Sustainable Production and In vitro Biodegradability of Edible Films

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Sustainable Production and In vitro Biodegradability of Edible Films from Yellow Passion Fruit Co-products via Continuous Casting Davi Renato Munhoz, Francys Kley Moreira, Joana Dias Bresolin, Marcela Piassi Bernardo, Cristina Paiva de Sousa, and Luiz H. C. Mattoso ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b01101 • Publication Date (Web): 10 Jun 2018 Downloaded from http://pubs.acs.org on June 10, 2018

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ACS Sustainable Chemistry & Engineering

Sustainable Production and In vitro Biodegradability of Edible Films from Yellow Passion Fruit Co-products via Continuous Casting Davi R. Munhoz†, Francys K.V. Moreira†‡*, Joana D. Bresolin†, Marcela P. Bernardo†, Cristina P. De Sousa§, Luiz H. C. Mattoso† † National Nanotechnology Laboratory for Agribusiness (LNNA), Embrapa Instrumentation, XV de Novembro Street, 13560-970, São Carlos (SP), Brazil. ‡Department of Materials Engineering (DEMa), Federal University of Sao Carlos (UFSCar), Rod. Washington Luis, Km 235, 13565-905, São Carlos (SP), Brazil. *[email protected] § Postgraduate Program in Biotechnology (PPG-Biotec), Federal University of Sao Carlos (UFSCar), Rod. Washington Luis, Km 235, 13565-905, São Carlos (SP), Brazil.

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KEYWORDS. Passion fruit pomace, valorization of wastes, packaging material, tensile strength, biodegradability.

ABSTRACT. Edible films made up of yellow passion fruit (YPF) rind and pectin as a matrixforming agent are proposed as a means of valorizing passion fruit processing wastes. YPF films were produced at pilot-scale using continuous casting from aqueous formulations covering pectin/rind and water/pulp mass ratios of 100/0 – 0/100. Successful obtaining of YPF films with systematic, tunable yellowish coloration was achieved at optimal temperature of 120 °C, leading to drying time of 7 min and productivity of 0.03 m2 film min-1. YPF pulp is found to plasticize the pectin matrix of the films and thus can replace glycerol or other synthetic plasticizers. Films with largest rind content (50 wt.%) showed mechanical strength comparable to that of PVC cling film (9 MPa vs. 5 MPa). The biodegradable, renewable character of YPF films was demonstrated upon exposure to Escherichia coli, Staphylococcus aureus, and to Bradyrhizobium diazoefficiens, a nitrogen-fixing symbiotic bacterium.


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Introduction Passiflora is a well-established industry that exploits various plant species for therapeutic, ornamental, cosmetics and food applications.1,2 Yellow passion fruit (YPF) (Passiflora edulis Sims f. flavicarpa degener), in particular, is the edible berry-like fruit grown worldwide due to its gelatinous yellow pulp which exhibits unique sensory properties.1,2,3 The YPF physiology further comprises the epicarp (yellowish peel), mesocarp (internal white tissue) and the pulpsurrounded black seeds.1,3 Brazil is currently the largest passion fruit producer worldwide, accounting for nearly 60 % of the global production4. The Brazilian production has reached 700,000 tons with an average yield of 14 tons ha-1 in 2016,5 as an outcome of intensive researches for enhancement of the whole passion fruit industry.1 Most of YPF crops are intended for pulp extraction which is processed into juices and other products in large-scale extraction plants, while the rinds and seeds have been regarded as processing wastes. A regular YPF fruit contains 26 – 40 % pulp, while the rind and seeds correspond to the remaining 60 – 74 %,6,7,8 representing an amount of unexploited biomass that exceeds 500,000 tons year-1. Exploitation of YPF co-products (rind and seeds) for obtaining higher added-value products has progressed much over the past years, including cellulose nanocrystals9 and pectin6,10 extraction from YPF peels, linoleic acid (ω-6)11 and piceatannol12 extraction from seeds, use of YPF rind flour in biodegradable films13,14 among others.3,15,16,17 Nevertheless, these efforts only addressed reuse of YPF co-products partially. It is still necessary to develop strategies by which YPF could be integrated as a whole into valued products.


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Edible film is a sustainable approach capable of transforming YPF co-products into applicable materials. Edible films may replace non-biodegradable materials in conventional packaging systems, which is advantageous at a sideway environmental glance.18,19 The first edible films made up of fruits puree were developed by batch casting with the perspective of recycling blemished fruits, thereby generating valued materials at a low cost.20 Since then, progress has been made towards the development of various fruit puree-based edible films.21-25 Edible films have also been developed from potato26 and apple27 peels via batch casting. In this sense, while transforming YPF co-products into edible films seems feasible and beneficial from the sustainable perspective, scaled-up production of such materials is still challenged by the lack of processing methods by which loss of YPF sensory attributes (aroma, color etc.) could be minimal.

Herein, we address the challenge of scaling up the production of wasted fruit-based edible films with maximum retention of sensory attributes by applying the continuous casting technique to turn YPF co-products into edible films in a soft and highly productive fashion. The scaled-up production of an edible film comprising fruit processing waste by continuous casting has not been reported yet and is advantageous over classical processing techniques due to its green aspect and safety for food applications. To extend the sustainable perspective, we first compared the co-products yields of ripe and overripe YPF to be linked with film productivity. Pectin was tested as a matrix-forming agent because it already makes up the YPF rind and can be easily solubilized without heating. The validity of our hypothesis is confirmed to be applicable when establishing qualitative and quantitative relations between coloration and mechanical properties of films with water/pulp and pectin/rind mass ratios of film-forming suspensions, and comparing the continuous casting productivity with earlier reports. The biodegradability of the YPF films


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was tested upon exposure to Escherichia coli, Staphylococcus aureus, and to Bradyrhizobium diazoefficiens, a nitrogen-fixing symbiotic bacterium.

Experimental Section Materials. Yellow passion fruits (YPFs) (P. edulis Sims. f. flavicarpa Degener) were purchased at local markets in Sao Carlos, Brazil. Ripe (r-) and overripe (o-) fruits were selected with basis on their visual aspect and stored at 25 °C prior to using. Citrus pectin with esterification degree (DM) of 74 % and average molecular weight mass (Mw) of 1.3 x 105 g mol-1 was purchased from CP Kelco (Limeira, SP, Brazil). High purity (~99%) glycerol (Nº CAS 56-81-5) was purchased from Synth (Diadema, SP, Brazil). Poly(vinyl chloride) (PVC) cling film was purchased from Wyda (Sorocaba, SP, Brazil). Deionized water (ρ > 18.2 MΩ cm) was obtained with a Barnstead Nanopure DiamondTM purification system (Thermo Fisher Scientific Inc, USA). Processing of yellow passion fruits. YPFs were weighted and processed in a food centrifuge to isolate rind and pulp from the seeds into two different portions. Pulp was weighted, tested for pH and soluble solids content (°Brix) and kept at -18 °C in a freezer to preserve its sensory attributes. Rind was dried overnight at 70 °C in an oven and afterwards weighted on a semianalytical scale. Then, the rind was powdered and put through a 150-mesh sieve to standardize its grain size. These steps allowed the pulp and rind, contents of r- and o-YPFs to be determined in triplicate. Pulp content was calculated as pulp(wt.%) = (mpulp mtotal-1)·100, where mpulp is the pulp mass and mtotal is the total YPF mass. Rind content was determined as rind(wt.%) = (mrind mtotal-1)·100, where mrind is the dried rind mass, respectively. Total moisture content (wt.%) of YPFs was also determined in triplicate by drying at 105°C until constant weight.


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Continuous edible film casting. Edible films were produced with pectin and rind at mass ratios of 100/0, 75/25, 50/50 and 0/100. The total solid content of aqueous suspensions for film casting was fixed at 6 wt.% using water, pulp or their mixture at 50/50 as a solvent. In brief, pectin, rind and solvent were added to a polyacetal vessel (Vtotal = 2.6 dm3) and vigorously homogenized at 25 °C and 20,000 rpm using a mechanical stirrer. Suspensions were degassed by applying vacuum at -400 mm Hg alongside mechanical stirring and immediately cast into monolayer films in a KTF-B labcoater machine (Werner Mathis AG, Zürich, Switzerland), depicted in Figure 1. The suspension was inserted in the coating device, Figure 1a, where the wet layer thickness was adjusted to 1.7 mm using a doctor blade-B and a comparative clocks pair (± 0.001 mm), Figures 1b-c. The wet layer was dried by conveyance at speed of 12 cm min-1 through a IR pre-dryer with 40 – 55 % emission potency, Figure 1d, and subsequent through an air circulating oven (total length of 80 cm) equilibrated at 120 °C, Figure 1e. Films were collected at the oven outlet and stored for further characterizations, Figures 1f. Characterizations. Sample morphology was examined by scanning electron microscopy (SEM) on a JEOL JSM 6701 microscope. Film samples were cryo-fractured into liquid N2 for crosssectional surface imaging using the secondary electron detector and accelerating voltages of 2 – 5 kV. Thermogravimetric (TG) and differential thermogravimetric (DTG) curves were recorded on a TA Instruments thermal analyzer Q500. Samples (8 – 10 mg) were heated up to 600 °C at 10 °C min−1 under dynamic air atmosphere (O2 20% and N2 80%) flowing at 60 mL min−1. Powder X-ray diffraction (PXRD) patterns were registered using a Shimadzu XRD 6000 diffractometer operating with Cu Kα radiation (λ = 1.5405 Å). PXRD patterns were recorded over 2θ range of 5 – 45º at scanning speed of 2º min−1. Color parameters of CIELab system (L* = lightness, a* coordinate = red-green component and b* coordinate = yellow-blue component)


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were determined in triplicate using a HunterLab colorimeter Miniscan XE. Water uptake (%) of films (3 cm x 3cm) was calculated as WH20 (wt.%) = (mt – m0)/m0, where mt is the sample mass at time t measured periodically up to 48 h conditioning at RH = 54 ± 2% and m0 is the initial sample mass dried at 105 °C. Tensile tests were conducted as per ASTM D882-09 using a universal machine EMIC DL3000 (EMIC Equipamento e Ensaios Ltda, PR, Brazil) equipped with a 10 kgf (98 N) load cell. Young’s modulus (E), true tensile strength (σT), and true elongation break (εB) of edible films were calculated from stress (σ) – strain (ε) curves. σT was determined as σT= σ λ, where σ is the engineering tensile strength and λ is the extension ratio defined as λ = L/L0, where L and L0 are the final and initial specimen lengths, respectively. εB was computed as εB = ln λ. Young’s modulus (E) was obtained through linear regression of σ – ε curves in the limit σ = ε = 0 ([dσ/dε]ε=0). Average film thickness was determined from ten random positions in each film using a digital micrometer (Mitutoyo Manufacturing, Japan). In vitro microbial susceptibility of YPF edible films was tested for Escherichia coli (ATCC 11229), Staphylococcus aureus (ATCC 6538), and Bradyrhizobium diazoefficiens CPAC 7 (SEMIA 5080) following the disc diffusion test.28 Biodegradation tests were performed under aerobic composting conditions over 40 days according to EN ISO 20200 with slight modifications. Full description of experimental methods is provided in Supporting Information.


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Figure 1. Schematics of continuous edible YPF film casting in a KTF-B labcoater machine. a, feed of the labcoater’s coating device with aqueous YPF suspension. b, wet layer formation in the coating device (two-dimensional velocity gradient (vmax = 12 cm min-1) assuming viscous flow between parallel plates is illustrated as an insert). c, overview of the coating device detailing the doctor blade-B and clock pair for wet layer thickness adjustment (l = 1.7 mm). d, pre-drying of the wet layer with IR radiation (40 – 55 % emission potency). e, Outcome of wet


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layer passage through 80 cm-long air circulating oven (120 °C). f, monolayer film collection at the oven outlet. Results and discussion

Ripe (r-YPFs) and overripe (o-YPFs) yellow passion fruits were tested for edible film production. Overall, r-YPFs exhibit a smooth and fully yellow colored peel, whereas o-YPFs differ largely due to their very wrinkly rind and browned rind. Thus, o-YPFs are often rejected by consumers at the purchasing time. Figure 1a illustrates the visual aspect of YPFs upon variation of ripeness degree. Table 1 reports the composition of r- and o-YPFs in terms of moisture, pulp, rind and pulp soluble solids contents. It is observed that the moisture content of r-YPFs is larger than that of oYPFs (p0.05). Such composition of YPF agrees with earlier reports.2,8,29 The compositional data shown in Table 1 indicate that the proportions of rind in r-YPFs and oYPFs are similar. Rind and pulp of YPFs were specifically used to obtain edible films by continuous casting. This means that r-YPFs and o-YPFs could be used regardless to achieve the


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same film productivity. The use of o-YPFs still remains more valuable due to their highest market rejection. Suitable mixing of o-YPF rind with pectin is achieved upon milling the rind to a thin flour (Figure S1 in Supporting Information). Table 1. Overall analysis of r-YPF and o-YPF. Parameter

Ripeness degree

Moisture content (wt.%)

r-YPF 88.3 ± 2.1a

o-YPF 74.8 ± 2.0b

Pulp (wt.%)

33.8 ± 2.7a

30.2 ± 4.2a

Pulp soluble solids content (ºBrix)

17.7 ± 0.8a

18.9 ± 1.0a

Pulp pH

3.7 ± 0.3a

3.4 ± 0.2a

Rind (wt.%)

7.0 ± 1.2a

7.0 ± 0.8a

*(mean ± standard deviation). Mean in the same row followed by the same letter are not statistically different at probability level of 5 %.

Scanning electron microscopy (SEM) and X-ray diffraction (XRD) were performed to characterize the extracted YPF rind (Figures 2b and 2c). Figure 2b reveals a fibrous aspect with micrometric fibers spread throughout the rind structure. In addition, the XRD pattern shown in Figure 2c confirms the presence of cellulose as denoted by the peaks at 15.7°, 22°, and 34.3° of 2θ, which corresponds to crystalline planes (110), (200) and (040) of cellulose I.9,30 These results show that the YPF rind is mainly composed of cellulose fibers. However, there is enough evidence that the YPF rind also has elevated pectin content, which is an amorphous polysaccharide, therefore, not detectable by XRD. YPF rind cellulose content is typically around 38 – 50 %, whereas the pectin content varies from 7 to 23 %.2,6,7,10


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Figure 2. a, Schematic illustration of YPF as a function of ripeness degree (u- unripe, r- ripe, ooverripe). b, SEM micrograph (300x, scale = 10 µm) of YPF rind after drying at 105 °C (magnified insert, 500x, scale = 10 µm). c, PXRD diffractogram of YPF rind after drying at 105


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°C with peaks indexed as cellulose I. d, TG (blue) and DTG (red) curves of YPF rind (heating rate of 10 °C min-1; oxidizing atmosphere (O2 20% and N2 80%). To set up the processing conditions for continuous casting, the thermal stability of the YPF rind was assessed in O2-rich atmosphere. The TG curve in Figure 2d, and the DTG curve in the insert, shows a mass loss (~6 %) between 25 – 150 °C ascribed to water evaporation, two overlapped mass losses (~56 %) between 160 – 400 °C with Tmax at 232 °C and 310 °C ascribed to the pectin31 and cellulose30 degradation, respectively, and a mass loss (32 %) between 410 – 550 °C attributed to the oxidation of the char formed in the previous steps.31 Two final thermal events are observed at 600 – 650 °C and 700 – 900 °C, which may be ascribed to decomposition of K salts and CaCO3, respectively.32,33,34 The final residue mass at 800 °C is approximately 0.5 %. TG/DTG curves show that the thermal degradation of YPF rind starts at 160 °C. A processing window of 100 – 150 °C for the continuous casting was then outlined, the upper limit of 150 °C determined by the initial degradation temperature of the YPF rind and the lower limit of 100 °C because water-based suspensions were used to produce the YPF edible films. The experimental screening showed that YPF suspensions comprising 0/100 and 25/75 pectin/rind ratios do not form films, which relates to the fact that the rind cellulosic structure is unable to dry as a continuous matrix (See Figure S2 in Supporting Information). Thus, the presence of rind in the suspensions hampers the film forming process, which is pointed out to be critical at 75 % (pectin/rind ratio 25/75). On the other hand, obtaining of YPF edible films is achieved for suspensions with pectin/rind ratio starting at 50/50 with pure water, pure pulp or their 50/50 mixture as a solvent. Pectin efficiently acts as a matrix-forming agent, impregnating the fibrous rind structure to form cohesive films.


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Figure 3 illustrates the visual aspect of these samples, including the neat pectin films (pectin/rind ratio 100/0). It can be seen a systematic color change with pectin/rind and water/pulp ratios as shown in Figure 3a. Color parameters (L* = lightness, a* coordinate = red-green component and b* coordinate = yellow-blue component) of films have been summarized in Table 2. The rind provides color to films, even without adding pulp to the suspensions, which is noticed for the increased yellowness (b* coordinate value) of film with 50/50 pectin/rind ratio (p

Sustainable Production and In vitro Biodegradability of Edible Films

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