the experimental variables (lignin and cellulose nanofiber contents), showing the desirability function, with respect to the films prepared, is shown in Figure 5. The goal for both the water barrier and mechanical optimization was to minimize the moisture absorption and improve the mechanical property; thus, the target value of the responses, as obtained from the obtained experimental results, was at the lowest and uppermost values, respectively. The result from the optimization showed that increases in the lignin and cellulose nanofiber contents, from 1 to 5 wt. %, significantly affected the desirability of the films. Therefore, it is obvious from the optimization result that the selected film with a desirability closer to the goal is the one that was produced with the blend of 4.81 wt. % lignin and 5.00 wt. % nanofiber at 76.80% desirability. The corresponding MAB, TS, YM and PE of the selected film, apparently the optimum sample, are 17.80%, 21.51 MPa, 25.76 MPa and 48.81%, respectively. This finding is similar to that of Wang et al. [22], who documented optimum values of 21.6 MPa and 21.6% for the TS and elongation, respectively, for PLA composites reinforced with L-CNF. Akbar et al. [27], however, obtained optimum values of 50 MPa for the TS, 2.15 MPa for the YM and 141.07% for elongation, during the optimization of polyvinyl alcohol nanocomposite films. Patpen et al. [24] also reported an optimum value of 46.207 MPa for the optimization of PLA-based biocomposites that were reinforced with cellulose obtained from durian peel. and established a non-linear relationship between TS and fiber content, indicating that the strength of the biocomposite somewhat decreased as the fiber content increased from 0 to 40 wt. %.

Optimization of Lignin-Cellulose Nanofiber-Filled Thermoplastic Starch Composite Film Production
The result of the optimization of the experimental variables (lignin and cellulose nanofiber contents), showing the desirability function, with respect to the films prepared, is shown in Figure 5. The goal for both the water barrier and mechanical optimization was to minimize the moisture absorption and improve the mechanical property; thus, the target value of the responses, as obtained from the obtained experimental results, was at the lowest and uppermost values, respectively. The result from the optimization showed that increases in the lignin and cellulose nanofiber contents, from 1 to 5 wt. %, significantly affected the desirability of the films. Therefore, it is obvious from the optimization result that the selected film with a desirability closer to the goal is the one that was produced with the blend of 4.81 wt. % lignin and 5.00 wt. % nanofiber at 76.80% desirability. The corresponding MAB, TS, YM and PE of the selected film, apparently the optimum sample, are 17.80%, 21.51 MPa, 25.76 MPa and 48.81%, respectively. This finding is similar to that of Wang et al. [22], who documented optimum values of 21.6 MPa and 21.6% for the TS and elongation, respectively, for PLA composites reinforced with L-CNF. Akbar et al. [27], however, obtained optimum values of 50 MPa for the TS, 2.15MPa for the YM and 141.07% for elongation, during the optimization of polyvinyl alcohol nanocomposite films. Patpen et al. [24] also reported an optimum value of 46.207 MPa for the optimization of PLAbased biocomposites that were reinforced with cellulose obtained from durian peel.
This study thus affirms that lignin and cellulose nanofiber addition to starch composite minimized MAB and improved the mechanical properties of the produced films because of the good filler/matrix interfacial adhesion, as evident by the SEM micrograph. Hence, the produced films can be applied to package food.  This study thus affirms that lignin and cellulose nanofiber addition to starch composite minimized MAB and improved the mechanical properties of the produced films because of the good filler/matrix interfacial adhesion, as evident by the SEM micrograph. Hence, the produced films can be applied to package food.

Plant Materials
Cassava (Manihot esculenta crantz) was purchased from a farm in Minna, Nigeria, whereas bamboo (Bambusa vulgaris schrad) was collected from the river banks of Gurara in Izom, Niger State. They were identified and authenticated by a botanist at the National Institute for Pharmaceutical Research and Development (NIPRD) Idu, Abuja. Specimen voucher numbers (NIPRD/H/6792 and NIPRD/H/6793), respectively, were placed at their herbarium for references in the future.

Preparation Lignin and Cellulose Nanofiber from Bamboo (Bambusa vulgaris Schrad)
Lignin was extracted from bamboo according to standard procedure, as reported by Yong et al. [28], Alemdar and Sain [29] and Ming-Fei et al. [30]. The bamboo stalks obtained were first sun-dried and then chipped into small pieces. The sun-dried pieces of bamboo were ground and screened to obtain a 40-60 µm mesh fraction. The ground bamboo stalk (20 g) was first soaked in NaOH (4% w/w) at room temperature for 24 h. It was then filtered and washed with distilled water (1 L) until it was free of alkali. The residue was re-dispersed in 1 L of distilled water, filtered again and treated with 10% (w/w) NaOH at 121 • C in an autoclave for 4 h. Furthermore, the residue was again washed in distilled water to free it of residual alkali (1 L) and filtered. Lignin was precipitated from the filtrate by acidifying it to pH 2 with H 2 SO 4 . The precipitates were separated from the mixture by filtration. The separated lignin was washed with water several times and then oven-dried at 40 • C. In order to obtain bamboo fiber, the supernatant liquid left after the alkali treatment was bleached in 8% (v/v) H 2 O 2 at room temperature for 24 h. Finally, the material was again washed and filtered as before to obtain bamboo fiber. The bamboo fiber was then converted to nanofiber using acid hydrolysis [31]. The bamboo fibers obtained after lignin removal were steeped in HCl (10% w/w) with ultrasonic agitation at 60 • C for 2 h using an Ultrasonicator (SB25-12DT, Scientz, Ningbo, China). The material was given a final wash and then placed in a high shear homogenizer (Heidolph DIAX 900, Burladingen, Germany for 15 min to produce bamboo nanofibers.

Experimental Design and Optimization of Starch Nanocomposite Film Production
Design Expert software (version 7.0, Start-Ease Inc., Minneapolis, MN, USA) was employed for the experimental design, while Response Surface Methodology (RSM) was used for optimizing the conditions required for preparing the starch nanocomposite films. As a result, 1 center-point Box-Wilson Central Composite Design (CCD) was utilized. After the designed experiment was performed, linear regression was used to obtain the results. The design consisted of 9 experimental runs. The RSM considered the effect of two variables: lignin content (wt. %) and cellulose nanofiber content (wt. %) used as fillers in the nanocomposite preparation with 5 levels each. The response functions measured were moisture absorption (MAB), tensile strength (TS), percent elongation (PE) and Young's modulus (YM). An analysis of variance (ANOVA) was employed to analyze the obtained data, in order to determine the interactions that exist between the process variables and the responses. Accurate and proper models were picked at p < 0.05 and had a significantcorrelation. The fitting model's quality was expressed by the coefficient of determination R 2 and adjusted R 2 . The factors' level with their codings are shown in Table 7. In the optimization selection, two factors (lignin and cellulose nanofiber contents) were considered in order to build desirability indices. The objective was to reduce the MAB while improving TS, PE and YM; therefore, the target value of the responses was lowest for MAB and highest for TS, PE and YM from the experimental results obtained.

Preparation of Lignin-Cellulose Nanofiber-Filled Thermoplastic Starch Composite Film
Cassava starch (2 g) granules were dispersed in 50 mL of distilled water and heat was applied at 70 • C for 20 min under constant stirring over a magnetic stirrer. Glycerol (50 wt. % based on dry cassava starch content) was added to the dispersion while the heating at 70 • C was continued under constant stirring speed for the next 2 min. Next, the lignin and cellulose nanofiber (varied wt. %, with respect to the dry cassava starch content, and based on the statistical formulation of the Central Composite Design adopted) were added to the dispersion under the same conditions for another 2 min. Before being introduced into the plasticized starch mixture, the cellulose nanofibers were sonicated for 10 min by using a 60 W rated Sonicator. The mixture of dispersion was then cast into a mold and oven-dried at 50 • C using a still-air oven for 18 h, in order to obtain dry lignin-cellulose nanofiber-filled thermoplastic starch composite films [32,33], whose average thickness was found to be 0.12 mm. The control (TPS) sample was also prepared using the same process mentioned above, except that there were no fillers added to it. All films were conditioned at 55 ± 5% RH and 25 ± 2 • C before testing their permeability and mechanical properties, as described by the ASTM standard D882-09 [34] and Detduangchan et al. [35]. The conditioning was performed by inserting the films into desiccators containing a saturated solution of Mg (NO 3 ) 2 •6H 2 O for 72 h. Part of the films were tested for water absorption, TS, PE and YM, and the rest, which were for other tests, were kept in plastic bags and inserted into desiccators.

Characterization of Lignin-Cellulose Nanofiber-Filled Thermoplastic Starch Composite Film Water-Absorption Test
The film pieces (20 mm × 20 mm) were pre-conditioned by drying in the oven at 50 • C for 24 h and then weighed to determine the dry weight. They were then immersed in a bath containing distilled water at room temperature. The film samples were removed from distilled water after intervals of 1, 3, 5, 7, 9 and 11 h and, after wiping off the excess water on their surfaces with tissue, their weights were determined. The water-absorption capability (WAC) was, thus, calculated using Equation (5) [32,36]: where: Wwet = Wet specimen weight and Wdry represents the dry specimen weight.

Mechanical Properties
The TS, PE and the YM values were determined with a universal testing machine (DBBMTCL-2500kg Testometric, Rochdale, UK), according to the ASTM D882 Standard [34]. Prior to testing, each sample was conditioned at a temperature of 25 • C and a relative humidity (RH) of 55% for 24 h. The average thickness of the samples was about 0.12 mm. The tensile test was carried out using 1.3 mm/min crosshead speed. Each of the determinations was obtained from triplicate specimens.

Scanning Electron Microscopy (SEM)
The morphological structures of the lignin-cellulose nanofiber-filled thermoplastic starch composite films were determined using SEM (Zeiss Auriga HRSEM) at an accelerating voltage of 15 kV. The as-prepared samples were, respectively, placed on a stub with a double-sided adhesive tape, after which they were coated with a thin layer of gold. The micrographs were captured using a magnification of 350 times the original specimen size [33,37].

Statistical Analysis
Design Expert Software (version 7.0) was employed to analyze the obtained data. The determination of interaction effects between the factors and a quadratic surface plot was generated using an analysis of variance (ANOVA). The model adequacy was examined using the ANOVA, a normal probability plot and a residual plot, according to the method described in the literature [38]. An F-Test was also employed to determine the model's statistical significance and the regression coefficients' significance.

Conclusions
In conclusion, the film with a desirability of~76.80%-which is closest to the objective function-and containing 4.81 wt. % lignin and 5.00 wt. % cellulose nanofiber, was selected as the optimum sample. The MAB, TS, YM and PE of the selected film were 17.80%, 21.51 MPa, 25.76 MPa and 48.81%, respectively. This film presented the maximum mechanical potency and minimal moisture-absorption capacity. The addition of lignin and cellulose nanofiber concurrent to the TPS matrix evidently caused a decrease in moisture absorption, while at the same time an improvement in the mechanical properties of the films. Consequently, the prepared films have the potential to be employed as films for packaging foods. Funding: This research received no external funding. Notwithstanding, E.C.E, by virtue of being part of ACE for Mycotoxin and Food Safety, Federal University of Technology, Minna, Nigeria, qualified for financial support to help defray, in part, the publication fee of the manuscript. Such support is usually extended to ACE member(s) whose manuscript(s) is/are accepted for publication in Q1 or Q2 journals, such as this one.
Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.