Storage stability of soy protein isolate films incorporated with mango kernel extract at different temperature

Food Hydrocolloids 87 (2019) 541–549

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Storage stability of soy protein isolate films incorporated with mango kernel extract at different temperature

T

Z.A. Maryam Adilaha, Z.A. Nur Hanania,b,∗ a b

Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor, Malaysia Halal Products Research Institute, Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor, Malaysia

A R T I C LE I N FO

A B S T R A C T

Keywords: Mango kernel extract Soy protein isolate Storage stability Active packaging Antioxidant film

This research investigated the storage stability of antioxidant films made from waste and by-products which are soy protein isolate (SPI) and mango kernel extract (MKE) stored at room temperature (25 °C), refrigeration temperature (4 °C) and frozen temperature (−18 °C) for 90 days. The thickness of the films was maintained from 0.050 to 0.058 mm until the 90th day. The colour properties of SPI films incorporated with MKE (SPI + MKE) were generally not significantly affected by time and temperature except for the b value. All the films turned darker over the storage time. There was no dominant factor between temperature and time for the mechanical properties; all the films showed an increase in tensile strength and Young's modulus, and a decrease in elongation. The antioxidant activity of the films was determined by the total phenolic content and radical scavenging activity of DPPH and ABTS. SPI + MKE film at 25 °C showed the highest antioxidant activity as compared to films stored at 4 °C and −18 °C in all the analyses, with the result being significant in DPPH and ABTS analyses. The film stored at 25 °C showed 26 to 50% higher (p > 0.05) TPC than films stored at 4 °C and −18 °C, respectively and had the highest antioxidant activity (54%) in ABTS analysis (p < 0.05). SPI + MKE film stored at 25 °C also showed only 1% depreciation of radical scavenging activity (RSA) throughout the storage time. The highest decrease (4%) in antioxidant activity was recorded for SPI + MKE film stored at −18 °C, although it was considered very low. This shows that the antioxidant activity of the films is stable for 90 days of storage.

1. Introduction

properties of food. Antioxidant films also provide a controlled and gradual release of the antioxidant, thus providing longer protection to the packaged product (Riquelme, Herrera, & Matiacevich, 2017). Antioxidant films can be developed by casting method and, generally, the content is made up of biopolymer, antioxidant and plasticizer. Polysaccharides, proteins and lipids are mostly used as the source of biopolymer (Liu, Meng, Liu, Kan, & Jin, 2017). Protein films are superior in terms of the gas barrier and mechanical properties at low moisture content as compared to polysaccharides and lipids, therefore they continue to be the preferred material for film production (Alves, Gonçalves, & Rocha, 2017; Azeredo & Waldron, 2016; Ou, Kwok, & Kang, 2004). Soy protein isolate (SPI) is made by defatted soybean which is the by-product of soybean oil industry (Maryam Adilah, Jamilah, & Nur Hanani, 2018). It contains a minimum of 90% protein and it is normally used in agriculture, adhesives and biotechnology. SPI is biodegradable, renewable and has a good film-forming ability (Wang, Kang, Zhang, Zhang, & Li, 2017).

Manufacturing of conventional food packaging materials may involve the addition of some chemicals such as phthalate esters, alkylphenols, 2,2-bis(4-hydroxyphenyl)propane, (bisphenol A or BPA) and di(2-ethylhexyl) adipate (Fasano, Bono-Blay, Cirillo, Montuori, & Lacorte, 2012). These chemicals may leach from the packaging into food products and due to their toxicity, the long-term exposure to human will raise safety concerns (Weng & Zheng, 2015). Thus, research on the new packaging materials and systems considered as safe are gaining interest today. These include the development of active and smart packaging based on biodegradable and green materials. Active packaging involves the incorporation of active compounds, such as antioxidant and antimicrobial agents that can interact with the head-space of packaging by absorption or release of the compounds (Bastante, Cardoso, Serrano, & de la Ossa, 2017). Antioxidant film is one of the most popular types of active packaging. Antioxidants incorporated into the polymer matrix do not interfere with the sensorial

∗ Corresponding author. Department of Food Technology, Faculty of Food Science and Technology, Universiti Putra Malaysia, 43400, UPM, Serdang, Selangor, Malaysia. E-mail address: [email protected] (Z.A. Nur Hanani).

https://doi.org/10.1016/j.foodhyd.2018.08.038 Received 12 June 2018; Received in revised form 21 August 2018; Accepted 21 August 2018 Available online 22 August 2018 0268-005X/ © 2018 Elsevier Ltd. All rights reserved.

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The green consumerism has prompted the usage of natural antioxidants over synthetic ones. Some of the research has used natural antioxidants such as clove essential oil (Ortiz, Salgado, Dufresne, & Mauri, 2018), licorice residue extract (Han, Yu, & Wang, 2018), pineneedle extract (Yu et al., 2018) and also mango kernel extract which was used in the previous research (Maryam Adilah et al., 2018). Mango kernel is the waste product of the mango industry that is currently not yet being fully utilized. Although mango kernel is the waste product, it contains high active compounds such as phenolic acids, flavonoids, gallotannins and ellagitannins (Torres-León, Rojas, Serna-Cock, Belmares-Cerda, & Aguilar, 2017; Torres-León et al., 2016) that contribute to its high antioxidant activity. According to the Japanese regulation, the usage of mango kernel in food is permissible (Saito, Kohno, Yoshizaki, & Niwano, 2008), thus, it is safe for consumption. The usage of mango kernel extract (MKE) as a source of natural antioxidants will provide a new alternative to fully utilize this waste product. Inevitably, packaging material should have the ability to protect the packaged product throughout its lifetime. The storage stability of the film either in terms of physical or functional properties should be maintained at a reasonable level to ensure the safety of the packaged product. However, aging process is inevitable to biopolymer due to physical changes (recrystallization process and migration of compounds such as water and glycerol) and chemical changes (oxidation of the protein sulfhydryl groups) (Anker, Stading, & Hermansson, 2001; Osés, Fernández-Pan, Mendoza, & Maté, 2009). The aging process also differs according to the temperature and time. Therefore, the objective of this study was to determine the stability of antioxidant films made from SPI and MKE stored at room, refrigeration and freezing temperatures (25 °C, 4 °C and −18 °C, respectively) for 90 days.

w based on SPI content) and soy lecithin as an emulsifier (25% w/w based on MKE content) were added and stirred at 50 °C for other 30 min. The mixture was then homogenized at 5000 rpm for 3 min using a homogenizer (Heidolph Instruments GmbH & Co., Schwabach, Germany). Then, 14 ml of the film solution was spread on polystyrene petri dish plate (14 × 14 cm2) and dried at 25 °C and 50% relative humidity (RH) for 24 h. The film solution without the addition of MKE was used to prepare the control film sample. 2.4. Storage of films The films were stored at three different temperature which are the ambient temperature (25 °C), refrigerating temperature (4 °C) and freezing temperature (−18 °C) at 40–60% RH. These temperatures were chosen as most of the packaged food in the industry are stored at these conditions. The films were stored for 90 days and analyses were conducted for every 10 days interval. 2.5. Film thickness The film thickness was determined with a digital micrometer (Mitutoyo Absolute, Tester Sangyo Co. Ltd., Japan). The thickness was measured in ten randomly selected locations on each film and then an average value was calculated. 2.6. Colour

2. Materials and methods

The colour of the films was measured using a MiniScan XE Plus Hunter colourimeter (Hunter Associates Laboratory, Inc., Reston, Virginia). The L, a and b values were determined to indicate white/ black, red/green and yellow/blue, respectively. The machine was calibrated using a standard white tile.

2.1. Chemicals

2.7. Mechanical properties

Soy protein isolate (SPI) with 92% protein content was purchased from MP Biomedicals (Solon, Ohio, USA). ABTS and Folin Ciocalteu reagent were supplied by Merck and Co. (Darmstadt, Germany). Potassium persulphate, sodium carbonate and gallic acid were purchased from R&M Chemicals (Selangor, Malaysia) whereas anhydrous glycerol (99.5% purity) was supplied by Systerm (Karlsruhe, Germany). DPPH was purchased from Tokyo Chemical Industry (Tokyo, Japan). Absolute ethanol (99.9% purity) and soy lecithin were purchased from John Kollin Corporation (Midlothian, UK) and Modernist Pantry (Eliot, Maine USA), respectively.

The mechanical properties were measured according to the method by Maryam Adilah et al. (2018) as adapted from Ili Balqis, Nor Khaizura, Russly, and Nur Hanani (2017). The mechanical properties were expressed in terms of tensile strength, elongation at break and Young's modulus and were determined using INSTRON 4302 Series IX Machine (Instron Co., Canton, Massachusetts, USA). The films were cut into rectangular strips (1.5 × 9 cm2) and were conditioned at 23 ± 2 °C and 50 ± 5% RH for 2 days. The film strip was stretched between the grips with 50 mm initial separation and 50 mm/min cross head speed. The tensile load used was 5 kN.

2.2. Extraction of mango kernel

2.8. Total phenolic content (TPC)

Mango seeds from Chokanan variety (maturity stage 5; fully ripened) were collected from mango juice producer in Serdang, Selangor. The mango kernel was extracted according to Maryam Adilah et al. (2018). The mango kernels were separated from the seeds and cleaned. The kernels were diced and dried at 50 °C for 24 h (Abdalla, Darwish, Ayad, & El-Hamahmy, 2007; Augustin & Ling, 1987). The dried kernel was ground using a blender (Tefal, Rumilly, France). Ethanol was added in 5:1 (v/w) ratio. The mixture was left in dark for 24 h with regular shaking. The residue was removed by using Whatmann no. 4 filter paper and the supernatant was evaporated at 40 °C. The MKE was used for the film preparation.

The total phenolic content was determined according to previous research by Maryam Adilah et al. (2018) using the method as adapted from Ruiz-Navajas, Viuda-Martos, Sendra, Perez-Alvarez, and Fernández-López (2013). Twenty-five mg of film sample was immersed in 3 ml ethanol to get the extract. After that, 0.3 ml of the extract was added to 2.5 ml Folin Ciocalteu reagent (10% v/v) followed by 2 ml 7.5% (w/v) sodium carbonate solution. Next, it was kept at 50 °C for 5 min. The absorption was measured at 760 nm using Genesys 10 UV–Vis spectrophotometer (Thermo Fisher Scientific, Madison, Wisconsin, USA). Gallic acid solutions (0–1000 ppm) were used to obtain the standard curve. The result was expressed as microgram gallic acid equivalent per gram film (μg GAE/g film).

2.3. Film preparation 2.9. DPPH radical scavenging assay The film was prepared according to Maryam Adilah et al. (2018) and Tongnuanchan, Benjakul, and Prodpran (2013). Distilled water was heated to 70 °C and 3.5% SPI was added and mixed for 30 min. Glycerol as a plasticizer (30% w/w based on SPI content), MKE (1, 3 and 5% w/

DPPH free radical scavenging assay was carried out according to previous researches (Maryam Adilah et al., 2018; Siripatrawan & Harte, 2010). Twenty-five mg of film sample was immersed in 3 ml ethanol to 542

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get the extract. Consequently, 3 ml film extract was added to 1 ml 0.1 mM ethanolic DPPH solution. It was then mixed thoroughly and was incubated in a dark room at normal room temperature for 30 min. The absorbance was measured at 517 nm. The DPPH radical scavenging activity was measured using the following equation (1):

Radical scavenging activity (%) (Abs DPPH − Abs sample extract) x 100 = Abs DPPH

value indicates blue or yellow. For the L value, the general trend showed that the temperature had no significant effect (p > 0.05) on both control films and SPI + MKE films until day 90. However, SPI + MKE films stored at all the temperatures under study showed significant (p < 0.05) lower L values (70–79) compared to their respective control films (83–86). The significant (p < 0.05) darker colour of SPI + MKE films, regardless of temperature, is contributed by the MKE that is yellowish-orange in colour. The MKE also has been reported to contain a high amount of phenolic compounds that can impart a darker colour to the film (Cheng, Wang, & Weng, 2015). The L value of the film, as affected by storage time, showed an interesting trend. The change in the L value was only significant (p < 0.05) for the control films as compared from day 1 and day 90, as shown in Table 2. However, there was no notable change in the SPI + MKE films from day 1 to day 90, regardless of the temperature. Due to the nature of film components, molecular changes and reorganization can be expected to take place over time depending on the matrix composition (Blanco-Pascual, Fernández-Martín, & Montero, 2014). Reorganization process makes the structure to become more compact, thus increasing its opacity and lowering its L value. Since MKE contains a high amount of fat (47.1%), the incorporation of MKE in SPI films might hinder or slows down the reorganization process compared to the control film as SPI + MKE films have lipid droplets embedded in the film matrix. Thus, the change in the L value of the SPI + MKE films compared to the control was less pronounced and was not significant (p > 0.05). The effect of temperature on the a values of the control and SPI + MKE films was not significant (p > 0.05) on the earlier part of the storage, as shown in Table 3. However, the control film stored at 25 °C showed a significant (p < 0.05) change of a value as compared to other films observed on the 90th day. This is probably because of higher rate occurrence of protein reorganization at 25 °C compared to 4 °C and −18 °C due to the shown in higher migration of water and glycerol from the film matrix. The a values of the SPI + MKE films remained unchanged at the 90th day compared to the 1st day. The a values of SPI + MKE films were significantly different (p < 0.05) to the control films at their respective temperatures. The control films showed negative a values (green) while the SPI + MKE films showed an inclination to the positive a values (red). The higher red colour of SPI + MKE films is controlled by the colour of MKE in the films. The effect of storage time on the a values of the films also showed a similar trend as seen for L values. Only the control films at 4 °C and −18 °C showed significant (p < 0.05) change of a values from day 1 to day 90. However, the control film stored at 25 °C did not show any significant change (p > 0.05) from day 1 to day 90, although the a value was higher than that of the films stored at 4 °C and −18 °C. On the other hand, the SPI + MKE films showed insignificant change of a values, most probably due to the lipids that act as a lubricant that prevents extensive protein reorganization, thus lowering the colour change. The effect of temperature on the b values of the film showed no

(1)

2.10. ABTS radical scavenging assay The ABTS radical scavenging assay was done with reference to Maryam Adilah et al. (2018). ABTS solution (7 mM) was mixed with 2.45 mM potassium persulfate solution in 1:1 ratio for 16 h. The solution was then diluted with ethanol until it reached the absorbance of 0.70 ± 0.02 at 734 nm. Consequently, 40 ml film extract was added to 3960 mL of the diluted solution. The absorbance was measured at 734 nm after incubation in dark for 6 min. Gallic acid solutions (0–1000 ppm) were used to obtain the standard curve. The result was expressed as microgram gallic acid equivalent per gram film (μg GAE/g film). 2.11. Statistical analysis One-way analysis of variance (ANOVA) and Tukey's multiple tests using Minitab 17 software (Minitab Inc., State College, Pennsylvania, USA) were used for statistical analyses. The significant level was set at p < 0.05. 3. Results and discussion 3.1. Film thickness The general trend showed that the thickness remains stable from day 1 up to the 90th day of storage for both control and SPI + MKE films as shown in Table 1. The thickness of the control films, regardless of temperature and storage time, remains at 0.050 to 0.058 mm. However, the SPI + MKE films showed higher thickness from 0.055 to 0.068 mm regardless of the temperature and storage time. The higher thickness of SPI + MKE films is probably due to the incorporation of extract that prevents the formation of ordered structure and, thus, imparts higher thickness to the film (Tongnuanchan, Benjakul, Prodpran, & Nilsuwan, 2015). 3.2. Colour Colour is an important parameter that shows visual changes of the film. The evolvement of the film's colour, as affected by the temperature and storage time, was determined using the Hunter Lab. The L value indicates darkness or lightness, a value indicates red or green and b

Table 1 Thickness of control and SPI + MKE films at different storage temperature and time. Thickness Storage time (Days) Control 25 °C Control 4 °C Control −18 °C SPI + MKE 25 °C SPI + MKE 4 °C SPI + MKE -18 °C

1

10 B,a

0.058 ± 0.001 0.058B,a ± 0.001 0.058B,a ± 0.001 0.067A,a ± 0.003 0.067A,a ± 0.003 0.067A,a ± 0.003

30 BC,a

60 A,a

0.057 ± 0.001 0.055C,a ± 0.002 0.059BC,a ± 0.002 0.067A,a ± 0.003 0.068A,a ± 0.002 0.061B,b ± 0.002

0.057 ± 0.000 0.055A,a ± 0.002 0.058A,ab ± 0.002 0.067A,a ± 0.005 0.067A,a ± 0.005 0.060A,bc ± 0.001

90 B,a

0.055 ± 0.002 0.053B,a ± 0.002 0.057AB,ab ± 0.002 0.061A,a ± 0.002 0.057AB,a ± 0.001 0.057AB,bc ± 0.001

0.058AB,a ± 0.004 0.055AB,a ± 0.003 0.051B,c ± 0.001 0.060A,a ± 0.005 0.057AB,a ± 0.001 0.055AB,c ± 0.002

Values are given as mean ± standard deviation. Means with different superscript letters are significantly different (p < 0.05): uppercase letters (effect of storage time on films); lowercase letters (effect of storage temperature on films). 543

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Table 2 L value of control and SPI + MKE films at different storage temperature and time. L values Storage time (Days) Control 25 °C Control 4 °C Control −18 °C SPI + MKE 25 °C SPI + MKE 4 °C SPI + MKE -18 °C

1

10 A,c

83.87 ± 0.45 83.87A,c ± 0.45 83.87A,b ± 0.45 78.50B,ab ± 0.78 78.50B,a ± 0.78 78.50B,a ± 0.78

30 A,b

60 A,a

84.93 ± 0.29 84.69A,bc ± 0.43 85.08A,a ± 0.10 78.50B,ab ± 0.22 78.67B,a ± 0.34 78.70B,a ± 0.60

86.02 ± 0.15 85.97A,a ± 0.47 85.96A,a ± 0.34 78.79B,a ± 0.19 78.88B,a ± 0.86 78.86B,a ± 0.58

90 A,ab

85.61 ± 0.23 85.79A,a ± 0.50 85.51A,a ± 0.58 75.92C,cd ± 0.28 79.01B,a ± 0.29 79.01B,a ± 0.07

86.20A,a ± 0.14 85.58 AB,ab ± 0.35 85.38A,a ± 0.43 76.53D,bcd ± 0.30 78.28C,a ± 0.13 78.54C,a ± 0.10

Values are given as mean ± standard deviation. Means with different superscript letters are significantly different (p < 0.05): uppercase letters (effect of storage time on films); lowercase letters (effect of storage temperature on films).

significant difference (p > 0.05) on the control films on day 1 and day 90 as shown in Table 4. Nonetheless, the SPI + MKE films showed a significant difference (p < 0.05) in b value as compared to their control films for day 1 and day 90. The effect of storage time indicated that all the control and SPI + MKE films showed significant (p < 0.05) lower b values at day 90 as compared to day 1 except for SPI + MKE films stored at 25 °C. Since the addition of MKE lowered the colour change, it is more beneficial to use SPI + MKE films. SPI + MKE films stored at room temperature are effective because of their colour retention ability at prolonged storage time, even after 90 days.

plasticizers to the surrounding and also to the packaged product (Ciannamea, Stefani, & Ruseckaite, 2015). The reduction of plasticizer level affected the film by decreasing the flexibility and promoting molecular rearrangement of the protein which was not favorable at high plasticizer level (Ciannamea, Espinosa, Stefani, & Ruseckaite, 2017). Similar result was reported on the storage stability of bean starch film that showed an increase in TS at the end of storage time (Lima et al., 2017). The temperature does not show any significant effect (p > 0.05) on the EAB of SPI + MKE films within the studied time range (Fig. 2). However, the lower the temperature, the higher the EAB value that was recorded throughout the storage time for SPI + MKE films. The storage time had a significant effect (p < 0.05) on the EAB of all films except for SPI + MKE films stored at −18 °C. Although the SPI + MKE films stored at this temperature did not show any significant difference (p > 0.05) from day 1 to day 90, it still maintained the highest value of EAB. The EAB value is highly correlated with the TS value. The high EAB value is normally accompanied by low TS value and vice versa. Since the TS value of all the films increased significantly (p < 0.05), the decrease in EAB was expected. However, the lower decrease of EAB for SPI + MKE films stored at −18 °C was due to the lower loss of bound water at low temperature. Therefore, the flexibility of the film was less affected, hence EAB value did not decrease significantly (p < 0.05). The YM generally was not significantly (p > 0.05) affected by the temperature for both control films and SPI + MKE films for the first 30 days of the storage (Fig. 3). However, the effect of temperature started to be significant (p < 0.05) from day 40 onwards for control films and SPI + MKE films. At day 90, the SPI + MKE films stored at 25 °C showed significantly (p < 0.05) higher YM value compared to the film at 4 °C and −18 °C. The SPI + MKE films showed no significant difference (p > 0.05) with their respective control films from day 1 to day 50. However, from day 60 onwards, the result was more prominent, and SPI + MKE films stored at 25 °C recorded the highest YM (3.5 MPa). YM shows the stiffness of the film. The higher the YM value, the stiffer the film is. YM is deduced from the slope of stress-strain curve,

3.3. Mechanical properties Food packaging is the final protection of the food product. Therefore, the packaging should have reasonable mechanical properties to ensure that the packaged product is well-protected. The mechanical properties, including tensile strength (TS), elongation at break (EAB) and Young's modulus (YM), were studied to determine the strength, flexibility and stiffness of the film. Fig. 1 shows that the temperature had a significant effect (p < 0.05) on TS only for the control films on day 90. The control film (2.6 MPa) stored at the highest temperature (25 °C) showed the most significant (p < 0.05) of TS by 12% compared to the control film (2.3 MPa) stored at the lowest temperature (−18 °C). It is also interesting to note that from day 40–70, there was a significant difference (p < 0.05) between films (both control and SPI + MKE films) stored at 25 °C and at −18 °C. The research by Srinivasa, Ravi, and Tharanathan (2007) showed that the increase of TS at a higher temperature also occurred in chitosan film. The higher temperature also promotes higher loss of bound water and glycerol that could decrease the flexibility, thereby increasing the TS. The 90 day-storage time significantly (p < 0.05) increased the TS of all films compared to day 1. The TS values recorded were between 1.9 and 2.6 MPa. The highest TS (2.6 MPa) was recorded for control films stored at 25 °C on day 90. This could be due to the aging process of the film which is associated with the leaching out of water and Table 3 a value of control and SPI + MKE films at different storage temperature and time. a values Storage time (Days)

1

10

Control 25 °C Control 4 °C Control −18 °C SPI + MKE 25 °C SPI + MKE 4 °C SPI + MKE -18 °C

−0.82 ± 0.15 −0.82B,a ± 0.15 −0.82B,a ± 0.15 2.05A,a ± 0.01 2.05A,a ± 0.01 2.05A,a ± 0.01 B,a

30

−1.15 ± 0.08 −1.11B,b ± 0.04 −1.14B,b ± 0.07 2.03A,a ± 0.07 2.15A,a ± 0.07 2.10A,a ± 0.11

60

−1.53 ± 0.02 −1.36C,c ± 0.01 −1.34C,cd ± 0.03 2.04B,a ± 0.01 2.25A,a ± 0.10 2.16 AB,a ± 0.05

B,a

D,a

90

−1.47 ± 0.01 −1.31B,c ± 0.03 −1.26B,bcd ± 0.01 2.11A,a ± 0.06 2.27A,a ± 0.24 1.99A,a ± 0.18 B,a

−1.59C,a ± 0.04 −1.38 BC,c ± 0.01 −1.16B,bc ± 0.13 2.10A,a ± 0.08 2.21A,a ± 0.10 2.20A,a ± 0.09

Values are given as mean ± standard deviation. Means with different superscript letters are significantly different (p < 0.05): uppercase letters (effect of storage time on films); lowercase letters (effect of storage temperature on films). 544

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Table 4 b value of control and SPI + MKE films at different storage temperature and time. b values Storage time (Days) Control 25 °C Control 4 °C Control −18 °C SPI + MKE 25 °C SPI + MKE 4 °C SPI + MKE -18 °C

1

10 A,a

12.97 ± 0.39 12.97A,a ± 0.39 12.97A,a ± 0.39 11.58B,a ± 0.47 11.58B,a ± 0.47 11.58B,a ± 0.47

30 A,ab

60 A,b

12.36 ± 0.38 11.75 AB,b ± 0.26 11.69 AB,b ± 0.41 11.50 AB,a ± 0.33 11.20B,ab ± 0.12 11.54 AB,ab ± 0.67

11.38 ± 0.41 10.28B,c ± 0.25 10.09B,c ± 0.08 11.51A,a ± 0.31 10.76 AB,ab ± 0.37 10.74 AB,abc ± 0.14

90 C,c

9.38 ± 0.25 9.27C,d ± 0.20 8.92C,d ± 0.14 11.42A,a ± 0.46 10.61 AB,b ± 0.55 10.41B,c ± 0.20

9.38 CD,c ± 0.18 9.20D,d ± 0.31 9.32 CD,cd ± 0.61 11.43A,a ± 0.05 10.54 AB,b ± 0.37 10.29 BC,c ± 0.33

Values are given as mean ± standard deviation. Means with different superscript letters are significantly different (p < 0.05): uppercase letters (effect of storage time on films); lowercase letters (effect of storage temperature on films).

protein interaction. On the other hand, at lower temperature, such as −18 °C, the rearrangement process is lower. Therefore, at −18 °C, the antioxidant is preserved because it is still bonded to the protein-structure. However, the phenolic compounds that are attached to the protein could not interact with the radical in the antioxidant analysis, thus this decreased the antioxidant activity of films at lower temperature. Since antioxidant film works by migration or releasing of the antioxidant from the film to the product, films at 25 °C are more suitable in this context.

which means that it is highly related to the tensile strength and the elongation at break. SPI + MKE films stored at 25 °C showed the highest TS and the lowest EAB values, implying that the film at 25 °C was the most rigid one. This is reflected in the YM result in which SPI + MKE films stored at 25 °C showed the highest percentage of increment (68%) of YM as compared to SPI + MKE films stored at 4 °C (59%) and SPI + MKE films stored at −18 °C (56%) at day 90 relative to day 1. The combined effect of the loss of bound water, diffusion of glycerol and higher rearrangement of protein network contributed to the higher TS, lower EAB and higher YM value of films stored at 25 °C.

3.5. DPPH radical scavenging assay 3.4. Total phenolic content The effect of temperature on radical scavenging activity (RSA) of DPPH was not significant (p > 0.05) for all the films (Fig. 5). However, the highest temperature showed higher RSA values. The RSA values of SPI + MKE films ranged from 83% to 94%. On the other hand, the control films also showed some RSA, albeit quite low, around 5 to 17%. The antioxidant activity of the control film is contributed by the amino acids in SPI, such as phenylalanine, tyrosine and tryptophan, that can react with the reagent through its phenolic side chains (Wang, Hu, Ma, & Wang, 2016). The RSA of SPI + MKE films at all the temperatures under study was higher than their respective control films due to the presence of MKE that possess a high antioxidant ability. The storage time showed insignificant decrease (p > 0.05) on the RSA of the SPI + MKE films stored at all the temperatures under study. SPI + MKE films only showed 1% depreciation in RSA after 90 days of storage. The highest decrease in RSA, shown by film stored at −18 °C, was only 4%. The decrease was from 86% to 83% RSA on day 1 and 90, respectively. The low percentage of antioxidant activity reduction from

TPC is highly related to the antioxidant properties of the film. Therefore, the condition that can maintain the TPC during storage time is needed to ensure the antioxidant activity of the film is still reasonable throughout the storage of the food product. The effect of temperature on the TPC of SPI + MKE films was significant (p < 0.05) on the later part of the storage, around day 80 to day 90. SPI + MKE films stored at 25 °C had significantly (p < 0.05) higher TPC values as compared to 4 °C and −18 °C (Fig. 4). The effect of storage time did not show any significant effect (p > 0.05) on day 90 compared to the initial TPC value for SPI + MKE films at all temperatures. However, at day 90, the films stored at 25 °C showed around 26 to 50% higher TPC values (p > 0.05) when compared to the films at 4 °C and −18 °C, respectively. Films at 25 °C recorded higher antioxidant activity than at other temperatures. This is because higher temperature promotes higher rearrangement process in the protein matrix, thus increasing the protein-

Fig. 1. Tensile strength of control and SPI + MKE films at different storage temperature and time. 545

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Fig. 2. Elongation at break of control and SPI + MKE films at different storage temperature and time.

Fig. 3. Young's modulus of control and SPI + MKE films at different storage temperature and time.

Fig. 4. Total phenolic content of control and SPI + MKE films at different storage temperature and time.

the first to last day of storage at all the temperatures under study showed that the functionality of antioxidant film was still high even after storage. The effect of storage time was insignificant (p > 0.05) in DPPH analysis, similar to the insignificant decrease of TPC, as previously discussed. This strengthened the fact that the activity of the film is

mostly contributed by the phenolic content, aside from other compounds in MKE, such as sesquiterpenoids, phytosterols and tocopherols, that play roles in antioxidant activity too (Dorta, Lobo, & González, 2013; Kabuki et al., 2000; Puravankara, Boghra, & Sharma, 2000).

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Fig. 5. DPPH radical scavenging assay of control and SPI + MKE films at different storage temperature and time.

Fig. 6. ABTS radical scavenging assay of control and SPI + MKE films at different storage temperature and time.

Fig. 7. Image of control and SPI + MKE films at different storage temperature and time.

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3.6. ABTS radical scavenging assay

References

The effect of temperature on the antioxidant activity of all films generally was not significantly (p > 0.05) affected until the end of storage time (Fig. 6). The higher the temperature, the higher gallic acid equivalent (GAE) was recorded. The highest GAE of 6.11 μg GAE/g film was recorded for SPI + MKE film stored at 25 °C. This value was 26 and 40% higher than that observed at 4 °C and −18 °C, respectively. During the film formation process, the SPI solution was heated to uncoil the globular protein structure to a certain extent. This was needed to improve the interaction between the protein and other compounds such as water, glycerol and MKE to form a film-forming solution. After drying the specimen, the protein could not return fully to their original structure due to the formation of new bonding with other compounds. The higher antioxidant recorded in films stored at 25 °C was associated with higher loss of bound water and plasticizer. This, in turn, encouraged the renaturation process (the return to the original structure) of the protein that released some of the previously bonded antioxidant, thus making them free to be extracted during the antioxidant analysis (Jongjareonrak, Benjakul, Visessanguan, & Tanaka, 2008). The effect of storage time on ABTS showed that decrease in antioxidant activity was significant (p < 0.05) for SPI + MKE films stored at all the temperatures under study. At the last day of storage, SPI + MKE film stored at 25 °C could retain more than 54% of the antioxidant compared to 41% and 33% recorded at 4 °C and −18 °C, respectively. As discussed in the previous section, the storage time affected the mechanical properties of the films due to the higher loss of water and glycerol from the structure. In addition, the higher reorganization process over time also contributed to the result. Therefore, the storage time was expected to increase the antioxidant activity of the film since higher rearrangement process increased the availability of antioxidant (Jongjareonrak et al., 2008). However, the result from TPC, DPPH and ABTS analyses showed the opposite. This is because there is the possibility of migration of the MKE from the film matrix to the surrounding, just as happened to the water and glycerol. It is also interesting to note that the control films at all the temperatures under study showed a reduction in the antioxidant activity, probably due to the change in the structure of the protein. Therefore, it can be deduced that the loss of antioxidant activity over time was also partially contributed by the film itself and not solely due to the loss of MKE. Fig. 7 shows the image of films before and after storage at different temperatures.

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4. Conclusion The storage stability of the films is highly affected by the storage temperature and time. Most of the changes in the physical and functionality of the films over the storage period are related to the rearrangement process in the film matrix due to the loss of bound water and glycerol. The physical properties of the films changed with temperature and time and this was closely observed in the depreciation in the lightness and elasticity of the resulting film. Besides, the tensile strength and Young's modulus results observed illustrate the impact of time and temperature on the films. The antioxidant properties of the films showed that the best storage temperature for the film was at 25 °C as the TPC of the SPI + MKE film showed 26 and 50% higher than those at 4 °C and −18 °C, respectively. The finding also showed that only 1% decrease in RSA was recorded for the SPI + MKE film at 25 °C for the 90th day. The highest decrease (4%) in antioxidant activity was recorded for SPI + MKE film stored at −18 °C. The storage stability test showed that the antioxidant film still exhibited reasonable physical and antioxidant properties after 90 days of storage.

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