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Preparation and characterization of active and intelligent films based on fish gelatin and haskap berries (Lonicera caerulea L.) extract
Food Packaging and Shelf Life 22 (2019) 100417
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Preparation and characterization of active and intelligent films based on fish gelatin and haskap berries (Lonicera caerulea L.) extract ⁎
Jing Liu, Huimin Yong, Yunpeng Liu, Yan Qin, Juan Kan , Jun Liu
T
⁎
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Active packing Gelatin Haskap berries Intelligent films
Haskap berries (Lonicera caerulea L.) are rich in polyphenols and possess potential health-promoting effects. In this study, polyphenols were extracted from haskap berries and further incorporated into fish gelatin (FG) to develop active and intelligent packaging films. Results showed anthocyanins and phenolic acids were the main polyphenols in haskap berry extract (HBE). Scanning electron microscopy showed the cross-sections of FG-HBE films were smooth and uniform. Fourier transform infrared spectroscopy revealed intermolecular hydrogen bonds formed between the hydroxyl groups of polyphenols in HBE and the amino/hydroxyl groups in FG. The crystallinity of FG film was enhanced by the addition of HBE. Moreover, the incorporation of HBE significantly increased the total color difference value, water vapor and UV–vis light barrier properties, tensile strength, and antioxidant ability of FG film. Due to high anthocyanin content in HBE, FG-HBE films were pH-sensitive and exhibited visible color changes as a function of pH. Notably, FG-HBE film containing 1 wt% of HBE was more appropriated to be used as the freshness indicator of shrimp. Our results suggest FG-HBE films can be applied as novel as antioxidant and intelligent food packaging films in food industry.
1. Introduction Due to increasing demands to extend the shelf life of food and protect environment, the petroleum-based packaging materials can be partially substituted by biopolymer-based packaging materials (Gan & Chow, 2018). Proteins and polysaccharides are the main biopolymers employed to develop biodegradable packaging films (Hassan, Chatha, Hussain, Zia, & Akhtar, 2018). Gelatin is an animal protein obtained by the controlled hydrolysis of fibrous insoluble collagen, which is generated as waste during animal slaughtering and processing (GómezGuillén, Giménez, López-Caballero, & Montero, 2011). Gelatin is also one of the most commonly used proteins for food packaging film production due to its abundance, good film forming ability and barrier properties against UV light, oxygen and lipids (Etxabide, Uranga, Guerrero, & de la Caba, 2017). Up to now, gelatins from diverse sources including bovine, porcine and fish have been used to develop food packaging films (Suderman, Isa, & Sarbon, 2018). Nonetheless, the water vapor barrier property of gelatin films is poor due to the hydrophilic nature of gelatin and the plasticizer (e.g. glycerol) used (Tongnuanchan, Benjakul, & Prodpran, 2014). Moreover, the mammalian gelatins are rejected by some consumers due to either sociocultural-religious or health-related concerns (Lin, Regenstein, Lv, Lu, & Jiang, 2017). Therefore, gelatins from aquatic sources (e.g. cold-water ⁎
and warm-water fishes) are possible alternatives to mammalian gelatins (Karim & Bhat, 2009). In recent years, gelatin-based films have been used as the carriers of natural bioactive additives (e.g. antioxidants and antimicrobials) to achieve active packaging functions (Adilah, Jamilah, Noranizan, & Hanani, 2018; Shahbazi, 2017). Polyphenols are the most abundant secondary metabolites widespread throughout the plant kingdom (Liu, Pu, Liu, Kan, & Jin, 2017). Due to their potent antioxidant and antimicrobial activities, polyphenol-rich plant extracts including tea polyphenols, longan seed extract, mango peel extract and blood orange peel extract have been incorporated into gelatin-based films (Adilah et al., 2018; Jridi et al., 2019; Liu et al., 2015; Sai-Ut, Benjakul, & Rawdkuen, 2015). The interactions between bioactive additives and gelatin matrix can provide controlled release of active ingredients over the storage period. Moreover, the incorporation of polyphenol-rich plant extracts can greatly improve the light/water vapor barrier and mechanical properties of gelatin-based films (Jridi et al., 2019). Therefore, polyphenol-rich gelatin-based films can be applied to protect food products from oxidation and microbial spoilage (Lee, Yang, & Song, 2016; Wang, Xia et al., 2019). Haskap berry (Lonicera caerulea L.), also called blue honeysuckle, is a plant native to Siberia, China and Japan (Chaovanalikit, Thompson, & Wrolstad, 2004). The fruits of this plant (haskap berries) are oval and
Corresponding authors. E-mail addresses: [email protected] (J. Kan), [email protected] (J. Liu).
https://doi.org/10.1016/j.fpsl.2019.100417 Received 2 July 2019; Received in revised form 4 October 2019; Accepted 8 October 2019 2214-2894/ © 2019 Elsevier Ltd. All rights reserved.
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3 wt% of HBE were designated as FG, FG-HBE I, FG-HBE II, FG-HBE III and FG-HBE IV films, respectively. All the films were stored in a desiccator with 50% RH at 25 °C for at least 48 h before tests.
covered with dark blue to purple skin (Becker & Szakiel, 2018). Haskap berries are rich in polyphenols, especially anthocyanins and phenolic acids (Bell & Williams, 2018). The total phenol content of haskap berries assessed by Folin-Ciocalteu assay ranges from 140.5 to 1142.0 mg gallic acid equivalent per 100 g of fresh weight (Celli, Ghanem, & Brooks, 2014). A number of studies have demonstrated the potential health-promoting effects (e.g. antioxidant, anti-microbial, antiatherosclerotic, anticarcinogenic and anti-inflammatory effects) of polyphenol-rich haskap berry extract (HBE) (Bell & Williams, 2018; Jurikova et al., 2012). Considering the abundant polyphenolic content, HBE is a promising addictive for active packaging films. Meanwhile, anthocyanins in HBE can undergo structural changes and exhibit color variations under different pH conditions. Since the process of food spoilage is usually accompanied by pH changes, anthocyanin-rich films can be used as intelligent packaging (i.e. pH indicators) to monitor the freshness of food (Liu et al., 2019; Qin et al., 2019). Till now, the development of active and intelligent packaging by using polyphenol-rich HBE has not been reported. In this study, active and intelligent packaging films were developed by incorporating polyphenol-rich HBE into fish gelatin (FG) matrix for the first time. The structures and physical properties of the developed FG-HBE films were characterized. In addition, the functional properties (i.e. antioxidant and pH-sensitive properties) of FG-HBE films were also evaluated. Finally, FG-HBE films were applied to indicate the freshness of shrimp.
2.4. Structural characterization of FG-HBE films 2.4.1. Scanning electron microscopy (SEM) Film sample was first immersed in liquid nitrogen and cryofractured. Then, the fractured sample was mounted on an aluminum stub and sputter coated with gold. The cross-sectional morphology of film sample was observed by GeminiSEM 300 (Carl Zeiss, Oberkochen, Germany) at 400× of magnification. 2.4.2. Fourier transform infrared (FT-IR) spectroscopy FT-IR spectrum was recorded in the range of 400–4000 cm−1 by Varian 670 spectrometer (Agilent Technologies, CA, USA) equipped with attenuated total reflectance accessory. 2.4.3. X-ray diffraction (XRD) The crystalline property of film sample was evaluated by D8 Advance X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) containing Ni-filtered Cu Kα radiation at 40 mA and 40 kV. The XRD pattern of film sample was recorded from 5° to 80° at the scanning rate of 0.1°/s. 2.5. Determination of the physical properties of FG-HBE films
2. Materials and methods 2.5.1. Color The color parameters (L, a and b) of film were recorded by SC-80C colorimeter (Kangguang Instrument Co., Ltd., Bejing, China) with a D65 illuminant. The total color difference (ΔE) and whitish index (WI) of film were calculated as follows (Liu, Meng, Liu, Kan, & Jin, 2017):
2.1. Materials and chemical reagents Haskap berries (L. caerulea) were purchased from the market of Yichun city (Heilongjiang, China). Gelatin from cold water fish skin (average molecular weight of 60 kDa and isoelectric point of 6) and 2,2diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma Chemical Co. (MO, USA). All other reagents were of analytical grade.
ΔE =
(L*−L)2 + (a*−a)2 + (b*−b)2
WI = 100 −
2.2. Extraction and characterization of polyphenols from haskap berries
(100 − L)2 + a2 + b2
(1) (2)
where L* (92.60), a* (−0.92) and b* (−2.24) were the color parameters of white plate.
The extraction of polyphenols from haskap berries was performed according to the method of Yong, Wang, Bai et al. (2019) with some modifications. In brief, the fruits of haskap berries (100 g) were extracted in 400 mL of 80% (v/v) ethanol solution containing 1% (v/v) of HCl at 4 °C for 24 h. The extraction process was repeated for three times. All the extract solutions were combined and centrifuged at 8000 × g for 20 min to remove debris. The obtained supernatant was concentrated at 35 °C by a rotary evaporator to remove ethanol and then vacuum dried to obtain polyphenol-rich HBE powder. The total phenol content in HBE was determined by Folin-Ciocalteu assay (Liu, Jia, Kan, & Jin, 2013). The anthocyanin content in HBE was determined by pHdifferential assay (Yong, Wang, Zhang et al., 2019). Finally, the composition of polyphenols in HBE was characterized by Agilent 1200 HPLC system (Agilent Technologies, CA, USA) equipped with 6460 triple quadrupole mass spectrometer according to the method of Wang, Yong et al. (2019).
2.5.2. Thickness The thickness of film was measured at ten randomly points on each film by Mitutoyo No. 293-766 digital micrometer (Tester Sangyo Co., Ltd., Tokyo, Japan) with precision of 0.001 mm. 2.5.3. Moisture content The moisture content was determined by drying the equilibrated film piece (3 cm × 3 cm) at 110 °C to a constant weight (Zhai et al., 2018):
Moisture content(%) =
(Mi − Mt ) × 100 Mi
(3)
where Mi and Mt were the initial and final weights of film piece, respectively. 2.5.4. Water vapor permeability (WVP) WVP of film was determined by the method of Yong, Wang, Zhang et al. (2019). Briefly, film piece (6 cm × 6 cm) was sealed on the test cup containing 40 g anhydrous silica gels. The cup was kept at 20 °C in a desiccator containing distilled water (100% RH). The weight of film sealed cup was tested every 24 h for 7 days.
2.3. Development of FG-HBE films FG-HBE films were developed according to the method of Uranga, Etxabide, Guerrero, and de la Caba (2018) with some modifications. Breifly, 3.5 g FG was dissolved in 100 mL of distilled water at 70 °C under magnetic stirring for 30 min. Then, 30 wt% of glycerol (1.05 g) and different contents of HBE (0, 0.5, 1, 2 and 3 wt%) on FG basis were added into FG solution. After stirring at 40 °C for 10 min, the filmforming solutions were degassed and then cast onto Plexiglas plates (24 cm × 24 cm). The plates were dried at 30 °C with 50% relative humidity (RH) for 48 h. The obtained films containing 0, 0.5, 1, 2 and
WVP =
W×x t × A × ΔP
(4)
where W was the increased weight of film sealed cup (g), x was film thickness (m), t was time (s), A was permeation area (m2), and ΔP was saturated water vapor pressure at 20 °C. 2
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Table 1 The composition of polyphenols in HBE by HPLC-MS analysis. Retention time (min)
Polyphenols
[M]+ (m/z)
Relative content (%)
4.45 5.98 6.19 7.90 8.62 11.50 11.87 12.48 14.48
Caffeic acid Cyanidin-3-O-glucoside Malvidin-3-O-(6-acetyl-glucoside) Kaempferol-3-O-glucoside Cyanidin-3-O-(6-malonyl-glucoside) Pelargonidin-3-O-(6-acetyl-glucoside) Peonidin-3-O-(6-acetyl-rutinoside) Ferulic acid Chlorogenic acid
181 449 531 448 535 475 650 195 355
1.48 41.85 2.62 15.79 6.77 0.72 7.69 6.19 16.89
2.8. Statistical analysis
2.5.5. Light transmittance The light transmittance of film was measured by Lambda 35 UV–vis spectrophotometer (PerkinElmer Ltd., MA, USA) at wavelength range from 200 to 800 nm.
Duncan test and one-way analysis of variance were carried out by SPSS 13.0 software package (SPSS Inc., IL, USA). Difference was considered as statistically significant if p < 0.05.
2.5.6. Mechanical property The tensile strength (TS) and elongation-at-break (EAB) of film sample (6 cm × 1 cm) were determined by using TMS-Pro texture analyzer (Food Technology Co., VA, USA) with the crosshead speed of 6 cm/min (Yong, Wang, Zhang et al., 2019).
3. Results and discussion 3.1. Characterization of polyphenols in HBE Polyphenols were extracted from haskap berries in acidic ethanol solution, which was followed by centrifugation, concentration and vacuum drying. The extraction yield of HBE was 5.28 wt% based on the fresh weight of haskap berries. The total phenol and anthocyanin contents in HBE were 435.19 g gallic acid equivalent/100 g extract and 264.46 g cyanidin-3-glucoside equivalent/100 g extract, respectively. The polyphenolic compositions in HBE were further characterized by HPLC-MS. As summarized in Table 1, nine different polyphenols were identified based on their mass spectra. Notably, anthocyanins and phenolic acids were the main polyphenols in HBE. Moreover, cyanidin3-glucoside is the principal anthocyanin in HBE. Celli, Ghanem, and Brooks (2015) extracted anthocyanins from haskap berries by using a ultrasound-assisted method, and found the anthocyanins in the extract were cyanidin-3,5-O-diglucoside (1.25 mg/g dry weight (DW)), cyanidin-3-O-glucoside (16.75 mg/g DW), cyanidin-3-O-rutinoside (0.65 mg/g DW), pelargonidin-3-O-glucoside (0.29 mg/g DW) and peonidin-3-O-glucoside (0.58 mg/g DW). Liu, Wu, Guo, Meng, and Chang (2018) reported the main active components in haskap berries were cyanidin-3-O-glucoside, (+)-catechin and chlorogenic acid, which accounted for 43.7%, 26.3% and 11.6% of total polyphenols, respectively. Wang et al. (2016) suggested the total phenol and anthocyanin contents were closely related to the variety of haskap berries. In addition, cyanidin-3-O-glucoside was the most prominent anthocyanin in all the tested varieties.
2.6. Determination of the functional properties of FG-HBE films 2.6.1. Antioxidant property The antioxidant property of film was evaluated by DPPH radical scavenging assay (Liu, Meng et al., 2017). Briefly, film sample (4–20 mg) was reacted with 4 mL of 100 μM DPPH methanol solution at 20 °C for 1 h in the dark. The absorbance of reaction solution was measured at 517 nm.
DPPH radical scavenging activity(%) =
A0 − A1 × 100 A0
(5)
where A0 and A1 were the absorbance of the blank and reaction solutions, respectively. 2.6.2. pH-sensitive property The pH-sensitive property of HBE was determined by dissolving HBE powder (2 mg) in 20 mL of different buffer solutions (pH 3–12). Then, the UV–vis spectra of HBE solutions were recorded by Lambda 35 UV–vis spectrophotometer (PerkinElmer Ltd., MA, USA) at wavelength range of 400–700 nm (Luchese, Garrido, Spada, Tessaro, & de la Caba, 2018). To determine the pH-sensitive property of FG-HBE films, film samples were immersed in different buffer solutions (pH 3–12) at room temperature for 5 min. The color changes of film were recorded by SC80C colorimeter (Kangguang Instrument Co., Ltd., Beijing, China).
3.2. Microstructure of FG-HBE films The cross-sectional microstructures of FG and FG-HBE films were investigated by SEM. As shown in Fig. 1, the cross-sections of FG and FG-HBE films were smooth and uniform, indicating the filmogenic components (e.g. FG, HBE and glycerol) were homogeneously mixed and well compatible with each other (Wang, Yong et al., 2019). It was hypothesized that the polyphenols in HBE could interact with FG through hydrogen bonds, yielding a compact and dense network (Zhang et al., 2010). The dense inner structures of FG-HBE films were benefit to improve their mechanical and barrier properties. Similar phenomena were observed in pig skin gelatin films containing polypheol-rich hibiscus extract that showed continuous structure and uniform film matrix (Peralta, Bitencourt-Cervi, Maciel, Yoshida, & Carvalho, 2019). However, Zhai et al. (2018) found the cross-sections of pig skin gelatingellan gum films became rough and heterogeneous when polypheolrich red radish extract was incorporated. This indicated the
2.7. Application of FG-HBE films for indicating shrimp spoilage FG-HBE films were used to indicate the freshness of shrimp according to the methods of Kang et al. (2018). First, 25 ± 2 g of fresh shrimp sample was placed in the Petri dish (11 cm in diameter). Then, FG-HBE film sample (2 cm × 2 cm) was stuck on the inside surface of polyethylene film which sealed the Petri dish. The shrimp was then stored at 25 °C for 48 h. During the whole process, the active film was not in contact with shrimp sample. The color change of film sample and the total volatile basic nitrogen (TVB-N) level of shrimp were recorded every 8 h. Briefly, 10 g of shrimp sample was homogenized in 100 mL of deionized water. Then, the TVB-N value of the homogenate was determined by a K9860 Kjeldahl distillation apparatus (Jinan Hanon Instruments Co., Ltd., Jinan, China). 3
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Fig. 1. The cross-sectional morphology of FG (A), FG-HBE I (B), FG-HBE II (C), FG-HBE III (D) and FG-HBE IV (E) films.
hydrogen bonds could result in dense network in FG-HBE films (Fig. 1). Similar changes were found in the FT-IR spectra of FG films incorporated with carvacrol and green tea extract (Neira, Martucci, Stejskal, & Ruseckaite, 2019; Wu et al., 2018).
microstructures of gelatin-based films were greatly influenced by the type of polypheol-rich plant extract added.
3.3. FT-IR spectra of FG-HBE films 3.4. XRD patterns of FG-HBE films
The intermolecular interactions between FG and HBE were revealed by FT-IR spectroscopy. As presented in Fig. 2, HBE exhibited the characteristic bands of polyphenols at 3349 cm−1(OHe stretching), 2962 cm−1(CHe stretching), 1638 cm−1 (C]O stretching), 1602 cm−1 (C]C stretching of aromatic ring) and 1018 cm−1(CHe deformation of aromatic ring) (Yong, Wang, Bai et al., 2019). FG film displayed several characteristic bands at 3284 cm−1(amide-A, NeH and OHe stretching coupled with hydrogen bonds), 2934 cm−1(amide-B, CHe stretching), 1634 cm−1 (amide I, C]O stretching), 1534 cm−1 (amide II, NHe bending and CeN stretching) and 1239 cm−1 (amide III, NHe and CeN in-plane bending of amide bond or eCH2 groups of glycine) (Theerawitayaart, Prodpran, Benjakul, & Sookchoo, 2019). When HBE was incorporated into FG film, the band at 3284 cm−1 was broadened and intensified. This was caused by the intermolecular hydrogen bonds formed between the hydroxyl groups of polyphenols in HBE and the amino/hydroxyl groups in FG. The newly formed intermolecular
As shown in Fig. 3, the XRD pattern of HBE exhibited a broad peak around 24.1°, demonstrating HBE was in the amorphous state. The XRD pattern of FG film exhibited two broad diffraction peaks at 8.8° and 21.0°, respectively. The diffraction peak around 8.8° was attributed to the triple-helical crystalline structure of collagen renatured in gelatin during drying, while the diffraction peak around 21.0° was assigned to the amorphous halo of protein (Benbettaïeb, Karbowiak, Brachais, & Debeaufort, 2016). FG-HBE films showed the same diffraction peaks as FG film, indicating HBE was well dispersed in film matrix. However, the diffraction peak intensities of FG-HBE films were remarkably higher than that of FG film, indicating that the incorporation of HBE into FG film produced more ordered structures through intermolecular hydrogen bonds. Similar enhancement in the diffraction peak intensities was found in chitosan films incorporated with polyphenol-rich eggplant 4
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Fig. 2. FT-IR spectra of HBE powder (a), and FG (b), FG-HBE I (c), FG-HBE II (d), FG-HBE III (e) and FG-HBE IV (f) films.
with the increase of HBE content. This indicated the color of FG-HBE films was mainly attributed to the presence of HBE. As listed in Table 2, FG-HBE films showed higher a and ΔE values than FG film (p < 0.05). However, the L and WI values of FG-HBE films were remarkably lower than those of FG film (p < 0.05). The change of these color indices suggested the tendency of FG-HBE films towards redness and darkness. Notably, the a and ΔE values of FG-HBE films significantly increased with the increase of HBE content (p < 0.05). However, the L and WI values of FG-HBE films remarkably decreased as HBE content increased (p < 0.05). These results indicated the color of FG-HBE films was greatly influenced by the content of HBE. Similar changes of color indices were reported in other polyphenol-rich films, such as chitosan-
extract (Yong, Wang, Zhang et al., 2019). However, Wang, Yong et al. (2019) observed the reduction in the crystallinity of chitosan films incorporated with polyphenol-rich black soybean extract, which was because the incorporated extract disrupted the ordered structures of polymeric matrix. These results suggested the crystallinity of polyphenol-rich films was closely related to the type of polyphenols added. 3.5. Colors of FG-HBE films It’s important for food packaging films to have a pleasant appearance. As shown in Fig. 4A, FG film without HBE addition was almost colorless. FG-HBE films showed the amaranth color, which deepened
Fig. 3. XRD patterns of HBE powder (a), and FG (b), FG-HBE I (c), FG-HBE II (d), FG-HBE III (e) and FG-HBE IV (f) films. 5
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Fig. 4. Physical appearances (A) and UV–vis light transmittance of FG and FG-HBE films. Table 2 Color values including L, a, b, ΔE and WI of FG and FG-HBE films. Films FG film FG-HBE FG-HBE FG-HBE FG-HBE
L
I film II film III film IV film
94.25 90.35 85.77 81.18 75.55
± ± ± ± ±
0.14a 0.95b 1.74c 1.31d 1.98e
a
b
−0.80 ± 0.02e 1.32 ± 0.46d 3.59 ± 0.89c 5.80 ± 0.63b 9.09 ± 1.08a
−1.68 −2.12 −1.48 −3.38 −4.47
± ± ± ± ±
0.22ab 0.11b 0.12a 0.84c 0.32d
ΔE
WI
1.47 ± 0.13e 3.43 ± 0.99d 8.49 ± 1.94c 13.58 ± 1.40b 20.18 ± 2.26a
93.95 90.92 85.24 80.00 73.53
± ± ± ± ±
0.12a 0.95b 1.89c 1.35d 2.23e
Values are given as mean ± SD (n = 3). Different letters in the same column indicate significantly different (p < 0.05).
polypheol-rich red cabbage extract. However, Peralta et al. (2019) found the thickness of pig skin gelatin film significantly increased when 25 wt% of polypheol-rich hibiscus extract was incorporated. Above results suggested the thickness of gelatin-based films was related to the type and the amount of polypheol-rich plant extract added.
purple sweet potato extract, chitosan-black soybean extract and FG-red cabbage extract films (Uranga, Etxabide, Guerrero, & de la Caba, 2018; Wang, Yong et al., 2019; Yong, Wang, Bai et al., 2019). 3.6. Thicknesses of FG-HBE films
3.7. Moisture contents of FG-HBE films
The thicknesses of FG and FG-HBE films are presented in Table 3. However, different films showed no significant difference in thickness (p > 0.05), which was because of low content of HBE in the film. Since the hydroxyl groups of polyphenols in HBE formed intermolecular hydrogen bonds with the amino/hydroxyl groups in FG, HBE was uniformly distributed in the space among FG film matrix. As a result, the thickness of FG film was not significantly affected by the incorporation of HBE. Musso, Salgado, and Mauri (2019) also reported that the thickness of bovine gelatin film was unaffected by the presence of
Moisture content represents the total water molecules occupied in the network of films. As shown in Table 3, the addition of HBE did not significantly change the moisture content of FG film (p < 0.05). On one hand, the presence of hydrophilic compounds in the HBE could increase the affinity of the films toward water molecules. On the other hand, the interactions between polyphenols and gelatin could decrease the availability of the hydrophilic groups in the films, thereby reducing
Table 3 Thicknesses, moisture contents and WVP of FG and FG-HBE films. Films FG film FG-HBE FG-HBE FG-HBE FG-HBE
I film II film III film IV film
Thickness (mm)
Moisture content (%)
WVP (× 10−11 g m−1 s−1 Pa−1)
0.052 0.051 0.052 0.052 0.052
13.33 13.80 13.80 13.49 13.47
8.33 7.14 6.99 6.27 5.96
± ± ± ± ±
0.004 0.002 0.004 0.003 0.001
± ± ± ± ±
0.23 0.10 0.38 0.31 0.16
± ± ± ± ±
0.66a 0.64b 0.33b 0.53bc 0.21c
Values are given as mean ± SD (n = 10 for film thickness, and n = 3 for moisture content and WVP). Different letters in the same column indicate significantly different (p < 0.05). 6
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the affinity of the films toward water molecules. Recently, Peralta et al. (2019) found the incorporation of polypheol-rich hibiscus extract remarkably enhanced the moisture content in pig skin gelatin film. However, the addition of hibiscus extract significantly reduced the moisture content in chitosan film. Musso, Salgado, and Mauri (2019) reported the moisture content of bovine gelatin film was significantly reduced by incorporating polypheol-rich red cabbage extract. Above results further suggested the moisture content of gelatin-based films was associated with the type of polymeric film matrix and polypheolrich plant extract. Notably, FG and FG-HBE films could be thoroughly dissolved in water in less than 10 min due to their hydrophilic property (data not shown). Thus, the water solubility of the films could not be determined. Although the films were water soluble, they could be used as the active packaging materials of water insoluble food products (e.g. edible oils).
Table 4 Mechanical properties including TS and EAB of FG and FG-HBE films. Films FG film FG-HBE FG-HBE FG-HBE FG-HBE
TS (MPa)
I film II film III film IV film
42.5 46.7 47.4 49.5 51.5
± ± ± ± ±
EAB (%) c
2.85 3.69b 4.24b 4.6ab 4.44a
2.96 2.87 3.10 3.49 3.69
± ± ± ± ±
0.38d 0.44d 0.37c 0.54b 0.44a
Values are given as mean ± SD (n = 6 for TS and EAB). Different letters in the same column indicate significantly different (p < 0.05).
2018). Rasid, Nazmi, Isa, and Sarbon (2018) also found the incorporation of polyphenol-rich C. asiatica extract increased the TS of gelatin films, which was caused by the interactions between the phenolic compounds in the extract and the amino acids of gelatin. On the other hand, the EAB of FG film was also improved by the incorporation of HBE. The slightly increased EAB in FG-HBE films was probably because polyphenols could act as plasticizers, which enhanced the flexibility of FG-HBE films (Bermúdez-Oria, Rodríguez-Gutiérrez, Vioque, Rubio-Senent, & Fernández-Bolaños, 2017). Similar enhancements in the EAB were observed when C. asiatica extract was incorporated into gelatin films, which was because the interactions between polyphenols and gelatin led to the formation of more cohesive and flexible film matrix (Rasid et al., 2018).
3.8. WVP of FG-HBE films WVP is an important barrier parameter for packaging films to protect food against water-induced spoilage. As shown in Table 3, the WVP of FG-HBE films was much lower than that of FG film (p < 0.05). In addition, the WVP of FG-HBE films gradually decrease with the increase of HBE content (p < 0.05). The decrease in the WVP of FG-HBE films was because the incorporation of HBE into FG film produced more compact network (Fig. 1). It has been reported that water vapor is more likely to diffuse through the amorphous regions of film matrix (Khan et al., 2012). Thus, the decrease in the WVP of FG-HBE films was also related to the decreased amorphous regions in these films. Our results were consistent with those of Rasid, Nazmi, Isa, and Sarbon (2018), who found the WVP of bovine gelatin film decreased by incorporating Centella asiatica urban extract due to reduced free volume in polymer matrix. However, Uranga et al. (2018) found the WVP of FG film was not affected by anthocyanins extracted from red cabbage. Recently, Zhai et al. (2018) reported that the WVP of pig skin gelatin-gellan gum film first decreased and then increased with increasing the content of polypheol-rich red radish extract. Above results suggested the WVP of gelatin-based films was related to the type and amount of polyphenolrich plant extract incorporated.
3.11. Antioxidant property of FG-HBE films Antioxidant property is vital for active packaging since antioxidant films can protect food from oxidation and degradation. In addition, the release of antioxidants from active packaging works better than direct addition of antioxidants into food (Uranga et al., 2018). The antioxidant property of FG and FG-HBE films were evaluated by DPPH radical scavenging assay. Since FG and FG-HBE films could not be dissolved in DPPH methanol solution, the films were left intact after the reaction. As shown in Fig. 5, FG film exhibited very low DPPH radical scavenging activity (p < 0.05). By contrast, the DPPH radical scavenging activity of FG film was significantly improved by incorporating HBE (p < 0.05). The improved DPPH radical scavenging activity in FG-HBE films was attributed to the potent antioxidant property of polyphenols in HBE (Rasid et al., 2018). In addition, the DPPH radical scavenging activity of FG-HBE films significantly increased with the increase of HBE content (p < 0.05). It should be noted that the antioxidant activity of FG-HBE films was closely related to the content of polyphenols related from the films (Liu, Liu et al., 2017). Our results suggested the films containing a higher content of HBE were easier to release polyphenols into the reaction solution. Moreover, polyphenol-rich FG-HBE films could be used as antioxidant packaging to prevent the packaged food from oxidative damages. Similar improvement in antioxidant property was observed in gelatin films containing polyphenol-rich C. asiatica extract (Rasid et al., 2018).
3.9. Light transmittance of FG-HBE films UV-vis light barrier property is a desired character of food packaging films since UV–vis light can cause oxidization, nutrient loss and off-flavor in food. UV–vis light transmittance of FG and FG-HBE films is shown in Fig. 4B. FG film showed good UV barrier property when the wavelength was smaller than 280 nm, which was attributed to the presence of UV-absorbing chromophore (e.g. aromatic amino acids and disulphide bonds) in FG (Peralta et al., 2019). Notably, the UV–vis light transmittance of FG film was greatly reduced by incorporating HBE, which was attributed the UV–vis absorption ability of the aromatic rings in polyphenols (Wang, Yong et al., 2019). Similar results were observed when polypheol-rich hibiscus extract was added into pig skin gelatin film (Peralta et al., 2019). The UV–vis light barrier property of FG-HBE films remarkably increased as increasing the content of HBE. Zhai et al. (2018) also reported the UV–vis light barrier property of pig skin gelatin-gellan gum films gradually increased with the increase of polypheol-rich red radish extract content.
3.12. pH-sensitive property of FG-HBE films In general, anthocyanin-rich plant extracts are pH-sensitive since anthocyanins can change their structures and colors in different pHs (Wang, Yong et al., 2019). In order to exam the pH-sensitive property of HBE, HBE powder was dissolved in different buffers (pH 3–12). As shown in Fig. 6A, the color of HBE solution changed from red-pink (pH 3–5) to chestnut (pH 6–8) to finally purple-purplish grey (pH 9–12). The color variation of HBE solution was attributed to the structural change of anthocyanins from flavylium cation to quinonoidal anhydrobase and anionic quinoidal base (Halász & Csóka, 2018). The UV–vis spectra of HBE in different buffers are illustrated in Fig. 6B. The UV–vis absorption peak of HBE solution appeared at 520 nm when the pH value was pH 3. With the increase of pH value, the intensity of UV–vis
3.10. Mechanical property of FG-HBE films TS and EAB are two important mechanical properties of food packaging films. As shown in Table 4, FG-HBE films showed higher TS than FG film (p < 0.05). In addition, the TS of FG-HBE films increased with the increase of HBE content (p < 0.05). This was because the intermolecular hydrogen bonds formed between HBE and FG made the films more compact and resistant to applied tensile stress (Zhai et al., 7
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Fig. 5. DPPH radical scavenging activity of FG and FG-HBE films. Each value represents mean ± SD of triplicates.
Fig. 6. Color changes (A) and UV–vis spectra (B) of HBE in different buffer solutions (pH 3–12).
Fig. 7. Color changes of FG-HBE films after being immersed in different buffer solutions (pH 3–12) for 5 min.
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Table 5 The change of TVBN levels in the shrimp and the color response of FG-HBE films at different storage stages.
Values are given as mean ± SD (n = 3). Different letters in the same column indicate significantly different (p < 0.05).
absorption peak first decreased and then gradually increased. Moreover, the UV–vis absorption peak shifted from 520 to 600 nm when pH value increased from 3 to 12. Similar phenomena were observed when other anthocyanin-rich plant extracts were solubilized in different buffers (Uranga et al., 2018; Wang, Yong et al., 2019; Yong, Wang, Zhang et al., 2019). The pH-sensitivity of FG films with and without HBE are presented in Fig. 7. Due to the presence of anthocyanins in HBE, FG-HBE films showed obvious pH-sensitive property. The color of FG-HBE films changed from amaranth to chestnut and greenish-blue with the increase of pH value, which was due to the structural transformation of anthocyanins at different pHs (Halász & Csóka, 2018). At the same pH levels, the color of FG-HBE films depended on the content of HBE. Our results suggested that FG-HBE films could be used as natural pH indicators. The pH-sensitivity was also found in many other anthocyanin-rich films, such as chitosan films incorporated with black soybean extract and bovine gelatin films added with red cabbage extract (Musso et al., 2019; Wang, Yong et al., 2019).
TVB-N level for edible shrimp is 20 mg/100 g. Thus, the shrimp was not fresh after storage at 25 °C for 24 h. Notably, only FG-HBE II film presented a remarkable color change from brown to green when the shrimp was not fresh at 24 h. This indicated FG-HBE II film was more appropriate to be used as the freshness indicator of shrimp. When FGHBE II film turned green, the shrimp was not edible. 4. Conclusion Antioxidant and pH-sensitive films were successfully developed by incorporating polyphenol-rich HBE into FG matrix. The incorporation of HBE remarkably enhanced the water vapor barrier property and TS of FG film, which was due to the compact inner structures of FG-HBE films. The increased UV–vis light barrier and antioxidant properties of FG-HBE films were attributed to the presence of polyphenols in HBE. The pH-sensitive property of FG-HBE films was ascribed to abundant anthocyanins in HBE. Notably, the physical and functional properties of FG-HBE films were closely related to the incorporation amount of HBE. In the future, FG-HBE films can be further used as active and intelligent films to extend the shelf life and monitor the freshness of food products.
3.13. Application of FG-HBE films for indicating shrimp spoilage
Acknowledgements
The spoilage of protein-rich animal foods is owing to microbial contamination. The microbial-induced spoilage of animal foods can produce several volatile amines (e.g. ammonia, dimethylamine and trimethylamine), thereby changing the pH status of foods (Liu et al., 2019; Qin et al., 2019). Since anthocyanins are pH-sensitive and can change their colors as a function of pH, anthocyanin-rich films can be used as intelligent packaging to indicate the freshness of protein-rich animal foods (e.g. pork, fish, milk and shrimp) (Kang et al., 2018; Liu et al., 2019; Zhai et al., 2018). In this study, pH-sensitive FG-HBE films were used to indicate the freshness of shrimp. As shown in Table 5, the TVB-N level of shrimp increased from 4.80 to 83.01 mg/100 g in 48 h, which was mainly due to ammonia, dimethylamine and trimethylamine produced in the decomposition of proteins by microbial and endogenous enzymes (Choi, Lee, Lacroix, & Han, 2017; Zhai et al., 2018). According to the Chinese Standard of GB 2740-1994, the limitation of
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Preparation and characterization of active and intelligent films based on fish gelatin and haskap berries (Lonicera caerulea L.) extract ⁎
Jing Liu, Huimin Yong, Yunpeng Liu, Yan Qin, Juan Kan , Jun Liu
T
⁎
College of Food Science and Engineering, Yangzhou University, Yangzhou 225127, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: Active packing Gelatin Haskap berries Intelligent films
Haskap berries (Lonicera caerulea L.) are rich in polyphenols and possess potential health-promoting effects. In this study, polyphenols were extracted from haskap berries and further incorporated into fish gelatin (FG) to develop active and intelligent packaging films. Results showed anthocyanins and phenolic acids were the main polyphenols in haskap berry extract (HBE). Scanning electron microscopy showed the cross-sections of FG-HBE films were smooth and uniform. Fourier transform infrared spectroscopy revealed intermolecular hydrogen bonds formed between the hydroxyl groups of polyphenols in HBE and the amino/hydroxyl groups in FG. The crystallinity of FG film was enhanced by the addition of HBE. Moreover, the incorporation of HBE significantly increased the total color difference value, water vapor and UV–vis light barrier properties, tensile strength, and antioxidant ability of FG film. Due to high anthocyanin content in HBE, FG-HBE films were pH-sensitive and exhibited visible color changes as a function of pH. Notably, FG-HBE film containing 1 wt% of HBE was more appropriated to be used as the freshness indicator of shrimp. Our results suggest FG-HBE films can be applied as novel as antioxidant and intelligent food packaging films in food industry.
1. Introduction Due to increasing demands to extend the shelf life of food and protect environment, the petroleum-based packaging materials can be partially substituted by biopolymer-based packaging materials (Gan & Chow, 2018). Proteins and polysaccharides are the main biopolymers employed to develop biodegradable packaging films (Hassan, Chatha, Hussain, Zia, & Akhtar, 2018). Gelatin is an animal protein obtained by the controlled hydrolysis of fibrous insoluble collagen, which is generated as waste during animal slaughtering and processing (GómezGuillén, Giménez, López-Caballero, & Montero, 2011). Gelatin is also one of the most commonly used proteins for food packaging film production due to its abundance, good film forming ability and barrier properties against UV light, oxygen and lipids (Etxabide, Uranga, Guerrero, & de la Caba, 2017). Up to now, gelatins from diverse sources including bovine, porcine and fish have been used to develop food packaging films (Suderman, Isa, & Sarbon, 2018). Nonetheless, the water vapor barrier property of gelatin films is poor due to the hydrophilic nature of gelatin and the plasticizer (e.g. glycerol) used (Tongnuanchan, Benjakul, & Prodpran, 2014). Moreover, the mammalian gelatins are rejected by some consumers due to either sociocultural-religious or health-related concerns (Lin, Regenstein, Lv, Lu, & Jiang, 2017). Therefore, gelatins from aquatic sources (e.g. cold-water ⁎
and warm-water fishes) are possible alternatives to mammalian gelatins (Karim & Bhat, 2009). In recent years, gelatin-based films have been used as the carriers of natural bioactive additives (e.g. antioxidants and antimicrobials) to achieve active packaging functions (Adilah, Jamilah, Noranizan, & Hanani, 2018; Shahbazi, 2017). Polyphenols are the most abundant secondary metabolites widespread throughout the plant kingdom (Liu, Pu, Liu, Kan, & Jin, 2017). Due to their potent antioxidant and antimicrobial activities, polyphenol-rich plant extracts including tea polyphenols, longan seed extract, mango peel extract and blood orange peel extract have been incorporated into gelatin-based films (Adilah et al., 2018; Jridi et al., 2019; Liu et al., 2015; Sai-Ut, Benjakul, & Rawdkuen, 2015). The interactions between bioactive additives and gelatin matrix can provide controlled release of active ingredients over the storage period. Moreover, the incorporation of polyphenol-rich plant extracts can greatly improve the light/water vapor barrier and mechanical properties of gelatin-based films (Jridi et al., 2019). Therefore, polyphenol-rich gelatin-based films can be applied to protect food products from oxidation and microbial spoilage (Lee, Yang, & Song, 2016; Wang, Xia et al., 2019). Haskap berry (Lonicera caerulea L.), also called blue honeysuckle, is a plant native to Siberia, China and Japan (Chaovanalikit, Thompson, & Wrolstad, 2004). The fruits of this plant (haskap berries) are oval and
Corresponding authors. E-mail addresses: [email protected] (J. Kan), [email protected] (J. Liu).
https://doi.org/10.1016/j.fpsl.2019.100417 Received 2 July 2019; Received in revised form 4 October 2019; Accepted 8 October 2019 2214-2894/ © 2019 Elsevier Ltd. All rights reserved.
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3 wt% of HBE were designated as FG, FG-HBE I, FG-HBE II, FG-HBE III and FG-HBE IV films, respectively. All the films were stored in a desiccator with 50% RH at 25 °C for at least 48 h before tests.
covered with dark blue to purple skin (Becker & Szakiel, 2018). Haskap berries are rich in polyphenols, especially anthocyanins and phenolic acids (Bell & Williams, 2018). The total phenol content of haskap berries assessed by Folin-Ciocalteu assay ranges from 140.5 to 1142.0 mg gallic acid equivalent per 100 g of fresh weight (Celli, Ghanem, & Brooks, 2014). A number of studies have demonstrated the potential health-promoting effects (e.g. antioxidant, anti-microbial, antiatherosclerotic, anticarcinogenic and anti-inflammatory effects) of polyphenol-rich haskap berry extract (HBE) (Bell & Williams, 2018; Jurikova et al., 2012). Considering the abundant polyphenolic content, HBE is a promising addictive for active packaging films. Meanwhile, anthocyanins in HBE can undergo structural changes and exhibit color variations under different pH conditions. Since the process of food spoilage is usually accompanied by pH changes, anthocyanin-rich films can be used as intelligent packaging (i.e. pH indicators) to monitor the freshness of food (Liu et al., 2019; Qin et al., 2019). Till now, the development of active and intelligent packaging by using polyphenol-rich HBE has not been reported. In this study, active and intelligent packaging films were developed by incorporating polyphenol-rich HBE into fish gelatin (FG) matrix for the first time. The structures and physical properties of the developed FG-HBE films were characterized. In addition, the functional properties (i.e. antioxidant and pH-sensitive properties) of FG-HBE films were also evaluated. Finally, FG-HBE films were applied to indicate the freshness of shrimp.
2.4. Structural characterization of FG-HBE films 2.4.1. Scanning electron microscopy (SEM) Film sample was first immersed in liquid nitrogen and cryofractured. Then, the fractured sample was mounted on an aluminum stub and sputter coated with gold. The cross-sectional morphology of film sample was observed by GeminiSEM 300 (Carl Zeiss, Oberkochen, Germany) at 400× of magnification. 2.4.2. Fourier transform infrared (FT-IR) spectroscopy FT-IR spectrum was recorded in the range of 400–4000 cm−1 by Varian 670 spectrometer (Agilent Technologies, CA, USA) equipped with attenuated total reflectance accessory. 2.4.3. X-ray diffraction (XRD) The crystalline property of film sample was evaluated by D8 Advance X-ray diffractometer (Bruker AXS GmbH, Karlsruhe, Germany) containing Ni-filtered Cu Kα radiation at 40 mA and 40 kV. The XRD pattern of film sample was recorded from 5° to 80° at the scanning rate of 0.1°/s. 2.5. Determination of the physical properties of FG-HBE films
2. Materials and methods 2.5.1. Color The color parameters (L, a and b) of film were recorded by SC-80C colorimeter (Kangguang Instrument Co., Ltd., Bejing, China) with a D65 illuminant. The total color difference (ΔE) and whitish index (WI) of film were calculated as follows (Liu, Meng, Liu, Kan, & Jin, 2017):
2.1. Materials and chemical reagents Haskap berries (L. caerulea) were purchased from the market of Yichun city (Heilongjiang, China). Gelatin from cold water fish skin (average molecular weight of 60 kDa and isoelectric point of 6) and 2,2diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma Chemical Co. (MO, USA). All other reagents were of analytical grade.
ΔE =
(L*−L)2 + (a*−a)2 + (b*−b)2
WI = 100 −
2.2. Extraction and characterization of polyphenols from haskap berries
(100 − L)2 + a2 + b2
(1) (2)
where L* (92.60), a* (−0.92) and b* (−2.24) were the color parameters of white plate.
The extraction of polyphenols from haskap berries was performed according to the method of Yong, Wang, Bai et al. (2019) with some modifications. In brief, the fruits of haskap berries (100 g) were extracted in 400 mL of 80% (v/v) ethanol solution containing 1% (v/v) of HCl at 4 °C for 24 h. The extraction process was repeated for three times. All the extract solutions were combined and centrifuged at 8000 × g for 20 min to remove debris. The obtained supernatant was concentrated at 35 °C by a rotary evaporator to remove ethanol and then vacuum dried to obtain polyphenol-rich HBE powder. The total phenol content in HBE was determined by Folin-Ciocalteu assay (Liu, Jia, Kan, & Jin, 2013). The anthocyanin content in HBE was determined by pHdifferential assay (Yong, Wang, Zhang et al., 2019). Finally, the composition of polyphenols in HBE was characterized by Agilent 1200 HPLC system (Agilent Technologies, CA, USA) equipped with 6460 triple quadrupole mass spectrometer according to the method of Wang, Yong et al. (2019).
2.5.2. Thickness The thickness of film was measured at ten randomly points on each film by Mitutoyo No. 293-766 digital micrometer (Tester Sangyo Co., Ltd., Tokyo, Japan) with precision of 0.001 mm. 2.5.3. Moisture content The moisture content was determined by drying the equilibrated film piece (3 cm × 3 cm) at 110 °C to a constant weight (Zhai et al., 2018):
Moisture content(%) =
(Mi − Mt ) × 100 Mi
(3)
where Mi and Mt were the initial and final weights of film piece, respectively. 2.5.4. Water vapor permeability (WVP) WVP of film was determined by the method of Yong, Wang, Zhang et al. (2019). Briefly, film piece (6 cm × 6 cm) was sealed on the test cup containing 40 g anhydrous silica gels. The cup was kept at 20 °C in a desiccator containing distilled water (100% RH). The weight of film sealed cup was tested every 24 h for 7 days.
2.3. Development of FG-HBE films FG-HBE films were developed according to the method of Uranga, Etxabide, Guerrero, and de la Caba (2018) with some modifications. Breifly, 3.5 g FG was dissolved in 100 mL of distilled water at 70 °C under magnetic stirring for 30 min. Then, 30 wt% of glycerol (1.05 g) and different contents of HBE (0, 0.5, 1, 2 and 3 wt%) on FG basis were added into FG solution. After stirring at 40 °C for 10 min, the filmforming solutions were degassed and then cast onto Plexiglas plates (24 cm × 24 cm). The plates were dried at 30 °C with 50% relative humidity (RH) for 48 h. The obtained films containing 0, 0.5, 1, 2 and
WVP =
W×x t × A × ΔP
(4)
where W was the increased weight of film sealed cup (g), x was film thickness (m), t was time (s), A was permeation area (m2), and ΔP was saturated water vapor pressure at 20 °C. 2
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Table 1 The composition of polyphenols in HBE by HPLC-MS analysis. Retention time (min)
Polyphenols
[M]+ (m/z)
Relative content (%)
4.45 5.98 6.19 7.90 8.62 11.50 11.87 12.48 14.48
Caffeic acid Cyanidin-3-O-glucoside Malvidin-3-O-(6-acetyl-glucoside) Kaempferol-3-O-glucoside Cyanidin-3-O-(6-malonyl-glucoside) Pelargonidin-3-O-(6-acetyl-glucoside) Peonidin-3-O-(6-acetyl-rutinoside) Ferulic acid Chlorogenic acid
181 449 531 448 535 475 650 195 355
1.48 41.85 2.62 15.79 6.77 0.72 7.69 6.19 16.89
2.8. Statistical analysis
2.5.5. Light transmittance The light transmittance of film was measured by Lambda 35 UV–vis spectrophotometer (PerkinElmer Ltd., MA, USA) at wavelength range from 200 to 800 nm.
Duncan test and one-way analysis of variance were carried out by SPSS 13.0 software package (SPSS Inc., IL, USA). Difference was considered as statistically significant if p < 0.05.
2.5.6. Mechanical property The tensile strength (TS) and elongation-at-break (EAB) of film sample (6 cm × 1 cm) were determined by using TMS-Pro texture analyzer (Food Technology Co., VA, USA) with the crosshead speed of 6 cm/min (Yong, Wang, Zhang et al., 2019).
3. Results and discussion 3.1. Characterization of polyphenols in HBE Polyphenols were extracted from haskap berries in acidic ethanol solution, which was followed by centrifugation, concentration and vacuum drying. The extraction yield of HBE was 5.28 wt% based on the fresh weight of haskap berries. The total phenol and anthocyanin contents in HBE were 435.19 g gallic acid equivalent/100 g extract and 264.46 g cyanidin-3-glucoside equivalent/100 g extract, respectively. The polyphenolic compositions in HBE were further characterized by HPLC-MS. As summarized in Table 1, nine different polyphenols were identified based on their mass spectra. Notably, anthocyanins and phenolic acids were the main polyphenols in HBE. Moreover, cyanidin3-glucoside is the principal anthocyanin in HBE. Celli, Ghanem, and Brooks (2015) extracted anthocyanins from haskap berries by using a ultrasound-assisted method, and found the anthocyanins in the extract were cyanidin-3,5-O-diglucoside (1.25 mg/g dry weight (DW)), cyanidin-3-O-glucoside (16.75 mg/g DW), cyanidin-3-O-rutinoside (0.65 mg/g DW), pelargonidin-3-O-glucoside (0.29 mg/g DW) and peonidin-3-O-glucoside (0.58 mg/g DW). Liu, Wu, Guo, Meng, and Chang (2018) reported the main active components in haskap berries were cyanidin-3-O-glucoside, (+)-catechin and chlorogenic acid, which accounted for 43.7%, 26.3% and 11.6% of total polyphenols, respectively. Wang et al. (2016) suggested the total phenol and anthocyanin contents were closely related to the variety of haskap berries. In addition, cyanidin-3-O-glucoside was the most prominent anthocyanin in all the tested varieties.
2.6. Determination of the functional properties of FG-HBE films 2.6.1. Antioxidant property The antioxidant property of film was evaluated by DPPH radical scavenging assay (Liu, Meng et al., 2017). Briefly, film sample (4–20 mg) was reacted with 4 mL of 100 μM DPPH methanol solution at 20 °C for 1 h in the dark. The absorbance of reaction solution was measured at 517 nm.
DPPH radical scavenging activity(%) =
A0 − A1 × 100 A0
(5)
where A0 and A1 were the absorbance of the blank and reaction solutions, respectively. 2.6.2. pH-sensitive property The pH-sensitive property of HBE was determined by dissolving HBE powder (2 mg) in 20 mL of different buffer solutions (pH 3–12). Then, the UV–vis spectra of HBE solutions were recorded by Lambda 35 UV–vis spectrophotometer (PerkinElmer Ltd., MA, USA) at wavelength range of 400–700 nm (Luchese, Garrido, Spada, Tessaro, & de la Caba, 2018). To determine the pH-sensitive property of FG-HBE films, film samples were immersed in different buffer solutions (pH 3–12) at room temperature for 5 min. The color changes of film were recorded by SC80C colorimeter (Kangguang Instrument Co., Ltd., Beijing, China).
3.2. Microstructure of FG-HBE films The cross-sectional microstructures of FG and FG-HBE films were investigated by SEM. As shown in Fig. 1, the cross-sections of FG and FG-HBE films were smooth and uniform, indicating the filmogenic components (e.g. FG, HBE and glycerol) were homogeneously mixed and well compatible with each other (Wang, Yong et al., 2019). It was hypothesized that the polyphenols in HBE could interact with FG through hydrogen bonds, yielding a compact and dense network (Zhang et al., 2010). The dense inner structures of FG-HBE films were benefit to improve their mechanical and barrier properties. Similar phenomena were observed in pig skin gelatin films containing polypheol-rich hibiscus extract that showed continuous structure and uniform film matrix (Peralta, Bitencourt-Cervi, Maciel, Yoshida, & Carvalho, 2019). However, Zhai et al. (2018) found the cross-sections of pig skin gelatingellan gum films became rough and heterogeneous when polypheolrich red radish extract was incorporated. This indicated the
2.7. Application of FG-HBE films for indicating shrimp spoilage FG-HBE films were used to indicate the freshness of shrimp according to the methods of Kang et al. (2018). First, 25 ± 2 g of fresh shrimp sample was placed in the Petri dish (11 cm in diameter). Then, FG-HBE film sample (2 cm × 2 cm) was stuck on the inside surface of polyethylene film which sealed the Petri dish. The shrimp was then stored at 25 °C for 48 h. During the whole process, the active film was not in contact with shrimp sample. The color change of film sample and the total volatile basic nitrogen (TVB-N) level of shrimp were recorded every 8 h. Briefly, 10 g of shrimp sample was homogenized in 100 mL of deionized water. Then, the TVB-N value of the homogenate was determined by a K9860 Kjeldahl distillation apparatus (Jinan Hanon Instruments Co., Ltd., Jinan, China). 3
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Fig. 1. The cross-sectional morphology of FG (A), FG-HBE I (B), FG-HBE II (C), FG-HBE III (D) and FG-HBE IV (E) films.
hydrogen bonds could result in dense network in FG-HBE films (Fig. 1). Similar changes were found in the FT-IR spectra of FG films incorporated with carvacrol and green tea extract (Neira, Martucci, Stejskal, & Ruseckaite, 2019; Wu et al., 2018).
microstructures of gelatin-based films were greatly influenced by the type of polypheol-rich plant extract added.
3.3. FT-IR spectra of FG-HBE films 3.4. XRD patterns of FG-HBE films
The intermolecular interactions between FG and HBE were revealed by FT-IR spectroscopy. As presented in Fig. 2, HBE exhibited the characteristic bands of polyphenols at 3349 cm−1(OHe stretching), 2962 cm−1(CHe stretching), 1638 cm−1 (C]O stretching), 1602 cm−1 (C]C stretching of aromatic ring) and 1018 cm−1(CHe deformation of aromatic ring) (Yong, Wang, Bai et al., 2019). FG film displayed several characteristic bands at 3284 cm−1(amide-A, NeH and OHe stretching coupled with hydrogen bonds), 2934 cm−1(amide-B, CHe stretching), 1634 cm−1 (amide I, C]O stretching), 1534 cm−1 (amide II, NHe bending and CeN stretching) and 1239 cm−1 (amide III, NHe and CeN in-plane bending of amide bond or eCH2 groups of glycine) (Theerawitayaart, Prodpran, Benjakul, & Sookchoo, 2019). When HBE was incorporated into FG film, the band at 3284 cm−1 was broadened and intensified. This was caused by the intermolecular hydrogen bonds formed between the hydroxyl groups of polyphenols in HBE and the amino/hydroxyl groups in FG. The newly formed intermolecular
As shown in Fig. 3, the XRD pattern of HBE exhibited a broad peak around 24.1°, demonstrating HBE was in the amorphous state. The XRD pattern of FG film exhibited two broad diffraction peaks at 8.8° and 21.0°, respectively. The diffraction peak around 8.8° was attributed to the triple-helical crystalline structure of collagen renatured in gelatin during drying, while the diffraction peak around 21.0° was assigned to the amorphous halo of protein (Benbettaïeb, Karbowiak, Brachais, & Debeaufort, 2016). FG-HBE films showed the same diffraction peaks as FG film, indicating HBE was well dispersed in film matrix. However, the diffraction peak intensities of FG-HBE films were remarkably higher than that of FG film, indicating that the incorporation of HBE into FG film produced more ordered structures through intermolecular hydrogen bonds. Similar enhancement in the diffraction peak intensities was found in chitosan films incorporated with polyphenol-rich eggplant 4
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Fig. 2. FT-IR spectra of HBE powder (a), and FG (b), FG-HBE I (c), FG-HBE II (d), FG-HBE III (e) and FG-HBE IV (f) films.
with the increase of HBE content. This indicated the color of FG-HBE films was mainly attributed to the presence of HBE. As listed in Table 2, FG-HBE films showed higher a and ΔE values than FG film (p < 0.05). However, the L and WI values of FG-HBE films were remarkably lower than those of FG film (p < 0.05). The change of these color indices suggested the tendency of FG-HBE films towards redness and darkness. Notably, the a and ΔE values of FG-HBE films significantly increased with the increase of HBE content (p < 0.05). However, the L and WI values of FG-HBE films remarkably decreased as HBE content increased (p < 0.05). These results indicated the color of FG-HBE films was greatly influenced by the content of HBE. Similar changes of color indices were reported in other polyphenol-rich films, such as chitosan-
extract (Yong, Wang, Zhang et al., 2019). However, Wang, Yong et al. (2019) observed the reduction in the crystallinity of chitosan films incorporated with polyphenol-rich black soybean extract, which was because the incorporated extract disrupted the ordered structures of polymeric matrix. These results suggested the crystallinity of polyphenol-rich films was closely related to the type of polyphenols added. 3.5. Colors of FG-HBE films It’s important for food packaging films to have a pleasant appearance. As shown in Fig. 4A, FG film without HBE addition was almost colorless. FG-HBE films showed the amaranth color, which deepened
Fig. 3. XRD patterns of HBE powder (a), and FG (b), FG-HBE I (c), FG-HBE II (d), FG-HBE III (e) and FG-HBE IV (f) films. 5
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Fig. 4. Physical appearances (A) and UV–vis light transmittance of FG and FG-HBE films. Table 2 Color values including L, a, b, ΔE and WI of FG and FG-HBE films. Films FG film FG-HBE FG-HBE FG-HBE FG-HBE
L
I film II film III film IV film
94.25 90.35 85.77 81.18 75.55
± ± ± ± ±
0.14a 0.95b 1.74c 1.31d 1.98e
a
b
−0.80 ± 0.02e 1.32 ± 0.46d 3.59 ± 0.89c 5.80 ± 0.63b 9.09 ± 1.08a
−1.68 −2.12 −1.48 −3.38 −4.47
± ± ± ± ±
0.22ab 0.11b 0.12a 0.84c 0.32d
ΔE
WI
1.47 ± 0.13e 3.43 ± 0.99d 8.49 ± 1.94c 13.58 ± 1.40b 20.18 ± 2.26a
93.95 90.92 85.24 80.00 73.53
± ± ± ± ±
0.12a 0.95b 1.89c 1.35d 2.23e
Values are given as mean ± SD (n = 3). Different letters in the same column indicate significantly different (p < 0.05).
polypheol-rich red cabbage extract. However, Peralta et al. (2019) found the thickness of pig skin gelatin film significantly increased when 25 wt% of polypheol-rich hibiscus extract was incorporated. Above results suggested the thickness of gelatin-based films was related to the type and the amount of polypheol-rich plant extract added.
purple sweet potato extract, chitosan-black soybean extract and FG-red cabbage extract films (Uranga, Etxabide, Guerrero, & de la Caba, 2018; Wang, Yong et al., 2019; Yong, Wang, Bai et al., 2019). 3.6. Thicknesses of FG-HBE films
3.7. Moisture contents of FG-HBE films
The thicknesses of FG and FG-HBE films are presented in Table 3. However, different films showed no significant difference in thickness (p > 0.05), which was because of low content of HBE in the film. Since the hydroxyl groups of polyphenols in HBE formed intermolecular hydrogen bonds with the amino/hydroxyl groups in FG, HBE was uniformly distributed in the space among FG film matrix. As a result, the thickness of FG film was not significantly affected by the incorporation of HBE. Musso, Salgado, and Mauri (2019) also reported that the thickness of bovine gelatin film was unaffected by the presence of
Moisture content represents the total water molecules occupied in the network of films. As shown in Table 3, the addition of HBE did not significantly change the moisture content of FG film (p < 0.05). On one hand, the presence of hydrophilic compounds in the HBE could increase the affinity of the films toward water molecules. On the other hand, the interactions between polyphenols and gelatin could decrease the availability of the hydrophilic groups in the films, thereby reducing
Table 3 Thicknesses, moisture contents and WVP of FG and FG-HBE films. Films FG film FG-HBE FG-HBE FG-HBE FG-HBE
I film II film III film IV film
Thickness (mm)
Moisture content (%)
WVP (× 10−11 g m−1 s−1 Pa−1)
0.052 0.051 0.052 0.052 0.052
13.33 13.80 13.80 13.49 13.47
8.33 7.14 6.99 6.27 5.96
± ± ± ± ±
0.004 0.002 0.004 0.003 0.001
± ± ± ± ±
0.23 0.10 0.38 0.31 0.16
± ± ± ± ±
0.66a 0.64b 0.33b 0.53bc 0.21c
Values are given as mean ± SD (n = 10 for film thickness, and n = 3 for moisture content and WVP). Different letters in the same column indicate significantly different (p < 0.05). 6
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the affinity of the films toward water molecules. Recently, Peralta et al. (2019) found the incorporation of polypheol-rich hibiscus extract remarkably enhanced the moisture content in pig skin gelatin film. However, the addition of hibiscus extract significantly reduced the moisture content in chitosan film. Musso, Salgado, and Mauri (2019) reported the moisture content of bovine gelatin film was significantly reduced by incorporating polypheol-rich red cabbage extract. Above results further suggested the moisture content of gelatin-based films was associated with the type of polymeric film matrix and polypheolrich plant extract. Notably, FG and FG-HBE films could be thoroughly dissolved in water in less than 10 min due to their hydrophilic property (data not shown). Thus, the water solubility of the films could not be determined. Although the films were water soluble, they could be used as the active packaging materials of water insoluble food products (e.g. edible oils).
Table 4 Mechanical properties including TS and EAB of FG and FG-HBE films. Films FG film FG-HBE FG-HBE FG-HBE FG-HBE
TS (MPa)
I film II film III film IV film
42.5 46.7 47.4 49.5 51.5
± ± ± ± ±
EAB (%) c
2.85 3.69b 4.24b 4.6ab 4.44a
2.96 2.87 3.10 3.49 3.69
± ± ± ± ±
0.38d 0.44d 0.37c 0.54b 0.44a
Values are given as mean ± SD (n = 6 for TS and EAB). Different letters in the same column indicate significantly different (p < 0.05).
2018). Rasid, Nazmi, Isa, and Sarbon (2018) also found the incorporation of polyphenol-rich C. asiatica extract increased the TS of gelatin films, which was caused by the interactions between the phenolic compounds in the extract and the amino acids of gelatin. On the other hand, the EAB of FG film was also improved by the incorporation of HBE. The slightly increased EAB in FG-HBE films was probably because polyphenols could act as plasticizers, which enhanced the flexibility of FG-HBE films (Bermúdez-Oria, Rodríguez-Gutiérrez, Vioque, Rubio-Senent, & Fernández-Bolaños, 2017). Similar enhancements in the EAB were observed when C. asiatica extract was incorporated into gelatin films, which was because the interactions between polyphenols and gelatin led to the formation of more cohesive and flexible film matrix (Rasid et al., 2018).
3.8. WVP of FG-HBE films WVP is an important barrier parameter for packaging films to protect food against water-induced spoilage. As shown in Table 3, the WVP of FG-HBE films was much lower than that of FG film (p < 0.05). In addition, the WVP of FG-HBE films gradually decrease with the increase of HBE content (p < 0.05). The decrease in the WVP of FG-HBE films was because the incorporation of HBE into FG film produced more compact network (Fig. 1). It has been reported that water vapor is more likely to diffuse through the amorphous regions of film matrix (Khan et al., 2012). Thus, the decrease in the WVP of FG-HBE films was also related to the decreased amorphous regions in these films. Our results were consistent with those of Rasid, Nazmi, Isa, and Sarbon (2018), who found the WVP of bovine gelatin film decreased by incorporating Centella asiatica urban extract due to reduced free volume in polymer matrix. However, Uranga et al. (2018) found the WVP of FG film was not affected by anthocyanins extracted from red cabbage. Recently, Zhai et al. (2018) reported that the WVP of pig skin gelatin-gellan gum film first decreased and then increased with increasing the content of polypheol-rich red radish extract. Above results suggested the WVP of gelatin-based films was related to the type and amount of polyphenolrich plant extract incorporated.
3.11. Antioxidant property of FG-HBE films Antioxidant property is vital for active packaging since antioxidant films can protect food from oxidation and degradation. In addition, the release of antioxidants from active packaging works better than direct addition of antioxidants into food (Uranga et al., 2018). The antioxidant property of FG and FG-HBE films were evaluated by DPPH radical scavenging assay. Since FG and FG-HBE films could not be dissolved in DPPH methanol solution, the films were left intact after the reaction. As shown in Fig. 5, FG film exhibited very low DPPH radical scavenging activity (p < 0.05). By contrast, the DPPH radical scavenging activity of FG film was significantly improved by incorporating HBE (p < 0.05). The improved DPPH radical scavenging activity in FG-HBE films was attributed to the potent antioxidant property of polyphenols in HBE (Rasid et al., 2018). In addition, the DPPH radical scavenging activity of FG-HBE films significantly increased with the increase of HBE content (p < 0.05). It should be noted that the antioxidant activity of FG-HBE films was closely related to the content of polyphenols related from the films (Liu, Liu et al., 2017). Our results suggested the films containing a higher content of HBE were easier to release polyphenols into the reaction solution. Moreover, polyphenol-rich FG-HBE films could be used as antioxidant packaging to prevent the packaged food from oxidative damages. Similar improvement in antioxidant property was observed in gelatin films containing polyphenol-rich C. asiatica extract (Rasid et al., 2018).
3.9. Light transmittance of FG-HBE films UV-vis light barrier property is a desired character of food packaging films since UV–vis light can cause oxidization, nutrient loss and off-flavor in food. UV–vis light transmittance of FG and FG-HBE films is shown in Fig. 4B. FG film showed good UV barrier property when the wavelength was smaller than 280 nm, which was attributed to the presence of UV-absorbing chromophore (e.g. aromatic amino acids and disulphide bonds) in FG (Peralta et al., 2019). Notably, the UV–vis light transmittance of FG film was greatly reduced by incorporating HBE, which was attributed the UV–vis absorption ability of the aromatic rings in polyphenols (Wang, Yong et al., 2019). Similar results were observed when polypheol-rich hibiscus extract was added into pig skin gelatin film (Peralta et al., 2019). The UV–vis light barrier property of FG-HBE films remarkably increased as increasing the content of HBE. Zhai et al. (2018) also reported the UV–vis light barrier property of pig skin gelatin-gellan gum films gradually increased with the increase of polypheol-rich red radish extract content.
3.12. pH-sensitive property of FG-HBE films In general, anthocyanin-rich plant extracts are pH-sensitive since anthocyanins can change their structures and colors in different pHs (Wang, Yong et al., 2019). In order to exam the pH-sensitive property of HBE, HBE powder was dissolved in different buffers (pH 3–12). As shown in Fig. 6A, the color of HBE solution changed from red-pink (pH 3–5) to chestnut (pH 6–8) to finally purple-purplish grey (pH 9–12). The color variation of HBE solution was attributed to the structural change of anthocyanins from flavylium cation to quinonoidal anhydrobase and anionic quinoidal base (Halász & Csóka, 2018). The UV–vis spectra of HBE in different buffers are illustrated in Fig. 6B. The UV–vis absorption peak of HBE solution appeared at 520 nm when the pH value was pH 3. With the increase of pH value, the intensity of UV–vis
3.10. Mechanical property of FG-HBE films TS and EAB are two important mechanical properties of food packaging films. As shown in Table 4, FG-HBE films showed higher TS than FG film (p < 0.05). In addition, the TS of FG-HBE films increased with the increase of HBE content (p < 0.05). This was because the intermolecular hydrogen bonds formed between HBE and FG made the films more compact and resistant to applied tensile stress (Zhai et al., 7
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Fig. 5. DPPH radical scavenging activity of FG and FG-HBE films. Each value represents mean ± SD of triplicates.
Fig. 6. Color changes (A) and UV–vis spectra (B) of HBE in different buffer solutions (pH 3–12).
Fig. 7. Color changes of FG-HBE films after being immersed in different buffer solutions (pH 3–12) for 5 min.
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Table 5 The change of TVBN levels in the shrimp and the color response of FG-HBE films at different storage stages.
Values are given as mean ± SD (n = 3). Different letters in the same column indicate significantly different (p < 0.05).
absorption peak first decreased and then gradually increased. Moreover, the UV–vis absorption peak shifted from 520 to 600 nm when pH value increased from 3 to 12. Similar phenomena were observed when other anthocyanin-rich plant extracts were solubilized in different buffers (Uranga et al., 2018; Wang, Yong et al., 2019; Yong, Wang, Zhang et al., 2019). The pH-sensitivity of FG films with and without HBE are presented in Fig. 7. Due to the presence of anthocyanins in HBE, FG-HBE films showed obvious pH-sensitive property. The color of FG-HBE films changed from amaranth to chestnut and greenish-blue with the increase of pH value, which was due to the structural transformation of anthocyanins at different pHs (Halász & Csóka, 2018). At the same pH levels, the color of FG-HBE films depended on the content of HBE. Our results suggested that FG-HBE films could be used as natural pH indicators. The pH-sensitivity was also found in many other anthocyanin-rich films, such as chitosan films incorporated with black soybean extract and bovine gelatin films added with red cabbage extract (Musso et al., 2019; Wang, Yong et al., 2019).
TVB-N level for edible shrimp is 20 mg/100 g. Thus, the shrimp was not fresh after storage at 25 °C for 24 h. Notably, only FG-HBE II film presented a remarkable color change from brown to green when the shrimp was not fresh at 24 h. This indicated FG-HBE II film was more appropriate to be used as the freshness indicator of shrimp. When FGHBE II film turned green, the shrimp was not edible. 4. Conclusion Antioxidant and pH-sensitive films were successfully developed by incorporating polyphenol-rich HBE into FG matrix. The incorporation of HBE remarkably enhanced the water vapor barrier property and TS of FG film, which was due to the compact inner structures of FG-HBE films. The increased UV–vis light barrier and antioxidant properties of FG-HBE films were attributed to the presence of polyphenols in HBE. The pH-sensitive property of FG-HBE films was ascribed to abundant anthocyanins in HBE. Notably, the physical and functional properties of FG-HBE films were closely related to the incorporation amount of HBE. In the future, FG-HBE films can be further used as active and intelligent films to extend the shelf life and monitor the freshness of food products.
3.13. Application of FG-HBE films for indicating shrimp spoilage
Acknowledgements
The spoilage of protein-rich animal foods is owing to microbial contamination. The microbial-induced spoilage of animal foods can produce several volatile amines (e.g. ammonia, dimethylamine and trimethylamine), thereby changing the pH status of foods (Liu et al., 2019; Qin et al., 2019). Since anthocyanins are pH-sensitive and can change their colors as a function of pH, anthocyanin-rich films can be used as intelligent packaging to indicate the freshness of protein-rich animal foods (e.g. pork, fish, milk and shrimp) (Kang et al., 2018; Liu et al., 2019; Zhai et al., 2018). In this study, pH-sensitive FG-HBE films were used to indicate the freshness of shrimp. As shown in Table 5, the TVB-N level of shrimp increased from 4.80 to 83.01 mg/100 g in 48 h, which was mainly due to ammonia, dimethylamine and trimethylamine produced in the decomposition of proteins by microbial and endogenous enzymes (Choi, Lee, Lacroix, & Han, 2017; Zhai et al., 2018). According to the Chinese Standard of GB 2740-1994, the limitation of
This work was supported by Grants-in-Aid for scientific research from the National Natural Science Foundation of China (No. 31571788), Natural Science Foundation of Jiangsu Province (No. BK20151310), and Qing Lan Project of Jiangsu Province. References Adilah, A. N., Jamilah, B., Noranizan, M. A., & Hanani, Z. N. (2018). Utilization of mango peel extracts on the biodegradable films for active packaging. Food Packaging and Shelf Life, 16, 1–7. Becker, R., & Szakiel, A. (2018). Phytochemical characteristics and potential therapeutic properties of blue honeysuckle Lonicera caerulea L. (Caprifoliaceae). Journal of Herbal Medicine. https://doi.org/10.1016/j.hermed.2018.10.002. Bell, L., & Williams, C. M. (2018). A pilot dose–response study of the acute effects of
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