Preparation of tuna skin collagen-chitosan composite film improved by sweep frequency pulsed ultrasound technology.

To produce a natural food packaging film from tuna skin collagen (TSC) and chitosan (CTS) and improve its mechanical and physicochemical properties, the sweep frequency pulsed ultrasound (SFPU) was introduced as a new technology and compared with the conventional method. The optimum preparation conditions of the SFPU-TSC-CTS film were sweep frequency of 28 ± 0.5 kHz, power density of 100 W/L, sweep frequency cycle of 100 ms, pulse duty ratio of 77%, and ultrasonic time of 10 min. Significant increases in the tensile strength (27.14%) and elongation at break (16.54%) and a significant decrease in the water vapor permeability (12.15%) were observed by sonication. Thus, a moderate SFPU treatment can significantly improve the moisture resistance and mechanical properties of the film. These enhancements were achieved by a more ordered and compact structure, a good crystallinity and a higher thermostability of SFPU-TSC-CTS film, which were verified by the Fourier transform infrared spectroscopy (FTIR), circular dichroism (CD), scanning electron microscopy (SEM), X-ray diffraction (XRD), and thermal stability indexes. Moreover, SFPU-TSC-CTS film also presented good antioxidant and antibacterial activities. Therefore, SFPU was an effective auxiliary technology for improving the quality of food packaging film and can be deeply explored.

Introduction

In recent years, edible film with biodegradable, non-toxic and eco-friendly characteristics is becoming an important branch of food packaging [1]. These films generally use food-derived substances (e.g. protein or polysaccharide) as the matrix. Through various intramolecular and intermolecular interactions, the food-derived matrix and the catalyzer can form a thin film, which can insulate water, oxygen and other environmental hazards [2]. The attention to developing biodegradable materials made from natural sources in the food packaging industry for replacing traditional plastic materials is increasing [3].

In this field, collagen and chitosan are the most common natural biopolymer for making degradable biofilms [4], [5]. Collagen possesses good film-forming properties due to its low antigenicity and good biocompatibility. The collagen-based film has good rigidity and strength, but the very limited application was reported due to its poor toughness and hydrophobicity. The chitosan-based film with good film-forming capacities, antioxidant and antimicrobial properties has broad prospects of fresh-keeping packaging, but it has a low elongation at break. Although films made from single material are of simple process technology, such films' mechanical properties and thermal stability are always disappointing [6]. Grafting collagen to chitosan as a composite film is expected to compensate for the disadvantages of each component and achieve the purpose of practical application [7]. Tuna skin is a by-product during tuna processing which is rich in high-value collagen [8]. It can be a potential raw material for film formation, but it is often discarded without good use. Therefore, our study is expected to make full use of it to prepare composite film in order to improve the added value of fish skin resources.

The development of ultrasound technology is widely used to promote the interactions between protein and polysaccharide molecules. It has achieved good results in the functional improvement of composite products [9], [10]. However, studies on ultrasound-promoted protein-polysaccharide grafting reactions generally focused on the single-frequency ultrasound; in addition, there were few reports on advanced ultrasound modes such as pulsed ultrasound and sweep frequency ultrasound. Pulsed ultrasound refers to alternating ultrasonic working time and intermittent time during ultrasonic treatment, which reduces the temperature rise of the reaction system compared with continuous ultrasound. Furthermore, pulsed ultrasound is more energy efficient. Under the pulsed mode, molecular homogenization and cell relaxation are facilitated, and hence the cross-linking reactions can be enhanced [11]. Sweep frequency ultrasound refers to the fluctuation of the ultrasonic frequency of a fixed range. Unlike fixed frequency ultrasound, sweep frequency ultrasound can match the natural frequency of the treated material, reaching a resonant frequency. This resonant frequency can promote the cross-linking of biopolymers [11]. It has been reported that sweep frequency pulsed ultrasound can indeed alter the structure and functional characteristics of macromolecules, for example, the intermolecular/intramolecular hydrogen bond binding, secondary structure, tertiary structure, microstructures and thermal stability of the protein were changed by sweep frequency pulsed ultrasound during the walnut protein extraction process [12]. The gelling properties of myofibrillar protein were increased by sweep frequency ultrasound treatment compared to the fixed frequency ultrasound [13]. Therefore, the applying of sweep frequency pulsed ultrasound technique in the preparation of composite film can make full use of the synergistic effect of sweep frequency and pulsed ultrasound. This would promote the protein-polysaccharide interaction and form a composite film with good compactness and mechanical properties. However, to the best of our knowledge, few previous studies have been reported on the application of sweep frequency pulsed ultrasound technique in the preparation of edible composite films.

Therefore, this study used the sweep frequency pulsed ultrasound (SFPU) technique to improve the preparation of tuna skin collagen-chitosan (TSC-CTS) composite film. Correspondingly, the effects of SFPU treatment on mechanical properties, water solubility (WS), water vapor permeability (WVP), and film transmittance were investigated. Furthermore, the effects of SFPU treatment on the structural properties, thermal stability, antioxidant activity and antibacterial activity of the film were also studied.

Materials and methods

Materials and reagents

Tuna skin was supplied from Ningbo Today Food Co. Ltd. (Zhejiang, China). Glacial acetic acid, pepsin, sodium chloride, chitosan, glycerol, sodium bromide and LB medium were obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Escherichia coli CICC 20658, Bacillus subtilis CICC 10732, Staphylococcus aureus CICC 10,201 and Listeria monocytogenes ATCC 19,111 were ordered from China Center of Industrial Culture Collection (Beijing, China). 1,1-diphenyl-2-picryl-hydrazyl (DPPH) was purchased from Sigma Company (St. Louis, MO, USA). All reagents were of analytical grade.

Methods

Extraction of tuna skin collagen (TSC)

Collagen was extracted from tuna skin according to a previously described method [14] with some modifications. The pretreated fish skin was transferred to 0.5 mol/L acetic acid solution at a ratio of 1:70 (w/v). Then, 0.06% (w/v) of 1800 U/g pepsin was added to the above-mentioned solution for enzymatic extraction 48 h at 4 ± 1 °C (pH = 2). The next step of salting out and purification is the same as that of Zhou et al. [14]. The freeze-dried TSC (yield of 70% and content of 90%) was stored in a 4 °C refrigerator.

Preparation of tuna skin collagen-chitosan (TSC-CTS) film

The TSC-CTS film was prepared according to a reported method [15] with some modifications. Briefly, 2 g of the extracted TSC was fully dissolved in 100 mL distilled water at 50 °C for 30 min and then filtered to obtain a pure TSC solution. Chitosan (CTS) solution was dissolved by using 2% (v/v) acetic acid to a concentration of 20 mg/mL, stirred at 50 °C for 30 min, and then equilibrated at room temperature (25 °C) for 24 h. Pure TSC and CTS solution were mixed at a ratio of 1:4 (v/v), and 25% glycerol (w/v) was added as the plasticizer. The mixture was stirred at room temperature for 10 min and then cross-linked in a water bath at 55 °C for 1 h. The reaction was terminated by rapid cooling in an ice water bath; TSC-CTS solution was promptly transferred to a disposable dish (90 mm in diameter) and then dried at 40 °C for 24 h. The dried TSC-CTS film was stored in a desiccator at room temperature with a relative humidity of 58% (adjusted with a saturated sodium bromide solution).

Optimization of the SFPU assisted preparation process

In the conventional method, TSC-CTS was cross-linked at 55 °C for 1 h (mentioned in section 2.2.2) to form a TSC-CTS film. Alternatively, the TSC-CTS solution was treated by the SFPU equipment (manufactured by Jiangsu University) under different conditions. After the SFPU treatment, the mixture was further cross-linked at 55 °C with a total time, including an ultrasonic time of 1 h. The non-ultrasonic treated TSC-CTS film and SFPU treated SFPU-TSC-CTS film were prepared under the same conditions, and the TSC-CTS film was selected as the control.

The defaulted parameters for the SFPU treatment were power density of 75 W/L, sweep frequency cycle of 100 ms, sweep frequency amplitude of ± 1.0 kHz, duty ratio of 77%, ultrasonic time of 10 min and temperature of 55 °C. In this study, we chose a range of variations for the optimization. It included ultrasonic frequency (22, 28, 33, 40 and 68 kHz), power density (50, 75, 100, 150 and 200 W/L), sweep frequency cycle (20, 100, 200, 300 and 500 ms), sweep frequency amplitude (0, ±0.5, ±1.0, ±1.5 and ± 2.0 kHz), duty ratio (20%, 30%, 50%, 77% and 90%), i.e. ultrasonic working time/intermittent time (5 s/20 s, 6 s/14 s, 10 s/10 s, 10 s/3 s and 24 s/3 s), and ultrasonic time (5, 10, 15, 20 and 30 min). The purpose of the optimization was to obtain the film with the best properties.

Determination of physicochemical properties

Determination of tensile strength (TS) and elongation at break (EAB)

The film was cut into strips of 20 × 60 mm, and the tensile strength (TS) and elongation at break (EAB) were measured by a texture analyzer (TA-XT2i, Stable Micro Systems Company, England) according to a previously reported method [16]. The stretching rate and gauge length were set to 60 mm/min and 40 mm, respectively. The TS (MPa) and EAB (%) was calculated by using Eq. (1), (2), respectively.

where, F is the maximum tension when the film breaks (N), d is the thickness of the film (mm), W is the width of the film (mm), L represents the line distance when the film breaks (mm), and L indicates the original line distance of film (mm).

Determination of water solubility (WS) and water vapor permeability (WVP)

Water solubility (WS, %) of the film was measured according to a previously reported method [17] and calculated by using the Eq. (3):

where, W represents the weight (g) of the dried film, W represents the weight (g) of the film after immersing it into 50 mL distilled water at room temperature for 24 h and then thoroughly drying again.

Water vapor permeability (WVP) of the film was determined accorded to a previously described method [17] with some modifications. The film was wrapped over a 50 mL beaker containing 20 mL distilled water and fastened with a rubber band. Subsequently, the beaker was placed in a desiccator containing silica gel which was incubated at 22 °C. The weight has been taken every 2 h for 6 times. WVP (g/(m·s·Pa)) was calculated by using Eq. (4):

where, x is the thickness of the film (m), A is utilization area of the films (18.08 × 10-4 m2), Δm is the mass increase (g), t is the time interval (s), and ΔP is the difference in water vapor pressure between the two sides of the film, ΔP = 3179 Pa (22 °C).

Determination of light transmittance

The film light transmittance was assayed using a previously reported method [18] with some modifications. Briefly, the film was cut into 40 × 10 mm and placed on one side of a 1 cm cuvette. The absorbance (Abs) was measured using a UV–Vis spectrophotometer (T6 New Century, General Analysis Beijing General Instrument Co., Ltd. Beijing, China) at 500 nm. The transmittance (%) was calculated by using Eq. (5):

Determination of structural and functional properties

Measurement of Fourier transform infrared spectroscopy (FTIR)

The FTIR spectra of the film were measured by using a Fourier transform infrared spectrometer (Nicolet is 50, Thermo Electron Corporation, America) with a KRS-5 ATR probe. Spectra were taken between 4000 and 500 cm−1 with 36 consecutive scans and a 4 cm−1 resolution.

Measurement of circular dichroism (CD)

Film solution (0.04 mg/mL) was purified through an ultrafiltration centrifuge tube to remove impurities such as acetic acid and glycerol. The ultrafiltration-treated film solution was scanned using a CD instrument (J-815, Jasco Corporation, Japan) in a 1 mm cuvette. The molar ellipticity between 190 and 240 nm was taken, and the secondary structure was analyzed using CDPro software (IBM Corporation, NY, USA).

Measurement of X-ray diffraction (XRD)

XRD patterns of the film were recorded at room temperature using an X-ray diffractometer (D8 ADVANCE, Bruker AXS Inc., Madison, WI, USA). The operating parameters were voltage 40 kV, current 40 mA, and scanning rate 3 min−1.

Measurement of the scanning electron microscope (SEM)

The surface microstructures of the film were acquired by using an SEM instrument (S-3400 N, Hitachi Corporation, Japan). The film was sprayed with gold. The accelerating voltage was 15 kV and the magnification was 800 times.

Measurement of thermal stability

The thermogravimetric experiments were performed on a synchronous thermal analyzer (STA449F3, Netzsch Corporation, Germany). The film (5 mg) was heated from 25 to 600 °C at 10 °C/min under N2 atmosphere (flow rate 70 mL/min).

Measurement of antioxidant activity

The antioxidant activity of the film was expressed by the DPPH radical scavenging activity, which was assayed according to a previously reported method [19] with some modifications. Approximately 50 mg film was chopped, dissolved in 2 mL methanol, and soaked for 3 h. Then, 0.5 mL of the soaked film sample was mixed with 2 mL DPPH ethanol solution (0.2 mmol/L) for 30 min in the dark. The absorbance of the sample solution (ABS), the mixture of 0.5 mL methanol with 2 mL DPPH ethanol solution (ABS), and the mixture of 0.5 mL soaked film sample with 2 mL ethanol which was as a blank (ABS) were measured at 517 nm. DPPH radical scavenging activity (%) was calculated by using the following Eq. (6):

Measurement of antibacterial activity

The antibacterial activity of the film was assayed using an inhibition zone method according to a previously reported method [19] with slight modifications. The bacterial test strain was inoculated on a sterile liquid medium and cultured at 37 °C using 120 r/min for 24 h in an incubator shaker (LRH-250, Shanghai Yiheng Scientific Instrument Co., Ltd., Shanghai, China). The activated bacterial suspension was transferred into sterile water and was fully shaken to prepare 106 to 107 CFU/mL initial bacterial concentration. One hundred microliters of the initial bacterial suspension were inoculated on the LB solid medium. The sterile filter paper (6 mm in diameter) with 10 μL film solution was placed on the LB solid medium. Then, the LB solid medium was cultured in an incubator at 37 °C for 24 h. Finally, the inhibition zone was measured. The filter paper with sterile water was used as a control.

Statistical analysis

All the samples were prepared in triplicate, and measurements were repeated six times. Data were presented as mean ± standard deviation. The statistical analysis was performed using SPSS19.0 software (IBM Corporation, NY, USA). The results were assessed for significance using Duncan's test at the level of p < 0.05.

Results and discussion

Effects of ultrasonic factors on the film preparation

Effect of ultrasonic frequency

Fig. 1A shows the tensile strength (TS) and elongation at break (EAB) of the film (SFPU-TSC-CTS) prepared by the SFPU method under different ultrasonic frequencies. TS and EAB are important performance indicators for packaging materials that require good mechanical properties to withstand the pressure. The mechanical property of the film refers to the intermolecular forces and crystal structures between the components [20]. When the ultrasonic frequency was increased from 22 to 68 kHz, the TS slightly increased and then decreased significantly (p < 0.05) and finally stabilized. The TS value reached a maximum of 18.38 MPa at 28 kHz. This trend was due to the different changes of cavitation caused by ultrasound at different frequencies [10]. Frequency is an important factor in determining ultrasound characteristics and is also a key factor influencing cavitation. The collapse of the bubbles from ultrasonic cavitation generated high pressure resulting in a high shear rate and strong micro-streaming, which can influence the interaction of protein and polysaccharide [10]. In the early stage, the ultrasonic cavitation effect was moderate with the increase of the ultrasonic frequency, which was beneficial to the formation and physical properties of protein-chitosan polymer [10], [21]. Therefore, the TS of the film was high at the very beginning. However, as the ultrasonic frequency continued to increase (>28 kHz), the excessive cavitation effect disrupted the formation of the collagen-chitosan polymer and destroyed the polymer structure [10], resulting in a significant decrease in the TS value. The trend of EAB was consistent with the effect of ultrasonic frequency on TS, with a maximum value of 58.20% at 28 kHz. The cavitation of ultrasound at a low frequency made the polymer structure more orderly, thereby increasing the EAB of the film. While the cavitation effect became larger with the continued growth of ultrasonic frequency, the film structure was excessively affected by ultrasound. In this way, the film toughness decreased, and hence the EAB showed a downward trend. After the ultrasonic frequency was continuously increased from 33 to 68 kHz, the degree of damage to the polymer structure by ultrasound was no longer increased, so the EAB remained unchanged. Qu et al. [10] found a similar trend that the binding degree of rapeseed protein and dextran reached the highest value at an ultrasonic frequency of 28 kHz and then decreased significantly with the continued growth of ultrasonic frequency from 28 to 50 kHz. Based on the mechanical indexes of TS and EAB, ultrasonic treatment with a frequency of 28 kHz was suitable, and ultrasonic treatment with moderate frequency was beneficial to improve the film mechanical properties.

Fig. 1

Effects of (A) ultrasonic frequency, (B) power density, (C) sweep frequency cycle, (D) sweep frequency amplitude, (E) duty ratio, (F) ultrasonic time on the tensile strength (TS), elongation at break (EAB), light transmittance, water solubility (WS), water vapor permeability (WVP) of SFPU-TSC-CTS films. Different letters above each line indicate significant differences (p < 0.05).

The effects of ultrasonic frequency on the WS, WVP and light transmittance of SFPU-TSC-CTS film are also shown in Fig. 1A. The WS can be used to evaluate the resistance of the film to external moisture. As shown in Fig. 1A, different ultrasonic frequencies had no significant effect (p > 0.05) on WS, and the values ranged from 28.36% to 29.37%. This might be due to the low water solubility of chitosan and collagen [4], [5]. Food quality may constantly change as water vapor penetrates through the food packaging. Therefore, the WVP of the food packaging film should be as low as possible to avoid the moisture exchange between the food and the external environment [2]. In Fig. 1A, the influence of different ultrasonic frequencies in WVP was significant (p < 0.05). As the increase of the ultrasonic frequency, the overall trend of WVP showed a significant decrease and then increased significantly (p < 0.05) and remained stabilized at the final stage. At 28 kHz, WVP reached a minimum value of 6.20 × 10-11 g/(m·s·Pa). The reason may be that the increased ultrasonic frequency (28 kHz) promoted the interaction of collagen and chitosan, making the film more compact. The water vapor penetration was reduced due to the compact film structure, resulting in a decreased WVP. However, when the ultrasonic frequency continued to increase (>28 kHz), WVP showed an increasing trend. This was because the structure of the film damaged dramatically due to the excessive ultrasonic cavitation effect, and the microstructure became loose [10], so the penetration and escape of water vapor on the film surface increased. When the ultrasonic frequency increased to 68 kHz, the WVP remained stable as the degree of damage to the structure by ultrasound was no longer increased. As shown in Fig. 1A, the light transmittance of the film at different ultrasonic frequencies was between 73.93% and 76.14%, with no significant difference (p > 0.05). Considering the results of WS, WVP and light transmittance indexes, an ultrasonic frequency of 28 kHz was selected, which was beneficial to improve the moisture resistance of the film.

All the above indexes consistently indicated that ultrasonic treatment with a moderate frequency was beneficial to enhance the mechanical properties and moisture resistance of the SFPU-TSC-CTS film. When the ultrasonic frequency was 28 kHz, the TS, EAB, WS, WVP and light transmittance were optimal. Therefore, an ultrasonic frequency of 28 kHz was applied for the subsequent tests.

Effect of power density

In order to evaluate the effect of powder density on the physicochemical performance of SFPU-TSC-CTS film, different power densities (50, 75, 100, 150 and 200 W/L) were applied.

As shown in Fig. 1B, with the ultrasonic power increased, the TS increased slightly and then decreased significantly (p < 0.05). The TS reached a maximum value of 18.36 MPa at the power density of 100 W/L. Ultrasonic power directly controls cavitation production [11], which was progressively enhanced with the rise of the power density in the early stage. This phenomenon facilitated the collagen-chitosan polymer formation, therefore, TS was slightly increased. However, as the power density continued to increase (>100 W/L), the excessive cavitation effect destroyed the molecular structure [11], resulting in a significant decrease in TS of the film. Similarly, the EAB of the film increased significantly with the rise of power density, reaching a maximum value of 58.73% at 100 W/L; subsequently, the EAB decreased significantly (p < 0.05). This was due to the enhancement of the cavitation effect with the increase of power density [11], [22]. The polymer structure became more ordered due to the slightly enhanced cavitation effect, thereby increasing the EAB of the film. However, the enormous energy released by the rupture of the cavitation bubble (generated under large power density) destroyed the polymer structure, resulting in a decrease in toughness. Thus, a decrease in the EAB value was observed at a high power density level. Overall, it was found that the most suitable power density was 100 W/L. Moderate ultrasonic power was beneficial for improving the mechanical properties of the film.

The effect of power density on the WS, WVP and light transmittance of the film are depicted in Fig. 1B. Variation of power density did not significantly affect the WS (p > 0.05). The WS value was in a range of 26.75%-27.57% that might be due to the compact structure formed by the cross-linking of chitosan and collagen. Fig. 1B indicated that different power densities had a significant effect on the WVP (p < 0.05), and the overall trend appeared to decrease initially and then increase with the increase of power densities. At a power density of 100 W/L, the WVP reached a minimum value of 6.39 × 10-11 g/(m·s·Pa). This reduction was due to changes in the cavitation effect caused by the changes in power density. The light transmittance of the film was between 75.62% and 77.27%, with no significant difference (p > 0.05). The influence of ultrasonic power density on WS, WVP and light transmittance was consistent with the influence of ultrasonic frequency. Combining the WS, WVP and light transmittance indexes, ultrasonic treatment with a power density of 100 W/L was suitable and conducive to improving the moisture resistance of the film.

Based on the above indicators, when power density was 100 W/L, the mechanical properties, WS, WVP, and light transmittance of SFPU-TSC-CTS films were optimal. Therefore, the power density of 100 W/L was applied for the subsequent tests.

Effect of sweep frequency cycle

The effect of different sweep frequency cycles on the mechanical properties of the SFPU-TSC-CTS film is shown in Fig. 1C. It can be seen that the TS firstly increased, and then decreased sharply, and finally stabilized with the increase of sweep frequency cycle. When the sweep frequency cycle was 200 ms, TS reached a maximum value of 18.55 MPa. The TS at the frequency of 100 ms was slightly lower than that at 200 ms, reaching 17.82 MPa; however, there was no significant difference (p > 0.05). In addition, the EAB reached a maximum value (57.59%) at a sweep frequency cycle of 100 ms, and its trend was similar to that of TS. It was concluded that different sweep frequency cycles had a significant effect on the film mechanical properties. A shorter sweep cycle means a faster change of sweep frequency, which produces a bigger vibration and strengthens the cavitation effect [11]. Moderate cavitation effect was beneficial to the formation of collagen-chitosan polymer. Therefore, the polymer structure was tighter, resulting in the increases in TS and EAB. However, as the sweep frequency cycle continued to increase, the enhanced destroying effect of ultrasound hindered the interaction of protein and polysaccharides and may also destroy the structure of polymer [11], which significantly reduced TS and EAB. Based on the above mechanical indexes, ultrasonic treatment with a sweep frequency cycle of 100 ms was the most suitable to improve the film mechanical properties.

The effect of sweep frequency cycle on the WS, WVP, and light transmittance of the film is shown in Fig. 1C. With the sweep frequency cycle increase, WS remained stable at 26.38%-27.39% with no significant changes (p > 0.05). With the augmentation of the sweep frequency cycle, WVP firstly decreased, then increased and finally reached an equilibrate stage. At the sweep frequency cycle of 100 ms, WVP reached a minimum value of 6.06 × 10-11 g/(m·s·Pa). As the sweep frequency cycle increased, the progressively enhanced ultrasound promoted the interaction of collagen and chitosan, making the film more compact, thereby reducing the penetration and escape of water vapor which performed as a reduction in WVP. The light transmittance values (74.81%-76.56%) of different samples did not show any significant differences (p > 0.05). The influence of the sweep frequency cycle on WS, WVP and light transmittance was consistent with the influence of ultrasonic frequency and power density. Combining the above WS, WVP and light transmittance indexes, ultrasonic treatment with a sweep frequency cycle of 100 ms was the most suitable, which was beneficial to improve the moisture resistance of the film.

Based on all the above indexes, the ultrasonic treatment of the moderate sweep frequency cycle was more conducive to improving the film mechanical properties and moisture resistance. When the sweep frequency cycle was 100 ms, the mechanical properties, WS, WVP and light transmittance were optimal. Therefore, the sweep frequency cycle of 100 ms was applied for the subsequent tests.

Effect of sweep frequency amplitude

Fig. 1D shows the influence of different sweep frequency amplitudes on the mechanical properties of the SFPU-TSC-CTS film. As depicted in Fig. 1D, sweep frequency amplitude caused a significant change in the TS and EAB. The alterations of TS and EAB due to changes in sweep frequency amplitude were almost the same as those caused by the changes in ultrasonic frequency, power density, and sweep frequency cycle. Qu et al. [11] reported that sweep frequency ultrasound indeed affected the strength of cavitation. As the sweep frequency amplitude increased, the progressively enhanced ultrasonic cavitation increased the cross-linking of the polymer, making a tighter polymer structure and therefore increasing the TS of the film. When the sweep frequency amplitude was ± 1.0 kHz, TS reached a maximum value of 18.85 MPa. When the sweep frequency amplitude was ± 0.5 kHz, TS was 18.30 MPa. The TS values obtained at sweep frequency amplitude of ± 1.0 kHz and ± 0.5 kHz had no significant difference (p > 0.05), but they were significantly higher than that at ± 0 kHz (i.e. fixed frequency ultrasound). When the frequency sweep range reaches a certain extent, the cavitation of sweep frequency ultrasound is stronger than that of fixed frequency ultrasound (0 kHz). It was because that the frequency in sweep frequency ultrasound is in a state of change and theoretically has more possibility of matching inherent frequency of cavitation nuclei. When the changed ultrasound frequency is consistent to the inherent frequency of cavitation nuclei, the sonochemical effect will reach the highest [23], [24]. Similarly, the EAB increased with the increase of the sweep frequency amplitude. When the sweep frequency amplitude was ± 0.5 kHz, EAB reached the maximum value of 58.77%. As the sweep frequency amplitude continued to increase, EAB decreased dramatically. It was concluded that the sweep frequency amplitude of ± 0.5 kHz can better match the natural frequency of the experimental object, thus achieving a better ultrasonic effect. Similarly, Gul et al. [25] reported that the ultrasonic amplitude had a significant effect on film preparation using hazelnut meal protein and clove essential oil. The film mechanical properties increased with the increase of ultrasonic amplitude. Combining the above mechanical indexes, it was most suitable for ultrasonic sweep frequency amplitude of ± 0.5 kHz. Suitable ultrasonic treatment was beneficial to improve the mechanical properties of the film.

The effect of sweep frequency amplitude on the WS, WVP and light transmittance is shown in Fig. 1D. With the increase of sweep frequency amplitude, WS changed from 26.48% to 27.07% without significant differences (p > 0.05). The WVP firstly decreased and then stabilized with the increase of the sweep frequency amplitude. When the sweep frequency amplitude increased from ± 0 kHz to ± 0.5 kHz, WVP was significantly reduced (p < 0.05) from 6.91 × 10-11 g/(m·s·Pa) to a minimum value of 6.09 × 10-11 g/(m·s·Pa). Sweep frequency ultrasound presented the best WVP than fixed frequency ultrasound (0 kHz). It was due to the stronger cavitation of sweep frequency ultrasound than fixed frequency ultrasound [11]. As shown in Fig. 1D, with the increase of sweep frequency amplitude, the light transmittance (75.20%-76.42%) did not change significantly (p > 0.05). The effect of sweep frequency amplitude on WS, WVP and light transmittance was consistent with those of ultrasonic frequency, power density and sweep frequency cycle. According to the results of WS, WVP and light transmittance, the optimal ultrasonic treatment with sweep frequency amplitude of ± 0.5 kHz was obtained, which was beneficial to improve the moisture resistance of the film.

Based on all the above indicators, the ultrasonic treatment of moderate sweep frequency was beneficial to enhancing the film mechanical properties and moisture resistance. When the sweep amplitude was ± 0.5 kHz, the mechanical properties, WS, WVP and light transmittance were optimal. Therefore, sweep frequency amplitude of ± 0.5 kHz was applied for the subsequent tests.

Effect of duty ratio

It can be seen from Fig. 1E that different duty ratios had significant effects on the TS and EAB of the SFPU-TSC-CTS film (p < 0.05). As the duty ratio increased, the TS and EAB values showed a consistent trend with that reported in the previous sections (i.e. studies on the effect of ultrasonic frequency, power density, sweep frequency cycle and sweep frequency amplitude on TS and EAB). When the duty ratio was increased from 20% to 77%, the ultrasonic cavitation effect was gradually enhanced, conducive to the TSC-CTS films cross-linking. Therefore, TS increased significantly, and the TS reached a maximum value of 18.12 MPa at a duty ratio of 77%. After that, the trend was reversed. The effect of ultrasonic duty ratio on EAB was consistent with that of TS. The EAB also reached a maximum value of 58.77% at a duty ratio of 77% due to the augmentation in pulse ratio, which increased the time of ultrasonic treatment resulting in a stronger sonochemical effect. The latter improved the interaction between collagen and chitosan molecules, and the composite film was closely cross-linked; thus, the mechanical properties were improved. When the duty ratio increased to a certain extent, an excessive cavitation effect hindered the cross-linking between collagen and chitosan, thus damaging the polymer structure [11]. Lin et al. [26] also reported that the thresholds of ultrasound cavitation were positively correlated with pulse duration. The most suitable ultrasonic treatment with a duty ratio of 77% was obtained according to the above mechanical indexes.

The effect of duty ratio on WS, WVP and light transmittance are shown in Fig. 1E. The duty ratio had no significant influence on the WS (p > 0.05), ranging from 26.37% to 26.85%. The WVP was significantly affected by ultrasonic duty ratio (p < 0.05), and the overall trend was first decreased and then increased. When the duty ratio of ultrasound increased from 20% to 50%, WVP remained stable. When the duty ratio continued to increase from 50% to 77%, WVP significantly decreased (p < 0 0.05), and the WVP reached the minimum value of 6.00 × 10-11 g/(m·s·Pa) at a duty ratio of 77%. When the duty ratio continued to increase to 90%, WVP increased significantly (p < 0.05). Different ultrasonic duty ratios had no significant effect on the light transmittance (75.72%-76.49%) (p > 0.05). The effect of WS, WVP and light transmittance of ultrasonic duty ratio was consistent with ultrasonic frequency, power density, sweep frequency cycle and sweep frequency amplitude. Based on the above WS, WVP and light transmittance indexes, the optimal ultrasonic treatment with a duty ratio of 77% was obtained, which was expected to improve the moisture resistance of the film.

According to all the above indexes, the ultrasonic treatment with a moderate duty ratio was beneficial to improve the mechanical properties and moisture resistance of the SFPU-TSC-CTS film. When the duty ratio was 77%, the mechanical properties, WS, WVP and light transmittance, were the best. Therefore, a duty ratio of 77% was applied for the following tests.

Effect of ultrasonic time

Fig. 1F shows that the different ultrasonic times had significant effects (p < 0.05) on the TS and EAB of the SFPU-TSC-CTS film. The film mechanical properties firstly increased and then decreased with the rise of ultrasonic time, which was basically consistent with the effects of ultrasonic frequency, power density, sweep frequency cycle, and amplitude and duty ratio on TS and EAB. When the ultrasonic time was increased from 5 to 10 min, TS significantly increased (p < 0.05), reaching the maximum value of 19.30 MPa at an ultrasonic time of 10 min. When the ultrasonic time continued to rise, the TS decreased significantly (p < 0.05). This may be because as the ultrasonic time increased, the enhanced cavitation effect induced augmentation of composite film cross-linking, changing its intermolecular force and crystal structure; hence, TS increased continuously. While as ultrasonic time continued to increase, ultrasound accumulated excessive thermal effects, which was not conducive to cross-linking of the composite film and reduced mechanical properties. The effect of different ultrasonic times on EAB was similar to that of TS. It can be seen from Fig. 1F that when the ultrasonic time was increased from 5 min to 10 min, the EAB was significantly increased (p < 0.05), and EAB reached a maximum value of 60.54% at 10 min. Eventually, as the ultrasound time increased, the EAB decreased significantly (p < 0.05). Qu et al. [10] also found that a short-term ultrasound-assisted technology promoted the cross-linking of rapeseed protein and polysaccharide. However, with the further increase of ultrasonic time, the promotion effect was weakened and finally became unobvious. Overall, it was found that the most suitable ultrasonic treatment time was 10 min.

The effect of ultrasonic time on the WS, WVP, and light transmittance is shown in Fig. 1F. Ultrasonic time had no significant effect on the WS (27.42%-28.03%) (p > 0.05). The WVP decreased first, then increased and finally remained unchanged with the increase of ultrasonic time. The WVP reached a minimum value of 5.93 × 10-11 g/(m·s·Pa) at an ultrasonic time of 10 min compared with other ultrasonic time (p < 0.05). With the increase of ultrasonic time, the change of light transmittance (75.90%-77.06%) was not significant (p > 0.05). The effect of ultrasonic time on WS, WVP and light transmittance was consistent with the effects of ultrasonic frequency, power density, sweep frequency cycle, amplitude and duty ratio. Based on the above indexes of WS, WVP and light transmittance, the optimal ultrasonic treatment was under an ultrasonic time of 10 min.

Based on all the above indexes, ultrasonic treatment at an appropriate time was more conducive to improving the film mechanical properties and moisture resistance. When the ultrasonic time was 10 min, the mechanical properties, WS, WVP and light transmittance were optimal. Therefore, the recommended ultrasound time was 10 min.

In general, the optimal conditions for the preparation of SFPU-TSC-CTS film were obtained. Under the optimal conditions, i.e., ultrasonic frequency of 28 kHz, power density of 100 W/L, sweep frequency cycle of 100 ms, sweep frequency amplitude of ± 0.5 kHz, duty ratio of 77% (10 s/3 s) and time of 10 min, the film was obtained with the best mechanical properties. TS was 19.30 MPa; EAB was 60.54%; WVP was 5.93 × 10-11 g/(m·s·Pa); WS was 27.59%, and light transmittance was 76.63%.

Effects of ultrasonic treatment on the performance of the film

The comparative analysis results of TSC-CTS and SFPU-TSC-CTS films are shown in Fig. 2. As shown in Fig. 2A, compared with the TSC-CTS film, the SFPU-TSC-CTS film showed a significant improvement in the TS (27.14%) and EAB (16.54%) (p < 0.05). As a proper ultrasonic treatment could enhance the interactions between collagen and chitosan, the molecular network structure of SFPU-TSC-CTS film was expected to be more compact and uniform [10]. Similarly, Wang et al. [27] applied ultrasound-assisted application to the binding reaction of mung bean protein isolate and glucose. They found that ultrasound promoted the glycosylation reaction of mung bean protein-glucose polymer. Li et al. [28], [29] also found that ultrasonic treatment can accelerate the grafting reaction between peanut protein isolate and polysaccharides. As shown in Fig. 2B, the ultrasonic treatment showed no significant effect on the WS (p > 0.05). This may be due to the low solubility of both chitosan and collagen in water [4], [5]. After ultrasonic treatment, WVP was significantly decreased by 12.15% (p < 0.05). This may be because proper ultrasound promoted the interaction of collagen with chitosan, increased the degree of cross-linking[10], made the SFPU-TSC-CTS film more compact, thereby reducing the penetration and escape of water vapor, and reducing the WVP. Gul et al. [25] studied film preparation using hazelnut powder protein and clove oil by the ultrasonic-assisted method. It was also found that the ultrasonic-assisted film had a more uniform structure, improved mechanical properties and reduced water vapor permeability. Ultrasonic treatment had no significant effect on the light transmittance (p > 0.05).

Fig. 2

(A) Tensile strength (TS) and elongation at break (EAB), and (B) light transmittance, water solubility (WS) and water vapor permeability (WVP) of TSC-CTS and SFPU-TSC-CTS films. Values with different lowercase letters above each column indicate that samples had significant differences (p < 0.05).

The SFPU treatment improved the film mechanical properties and moisture resistance based on the above comparative analysis results. The overall performance yielded significant increases in TS and EAB, and a significant decrease in WVP. The film still maintained excellent WS and light transmittance after ultrasonic treatment.

Effects of ultrasonic treatment on structural properties of the film

FTIR analysis

As one of the effective techniques for characterizing the interaction between polymer molecules, FTIR is an important method to reflect the cross-linking between collagen and chitosan and the ultrasonic effect on the structural properties of the film. The main intermolecular interaction of the molecule is the hydrogen bonding among carboxyl, amino, and hydroxyl groups present in the components of the film. Additionally, ionic interactions between the oppositely charged groups of protonated amino groups in chitosan and anionic groups in collagen occurred [7].

As shown in Fig. 3A, the amide A band in collagen from the TSC-CTS and SFPU-TSC-CTS films appeared at 3289 cm−1 and 3285 cm−1, respectively. This band corresponded to the stretching vibration of the hydroxyl group and the amino group [30]. It was found that the peak strength of the SFPU-TSC-CTS film shifted to the lower wavenumber, indicating that sonication enhanced the force of the intermolecular/internal hydrogen bonding of the protein-polysaccharide polymer [10]. The amides I, II and III bands were observed in the TSC-CTS film at 1648, 1558 and 1411 cm−1 [31]. Correspondingly, the peaks of SFPU-TSC-CTS film appeared at 1645, 1555 and 1409 cm−1. Compared to the conventional film, the peaks of SFPU-TSC-CTS film slightly moved and the strengths slightly increased. This indicated that the ultrasonic treatment enhanced the cross-linking between collagen and chitosan, making the polymer structure more compact. In addition, the absorption intensity of SFPU-TSC-CTS film at wavenumber of 1074 cm−1, related to C-N covalent bond, was higher than that of conventional film, indicating that the ultrasonic treatment enhanced the covalent binding ability between collagen and chitosan. This was beneficial to increase the degree of cross-linking of the polymer [10].

Fig. 3

(A) FTIR, (B) CD spectra, (C) XRD diffraction curves of TSC-CTS and SFPU-TSC-CTS films.

Overall, SFPU treatment enhanced the hydrogen bonding force and covalent binding ability between tuna skin collagen and chitosan. This could explain the significant improvements in the film mechanical properties and moisture resistance after ultrasonic treatment (Section 3.1.7).

CD analysis

The effect of ultrasonic treatment on the secondary structure of the film was investigated by CD. The absorption range of the peptide bond in the far ultraviolet region and the chain conformation of polymer are presented in Fig. 3B. The negative absorption bands of TSC-CTS and SFPU-TSC-CTS films appeared at 198 nm and 199 nm, respectively. In addition, the difference in the position and shape of the negative absorption bands reflected the secondary structural changes. The secondary structure contents are calculated, and the data are listed in Table 1. Compared with the TSC-CTS film, the α-helix of the SFPU-TSC-CTS film increased by 10.20%; the β-turn and random coil decreased by 6.50% and 8.90%, respectively. The increased α-helix content and decreased β-turn and random coil contents indicated that ultrasonic treatment caused a polymer molecular structure switch. This switch was from a disordered state to an ordered state and the secondary structure SFPU-TSC-CTS film became more order. Qu et al. [10] found that ultrasonic treatment significantly changed the secondary structure of rapeseed protein isolate-dextran composite. Vivian et al. [32] also found an increase of α-helix with whey protein after ultrasonic treatment. This conclusion of CD analysis was consistent with the FTIR result in section 3.2.1, which confirmed that the ultrasonic treatment changed the film structure, making the structure more order, thereby significantly improving the mechanical properties and moisture resistance of the SFPU-TSC-CTS film (Section 3.1.7).

Table 1

Secondary structure contents of TSC-CTS and SFPU-TSC-CTS films.

Sample	α-helix (%)	β-sheet (%)	β-turn (%)	Random coil (%)
TSC-CTS	4.20 ± 0.06b	63.20 ± 0.13a	23.50 ± 0.03a	10.40 ± 0.02a
SFPU-TSC-CTS	14.40 ± 0.03a (↑10.20)*	64.80 ± 0.16a	17.00 ± 0.04b (↓6.50)	1.50 ± 0.07b (↓8.90)

Values with different lowercase letters above each column indicate that samples had significant differences (p < 0.05).

The values in parenthesis are the change of secondary structure content of SFPU-TSC-CTS film compared to that of TSC-CTS film.

XRD analysis

The XRD pattern of TSC-CTS and SFPU-TSC-CTS films which determined the effect of ultrasonic treatment on the crystallinity of the film, is shown in Fig. 3C. In the XRD pattern, the diffraction angle, diffraction intensity and crystallographic level of the substance are represented by the peak position, peak height and peak width, respectively. The broad and scattered diffraction peak indicates the small crystallinity of the material. On the contrary, a narrow diffraction peak with high intensity indicates a large crystallinity of the material. The TSC-CTS film showed a scattered diffraction peak at 2θ = 19.85°, and the diffraction peak was slightly wider, indicating the amorphous structure of the film [33]. The diffraction peak of the SFPU-TSC-CTS film appeared at 2θ = 20.62°, and the diffraction angle slightly shifted to the right. The right shift of the diffraction angle indicated that the sonication increased the structural order of the film. This may be due to stronger molecular interactions between collagen and chitosan under ultrasound, which altered composite structure. This conclusion of the XRD analysis was consistent with the results of FTIR and CD (3.2.1, 3.2.2), which further confirmed that the ultrasonic treatment made the film structure more order. Together, these structural changes improved the mechanical properties and moisture resistance of the SFPU-TSC-CTS film (Section 3.1.7).

SEM analysis

SEM was used to investigate the effect of ultrasonic treatment on the apparent structure of the film (Fig. 4). It was found that the cross-sections of TSC-CTS and SFPU-TSC-CTS films were both smooth and uniform. This indicated that tuna skin collagen had good compatibility with chitosan, which was also verified by the high film transmittance (∼76%) (Section 3.1.7). A significant difference was observed after ultrasonic treatment. The cross-sectional surface of the TSC-CTS film had some bubbles and pore structures. By contrast, the cross-section of the SFPU-TSC-CTS film was more continuous, and the structure was denser without bubbles. This phenomenon indicated that ultrasonic treatment improved the compatibility of collagen and chitosan composite. The continuous and dense structure can block the transmission of water molecules, which could explain the lower WVP and the higher TS and EAB of the SFPU-TSC-CTS film than those of the TSC-CTS film (Section 3.1.7). Gul et al. [25] obtained similar results that the microstructure of the film with ultrasonic treatment became more uniform and dense, resulting in a great decrease in WVP of the film. In conclusion, the SEM results showed that sonication achieved a more compact structure of the film. This result was in agreement with the biocompatibility of each component of the film-forming composition from the results of FTIR, CD and XRD (3.2.1, 3.2.2, 3.2.3).

Fig. 4

Scanning electron microscope images of (A) TSC-CTS and (B) SFU-TSC-CTS films.

Taking these findings into account, sonication achieved a more ordered structure of chitosan/collagen film. It could well explain the causes of ultrasound promotion in mechanical properties and moisture resistance of the film.

Effect of ultrasonic treatment on thermal stability of the film

Thermogravimetric analysis (TGA) is a widely used technique to characterize polymeric materials and determine their thermal stability [34]. From 30 to 600 °C, three thermal degradation processes were observed for the TSC-CTS and SFPU-TSC-CTS films (Fig. 5). The first stage of weight loss occurred between 30 and 160 °C. This stage is mainly related to water evaporation from the films. Derivative thermogravimetry (DTG) curves indicated that the moisture loss of the TSC-CTS and SFPU-TSC-CTS films reached the maximum at 132 °C and 143 °C, respectively (Fig. 5B). It is known that H-bonds link these water molecules to glycerol and polymer [19]. The second and larger stage ranged from 160 to 270 °C. The decomposition at this stage was related to the non-covalent bonds include intermolecular and intramolecular hydrogen bonds, electrostatic interactions and hydrophobic interactions. Except for non-covalent bonds, the covalent bonds in the amino acid composition, such as C-N, C(O)–NH and C(O)–NH2 may be broken by a high temperature. Thermal decomposition of chitosan molecules mainly refers to the cleavage and degradation of main chain glycoside bonds [6]. Fig. 5B showed that the thermal decomposition temperature (Td) of the SFPU-TSC-CTS film was 19 °C higher than of the TSC-CTS film. Therefore, ultrasonic treatment significantly improved the thermal stability of the film. This was because of the cross-linking acceleration of collagen and chitosan under the effect of ultrasound, and the structure of the film became more ordered and difficult to decompose, resulting in better thermal stability. This more ordered structure had been confirmed by the results of FTIR, CD, XRD and SEM (3.2.1, 3.2.2, 3.2.3, 3.2.4). The film lost weight rapidly in the third stage of thermal degradation (270–600 °C) due to the oxidative decomposition of small molecules. The maximum thermal decomposition rates of the TSC-CTS and SFPU-TSC-CTS films were obtained at thermal decomposition temperatures (Td) of 302 °C and 301 °C, respectively. There was no significant difference in the maximum thermal decomposition rates between the two films. In conclusion, the thermal stability of the ultrasound treated film was superior to that of the conventional film. These results are consistent with the conclusion of literature that sonication improved the thermal stability of zein-chitosan complex polymer [21].

Fig. 5

Thermal stability of TSC-CTS and SFPU-TSC-CTS films: (A) TGA and (B) DTG.

Effect of ultrasonic treatment on antioxidant and antibacterial activities of the film

DPPH scavenging activity analysis

The effect of ultrasonic treatment on the antioxidant activity of the film was investigated by measuring the DPPH radical scavenging activity (Fig. 6). It can be seen from Fig. 6 that all films showed certain antiradical activity. The DPPH radical scavenging activity of the TSC-CTS and SFPU-TSC-CTS films was 3.87% and 4.12%, respectively. There was no significant difference in the antioxidant activity between the ultrasonic film and the conventional film (p > 0.05), indicating that the ultrasonic treatment did not significantly improve the antioxidant activity of the film. The food packaging film with certain antioxidant properties can effectively weaken spoilage, prolong the shelf life and maintain the good quality of food [35].

Fig. 6

Antioxidant activity of TSC-CTS and SFPU-TSC-CTS films. Values with different lowercase letters above each column indicate that samples had significant differences (p < 0.05).

Antibacterial activity analysis

The tuna skin collagen-chitosan film we developed had been applied in pork preservation and we found that it effectively delayed the decay and deterioration, and was helpful to extend the shelf life of pork by inhibiting the increase pH, TVB-N and total bacteria count [35]. Therefore, four common pathogenic microbial species in pork (e.g. Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Listeria monocytogenes) were further screened for further study. The inhibition zone test was used to investigate the effect of ultrasonic treatment on the antibacterial activity of the film (Fig. 7), and data is presented in Table 2. It can be seen from Table 2 that the TSC-CTS and SFPU-TSC-CTS films both showed significant high inhibition abilities on the four tested bacteria compared to the control (p < 0.05). The order of inhibition effect was B. subtilis > E. coli > S. aureus > L. monocytogenes. The antibacterial activity of the tuna skin collagen-chitosan film might be attributed to the electrostatic interactions between the bacterial surface and the surface of the film [36]. The film-forming solution contained a positively charged amino group (from collagen), targeting the negatively charged microbial cell membrane. The microbial cell membrane could be damaged due to this binding and neutralizing process, which cause the leakage of endoplasm and cell death [37]. Moreover, chitosan has also been shown to be antibacterial [38]. Interestingly, there was no significant difference in the antibacterial effect of the film before and after ultrasonic treatment on the four strains (p > 0.05), which indicated that the ultrasonic treatment did not significantly improve the antibacterial activity of the film. The above results further confirmed our previous conclusion [35] that the tuna skin collagen-chitosan film can effectively inhibit bacteria, including some pathogenic bacteria can also be killed very well.

Fig. 7

Antimicrobial ability of (1) control, (2) TSC-CTS and (3) SFPU-TSC-CTS films: (A) E. coli, (B) B. subtilis, (C) S. aureus, (D) L. monocytogenes.

Table 2

Inhibitory zone diameters of control, TSC-CTS and SFPU-TSC-CTS films.

Sample	E. coli (mm)	S. aureus (mm)	B. subtilis (mm)	L. monocytogenes (mm)
Control	6.01 ± 0.02b	6.02 ± 0.03b	6.03 ± 0.08b	6.01 ± 0.04b
TSC-CTS	7.69 ± 0.08a	7.43 ± 0.04a	7.84 ± 0.17a	7.19 ± 0.05a
SFPU-TSC-CTS	7.78 ± 0.10a	7.37 ± 0.07a	7.96 ± 0.13a	7.13 ± 0.04a

Values with different lowercase letters above each column indicate that samples had significant differences (p < 0.05).

Conclusions and future work

Tuna skin collagen (TSC) and chitosan (CTS) was proved to be a good potential matrix to form an edible film in this study. In addition, an effective auxiliary technology of sweep frequency pulsed ultrasound (SFPU) was introduced to the TSC-CTS composite film preparation process. The preparation parameters of SFPU-TSC-CTS film were optimized to be sweep frequency of 28 ± 0.5 kHz, power density of 100 W/L, sweep frequency cycle of 100 ms, pulse duty ratio of 77% and ultrasonic time of 10 min. Under these conditions, the SFPU-TSC-CTS film presented a good TS value of 19.30 MPa, EAB value of 60.54%, WVP value of 5.93 × 10-11 g/(m·s·Pa), WS value of 27.59%, and light transmittance value of 76.63%. Compared with non-ultrasonic treated TSC-CTS film, SFPU-TSC-CTS film showed significant increases of TS (27.14%) and EAB (16.54%) and a significant decrease of WVP (12.15%). It was concluded that a moderate SFPU treatment significantly promoted the TSC-CTS interaction and improved the moisture resistance and mechanical properties of the film, which will be conducive to the development of high mechanical properties of the food packaging film. Furthermore, the causes of ultrasound promotion were investigated. FTIR results indicated that ultrasound enhanced the hydrogen bonding between chitosan and collagen molecules. XRD curves showed that ultrasound increased the crystallinity of the film and made the film tougher. CD results showed that the secondary structure of the film was changed to be more orderly by ultrasonic treatment with the significant increase of α-helix and decreases of β-turn and random coil. SEM found that the apparent structure of the SFPU-TSC-CTS film was more compact after ultrasonic treatment. The results of FTIR, XRD, CD and SEM consistently indicated that SFPU ultrasonic effect altered the structure of the film, leading to better mechanical properties and higher thermal stability. Moreover, SFPU treated film also showed good antioxidant and antibacterial activities, which further confirmed our previously published results that it can effectively delayed the decay and deterioration and extend the shelf life of pork, including some pathogenic bacteria can also be killed very well. In conclusion, SFPU treatment was an effective auxiliary technology in the preparation of the composite film. If the related more film function can be further studied, it will facilitate the application of ultrasound in a edible food packaging film preparation from the industrial and agricultural by-products.

CRediT authorship contribution statement

Wenjuan Qu: Data curation, Investigation, Writing – original draft. Tiantian Guo: Data curation, Investigation, Writing – original draft. Xinxin Zhang: Data curation, Investigation, Writing – original draft. Yuting Jin: Data curation, Investigation, Writing – original draft. Bo Wang: Writing – review & editing. Hafida Wahia: Writing – review & editing. Haile Ma: Supervision.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.