Sugar kelp Saccharina latissima extract as an innovative ingredient for chitosan films: Case study as cheese slice separators

Food Hydrocolloids 149 (2024) 109571 Available online 23 November 2023 0268-005X/© 2023 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). Sugar kelp Saccharina latissima extract as an innovative ingredient for chitosan films: Case study as cheese slice separators María Fl´ orez, Patricia Lopez-Sanchez, Manuel V´ azquez * , Patricia Caz´ on Department of Analytical Chemistry, Faculty of Veterinary, University of Santiago de Compostela, Campus Terra, 27002, Lugo, Spain ARTICLE INFO Keywords: Film Saccharina latissima Chitosan Antioxidant activity Lipid oxidation Separators ABSTRACT Chitosan films enriched with aqueous extract of Sugar kelp Saccharina latissima (SLE) were developed and characterized by measuring the equilibrium moisture, moisture content, water vapor permeability, mechanical properties, antioxidant properties, optical properties, surface morphology and thermostability. The developed films were tested as separators with antioxidant properties for sliced Havarti cheese. The addition of SLE showed a decrease in mechanical properties such as tensile strength from 48.99 to 36.16 MPa and Young’s Modulus from 929.49 to 743.71 MPa compared with pure chitosan films. The resulting chitosan-SLE films showed a high freeradical scavenging activity against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2 ′ -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ). Moreover, the presence of SLE in the chitosan matrix significantly improved the UV-barrier of the samples. When the chitosan-SLE films were employed as separators for cheese, a significant reduction of 52.8% in cheese lipid oxidation was observed after 45 days of refrigerated storage compared to the control group using chitosan films. Hence, an environmentally sustainable alternative to synthetic and non-biodegradable slice separators has been successfully developed using chitosan, a by-product of the fishing industry, fortified with an extract from sugar kelp, a commonly found brown algae. 1. Introduction Abundant active compounds can be found in nature within plants, animals, and soils. These compounds are of increasing importance in the food industry, either as functional ingredients or as part of active and intelligent packaging. Packaging is a key factor in the food industry, since it must protect the food from chemical, physical and biological hazards throughout the food chain as well as help extend food shelf life, contributing to a reduction of food waste (Fl´ orez, Guerra-Rodríguez, Caz´ on, & V´ azquez, 2022a, b, c). However, this industry is dominated by non-biodegradable synthetic polymers derived from petroleum (Mir, Dar, Wani, & Shah, 2018). The massive use of these materials has led to an uncontrolled accumulation of plastic waste in landfills and oceans, microplastics and toxic plastic additives accumulating in the food chain (Perera, Sharma, Pradhan, Jaiswal, & Jaiswal, 2021). Due to the disadvantages derived from the use of synthetic polymers, the application of biodegradable polymers is the most promising solution. The UNE-EN 13432:2001 standard defines the term biodegradable as the “capacity of a material to decompose into carbon dioxide, methane, water, inorganic compounds and biomass”. The predominant mechanism is the enzymatic action of microorganisms, as biodegradable materials are usually compostable. Chitosan, a non-toxic, biocompatible polysaccharide with outstanding filmogenic properties, has been investigated for use as a food packaging film (Kumar, Rahman, Ranwa, Kumar, & Kumar, 2020; Siripatrawan & Harte, 2010). Chitosan films, which are frequently made using the casting method, could be a sustainable substitute to eco-unfriendly synthetic plastics (Homez-Jara et al., 2018). These films can become active packaging after the incorporation of certain components capable of enhancing antioxidant and/or antimicrobial properties, thus promoting the ability to protect the food (Lunkov, Ilyina, & Varlamov, 2018). To date, numerous studies have investigated the impact of these added extracts on the techno-functional characteristics of the films. Natural plant and herbal extracts that could replace certain synthetic additives have been extensively studied. They serve as a rich source of antioxidant and active compounds, including polyphenols, flavonoids, and various others (Yilmaz & Toledo, 2006). Recent studies have been closely examining chitosan-based films incorporated with various extracts, such as Santalum album essential oil (Fl´ orez et al., 2022a, b, c), extracts from Pistacia terebinthus stems, leaves, and seeds * Corresponding author. E-mail address: [email protected] (M. V´ azquez). Contents lists available at ScienceDirect Food Hydrocolloids journal homepage: www.elsevier.com/locate/foodhyd https://doi.org/10.1016/j.foodhyd.2023.109571 Received 19 October 2023; Received in revised form 2 November 2023; Accepted 15 November 2023 Food Hydrocolloids 149 (2024) 109571 2 (Kaya et al., 2018) and algal extracts from Codium tomentosum (Augusto et al., 2018), among others. Seaweeds are multicellular, macroscopic, benthic organisms characterized by rapid growth, which results in an accumulation of biomass. They have great potential as a renewable resource because they grow in a diverse environment and their development does not depend on arable land (Zerrouki & Henni, 2019). Seaweeds are primarily classified into three groups based on their pigmentation: green algae (e.g., Cladophora, Ulva and Monostroma species), red algae (e.g., Poryphyra capensis, Aeodes orbitosa and Notogenia stiriata species) and brown algae (e.g., Saccharinna latissima, Laminaria pallida, Fucus and Zonaria species). The common point between them is that they have a high carbohydrate content (around 40% of their total mass). However, each type of algae produces a polysaccharide in greater proportion than the others. Green algae species produce the sulfated polysaccharide Ulvan. In addition to proteins and polysaccharides, algae are a sustainable source of high value natural bioactive compounds such as polyunsaturated fatty acids (PUFA) and phytosterols. Some amino acids and peptides can exhibit antioxidant activity, in addition to other functions such as immunostimulants, antihypertensives or anticoagulants (Becker, 2007; Paiva, Lima, Patarra, Neto, & Baptista, 2014). Seaweed and seaweed extracts are commonly used in agriculture, in the pharmaceutical industry, and in the biofuel industries. Furthermore nowadays, seaweeds are in great demand in the food industry, increasing their consumption by about 176% since 1995 (Araújo et al., 2021; Cai et al., 2021). Due to the previously commented composition of these organisms, algae are a very interesting raw material to produce active and intelligent packaging, edible coatings and films or sachets. To enhance the thermal, barrier, mechanical, antioxidant, and antimicrobial properties of biopackaging materials, algae are often combined with other substances such as polysaccharides, nanoparticles, essential oils, and plant extracts (Abdul Khalil et al., 2017). Phenolic compounds such as tocopherol (vitamin E), ascorbic acid (vitamin C), carotenoids like fucoxanthin and β-carotene, or phycobiliproteins are among the bioactive compounds with antioxidant function found in seaweed (Rodríguez-Bernaldo de Quir´ os, Lage-Yusty, & L´ opez-Hern´ andez, 2010). Furthermore, owing to its non-toxic and harmless attributes, seaweed extracts have been used in food coatings and films (Perera et al., 2021). For instance, Fucus vesiculosus was used with whey protein to create a film capable of inhibit lipid oxidation in chicken breasts (Andrade et al., 2021). A film based on seaweed and neem Azadirachta indica extract exhibited excellent antimicrobial activity against Staphylococcus aureus and Bacillus subtilis (Oyekanmi et al., 2021). The objective of this study was to develop an active packaging material with antioxidant capacity based on chitosan films enriched with S. Latissima aqueous extract (Ch_SLE). The effect of SLE at different ratio on the equilibrium moisture, moisture content, water vapor permeability, mechanical properties, antioxidant properties, and optical properties was analysed. The Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and simultaneous thermal analysis (TGA/DSC) were carried out to evaluate the surface morphology, thermostability and compatibility of the samples, respectively. The novel Ch_SLE film was tested as a separator for vacuumpacked Havarti cheese slices. The active properties of the developed separator were evaluated by measuring the antioxidant properties of the cheese and the films and the lipid oxidation of the cheese up to 45 days of refrigerated storage. 2. Experimental 2.1. Materials Chitosan (Mw 100,000-300000 and CAS number 9012-76-4) was purchased from Acros organics (Geel, Belgium). Acetic acid (CAS number 64-19-7) and glycerol (CAS number 56-81-5) provided by Scharlau Microbiology (Barcelona, Spain). They were used to prepare the chitosan film-forming solution. Polyamide/Polyethylene (PA/PE) films (Plastinal S.L., Arrubal, La Rioja, Spain) were used as a control film. Dried S. Latissima was purchased from KosterAlg (Gothenburg, Sweden). Havarti cheese tested was purchased from IFA RETAIL SA (Madrid, Spain). To provide homogeneous samples, the cheese slides were all taken from the same batch. 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH) and 2,2 ′ -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ) were provided by Alfa Aesar (Haverhill, MA, USA). FolinCiocalteu phenol reagent was provided by Panreac (Barcelona, Spain). For the analysis of lipid oxidation, 4,6-dihydroxy-2-mercaptopyrimidine (TBA) was provided by Thermo Fisher Scientific (Waltham, MA, USA) and trichloroacetic acid was provided by Sigma-Aldrich (San Luis, MO, USA). 2.2. Preparation of sugar kelp S. latissima extract The dried leaves of Sugar kelp S. latissima were ground with an electric grinder to obtain a powder. This powder was stored in airtight plastic jars at room temperature (20-25 ◦C). The residual moisture was 10.9% and the water activity was 0.426. The aqueous extraction was performed following the steps described in detail elsewhere (Fl´ orez et al., 2022a, b, c). In brief, 15 ml of distilled water were added to 1 g of sugar kelp powder in a screw-capped tube. The tubes were placed in an ultrasonic bath at 65 ◦C for 3.15 h. The suspension was filtered under vacuum conditions using Whatman nº 1 filter paper to remove any solid in the aqueous extract. The extract was placed in the refrigerator for 24 h and afterwards it was filtered again and finally subjected to a centrifugation (9050 g, 20 min, 5 ◦C). The Saccharina latissima extract (SLE) was stored in glass bottles in the dark at 5 ◦C until use. 2.3. Preparation of films Chitosan films were made by dissolving 2% (w/w) chitosan with 1% (w/v) acetic acid in an aqueous solution. The stock solution was left at room temperature overnight under agitation. Glycerol at a concentration of 0.1% (w/v) was added as a plasticizer. SLE was gradually added to the chitosan solution in the range of 0, 10 and 20% (v/v) by homogenizing at 15,000 rpm for 2 min (Ultra Turrax®, IKA, Staufen, Germany). The final concentration of chitosan in the Ch_SLE filmogenic solution was 1% (w/w). An ultrasonic bath was used for 15 min to remove the filmogenic solution’s bubbles. The resulting film-forming solution was poured (40 ml) in a 210 mm diameter Petri dish. After 48 h of solvent evaporation at room temperature, the Ch_SLE films were removed. The films were cut to a precise size and stored at specific conditions for 5 days depending on the test. The thickness (mm) was measured at five random locations using a Thickness Meter ET115S (Etari GmbH, Stuttgart, Germany). 2.4. Scanning electron microscope (SEM) and fourier transform infrared spectroscopy (FT-IR) SEM images were taken to investigate and characterize the morphology of Ch_SLE films. Dry and gold-coated samples were observed using a high-vacuum microscope (JEOL JSM-6360LV, Jeol Ltd, Tokyo, Japan) operating at an accelerating voltage of 20 kV. To determine the presence of specific chemical groups and their cross-linking in the films, the FT-IR-6800 (Jasco Inc., Japan) was used. The samples were conditioned for 48 h at 21±1 ◦C and at 65 ±2% relative humidity. FT-IR spectra were captured with a spectral resolution of 4 cm −1 and in the 4000-800 cm −1 range. 2.5. Equilibrium moisture and water vapor permeability The equilibrium moisture content (%W) was calculated using the gravimetric method by comparing the weights of dried samples that had been conditioned at a relative humidity (%RH) of 33% (Caz´ on, V´ azquez, M. Fl´ orez et al. Food Hydrocolloids 149 (2024) 109571 3 & Velazquez, 2019). The water vapor permeability (WVP) was assessed using the ASTM Standard Test Method E96 (https://www.astm.org/e00 96_e0096m-22a.html). The WVP of the samples was calculated at 30 ◦C and 50% RH. The test was conducted for a minimum of 4 h, which was enough time for the vapor flux to reach a dynamic equilibrium. 9 cm 2 film samples were kept in desiccators with silica gel for five days in order to determine the water retention (%W) using the gravimetric technique. Dry samples were weighed (W dry ) to the nearest 0.0001 g using a precision balance. The samples were then stored at 25 ◦C for 24 h while immersed in an airtight container filled with distilled water (100 ml). The excess surface water was drained from the swollen samples using filter paper. Wet samples were weighed (W wet ). Each test was performed in triplicate (Caz´ on et al., 2019). 2.6. Mechanical properties analysis The Ch_SLE films were subjected to tensile and puncture tests using a texturometer (TA-XT plus, Stable Micro System, UK). The percentage of elongation of break (%E, %), Youngs’ Modulus (YM, MPa) and tensile strength (TS, MPa) were all calculated using the tensile test. The samples (15 ×100 mm) were clamped between the texturometer’s grips using the ASTM standard technique D-882, with an initial separation of 40 mm and the load rate set to 1 mm/s. Seven replicas from each batch were evaluated (Caz´ on, V´ azquez, & Velazquez, 2018). Burst strength (BS), also known as puncture force, and distance to burst (DB), also referred to puncture deformation, were determined during the puncture test. A film holder (Reference HDP/FSR, Stable Micro System, UK) was used to hold the sample (30 ×30 mm) throughout the test. The force (g) and strain (mm) up to rupture were measured using a cylindrical probe with a diameter of 3 mm and moving at a speed of 1 mm/s. 2.7. Antioxidant properties of sugar kelp S. Latissima extract, films and cheese samples The 2,2-diphenyl-1-picrylhydrazyl radicals (DPPH) and 2,2 ′ -azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS + ) free radical scavenging were used to evaluate the antioxidant capacity of the seaweed extract. For the determination of antioxidants in the SLE, the pure aqueous seaweed extract at a 1/10 dilution was used. Regarding the determination of antioxidant capacity of the films, the methanolic extracts of the films were made by combining 1 g of film with 24 ml of methanol and stirring the mixture overnight in the dark. On the other hand, a cheese extract was prepared adding 9 ml of distilled water to 2 g of Havarti cheese. The mixture was homogenised in a vortex for 1.5 min before being filtered through Whatman nº1 filter paper (Bottesini et al., 2013). Using a UV-VIS spectrophotometer V-670 (Jasco Inc., Japan), the radical scavenging activity of DPPH was studied at 515 nm and the radical cation scavenging activity of ABTS + was examined at 734 nm (Rutkowska et al., 2020). The results were expressed as %. 2.8. Optical and thermal (TGA/DSC) properties of films Using a spectrophotometer V-670 (Jasco Inc., Japan), the UV-VIS spectra of the film samples were measured. The optical characteristics of the samples were determined following previously reported methods (Caz´ on et al., 2019). Differential scanning calorimetry and thermogravimetry equipment (Metter Toledo, Switzerland) were used to analyse the films’ thermal stability behaviour. To conduct the test, samples were heated at a rate of 10 ◦C/min from 50 to 400 ◦C in a nitrogen environment (50 ml/min). The samples were put in hermetically sealed pans of aluminium. 2.9. Application as cheese slice separators of havarti cheese The cheese slices were separated using three different kinds of films: 1) pure chitosan film (control), 2) chitosan enriched with SLE (10%) and 3) chitosan enriched with SLE (20%). The cheese slices with the separators were placed in polyamide/polyethylene vacuum bags and sealed with a vacuum heat-sealer (Model Plus F 380, Germany). The samples were stored at 5 ◦C for 45 days. The temperature and time settings were selected based on previous research that analysed cheese properties during 45 days of refrigerated storage (Küçük, Çelik, Mazi, & Türe, 2020; Modzelewska-Kapituła, Kłębukowska, & Kornacki, 2007). The DPPH and ABTS + parameters in the cheese packaged with the different separator formulations, as well as the films themselves, were examined at 0, 15, 30 and 45 days of storage. TBARS values in the cheese samples were analysed by spectrophotometric analysis (Lee, Yang, & Song, 2016). An amount of 2 g of cheese was mixed with 10 ml of 7.5% trichloroacetic acid under stirring for 1 h. The cheese-trichloroacetic mixture was filtered through Whatman nº1 filter paper. In a test tube, 5 ml of the filtered cheese extract was mixed with 5 ml of thiobarbituric acid (TBA) 0.02 M. The mixture was heated at 95 ◦C for 45 min in a hot water bath before cooling. The absorbance was measured at 539 nm after the centrifugation. Three duplicate analyses were performed. TBARS was expressed as malonaldehyde (MDA)/kg cheese. 2.10. Statistical analysis Statistical analysis of the results was conducted using Microsoft Excel® software, employing one-way analysis of variance (ANOVA). Subsequently, the Tukey Post Hoc test was utilized to explore differences with various confidence intervals, with statistical significance set at p < 0.05. Furthermore, the results were also analysed using Design Expert 11® software (Stat-Ease, Minneapolis, MN, USA). 3. Results and discussion 3.1. Film surface morphology analysis The chitosan films showed a compact and pore-free surface morphology. Fig. 1 shows the SEM images of the bottom, top and cross sections. The bottom SEM images are those that represent the face that is in contact with the Petri dish. In the Ch_SLE20 films, small particles can be observed on the surface of the film, indicating that the seaweed extract was not dissolved completely. Similar behaviour was observed when pine needle extract was added to chitosan films (Kadam, Singh, & Gaikwad, 2021). On the other hand, some lines are as well observed on the surface, which are attributed to possible scratches on the plastic surface of the Petri dish. The top SEM images are those that relate to the film’s surface side that is in contact with the environment. As in the previous case, the solution was well mixed resulting in a homogeneous film, except when the SLE concentration was 20% (Ch_SLE20). An agglomeration of particles belonging to the seaweed extract can be observed on the surface. The cross-section SEM images are those showing the inside of the film. The addition of a 10% concentration of SLE resulted in a film structure similar to that of the chitosan film without SLE. However, with a 20% concentration, the film exhibited unevenness, particularly on the surface and at the base. Similarly, films enriched with pine needle extract at high concentration showed alterations in the surface microstructure in SEM images (Kadam et al., 2021). 3.2. Fourier transform infrared spectroscopy (FT-IR) FT-IR was used to investigate the SLE and chitosan intermolecular interaction, which is mainly connected to the physical performance of the films (Zhang et al., 2018). Fig. 2 displays the FT-IR spectra on the films between 4000 and 800 cm −1 , with pure chitosan film (red line), with 10% SLE concentration (green line), and with 20% SLE (yellow line). Notable differences in the SLE enriched films can be observed, showing a lower absorbance compared to the chitosan films without M. Fl´ orez et al. Food Hydrocolloids 149 (2024) 109571 4 Fig. 1. Scanning electron microscopy images of the top, bottom and cross section of chitosan films with glycerol (0.1%) and SLE (0, 10, 20%). SLE is Sugar Kelp Saccharina Latissima extract. Fig. 2. FT-IR Spectra of chitosan films with glycerol (0.1%) and SLE (0, 10, 20%). SLE is Sugar Kelp Saccharina Latissima extract. M. Fl´ orez et al. Food Hydrocolloids 149 (2024) 109571 5 SLE. The first characteristic peak was at 2950 cm −1 , related to the hydroxyl groups (-OH) of the chitosan. The presence of SLE showed that the film weakened and decreased the absorbance values of the films. The higher the concentration of SLE in the matrix, the lower the -OH groups (Rai, Dutta, & Mehrotra, 2017). Below are visible the three characteristic peaks of chitosan (amide I: 1650 cm −1 , amide II: 1541 cm −1 and amide III: 1407 cm −1 ), which are caused by the deacetylation of chitin (Bilican et al., 2020). The peak at 1346 cm −1 is related to N-H bending vibrations (Qiao, Ma, Zhang, & Yao, 2017), while the next characteristic peak at 1150 cm −1 is attributed to C-O stretching in the chitosan ring structure (Priyadarshi, Sauraj Kumar, & Negi, 2018). The range between 1080 and 1000 cm −1 is dominated by strong stretching vibrations of C-O, C-N and C-C (Valentin, Bonelli, Garrone, Di Renzo, & Quidnard, 2007). As well, the bending of the C-H bonds in the chitosan structure are explained by the absorption spectra in the range of 1200 to 800 cm −1 (Liu, Adhikari, Guo, & Adhikari, 2013). Overall, the neat chitosan film spectra of the Ch_SLE film spectra showed a relatively similar pattern of bands. This indicates the good miscibility of SLE with the polymeric matrix. Only a lower wavelet shift has occurred at certain peaks with increasing SLE concentration. This could be due to the presence of SLE, which when mixed with the glycerol-chitosan matrix, can alter the availability of the OH group within the polymer network (Riaz et al., 2020). These results are consistent with those obtained when Chinese chive root extract (Riaz et al., 2020) or Herba Lophatheri extract (Wang et al., 2019) were added to chitosan films. 3.3. Evaluation of the equilibrium moisture content and water vapor permeability The equilibrium moisture %W describes the film’s ability to absorb water, which in turn is transferred to the inner product. Knowledge of the equilibrium moisture is crucial for forecasting stability and quality changes in food products throughout packaging and storage (Srinivasa, Ramesh, & Tharanathan, 2007). The %W values ranged from 0.85 to 1.2% (Table 1). Data was fitted to a quadratic model. The p-values of the model indicated that the quadratic effect of SLE (A 2 ) was the only significative term (p <0.01) (Table S1). However, the fit statistics results indicated a low r 2 of 0.65 to can obtain a feasible model (Table S2). The values of %W of the films decreased when SLE was added at 10% compared with the control with pure chitosan. When the extract concentration was 20%, the W% values increased. This may be due to the fact that algae are characterized by a hydroscopic nature, which could increase the moisture absorption capacity of the films at the high concentration (Kanatt et al., 2015). These variations were statistically significant as shown by the Tukey test. Another of the most significant factors affecting the quality of packaged foods is the WVP of the film (Xue Mei et al., 2020). WVP is used to describe a film’s capacity to transfer moisture between a product and the environment (Hermawan et al., 2019). WVP values ranged from 3.41⋅10 −11 to 5.46⋅10 −11 g/m⋅s⋅Pa. In this case, data were fitted to a linear model. The fit statistics r 2 was 0.70 for WVP. Film permeability increased with increasing SLE concentration. The WVP behaviour of the pure chitosan film is due to the presence of amino and hydroxyl groups, which act as water molecule-binding sites in the chitosan chain (Bourtoom & Chinnan, 2008). However, the increase in WVP could be explained due to hydrophilic nature of seaweed. The high presence of free hydroxyl groups in algae could increase the interaction between algae and water molecules, increasing the speed of water transmission through the film (Khalil et al., 2018). On the other hand, the highest WVP value was obtained at 20% SLE, and this could be due to the agglomeration of the extract particles in the matrix, as shown in the SEM analysis. This hydrophilic character of the films was also observed when Fucus vesiculosus (Andrade et al., 2021) and Kappaphycus alvarezii (Khalil et al., 2018) seaweeds were added to whey protein or corn starch films, respectively. 3.4. Evaluation of the mechanical properties The results of the tensile and puncture tests to characterize mechanical properties are shown in Table 1. These analyses are important to identify the mechanical characteristics of biopolymer films, since it may be used to assess film stability and resistance to uphold load during storage or use (Khalil et al., 2018). The results of the tensile test for the films ranged from 36.16 to 48.99 MPa for TS. Data was well fitted to a linear model. In this property, the F-value and the p-value of the model were 65.43 and <0.0001, respectively (Table S1). Because the p-value is <0.05, the model was considered significant. The fit statistics values showed a r 2 of 0.90 (Table S2). Equation (1) predicts the TS values of the Ch_SLE films in relation to SLE concentration. The TS prediction is shown in Fig. 3A. TS (MPa) =50.56 - 0.57 ⋅ SLE (%) (1) It is evident that with an increased concentration of SLE in the matrix, there is a more pronounced reduction in tensile strength. The heterogeneous dispersion and aggregation of SLE may have diminished the mechanical properties of the polymeric matrix. This leads to the formation of a heterogeneous network and a loss of cohesion in the films, as evident in SEM images (Deshmukh et al., 2021). According to the analysis of elongation, the films showed %E values ranged from 4.03 to 7.4 %. In this case, the data did not fit any mathematical model. This is because there is no large difference between the results. Thus, the mean value of the data suggests that the addition of SLE to the formulation does not impact the %E value. Young’s modulus (YM) values ranged from 743.71 to 973.63 MPa. Data were fitted to a quadratic model. The statistical analysis indicated that the model had an F-value of 70.94 and a p-value <0.0001. This means that the model is statistically significant. The most significant factor affecting Young’s modulus was the linear effect of SLE concentration (F-value =80.12), followed by the quadratic effect of SLE (F-value =61.76). The r 2 was 0.95. The values of YM of the Ch_SLE films samples are predicted by equation (2). YM (MPa) =951.47 +23.32 ⋅ SLE (%) - 1.73 ⋅ SLE 2 (%) (2) The YM prediction is shown in Fig. 3B. Based on the results, with a low SLE content (10%), the extract can be well dispersed in the chitosanbased matrix, which slightly increases the stiffness of the film. However, Table 1 Physical and mechanical properties of chitosan films with Saccharina Latissima extract. Film samples %W WVP TS %E % g/m⋅s⋅Pa MPa % Ch 1.18 ± 0.11 ab 3.41⋅10 −11 ± 7.27⋅10 −13 a 48.99 ± 1.20 a 6.24 ± 3.44 a Ch_SLE10 0.85 ± 0.18 a 4.00⋅10 −11 ± 5.77⋅10 −12 a 48.06 ± 3.03 a 4.03 ± 1.47 a Ch_SLE20 1.20 ± 0.13 b 5.46⋅10 −11 ± 8.99⋅10 −12 ba 36.16 ± 4.24 b 7.40 ± 6.67 a Film samples YM BS DB MPa g mm Ch 929.49 ±105.19 a 2343.64 ±449.02 a 3.06 ±0.52 a Ch_SLE10 973.63 ±105.84 a 3210.90 ±393.97 b 4.01 ±0.40 a Ch_SLE20 743.71 ±67.82 b 3716.79 ±523.70 bc 5.61 ±0.28 b CH – chitosan; SLE – Saccharina Latissima extract; %W - equilibrium moisture content; WVP - water vapor permeability; TS - tensile strength; %E −percentage of elongation to break; YM - Young’s modulus; BS - burst strength (puncture properties); DB - distance to burst (puncture properties). Values are expressed as mean ±standard deviation (SD). Different letters in the same column indicate significant differences (p <0.05). M. Fl´ orez et al. Food Hydrocolloids 149 (2024) 109571 6 with a higher content (20%), the extract is not well distributed, leading to a decrease in the values of this mechanical property. The same trend was observed when cellulose-based films with Ulva ohnoi extracts were studied (Saedi, Kim, Shokri, Kim, & Shin, 2023). Puncture resistance is another crucial mechanical property of packaging materials. In many applications, penetration can damage the packaging, reduce its barrier properties, and result in a loss of package integrity (Kanatt, Rao, Chawla, & Sharma, 2012). According to the puncture test, there was an increase in BS and DB with the SLE concentration (Table 1). BS values ranged from 2343.64 to 3716.79 g. Data were all fitted to a linear model. The F-value of the model was of 14.91 and the p-value of 0.0062. As p-value <0.05, it can be determined that the model is statistically significant (Table S1) although the r 2 value was low (0.68). BS values were slightly (p <0.05) higher in Ch_SLE films than in pure chitosan films, this may be related to more effective plasticization (Blanco-Pascual, Montero, & G´ omez-Guill´ en, 2014). The DB values of the Ch_SLE films under puncture forces ranged from 3.06 to 5.61 mm. A linear model was fitted with a significant F-value of 55.30 (p <0.0001) and an r 2 value of 0.88. Overall, an increase in the properties of BS and DB was observed with the SLE concentration. The same trend was observed in the literature (Sivarooban, Hettiarachchy, & Johnson, 2008; Wang, Marcone, Barbut, & Lim, 2012a). Hydrogen bonds between compounds present in plant extracts and polysaccharide molecules have been shown to increase the molecular interactions of biopolymers. As a result, the addition of plant extracts induced alterations in physical properties, including BS and DB (Wang, Marcone, Barbut, & Lim, 2012b). 3.5. Evaluation of antioxidant properties of the extracts The antioxidant capacity of the seaweed extract was analysed by the DPPH and ABTS + . The 1/10 diluted extracts gave DPPH results of 85.70 ±0.49 % and ABTS + of 85.88 ±0.84 %. Based on these results, it can be determined that the SLE possesses powerful antioxidant capacity. The primary antioxidant substances found in macroalgae are polysaccharides, polyphenols, tocopherols, carotenoids and peptides (Jacobsen, Sørensen, Holdt, Akoh, & Hermund, 2019). Phenolic substances, such as flavonoids or phenolic acids, are another source of antioxidant action in seaweeds. Phlorotannins are the main class of phenolic compounds found in brown seaweed such as Sugar kelp Saccharina latissima (Cotas et al., 2020). Further antioxidant capabilities can be enhanced by tocopherols, carotenoids (primarily fucoxanthin in brown seaweed), peptides, and amino acids (Pe˜ nalver et al., 2020). Specifically, fucoidan and carotenoids are reported to be present in significant concentrations in Saccharina species, whereas phenolic compounds like phlorotannin are present in relatively low concentrations (Jacobsen et al., 2019). Furthermore, algae-derived substances like Fucus vesiculosus ethyl acetate extract have been demonstrated to act as natural antioxidants, limiting lipid oxidation (J´ onsd´ ottir et al., 2016). On the other hand, the extraction method represents a crucial factor for achieving high antioxidant values in the extracts. Multiple studies have optimized extraction conditions to increase the yield, and ultrasound extraction has proven to be the most effective for recovering phenols and antioxidants from seaweed (Generali´ c Mekini´ c et al., 2019; Ummat et al., 2020). The use of water as a solvent allowed higher antioxidant values to be obtained because more polar solvents can more effectively release the polysaccharides from the cell wall, promoting the antioxidant effect (Mildenberger, Stangeland, & Rebours, 2022). Furthermore, the water extraction process is more economical and environmentally friendly than using solvents like methanol or ethanol. Fig. 3. Response graph of the predicted effect of SLE concentration on the A) Tensile strength (TS), B) Young’s Modulus (YM). SLE is Sugar Kelp Saccharina Latissima extract. Table 2 Antioxidant properties of the developed chitosan-based films with Saccharina Latissima extract. Samples DPPH ABTS + (%) (%) Ch 1.68 ±0.37 a 1.86 ±1.04 a Ch_SLE10 10.72 ±0.96 b 56.30 ±2.51 b Ch_SLE20 18.80 ±0.50 c 90.81 ±1.45 c Ch – chitosan; SLE – Saccharina latissima extract. Values are expressed as mean ±standard deviation (SD). Different letters in the same column indicate significant differences (p <0.05). M. Fl´ orez et al. Food Hydrocolloids 149 (2024) 109571 7 3.6. Evaluation of antioxidant properties of the films The antioxidant capacity of the films was also assessed (Table 2). Compared to pure chitosan matrix, films with SLE added demonstrated significant free-radical scavenging action, with DPPH values ranging from 1.68 in pure chitosan films to 18.8% in chitosan films with 20% of SLE. The data were well fitted to a significant linear model with an Fvalue of 1001 (p <0.0001) and an r 2 of 0.99 (Table S3). Equation (3) forecasts the DPPH values of the films as a function of SLE concentration. The DPPH prediction is shown in Fig. 4A. DPPH =1.84 +0.85 ⋅ SLE (%) (3) On the other hand, ABTS + values of the film samples ranged from 1.86% in pure chitosan films to 90.81% in chitosan films with 20% of SLE. A remarkable improvement in ABTS + activity was observed in Ch_SLE films. The quadratic model fit the data well and was statistically significant, with an F-value of 1906 (p <0.0001). The F-values indicated that the linear effect of SLE concentration (F-value =3750) had the most significant influence on ABTS + values, followed by the quadratic effect (F-value =62.77) (Table S3). The value of r 2 was 0.99. Equation (4) forecasts the ABTS + values as a function of SLE concentration. The ABTS + prediction is shown in Fig. 4B. ABTS + =1.86 +6.44 ⋅ SLE (%) - 0.09 ⋅ SLE 2 (%) (4) The improvement of antioxidant properties was as well observed when Himanthalia elongata and Palmaria palmata seaweed water extracts were added to chitosan films, thereby extending the shelf life of fresh fish burgers (Albertos, Martin-Diana, Bur´ on, & Rico, 2019). 3.7. Evaluation of the optical properties of the films The UV-barrier properties of the Ch_SLE films were determined using the UV transmittance values. The colour and opacity of the films were determined using the transmittance values in the visible region. Table 3 shows the average percentage of transmittance (%T) of the pure chitosan and Ch_SLE films in the UV-C (200–280 nm), UV-B (280–315 nm) and UV-A (315–400 nm) regions. Fig. 5 shows the UV profile spectra of the films. Ch_SLE films showed %T values in the UV-C ranged from 6.26 to 21.15%. The model F-value for UV-C was 475.44 and p-value <0.0001, which means that the model was significant (Table S4). The results of the fit statistics showed a r 2 of 0.99 (Table S5). Equation (5) forecasts the UV-C response as a function of SLE concentration. UV-C =21.47 - 1.93⋅ SLE (%) +0.05 ⋅ SLE 2 (%) (5) On the other hand, the UV-B property showed values from 18.80 to 46.17%. The F-value of the model was 424.83 and the p-value was less than 0.05. Therefore, the model was significant, with an r 2 of 0.99 (Table S5). The UV-B response is predicted as a function of SLE concentration by Equation (6). UV-B =45.56 - 3.40 ⋅ SLE (%) +0.10 ⋅ SLE 2 (%) (6) The UV-A property had values between 38.85 and 59.54%. Data relating the UVThe mathematical model was significant since its pFig. 4. Response graph of the predicted effect of SLE concentration on A) DPPH • and B) ABTS •+ of the films. SLE is Sugar Kelp Saccharina Latissima extract. Table 3 Optical properties and color parameters of the chitosan-based films. Film samples UV-C UV-B UV-A Transparency Opacity %T %T %T Ch 21.15 ± 1.12 46.17 ± 2.09 59.54 ± 1.54 47.43 3.35 Ch_SLE10 7.85 ± 0.54 21.59 ± 0.98 40.75 ± 0.03 48.23 4.96 Ch_SLE20 6.26 ± 0.91 18.80 ± 13.46 38.85 ± 0.49 41.57 4.37 Film samples L* a* b* Ch 89.19 ±0.03 −0.13 ±0.00 a 1.91 ±0.00 a Ch_SLE10 86.11 ±1.34 −0.32 ±0.11 a 5.73 ±0.15 b Ch_SLE20 86.15 ±2.38 −0.65 ±0.01 b 6.55 ±1.48 bc CH – chitosan; SLE – Saccharina Latissima extract. UV-C (200–280 nm). UV-B (280–315 nm). UV-A (315–400 nm). %T - percentage of transmittance. L*. lightness: black =0 and white =100; a*. green =-a* and red = +a*); b*. blue =-b* and yellow = +b*. Values are expressed as mean ±standard deviation (SD). Different letters in the same column indicate significant differences (p <0.05). M. Fl´ orez et al. Food Hydrocolloids 149 (2024) 109571 8 value was <0.0001 and the F-value was 521.88. The fit statistics results indicated a r 2 of 0.99 (Table S5). The UV-A response is predicted as a function of SLE concentration by Equation (7). UV-A =59.99 - 2.80 ⋅ SLE (%) +0.08 ⋅ SLE 2 (%) (7) Regarding the UV properties of the films, the values decreased with the increase in SLE. These results lead to the conclusion that the presence of the seaweed extract in the composition improves the UV blocking capacity of the formulated films. Transparency and opacity of the films are crucial factors to be studied. Table 3 shows that the values of transparency in the film samples ranged from 41.57 to 48.23. The obtained mathematical model for transparency was significative, indicating that the transparency of the films depended on the SLE content. The model had an F-value of 508.69 (p <0.0001) (Table S4) with an r 2 of 0.99 (Table S5). In terms of the real factor of SLE concentration in films, Equation (8) forecasts the transparency response. Transparency =47.39 +0.50 ⋅ SLE (%) - 0.04 ⋅ SLE 2 (%) (8) Fig. 6A displays the graphical representation of the transparency of the films as determined by Equation (8). Transparency’s film is a significant factor in customer acceptability since it allows for the inspection of food freshness (Min et al., 2020). Generally, a lower transparency implies higher opacity. In this case, the transparency values of Ch_SLE films decreased slightly compared to pure chitosan films. Regarding the opacity, the values ranged from 3.35 to 4.96. The mathematical model was significant, showing a F-value of 135.49 (pvalue <0.0001) with an r 2 of 0.97. Equation (9) predicts the opacity response as a function of SLE concentration. Fig. 6B illustrates the plot depicting the opacity of the Ch_SLE film. Opacity =3.373 +0.242 ⋅ SLE (%) - 0.009 ⋅ SLE 2 (%) (9) Opacity is a critical factor in film applications, particularly in packaging, as it shields food from light damage caused by sunlight, fluorescent, or incandescent light by controlling the passage of light rays through the material (Martins, Cerqueira, Bourbon, Pinheiro, & Vicente, 2011). It was observed that the opacity values of chitosan films increased with SLE concentration. Similar results were observed when Kappaphycus alvarezii seaweed was added to corn starch films (Khalil et al., 2018). Table 3 shows the Ch_SLE films’ CIE coordinates. Data were all fitted to a linear model. Lightness (L*) ranged from 86.11 to 89.19. The Fvalue of the model was 10.05 (p-value 0.0157). However, the r 2 was 0.58. Concerning the values of a*, these ranged from −0.13 to −0.65. The model obtained was significant, F-value of 108.20 (p-value <0.0001) and r 2 of 0.93. Equation (10) predicts a* values as a function of SLE concentration. Fig. 6C shows the graphical representation of a* values of the Ch_SLE films. a* = − 0.11 - 0.02 ⋅ SLE (%) (10) Values of b* ranging from 1.91 to 6.55, meaning that the films had a trend toward yellow when SLE was added. The mathematical model had an F-value of 48.55 (p-value of 0.0002), indicating that the model was significant, with an r 2 of 0.94. Equation (11) predicts the b* values of the Ch_SLE films. Fig. 6D shows the graphical representation of b* values of the Ch_SLE films. b* =1.91 +0.53 ⋅ SLE (%) - 0.01 ⋅ SLE 2 (%) (11) Regarding the overall CIE coordinates, the a* values decreased with the SLE concentration indicating a slight tendency to redness, while the b* values increased, meaning that the films appeared more yellowish when SLE was added to the chitosan films. Similar behaviour was observed when the whole red algae Kappaphycus alvarezii was added to starch films (Khalil et al., 2018). 3.8. Evaluation of the thermal properties (TGA/DSC) The effects of SLE on the thermal physical property of the Ch_SLE films were studied using TGA/DSC. The thermograms of chitosan films, pure or enriched with SLE from 10 to 20% of concentration are displayed in Fig. 7. In TGA, the weight change with relation to temperature under constant heating rate or time, under isothermal conditions and in a controlled environment can be used to assess the material’s thermal stability (Perera et al., 2021). The weight loss in the range 50–125 ◦C was due to the evaporation of the residual acetic acid and water (free and bound) (Moura et al., 2015). The decomposition of amine units and oxygenated functional groups may be responsible for the second step of weight loss, which occurred between 150 and 300 ◦C (Martínez-Camacho et al., 2010). Degradation of chitosan usually takes place at 280 ◦C, although previous studies have shown higher temperatures for this degradation (290 ◦C) (Leceta, Pe˜ nalba, Arana, Guerrero, & De La Caba, 2015). Seaweed degradation also occurs in this temperature range, as well this step is associated with degradative processes, such as Fig. 5. UV Spectra profile of chitosan (Ch) films with glycerol (0.1%) and SL (0, 10, 20%). SLE is Sugar Kelp Saccharina Latissima extract. M. Fl´ orez et al. Food Hydrocolloids 149 (2024) 109571 9 depolymerization and decomposition of polymeric units (Albertos et al., 2019; Peniche-Covas, Argüelles-Monal, & San Rom´ an, 1993). The third and final step of mass loss continue from 300 to 350 ◦C due to the degradation of –CH 2 OH and the denaturation of the polymeric organization of the biopolymer (Moura et al., 2015; Zawadzki & Kaczmarek, 2010). The results show that the weight loss in both the pure chitosan films and those with SLE added to the matrix followed a very similar trend. The total weight losses of the film samples being 33.86%, 34.24% and 35.81% in films with SLE of 0, 10, 20%, respectively. These results suggest that the addition of seaweed extract to the chitosan matrix did not alter the thermal properties of the film. In fact, an improvement of this property was observed when seaweed and seaweed water extracts such as Himanthalia elongate and Palmaria palmata were added to the matrix (Albertos et al., 2019). Regarding the DSC, it measures the amount of heat required to raise the temperature of the material as a function of the time (Hasan et al., 2019). Films exhibited exothermic peaks between 50 and 125–150 ◦C, relative to the evaporation of acetic acid and water respectively (Moura et al., 2015). As well, during these peaks the loosing of chitosan chains occurred (Zhang, Liu, Sun, Wang, & Li, 2020). The graph of pure Fig. 6. Response graph of the predicted effect of SLE concentration on the A) Transparency, B) Opacity, C) a* and D) b* values. SLE is Sugar Kelp Saccharina Latissima extract. M. Fl´ orez et al.