Incorporation of Natural Blueberry, Red Grapes and Parsley Extract by-Products into the Production of Chitosan Edible Films

polymers Article Incorporation of Natural Blueberry, Red Grapes and Parsley Extract By-Products into the Production of Chitosan Edible Films Simona Dordevic 1,* , Dani Dordevic 1, Petr Sedlacek 2, Michal Kalina 2, Karolina Tesikova 1, Bojan Antonic 1, Bohuslava Tremlova 1, Jakub Treml 3, Marcela Nejezchlebova 3, Lukas Vapenka 4, Ales Rajchl 4and Monika Bulakova 3   Citation: Dordevic, S.; Dordevic, D.; Sedlacek, P.; Kalina, M.; Tesikova, K.; Antonic, B.; Tremlova, B.; Treml, J.; Nejezchlebova, M.; Vapenka, L.; et al. Incorporation of Natural Blueberry, Red Grapes and Parsley Extract By-Products into the Production of Chitosan Edible Films. Polymers 2021, 13, 3388. https://doi.org/10.3390/ polym13193388 Academic Editor: Ana Beltrán Sanahuja Received: 15 September 2021 Accepted: 27 September 2021 Published: 1 October 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Department of Plant Origin Food Sciences, Faculty of Veterinary Hygiene and Ecology, University of Veterinary Sciences Brno, Palackeho tr. 1946/1, 61242 Brno, Czech Republic; [email protected] (D.D.); [email protected] (K.T.); [email protected] (B.A.); [email protected] (B.T.) 2Faculty of Chemistry, Brno University of Technology, Purkynova 118, 61200 Brno, Czech Republic; [email protected] (P.S.); [email protected] (M.K.) 3Department of Molecular Pharmacy, Faculty of Pharmacy, Masaryk University, Palackeho tr. 1946/1, 61200 Brno, Czech Republic; [email protected] (J.T.); [email protected] (M.N.); [email protected] (M.B.) 4Department of Food Preservation, University of Chemistry and Technology Prague, Technicka 5, 16628 Prague, Czech Republic; [email protected] (L.V.); [email protected] (A.R.) *Correspondence: dor[email protected]; Tel.: +420-077-460-7936 Abstract: The aim of the research was to produce edible packaging based on chitosan with the addition of various concentrations of extracts of blueberry, red grape and parsley marcs. Packaging was made from extrudate extracts, which were subsequently analyzed by physicochemical methods: zeta-potential, gas barrier properties, thickness, water content, solubility, swelling degree, textural properties, total polyphenol content (TPC), polyphenols by high pressure liquid chromatography (HPLC), antioxidant activity, attenuated total reflectance Fourier-Transform spectroscopy (FTIR), antimicrobial activity and determination of migration of bioactive substances. The results indicate that a higher content of plant extracts have a statistically significant (p< 0.05) influence on properties of experimentally produced edible films. Edible films produced with the highest concentrations of red grapes marc extracts showed the most advantageous properties since antimicrobial activity against E. coli were the highest in this kind of produced film. The physical properties of edible films were also improved by the addition of extracts; gas permeability toward oxygen can be defined as advantageous, as can swelling degree, which decreased with higher concentrations of extracts. The research emphasized the possibility to use plant foodstuffs by-products in the production of edible/biodegradable films, helping in the overall sustainability and eco-friendliness of food/package production. Keywords: antioxidant activity; FTIR; barrier properties; antimicrobial properties 1. Introduction The production of edible packaging based on polysaccharides has been developing greatly in recent years. With the use of edible—and therefore degradable—materials, it is possible to reduce the production of waste significantly [ 1 ], which is in line with the modern trends of a sustainable economy. There are many possibilities how to prepare the edible packaging and there are a lot of different compositions that can be used. The research concerning edible/biodegradable packaging can be focused on packaging production itself or it can be focused on edible/biodegradable packaging application on different food commodities. Recent publication was focused on the preparation of edible packaging from whey protein isolate nanofibers and carvacrol; the experimentally produced packing was Polymers 2021,13, 3388. https://doi.org/10.3390/polym13193388 https://www.mdpi.com/journal/polymers Polymers 2021,13, 3388 2 of 21 applied on duck egg yolk and shelf life was monitored [ 2 ]. In another study, the packaging based on microalgal exopolysaccharides with the addition of red seaweed extract were applied on shrimps [ 3 ]. The technology of preparation of films significantly affected the properties of edible/biodegradable packaging; the different preheating temperatures were studied for the preparation of films based on soy protein isolate and soy oil that led to the changes of hydrophobicity [4]. Chitosan (N-deacetylated derivative of chitin) belongs to the film-forming components that are most often proposed for use in the production of edible packaging. As the second most abundant polysaccharide in nature, chitosan has become commonly used in various industries in the last decades, and many studies dealing with its further application potential, based, among others, also on its film-forming properties, have been published [ 5 ]. Its advantage is that it is a natural polysaccharide; thus, in contrast to synthetic polymers, it is biocompatible and biodegradable. Chitosan is also non-toxic, and it possesses antimicrobial properties [ 6 , 7 ]. Examples of chitosan use are its incorporation in wound healing products [8] and in water purification to remove mercury [9]. Byproducts from the food production are most often incorporated in some low value products, for example, in feed production. However, as these products are still rich in various bioactive substances, it seems more reasonable to utilize them as active ingredients, providing the material with added value, such as in the production of edible packaging. This way, the packaging material can be provided with highly valued properties such as the shelf life improvement of packaged food, oxidation and dehydration prevention, etc. These additional properties occur mainly due to the ability of the packaging material to release the bioactive components to the packaged food in a gradual and controlled manner. Apart from the active performance, edible packaging can also have intelligent properties, where the packaging can act as an indicator of a change in the condition of the packaged food (an example might be a change in the color of the packaging during food spoilage) [ 10 – 12 ]. Numerous works have already dealt with a use of the food industry byproducts in the production of edible packaging. For instance, the use of protein and pectin extracted from pumpkin by-products (seeds and skins) has been investigated [ 13 ], as well as the incorporation of hydrolyzed gelatin obtained from carp skin, which may show some antioxidant activity [14,15], same as the use of mango seed core extract [16]. There are also other food industry byproducts that can be utilized in the production of food packaging. Blueberries, for instance, represent a very good source of anthocyanins that can be used in the food industry as a substitute for synthetic dyes, because dyes found in nature are safer compared to synthetic dyes. Among the most interesting properties of anthocyanins are their color change in environments with different pH and their antioxidant properties [ 17 ]. These substances could also replace the currently commonly used synthetic antioxidants BHA (butylhydroxyanisole) and BHT (butylhydroxytoluene) since they have adverse effects on enzymes found in the human body [ 18 ]. Red grapes are known for their high content of polyphenols. The group of flavan-3-ols found in red grapes includes catechin, epicatechin, gallocatechin and epigallocatechin [ 19 ]. In general most polyphenols in grapes are found in the skin and seeds [ 20 ]. Parsley has antibacterial, antiviral, anti-inflammatory and antioxidant properties [ 21 ], and it contains flavonoids and coumarins [22]. In the previous research the sensory analysis, color characterization, microscopy and biodegradability were studied [ 23 ]. The aim of the research was to experimentally produce edible packaging with the incorporation of plant byproducts (blueberries, red grapes and parsley marc) and to evaluate their physical and chemical properties; experimentally produced films are comprehensively analyzed, and based on these results potential applications can be found. Polymers 2021,13, 3388 3 of 21 2. Materials and Methods 2.1. Materials Low molecular weight chitosan (50,000–190,000 Da), as well as other chemicals needed to perform the analyses, were purchased from Sigma-Aldrich (St. Louis, MO, USA) Parsley (Petroselinum crispum (Mill.) A. W. Hill) was grown in the Czech Republic, blueberries (Vaccinium myrtillus L.) were grown in Spain and seedless red grapes (Vitis Vinifera L. Crimson Seedless variety) were grown in Chile. All the above-mentioned plant raw materials were purchased in the Tesco store in Brno, in the Czech Republic. 2.2. The Extract Preparation Extracts were prepared from the byproducts after preparation of juices; the residual waste after juicing was collected. The first step was the juicing of raw materials, the resulting waste containing part of the pulp and husks was obtained. A total of 10 g of the by-product thus obtained were weighed into a beaker and poured into 100 mL of hot distilled water (100 ◦ C) and infused for 10 min and then the extract was filtered and used to produce edible packaging. 2.3. Edible Packaging Preparation The preparation of edible films (Table 1) included following: 1.5 g of low molecular weight chitosan was weighed into a 250 mL beaker and subsequently dissolved in 1% lactic acid. The amount of 1% lactic acid solution varied depending on the addition of the extract (the amount of lactic acid was replaced by the extract). An amount of 135 mL of 1% lactic acid was used in the samples without extract addition. The samples were then transferred to magnetic stirrers where they were stirred for 15 min at 50 ◦ C and at 500 rpm. The fresh prepared plant extract was then added at concentrations of 5%, 10% and 20% (w/w), followed by stirring for five minutes and the addition of glycerol as a plasticizer. After five minutes, the film-forming solution was poured into 150 mm diameter Petri dishes and left to dry for 48 h. Table 1. Composition of prepared films. Sample Composition CHL1.5 g chitosan + 1% lactic acid + glycerol 5CHLBO 1.5 g chitosan + 1% lactic acid + 5% blueberry extract + glycerol 10CHLBO 1.5 g chitosan + 1% lactic acid + 10% blueberry extract + glycerol 20CHLBO 1.5 g chitosan + 1% lactic acid + 20% blueberry extract + glycerol 5CHLPE 1.5 g chitosan + 1% lactic acid + 5% parsley extract + glycerol 10CHLPE 1.5 g chitosan + 1% lactic acid + 10% parsley extract + glycerol 20CHLPE 1.5 g chitosan + 1% lactic acid + 20% parsley extract + glycerol 5CHLHR 1.5 g chitosan + 1% lactic acid + 5% red grapes extract + glycerol 10CHLHR 1.5 g chitosan + 1% lactic acid + 10% red grapes extract + glycerol 20CHLHR 1.5 g chitosan + 1% lactic acid + 20% red grapes extract + glycerol 2.4. Stability of the Film-Forming Solutions Zeta Potential The zeta potential of chitosan in all above mentioned film-forming solutions was determined by Zetasizer Nano ZS (Malvern Panalytical, UK) by means of the electrophoretic light scattering method. The values of zeta potential were determined through the measurement of the electrophoretic mobility of particles in used dispersion media after the application of an external electric field. For purposes of the analyses, approximately 1 mL of individual liquid film-forming solutions were transferred into the spectroscopic cuvette (optical glass, 12 × 12 × 45 mm, PSC 1115, Malvern Panalytical, UK), and subsequently the universal dip cell (Zen 1002, Malvern Panalytical, UK) was immersed to be able to apply the external electric field (used effective voltage 4.890 ± 0.011 V). The analysis of Polymers 2021,13, 3388 4 of 21 one sample was performed in 5 repeated measurements (each measurement represents an average value of 12 scans). 2.5. Basic Morphological and Textural Properties of the Films 2.5.1. Film Thickness Film thickness was measured using a Mitutoyo M310-25 micrometer (Kawasaki, Japan) at 5 different locations. 2.5.2. Textural Properties Strength (MPa) and breaking strain (%) were measured using a TA.XT plus texturometer (Godalming, UK) by the ASTM International Test Method—ASTM D882-02. The produced packages were cut into rectangles measuring 1 × 5 cm and each measurement was performed 5 times. 2.5.3. Gas Barrier Properties Water vapor transmission rate (WVTR) was determined by gravimetrical method according to DIN 53 122 standard at 23 ◦ C and relative humidity of 85%. Five parallel samples were tested for each packaging material. Oxygen permeability was determined using OxTran 2/20 MH measuring system (MOCON Inc., Minneapolis, MN, USA) according to ASTM D3985—17 standard at 23 ◦ C and relative humidity of 0%. Two parallel samples were tested for each packaging film. 2.6. Basic Compositional and Structural Analysis 2.6.1. Water Content, Solubility and Swelling Degree The determination was performed according to the slightly modified method published by Souza et al. [ 24 ]. The film samples were cut into 2 × 2 cm squares and then weighed on an analytical balance (KERN, Germany), the weight was marked as W1. Subsequently, the films were placed in an oven (Ecoccel 55) for 2 h at 105 ◦ C and then reweighed (W2). Subsequently, the samples were placed in beakers containing 25 mL of water and, after 24 h at room temperature, dried and reweighed (W3). Next, they were transferred to an oven for 24 h at 105 ◦ C and then weighed (W4). Replicates (n= 6) were prepared for each sample. The results were obtained from the following equations: Water content (%) = [(W −W2)/W1)] ×100 Solubility (%) = [(W2 −W4)/W2] ×100 Swelling degree (%) = [(W3 −W2)/W2] ×100 (1) 2.6.2. Attenuated Total Reflectance Fourier-Transform spectroscopy Fourier transform infrared (FTIR) spectra of the prepared films were with an iS50 FTIR spectrometer (Thermo Scientific, Waltham, MA, USA). All measurements were taken from a surface of a film at ambient temperature (in an air-conditioned room) with the built-in single-reflection diamond attenuated total reflectance (ATR) crystal. An individual absorption spectrum was collected as an average of 16 scans with a resolution of 4 cm −1 (data spacing 0.5 cm −1 ). Each film was analyzed at 6 randomly distributed spots on its surface (3 spots on each—back and front—side of the film), FTIR spectra are provided in respective figures, representing an average of spectra collected for an individual sample. The whole-spectra PCA analysis was performed using a standard multivariate principle component program written in-house using MATLAB software ( MathWorks, Natick, MA, USA ) at Institute of Scientific Instruments, Czech Academy of Sciences [25]. 2.7. Content of the Antioxidant Compounds in the Films 2.7.1. Attenuated Total Reflectance Fourier-Transform Spectroscopy The total polyphenol content was measured using the Folin–Ciocalteu method described by Tomadoni et al. [ 26 ] with slight modifications. An amount of 1 g of the edible Polymers 2021,13, 3388 5 of 21 packaging was weighed into a beaker and then 40 mL of distilled water was added. The samples were stirred for 10 min and then 1 mL was taken into a 25 mL volumetric flask, 5 mL of Folin–Ciocalteu solution (diluted 1:10 by volume) and 4 mL of 7.5% Na 2 CO 3 were added to the sample. The samples were incubated in the dark for 30 min. The absorbance was measured at 765 nm against a blank (1 mL of the sample was replaced by 1 ml of distilled water). The results were expressed as the content of gallic acid per gram of the sample. Each sample was measured in triplicate. 2.7.2. HPLC—Polyphenolic Compounds Determination HPLC chromatograph, 1260 Infinity high performance liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA) was used to determine polyphenolic compounds in the experimentally produced edible packaging. The method of Gómez-Estac et al. [ 27 ] with slight modifications was used. The mobile phase consisted of 1% phosphoric acid (A) and acetonitrile (B) in the following composition: 80% of A and 20% of B for 20th min, 70% of A and 30% of B from 20th to 25th min, 60% of A and 40% of B from 25th to 40th min. The separation was performed on a Zorbax SB-C18 4.6 × 250 mm column (Agilent Technologies, Santa Clara, CA, USA) (the temperature was 25 ◦ C) and detection was performed on a DAD array detector (detection wavelength was 324.5 nm) The injection volume was 10 µ L. Each sample was measured in triplicate. The software used for HPLC chromatohraph was Agilent ChemStation. 2.8. Evaluation of the Antioxidant Properties of the Films 2.8.1. FRAP (Ferric Reducing Antioxidant Power) The FRAP was conducted by Behbahani et al. [28]. An amount of 0.1 g of the sample was weighed, to which 20 mL of 75% methanol was added, and the samples were then sonicated in a water bath for 30 min. Subsequently, 180 µ L of the extract was pipetted into dark vials, to which 300 µ L of distilled water and 3.6 mL of working solution (acetate buffer, TPTZ (2,4,6-Tripyridyl-S-triazine) and FeCl 3 ) were added. The samples were further incubated for 8 min in the dark. Absorbance was measured at 593 nm against a blank sample (distilled water + working solution). Trolox was used to prepare a calibration curve and the results were expressed as µ mol of Trolox per gram of sample. Each sample was measured in triplicate. 2.8.2. ABTS (2,20-Azino-Bis(3-Ethylbenzothiazoline-6-Sulfonic Acid)) The ABTS method was conducted according to Thaipong et al. [ 29 ] with slight modification. An amount of 0.1 g of the sample was weighed into dark vials, to which 20 mL of ethanol was added, and the samples were sonicated for 30 min. Then, 12 to 16 h before the measurement, 10 mL of 0.007M ABTS solution was mixed with 10 mL of 0.00245 M potassium persulphate solution. The solution was diluted before the measurement so that its final absorbance at 735 nm was 0.7. Then, 1980 µ L of ABTS solution was mixed with 20 µ L of the prepared package extract. The samples were incubated for 5 min in the dark and then the absorbance at 735 nm was measured. Each sample was measured in triplicate. The results were calculated according to the following formula: ABTS [%] = [(AbsABTS-Abssample)/AbsABTS]×100 (2) AbsABTS—absorbance of ABTS solution (−). Abs sample—absorbance of sample (−). 2.8.3. DPPH (2,2-Diphenyl-1-Picrylhydrazyl) The DPPH method was conducted according to Adilah et al. [ 30 ], with slight modifications. The 0.1 g of the film sample was weighted and 20 mL of ethanol was added, the samples were sonicated for 30 min, then the extracts were filtrated, 3 mL of extract and 1 mL of 0.1 mM DPPH solution in ethanol were mixed. The samples were incubated at laboratory temperature in the dark for 30 min and the absorbance was measured at Polymers 2021,13, 3388 6 of 21 517 nm by spectrophotometer (CE7210 DIET-QUEST, Cambridge, England). Each sample was measured in triplicate. The scavenging activity of DPPH was calculated according to the following formula: DPPHscavenging activity [%] = [(AbsDPPH −Abssample)/AbsDPPH]×100 (3) AbsDPPH—absorbance of DPPH solution (−), Abs sample—absorbance of sample (−). 2.9. Antimicrobial Properties of Films Edible films were exposed to UV radiation (wavelength 260 nm) due to physical disinfection. Subsequently, disks (edible films) with a diameter of 5 mm were cut in an aseptic environment. A modified disk diffusion method according to EUCAST (European Committee on Antimicrobial Susceptibility Testing) was used to determine the antimicrobial resistance of edible films. Staphylococcus aureus subsp. aureus (methicillin resistant strain) CCM 7110 and Escherichia coli CCM 3954 were cultivated with edible coatings, solid medium according to Mueller and Hinton (MUELLER-HINTON broth, Agar for microbiology, Sigma-Aldrich) was used and solid medium was used for culturing Candida albicans CCM 8261 with edible coatings, malt broth (Malt Extract Broth, Agar for microbiology, Sigma-Aldrich). The inoculum concentration was adjusted to approximately 1−2×108CFU/mL , corresponding to 0.5 degree McFarland turbidity standard. An amount of 1 mL of inoculum was spread on the surface of the agar and, after drying, 4 discs from edible films were placed on a 9 cm diameter dish. Each set of films (LBO, LPE and LHR) was tested on all three microorganisms and each plate included a CH L control in addition to the individual discs set. The inoculated plates were incubated for 18 h at 35–37 ◦C . A positive result was considered in the case when the microorganism did not outgrow the disc of the edible shell or even created an inhibition zone around the disc in which the tested microorganism did not grow. Depending on the used medium, in some cases the edible casing flowed during the cultivation at 35–37 ◦C. Reference strains of microorganisms Staphylococcus aureus subsp. aureus (MRSA) CCM 7110, Escherichia coli CCM 3954 and Candida albicans CCM 8261 were obtained from the Czech Collection of Microorganisms, the Department of Experimental Biology, Faculty of Science, Masaryk University. 2.10. Determination of Migration of Bioactive Compounds The films were cut into 1 × 1 cm squares and immersed in 2.5 mL of a 10% aqueous ethanol solution, which was chosen as simulant A according to the Regulation No. 10/2011 on plastic materials and other materials intended to come into contact with food [ 31 ]. The samples were then incubated for 10 days at 40 ◦ C, conditions for using the food packaging from 3 to 30 days at 20–40 ◦ C. The samples were then analyzed on the following analysis: FRAP, DPPH, ABTS and the total content of polyphenols, which were chosen as indicators for the migration of substances that may affect the shelf life of packaged foods. 2.11. Statistical Analysis All results in tables present mean values ± standard deviations. Statistical significance of p< 0.05 was determined by a one-sample ANOVA test using parametric Tukey’s test (when Leven’s test showed p> 0.05) and non-parametric Games–Howell post hoc test (when Leven’s test showed p< 0.05). IBM SPSS software was used for statistical processing. 3. Results and Discussion 3.1. Stability of the Film-Forming Solutions The behavior of chitosan in studied film-forming solutions was initially analyzed through the determination of average zeta potential in the individual used samples. The results are shown in Table 2. Chitosan as a representative of polycationic polysaccharides [ 32 ] formed positively charged particles in all used dispersion media. The explanation is Polymers 2021,13, 3388 7 of 21 straightforward, the positive charge of observed particles is caused by the protonation of chitosan amino groups in used acidic solutions (caused mainly by the presence of 1% lactic acid) as the isoelectric point of chitosan is present at pH around 6; the pH of film forming solutions was 3.20 ± 0.04 [ 6 ]. The concentration of chitosan in the samples was constant. It was found that even with a change in the dispersion medium (ratio of lactic acid solution and plant extract) the values of zeta potential of the particles were not affected. The determined values of zeta potential showed statistically significant differences in some samples (p< 0.05), but it must be emphasized that the results are very similar numerically. This conclusion is supported also by similar measured conductivities of all the samples, indicating the comparable content of dissolved low-molecular ions in the individual used dispersion media but, primarily, also the similar colloidal behavior of chitosan in these media. Table 2. Zeta-potential of film forming solution. Sample Zeta Potential (mV) Conductivity (mS/cm) CHL33.84 ±0.72 2.008 ±0.005 ac 5CHLBO 34.58 ±2.14 ac 1.980 ±0.020 c 10CHLBO 32.26 ±1.07 abe 1.808 ±0.008 b 20CHLBO 31.44 ±1.36 abe 1.922 ±0.008 d 5CHLPE 34.52 ±1.36 acd 2.050 ±0.001 ef 10CHLPE 30.62 ±2.09 be 2.000 ±0.007 ac 20CHLPE 36.68 ±0.32 c2.084 ±0.006 g 5CHLHR 31.98 ±1.83 abe 1.920 ±0.008 d 10CHLHR 32.22 ±2.02 abe 1.996 ±0.011 ac 20CHLHR 31.30 ±2.32 de 2.029 ±0.022 af Letters in superscript indicate statistically significant (p< 0.05) differences between rows. Another important fact which can be observed from the zeta potential measurement is the stability of the prepared colloidal dispersion. All the determined values of zeta potential are above 30 mV, which indicate high stability of chitosan particles against mutual aggregation of particles for all the used dispersion media. Besides the mentioned high stability of described sample, these prepared systems can also be considered as welldispersed systems from the colloidal point of view [33]. 3.2. Basic Morphological and Textural Properties of the Films The thickness of films is given in Table 3. No net decrease or increase in thickness was found in the samples with increasing addition of the extract, these values fluctuated in the samples. The results also do not show statistically significant differences (p> 0.05), so it can be said that the increasing percentage of extract did not affect the thickness of the resulting package. In the case of comparison with previous studies, there was both a significant increase in thickness values after the addition of plant extracts [ 15 , 34 ] but there were also findings that resulted in a decrease [35,36]. Table 3. Thickness of chitosan films. Sample Thickness (mm) CHL0.174 ±0.013 5CHLBO 0.176 ±0.046 10CHLBO 0.172 ±0.040 20CHLBO 0.214 ±0.022 5CHLPE 0.196 ±0.030 10CHLPE 0.188 ±0.019 20CHLPE 0.180 ±0.017 5CHLHR 0.206 ±0.055 10CHLHR 0.186 ±0.019 20CHLHR 0.210 ±0.029 Polymers 2021,13, 3388 8 of 21 The results of textural properties are shown in Table 4. The interaction of hydrocolloids and other additives such as plasticizers, water and antimicrobial substances has the greatest influence on the textural properties of edible coatings [ 37 ]. The 5CH LBO , 5CH LPE , 5CH LHR and 10CH LHR samples are statistically significantly different (p< 0.05) from the 20CH LPE sample that had the highest strength (0.10 ± 0.02 MPa). No statistically significant difference (p> 0.05) was found between the other samples. The comparison of results with the control sample CH L did not resulted in statistically significant differences ( p> 0.05 ) between the samples with the addition of extracts. The increase in strength can be caused by interactions between plant extracts that contain phenolic acids and their esters. These compounds can react with the hydrophilic groups present in the chitosan matrix, and this interaction can lead to stronger adhesion between the plant extracts and the chitosan molecules. These interactions can cause an increase in strength [ 38 ]. In previously published articles, it has been found that some additives incorporated into the chitosan matrix both increase and decrease the strength of the prepared packages. The explanation is similar to that described above: the increase in strength is due to stronger interactions between additives and chitosan, and the decrease in strength is due to weak interactions [39]. Table 4. Textural properties expressed as strength (MPa) and breaking strain (%). Sample Strength (MPa) Breaking Strain (%) CHL0.06 ±0.04 122.34 ±11.89 acd 5CHLBO 0.04 ±0.01 a116.01 ±15.25 ad 10CHLBO 0.09 ±0.03 107.72 ±9.89 a 20CHLBO 0.06 ±0.01 102.08 ±7.26 a 5CHLPE 0.04 ±0.01 a136.67 ±18.18 10CHLPE 0.08 ±0.03 113.60 ±30.36 20CHLPE 0.10 ±0.02 b149.16 ±3.54 ce 5CHLHR 0.04 ±0.01 a142.99 ±13.18 def 10CHLHR 0.05 ±0.01 a182.41 ±17.94 be 20CHLHR 0.09 ±0.04 118.10 ±9.80 af Letters in superscript indicate statistically significant (p< 0.05) differences between rows. The prepared chitosan packaging was characterized by high flexibility, which was observed by the handling of the packaging itself and subsequently confirmed by measuring the flexibility, where the results are presented in Table 2. Flexibility decreased with the addition of the extract. Compared to the CH L sample, the lowest value of elasticity was found in the 20CH LBO sample, but no statistically significant difference was found ( p> 0.05 ). The results correspond to the results of the force; due to the interaction between phenolic acids and chitosan, the flexibility is not so high in most samples [ 38 ]. It has also been found, when compared with the results of measuring the strength and elasticity of packages made of κ -carrageenan and ι -carrageenan with the addition of lapacho tea extract, that chitosan packages are more flexible and also have a lower strength value [40]. The results of gas barrier properties are summarized in Table 5. Water vapor transmission rate of all tested samples was higher than 1000 g/m 2 day, which is a relatively high value that limits the use of this material for packaging products that need to be protected from moisture. However, this value is similar for chitosan-based materials [ 41 – 43 ]. Similar values of WVTR were measured for bleached Kraft paper [ 44 ]. The results also showed that the addition of plant extracts increased the WVTR value of the chitosan-based material by an average of: 3.84 ± 1.14% for 5% addition of blueberry extract, 9.63 ± 0.23% for 5% addition of parsley extract, 8.05 ± 1.64% for 5% addition of red grapes extract. The lowest average value of WVTR for the material with the addition of plant extract 1270.0 ±25.3 g/m2 day was achieved for film with 5% addition of blueberry extract, the highest average value of 1724.6 ± 30.8 g/m 2 day was achieved for material with 20% addition of red grapes extract. Polymers 2021,13, 3388 9 of 21 Table 5. Gas barrier properties expressed as water vapor transmission rate and oxygen permeability. Sample Water Vapor Permeability (23 ◦C, 85 % RH) (g/m2d) Oxygen Transmission Rate (23 ◦C, 0 % RH) (mL/m2d 0.1 MPa) CHL1235.8 ±42.1 a15.1 ±0.8 5CHLBO 1270.0 ±25.3 a11.4 ±1.1 10CHLBO 1327.4 ±57.6 ac 8.7 ±0.7 20CHLBO 1485.1 ±72.8 de 4.1 ±1.0 5CHLPE 1356.2 ±37.6 acde 12.6 ±0.5 10CHLPE 1467.3 ±50.9 e9.8 ±1.3 20CHLPE 1719.4 ±22.3 b6.5 ±0.5 5CHLHR 1318.8 ±66.3 ac 13.7 ±1.4 10CHLHR 1422.4 ±47.0 cde 10.3 ±0.4 20CHLHR 1724.6 ±30.8 b5.2 ±0.9 Letters in superscript indicate statistically significant (p< 0.05) differences between rows. Changes of oxygen permeability caused by the addition of plant extract are opposite to the changes of WVTR according to the plant extract addition. The reduction in oxygen permeability compared to chitosan films without the addition of plant extract was on average 21.30 ± 3.15% for 5% addition of blueberry extract, 16.11 ± 1.70% for 5% addition of parsley extract and 13.85 ± 3.97% for 5% addition of red grapes extract. The lowest average value of oxygen permeability of the material with the addition of plant extract 4.1 mL/m 2 day 0.1 MPa was measured for the material with 20% addition of blueberry extract, the highest average value of 13.7 mL/m 2 day 0.1 MPa was achieved for a film with 5% addition of red grapes extract. The resulting values are relatively low and correspond to permeability values for commonly used packaging materials based on, e.g., polyamide, polyethylene terephthalate [45]. Similar and completely different effects of the addition of plant extracts on the barrier properties of final materials have been published [ 46 , 47 ]. The increase in permeability may be caused by the destabilization of the original chitosan matrix by extract components that may act as plasticizers [ 48 ]. The reduction in permeability is then explained mainly by the possibility of crosslinking between components of the extracts and the polymer matrix [ 49 ]. For the purposes of wider use of this material in food packaging, it would be good to combine the material with barrier materials preventing the penetration of moisture into the packaged product. An example may be the incorporation of waxes, oils, etc., directly into the material [ 47 , 50 ]. A disadvantage of this method may be a significant reduction in oxygen transmission rate [48]. 3.3. Basic Compositional and Structural Analysis Data for water content, solubility and swelling degree are summarized in Table 6. In the case of water content, a decrease in water content was observed in the samples after the addition of the extracts, but significant (p< 0.05) differences in comparison with the control sample (CH L ) were noticed among the following samples: 10CH LBO , 5CH LPE and 20CH LPE . The reduction of the water content in the packaging with the addition of extracts is due to the formation of hydrogen bonds, which in turn reduce the availability of hydroxyl groups and amino groups and thus limit the interaction of chitosan with water [51,52]. Polymers 2021,13, 3388 16 of 21 crobial activity of red grapes skin extract was evaluated and an antimicrobial efficiency was reported against all tested microorganisms [ 68 , 69 ]. The blueberry extracts were also analyzed in previous research. The reported antimicrobial activity was unfortunately not confirmed by our results. It should be stressed, however, that the same happened with the analysis of the total polyphenol content and antioxidant activity results. We assume that the results were probably not as high due to the lower level of active compounds in samples with the addition of by-products extracts compared with the samples with the addition of whole fruits/vegetable extracts. The antimicrobial properties of plants are caused mainly by the presence of phenolic compounds [70]. Figure 3. The results of antimicrobial activity of chitosan edible films. 3.7. Release of the Active Components from the Films According to the legislation, simulant A was chosen to determine the migration values of active substances, it is a simulation of the transition of active substances into foods with a hydrophilic character which, according to EU Regulation No. 10/2011, includes the following food commodities with the hydrophilic character: nuts in paste or cream, fresh vegetables, fish, fresh meat and processed meat products, fried potatoes, donuts, preparations for making soups, etc. The results of migration tests for manufactured packaging are shown in Table 11 (polyphenol content) and Table 12 (antioxidant properties). The values of the results of polyphenols showed that there is a migration of polyphenolic substances, which was confirmed by the analysis of antioxidant activity, where higher Polymers 2021,13, 3388 17 of 21 values were measured with the addition of extracts from by-products of blueberries, parsley and red grapes. Table 11. Migration—total polyphenols content. Sample TPC (mg Gallic Acid/mL) TPC (mg Gallic Acid/g) CHL0.002 ±0.000 a0.12 ±0.01 a 5CHLBO 0.005 ±0.000 c0.47 ±0.00 b 10CHLBO 0.008 ±0.000 d0.49 ±0.00 b 20CHLBO 0.010 ±0.000 e0.85 ±0.00 cf 5CHLPE 0.004 ±0.000 b0.09 ±0.00 a 10CHLPE 0.010 ±0.000 f0.32 ±0.07 abde 20CHLPE 0.011 ±0.000 g0.47 ±0.02 bd 5CHLHR 0.011 ±0.000 e0.43 ±0.00 d 10CHLHR 0.012 ±0.000 g0.72 ±0.00 eg 20CHLHR 0.016 ±0.000 h0.96 ±0.05 fg Letters in superscript indicate statistically significant (p< 0.05) differences between rows Table 12. Migration–antioxidant activity. Sample FRAP (µmol Trolox/mL) ABTS (%) DPPH (%) CHL0.0112 ±0.0004 a2.60 ±0.40 a34.22 ±1.25 a 5CHLBO 0.0111 ±0.0002 a2.70 ±0.04 c51.62 ±3.00 cadg 10CHLBO 0.0137 ±0.0005 c3.10 ±0.08 d54.95 ±0.50 d 20CHLBO 0.0210 ±0.0002 d3.81 ±0.19 e70.07 ±0.97 b 5CHLPE 0.0163 ±0.0002 e3.73 ±0.04 e34.45 ±2.56 ead 10CHLPE 0.0152 ±0.0002 b4.86 ±0.05 b53.33 ±0.34 fcd 20CHLPE 0.0170 ±0.0001 e4.94 ±0.05 b58.75 ±0.06 g 5CHLHR 0.0153 ±0.0005 b2.67 ±0.08 c41.48 ±3.00 acd 10CHLHR 0.0129 ±0.0005 cb 3.24 ±0.08 d52.00 ±7.60 20CHLHR 0.0248 ±0.0004 f4.35 ±0.05 f53.15 ±3.64 gcdb Letters in superscript indicate statistically significant (p< 0.05) differences between rows. For polyphenols, the highest migration value was recorded for the 20CH LHR sample (0.016 ± 0.000 mg gallic acid/mL), which is statistically significantly (p< 0.05) different from all other measurements of polyphenol migration. In determining the antioxidant activity, the samples 20CH LHR , 20CH LBO and 20CH LPE achieved the best results, as described above, due to the higher concentration of extracts used for the production, so these packages can be characterized as most suitable for subsequent application to packaged foods. In general, when migrating substances from packaging to food, it is more likely to avoid migration in the case of film-forming components—e.g., migration of monomers of PET materials, etc., that are undesirable and must meet limits [ 71 ]. In contrast, the migration of active components such as polyphenols and antioxidants is desirable because they can contribute to improving the properties of packaged foods, prolong their shelf life, prevent oxidation, and thus function as a package with active properties [12]. Non-correlating results were found for antioxidant activity, i.e., in different methods (ABTS, FRAP and DPPH) the same sample did not always show the highest value of antioxidant activity, for these samples the effect of solvent is excluded, as the same solvent was used everywhere to detect migration of active substances, but the results are influenced by the conditions and the course of reactions of individual methods. For the FRAP method, the analysis is performed at low pH values (3.6) compared to the DPPH and ABTS methods, where the pH value was not adjusted. In the ABTS method, color loss is determined spectrophotometrically after the addition of an antioxidant to the blue-green chromophore ABTS ·+ , so the antioxidant reduces ABTS ·+ to ABTS and decolorizes it [ 72 ]. The DPPH method uses a stable free radical, and in the presence of an antioxidant compound it can donate a hydrogen atom; lead to a reduction and decolorization of the dark purple solution; Polymers 2021,13, 3388 18 of 21 in the case of DPPH this radical does not always react with the same compounds as in the case of ABTS; and the stability of the ABTS solution is also much lower than that of DPPH [72,73]. 4. Conclusions The research showed that a film-forming solution composed of chitosan, lactic acid solution and the addition of compacted extracts prepared from plant by-products is stable in terms of zeta-potential determination. The aggregation of particles is not present, which should affect the textural properties of prepared films. Based on the results of gas barrier properties, the water vapor transmission rate increased with the addition of extracts and an opposite trend was found in measuring of oxygen permeability; based on good oxygen barrier properties the prepared films have similar values as commonly used synthetic packaging materials. Furthermore, it was found that the value of swelling degree was significantly affected by the presence of polyphenolic substances, and thus in samples with extracts of blueberry and red grape marc, the samples with the highest value of polyphenols had the lowest value of swelling degree; a good example for the protection of packaged foodstuff against water. The FTIR results analyzed by advanced processing showed the decrease in the signal of lactic acid and lactate and increase in signal specific for every plant used for extract preparation. Another interesting finding was determined by antimicrobial analysis, the films with the addition of extract from red grapes showed the formation of inhibition zones during incubation with E. coli. In the case of migratory techniques, the transition of bioactive compounds was confirmed, meaning that the experimentally produced edible packaging can be defined as active. It must be emphasized that the content of polyphenols and antioxidant activity did not show high values compared to previous research, but this can be further investigated in the continuation of research, where the method of preparation of extracts can be modified to increase the amount of these active substances and also apply packaging directly to food and determine their effect on the shelf life of food. Author Contributions: Methodology, D.D., P.S., M.K., A.R., L.V., M.N. and J.T.; formal analysis, S.D., B.A., K.T., M.B., D.D., P.S., L.V., M.K.; writing—original draft preparation, S.D., D.D., P.S., M.K., L.V., A.R., M.N., J.T.; writing—review and editing, D.D., S.D.; supervision, B.T., D.D. All authors have read and agreed to the published version of the manuscript. Funding: The research was supported by project FVHE/Tremlová/ITA2020 from University of Veterinary Sciences, Brno, Czech Republic. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Conflicts of Interest: The authors declare no conflict of interest. References 1. Salgado, P.R.; Ortiz, C.M.; Musso, Y.S.; Di Giorgio, L.; Mauri, A.N. Edible films and coatings containing bioactives. Curr. Opin. Food Sci. 2015,5, 86–92. [CrossRef] 2. Wang, Q.; Liu, W.; Tian, B.; Li, D.; Liu, C.; Jiang, B.; Feng, Z. 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