Introduction

In recent years, the interest in using natural and renewable polymers has been increasing in different areas of application, especially in developing substitutes for synthetic films. Currently, among polymers obtained from renewable sources, starch is the most popular because it can be obtained from a great variety of crops [1], and it is the most promising natural polymer for the production of biodegradable plastic film. The use of starch for biodegradable film production is based on the physical, chemical and functional properties of one of its components (amylose), which is responsible for gelation and film formation capacity [2]. These films present oxygen and grease barriers, and are isotropic, odorless, tasteless, colorless, non-toxic and biologically degradable [3]; however, there are some limitations since they have poor tensile properties and high water vapor permeability [4]. Several strategies have been investigated to minimize or even overcome these drawbacks. These strategies involve the use of non-conventional starch sources [5–7], chemical modification [8–10], mixing with synthetic and polyester polymers [9–11], and the use of different nanoparticles such as cellulose nanocrystals mixed with starch [9–14].

Chayote (Sechium edule Sw.) tubers are a perennial climbing plant that is native to Mexico. This plant produces a fruit similar to squash that may be grown as a food crop. Chayote plants also produce tubers that are rich in starch and may provide a local or regional source of starch. Hernandez-Uribe et al. [7] compared the properties of chayote tuber starch with potato starch and reported differences in peak viscosity, gelatinization temperature, and molecular weight. There is a lack of cellulose nanocrystal/starch composites of particular research interest due to the beneficial effect of CN on tensile strength, Young’s modulus, water resistance and water vapor permeability using a wide array of starches [11, 12, 14–18]. In this same sense, Tang and Alavi [11] and Souza et al. [14] reported a decrease in the water vapor permeability up to 70 % and an increase in the mechanical properties up to 50 % of cellulose nanocrystal/starch films when were compared with the control starch film. Calorimetric studies reported an increase in the Tg and a decrease in the ΔH value when the concentration of cellulose nanocrystals increased in the composite film, improving its thermal stability [12, 19]. On the other hand, the rate of biodegradation is a very important issue in this kind of film (starch/nanocrystals). Spiridon et al. [20] and Tang et al. [21], reported that the rate of biodegradation of films depends on the type and content of nanoparticle. They observed a reduction in the biodegradation percentage when the concentrations of nanoparticle in the film increased. Vargas et al. [22] studied the effect of the storage conditions on the rate of degradation of starch films, reporting an increase in the rate of biodegradation when the samples were stored at −20 and 80 °C. This pattern was attributed to partial starch depolymerization in the matrix film.

The objective of this work was to evaluate the influence of storage conditions at different temperatures and relativity humidity (RH) in films made with chayotextle starch mixed with cellulose and nanocellulose crystals and determines the effect on the mechanical and thermal characteristics, structural properties, and biodegradation rate.

Experimental

Materials

Chayote (S. edule Sw.) tubers were collected in Tulancingo, Hidalgo, Mexico. Cellulose fiber with catalog number S3504 and Type 20, 20 μm, was purchased from Sigma-Aldrich of Mexico (Toluca, State of Mexico, Mexico). Glycerol with catalog number 911, lot number 205912 and HSO4 with 95.98 % of purity, lot number 341354 were purchased from HYCEL and Reasol (Mexico, D.F.), respectively. Pregelatinized wheat starch was purchased (MGP1, MGP, Atchison, KS) and used as a reference in respirometry tests. Various salts (MgCl2, NaBr, SrCl2, and BaCl2) (Reasol, Mexico City, Mexico) were acquired for use in RH tests.

Starch Isolation

Chayote (S. edule Sw.) tubers were collected in Tulancingo, Hidalgo, Mexico. The starch was isolated according to the methodology used by Flores-Gorosquera et al. [23]. The tubers were cut into 2 × 2 cm cubes and immediately macerated at low speed in a blender (500 g root: 500 g tap water) for 2 min. The homogenate was consecutively sieved and washed through screens number 50, 100, 200, 270 and 325 US mesh, until the washing water was clean and was pelleted overnight and decanted. This material was dried in convection oven at 35 °C overnight. Dry starch was ground in a blender and passed through a standard 100 mesh sieve and stored in sealed container until required.

Preparation of Cellulose Nanoparticle

Cellulose was subjected to acid hydrolysis for producing the cellulose nanoparticle according to the procedure of Teixeira et al. [12]. The cellulose nanoparticle obtained was ground using a laboratory blender and sieved (100 U.S. mesh). The size of the nanoparticles obtained was between 5 and 15 nm reported in previous study by Aila-Suarez et al. [24].

Film Preparation

Films were prepared in aqueous solution containing 4 % of chayote tubers starch (w/w, dry basis), 2 % of glycerol (w/w), cellulose (0.8 %, w/w) or cellulose nanoparticle (1.2 %, w/w) and water (170 mL). For identification of the films, the following abbreviations are used: CS-08C and CS-1.2CN, respectively. The dispersions were prepared by first homogenizing (Tissue Tearor homogenizer, model 985370-395, Mexico) the cellulose or cellulose nanocrystals was homogenized in 70 mL of H2O (25 °C) at 11,000 rpm for 5 min. In another container starch and glycerol were homogenized with 100 mL of water at 40 °C. The homogenates were combined in a mini-reactor (300 mL), stirred at 125 rpm (Lightnin Lab Master SPX-Mixer), and heated to 90 °C for 10 min. The films were prepared by casting and the suspensions were poured in a glass container (11 × 11 cm of length and width respectively, with 3 mm thickness) and dried at 40 °C in an oven for 24 h. Afterward, the films were removed from the glass container and stored at 25 ± 2 °C.

Storage of the Films

The films were stored for 3 and 5 days at different relative humidity (RH) and temperature. The storage conditions were 33, 57, 69 and 90 % RH (using saturated salt solutions of MgCl2, NaBr, SrCl2, and BaCl2, respectively) at 25 °C, and at different temperatures (−80, 4, 25 and 70 °C) at RH of 57 % (ASTM D 638 M-93).

Mechanical Properties

The mechanical measurements consisted of a test to determine the Elastic modulus (E), tensile strength (Φ U ) and percentage elongation at break (%El). The mechanical properties were obtained from force versus deformation curves according to the ASTM standard method D882-95a [25] with a texture analyzer equipped with 50 kg load cell (TA-HDi) (Stable Micro Systems, Haslernere, UK, and Texture Technologies Corp., Scarsdale, NY). The samples were prepared using the instructions of the official method ASTM D 638 M–93 [26] and they were maintained for 3 and 5 days in desiccators with the saturated saline solution. The separation among the gauges was 6 cm. The ends of the film were fixed in each of the subjection gauges. The speed of deformation was 1 mm/s. The thickness of the films was assessed with a digital micrometer (Truper with a sensibility of 0.001 mm) in 5 random positions of the film. This average value was used to calculate the cross-sectional area of the films (the area was equal to the thickness multiplied by the width of each film).

X-ray Diffraction (XRD)

Samples were milled to fine consistency (average particle size of 5 μm), and were analyzed between 2θ = 1.6–60° at a scan speed of 0.015°/s in an X-ray diffractometer (Philips X’pert (MPD), using Cu Kα radiation (λ = 0.1543 nm), 60 kV and 30 mA.

Thermogravimetric Analysis

The thermogravimetric analysis (TGA) was performed with a HI-Res TGA 2950, (TA instruments). The sample (10.0 ± 1.0 mg) was heated from 25 to 550 °C under a Nitrogen atmosphere with a flow rate of 20 mL min−1. The heating rate was 20 °C min−1.

Biodegradability Test

Samples were milled to fine consistency (average particle size is 1 μm) using a hammer mill. About 0.2 g of each sample was mixed with 20 g of compost with 60 % moisture content (Garden Basics Topsoil, Swiss Farm Products, and the Vegas, NV) in a 250 mL reaction chamber. Sample chambers were connected to an automated and fully computerized closed-circuit Micro-Oxymax Respirometer System (Columbus Instruments, Columbus, OH) equipped with an expansion interface and a condenser. Experiments were carried out at room temperature (23 ± 0.2 °C) over a period of 30 days. The respirometer was programmed to measure CO2 evolution from each sample every 5 h. Each sample was run in triplicate and averaged (<2 % variability). The two control samples consisted of compost alone that provided a measurement of the background CO2 evolution [27] and a sample containing pregelatinized wheat starch (PWS).

Results and Discussion

Mechanical Properties

Tables 1 and 2 show the effect of the RH on the mechanical properties in films of chayotextle starch mixed with cellulose and cellulose nanoparticles. A statistical analysis (α = 0.05) showed that the storage time (3 and 5 days) did not affect the mechanical properties of the films. The storage time was too short to produce changes in the arrangement of the polymers in the composite film, affecting the mechanical properties [28]. Composite films containing CN had higher elastic module (E), tensile strength (Φ U ) and elongation at break (%El) than films blended with C. Similar findings were reported by Bras et al. [29], Chang et al. [13], De Teixeira et al. [30], and Siqueira et al. [31], who mentioned that the change in mechanical properties was due to the superior reinforcing effect of the CN as a result of the increase in the contact area and hydrogen bonding between the surface of the CN and the starch matrix (amylose and amylopectin), which produce a continuous rigid network of cellulose nanofibers linked by hydrogen bridges, thus increasing the mechanical properties of the composite films.

Table 1 Effect of the relative humidity on the mechanical properties in CS-0.8C films
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Table 2 Effect of the relative humidity on the mechanical properties in CS-1.2CN films
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The E and Φ U values in the film with C increased when the RH increased from 33 to 70 %, and both values reached a plateau at 90 % RH. This pattern was due to the fact that the maximum antiplasticizing effect in the composite was produced at 70 % of RH. The E value did not change when the films were stored at 33 and 70 % RH, and this value decreased at the highest RH tested (90 %). This reduction in the E value could be related to an antiplasticizing effect from the humidity of the environment, which produced a softer film matrix [28, 32]. A similar pattern was found in the film composites with CN.

Tables 3 and 4 show the effect of the temperature on the mechanical properties. The films with C showed different behavior for each mechanical property. The E value was not affected when the films were stored at −80.4, and 25 °C, but an increase was determined in the film stored at 70 °C. High temperatures produced the loss of plasticizer, reducing mobility and provoking interactions among the polymer chains. It has been suggested that under such conditions polymeric chains in the starch molecule can undergo a reorganization, causing the composite to harden [33, 34].

Table 3 Effect of the temperature on the mechanical properties in CS-0.8C films
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Table 4 Effect of the temperature on the mechanical properties in CS-1.2CN film
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Φ U value showed an increase among the composite film stored at −80 and 4 °C, but no change was found in the films stored at higher temperatures (25 and 70 °C). On the other hand, %El value was not affected by storage temperature because a similar value was obtained at the four temperatures tested.

However, composite films with CN showed a slightly different behavior in the E value compared with its counterpart with C because the former showed an increase in the E value in the films stored at 4 and 25 °C compared with those stored at −80 °C, and the highest E value was determined in the film stored at 70 °C. Φ U value in the composite film with CN presented a similar pattern to its counterpart with C, but higher values were determined in the former sample. Similar to the composite films with C, %El value was not modified in the composite films with the storage temperature.

X-ray Diffraction (XRD)

The CS-1.2CN composite film was selected for the next studies (X-ray diffraction, thermogravimetric, and biodegradability) since it presented higher mechanical properties than its counterpart with C. The native chayote starch showed a B-type X-ray diffraction pattern (Fig. 1a) characteristic of a tuber [35]. The diffraction pattern showed peaks around 2θ = 17°, 19.6°, and 22.5°. The peak at 2θ = 17° is characteristic of amylopectin crystal (B-type crystallization). The X-ray diffraction pattern in composites (b, c, d, and e in Fig. 1a) showed peaks around 19.6° and 22.5°, which are characteristic of the VH-type. The VH-type consists of amylose recrystallization induced by lysophospholipids and complex-forming agents such as isopropanol and glycerol [12]. Van Soest and Vliegenthart [36], reporting the same peak (2θ = 19°) attributed to the crystallinity caused by the rapid recrystallization of the single-helical amylose structure during cooling after processing. There are studied reporting that the VH structure forms below 180 °C is frequently found in starch-based films made with more than 10 % water [37]. The peak around 2θ = 25° is attributed to the CN crystallinity in blended starch films. Teixeira et al. [12] reported a peak at 2θ = 26°, observing that this peak increased with the cellulose cassava bagasse nanofibrils which increased in the starch cassava films. In this same sense, a slight reduction in the peak intensity was observed in the conditioned films to 70 °C. This reduction in crystallization rate could be linked to the low water content due to the high storage temperatures. High storage temperatures produce water evaporation, decreasing the crystallites amount in the film. Muller et al. [38] and Riandla et al. [39] reported that this behavior is characteristic with low water content, and they also reported that the crystallization rate increased with higher water content.

Fig. 1
figure 1

X-ray diffraction patterns of CS-1.2CN films. a Effect of temperature; (a) native chayotextle starch, (b) −80 °C, (c) 4 °C, (d) 25 °C, (e) 70 °C. b Effect of RH; (a) chayotextle starch, (b) 33 %, (c) 70 %, (d) 90 %

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The Fig. 1b shows the X-ray diffraction of the composite films at different RH. Similar peaks were observed, as those presented by the effect of storage temperature, indicating that the storage conditions (temperature and RH) did not affect the re-arrangement of polymers in the film matrix.

Thermogravimetric Analysis (TGA)

Figure 2 shows three main steps in the TGA curves. The first step corresponds to the loss of water. The second step is due to the decomposition of the glycerol-rich phase, which also contains starch, while the third step corresponds to the oxidation of the partially decomposed starch [40]. In both cases (RH and temperature effect), the TGA curves show an initial drop between 80 and 210 °C, which corresponds to a mass loss of absorbed moisture. After this, in the temperature range between 210 and 300 °C a rapid loss matter occurs due to the decomposition in the glycerol-rich phase. The third step is observed between 300 and 420 °C when the loss mass rate decreased, which is attributed to the decomposition of starch and CN. Some studies have reported that the addition of cellulose nanoparticles or clay nanocomposites may increase the thermal stability (at a higher temperature to achieve the same weight loss), which can be ascribed to the better thermal stability of CN as compared to plasticized starch, and the good interaction between CN and plasticized starch [13, 41]. In this study, the CN introduction to the matrix film could be giving better stability to the thermal storage effect because no change was observed in the rate of mass loss from the effect of the temperature.

Fig. 2
figure 2

a TGA curves of temperature effect in CS-1.2CN films. b TGA curves of relative humidity effect in CS-1.2CN films

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Figure 2a shows a characteristic pattern as reported by Wilhelm et al. [40]. This indicates that the temperature conditions in the films do not influence the rate of mass loss. In Fig. 2b, it was observed that the films with 90 % of RH needed 222 °C to reach a 78 % of loss mass, while the films with 70 and 33 % of RH needed 219 and 240 °C, respectively. This increase in the temperature with the reduction in RH can be attributed to the addition of plasticizer (water or glycerol), decreasing the interaction (both intra and inter molecular links) of the starch–starch chains, weakening the hydrogen bonding formed between the hydroxyl groups of starch chains and the plasticizers molecules. As a consequence, it becomes easier for the film to degrade due to the presence of a higher water content [17].

Biodegradability Tests

Biodegradation test have revealed that with both storage conditions (temperature and RH), the films initially showed a fast degradation for about 8 days, reaching a maximum of 80 % in loss mass (an indicator of biodegradation) after 25 days (Fig. 3). Both storage parameters produced an increase in the biodegradation rate when these are compared with the reference (MPG1), but no difference in the degradation percentage was found at the different temperatures and RH tested. Although the degradation rate of the chayote starch films was higher than in pre-gelatinized starch, the time could be too short to produce a re-arrangement of the polymers in the films for observation of the changes in the degradation rate. These results agree with those obtained with X-ray diffraction and TGA because changes in crystallinity level and increase in the loss mass (%) in the films were not observed. It has been reported that an increase in the degree of crystallinity reduces the degradation rate in polymer blends [42–44]. On the other hand, the susceptibility of the composites to biodegradation depends largely on the moisture content [44, 45] and the fiber nanometric size [41, 42].

Fig. 3
figure 3

a Effect of the temperature on the degradation rate in CS-1.2CN films. b Effect of the relative humidity on the degradation rate in CS-1.2CN films. MGP1 = pre-gelatinized wheat starch

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Vargas et al. [22] reported a higher degradation rate in samples pre-exposed at −20 and 80 °C, causing a depolymerization and reduction in the molar mass of the polysaccharide. This can facilitate the breakdown of the matrix making it more accessible and prone to degradation by microbes and their hydrolytic enzymes [46, 47]. The importance of a fast degradation of the films elaborated with biodegradable polymeric material lies in minimizing the adverse environmental effects of the product disposal.

Conclusion

Mechanical properties are strongly influenced by fiber size in C versus CN composite films. Films blended with CN showed an increase in E and Φ U values. The amount of time in storage did not impact the mechanical properties. However, storage temperature and relative humidity did have an impact on mechanical properties. B-type crystalline structure of native starch was observed and it was transformed into VH-type structure due to complex formation with glycerol. The thermogravimetric curves showed that temperature and relativity humidity did not affect the thermal stability of the composite films. The degradation rate was higher for the films compared with that of the starch control. It may be that CN will also be effective in improving the mechanical properties of other natural polysaccharide matrices. This could lead to an increase in the use of CN in the food packaging industry and the use of non-conventional sources of starch (chayotextle).