Robust microfluidic construction of konjac glucomannan-based micro-films for active food packaging

International Journal of Biological Macromolecules 137 (2019) 982–991

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules journal homepage: http://www.elsevier.com/locate/ijbiomac

Robust microfluidic construction of konjac glucomannan-based micro-films for active food packaging Wanmei Lin 1, Yongsheng Ni 1, Dengyi Liu, Yingning Yao, Jie Pang ⁎ College of Food Science, Fujian Agriculture and Forestry University, Fuzhou 350002, China

a r t i c l e

i n f o

Article history: Received 12 March 2019 Received in revised form 2 July 2019 Accepted 7 July 2019 Available online 08 July 2019 Keywords: Konjac glucomannan Hydrophilicity Microfluidic spinning technology Micro-films Antimicrobial food packaging

a b s t r a c t The objective of this study was to develop a novel active food packaging with high performance by using natural active compound. Here, a facile and green strategy was employed to construct the konjac glucomannan/ polylactic acid/trans-cinnamic acid micro-films (KPTMF) via microfluidic spinning technology (MST). MST, a mild preparation approach, could remain the activities of natural compound during processing. Konjac glucomannan could drive the release of trans-cinnamic by using its hydrophilicity under our careful design. The results of fourier-transform infrared spectra and infrared images revealed that konjac glucomannan, polylactic acid, and trans-cinnamic acid had good compatibility through hydrogen bonds in the micro-films, which was consistent with the X-ray diffraction results. Also, the good swelling degree (81.36 ± 5.79%) of KPTMF could promote the release of trans-cinnamic. Besides, the KPTMF had excellent mechanical properties (Tensile strength: 14.09 ± 2.97 MPa, Elongation at break: 3.12 ± 0.57%), thermal stabilities and hydrophobicity (Water vapor permeability: 4.81 × 10−6 g/(m·h·kPa), Water contact angle: 99.2°). The obtained micro-films with large specific surface areas exhibited great antibacterial activities against Staphylococcus aureus and Escherichia coli, which suggested the potential applications in active food packaging. © 2019 Published by Elsevier B.V.

1. Introduction In the context of white pollution and food safety problems that caused by petrochemical-based nonbiodegradable packaging materials, a tremendous growth of research interest has shifted to natural active food packaging [1]. Active food packaging films based on natural and biodegradable materials could not only reduce the white pollution but also prevent all kinds of foodborne diseases [2]. Konjac glucomannan (KGM), composed of β-1,4-linked hydrophilic glucose and mannose, was chosen as a representative material [3–5]. This green macromolecular polysaccharide is natural, biodegradable, nontoxic, low-cost and has wide applications in biodegradable or edible films. For instance, Zhang's group reported the konjac glucomannan/carrageenan/nanoSiO2 coatings [6]. Wu's group prepared the konjac glucomannan-ethyl cellulose blends films [7]. Unfortunately, current KGM-based packaging films are faced with the challenges of poor antibacterial activities, low mechanical strength, and difficulty in short-term mass preparation. This primarily originated from the limitations of processing technology of KGM-based films. Recently, many approaches have been proposed for constructing active food packaging films. For instance, Lei's group reported pectin⁎ Corresponding author. E-mail address: [email protected] (J. Pang). 1 These authors contributed equally to this work.

https://doi.org/10.1016/j.ijbiomac.2019.07.045 0141-8130/© 2019 Published by Elsevier B.V.

konjac glucomannan composite edible films incorporated with tea polyphenol using the casting method [8]. Though the preparation process is facile and green, the obtained films lack large specific area, which may greatly influence the activities of the loaded bioactive compound. And Aydogdu's group prepared gallic acid loaded hydroxypropyl methylcellulose nanofibers for active food packaging via electrospinning [9]. Though the resultant films owned large specific area, the active substance may have relatively low bioactive activities after electrospinning because they are usually volatile and sensitive to high voltage and temperature. Here, microfluidic spinning technology (MST) was applied to prepare the active food packaging films. This technology is widely studied owing to its simplicity, safety, diversity, low cost and capability of scalable manufacturing [10]. For example, Ryan's group reported the fluorescent microfibers generated by microfluidic-spinning-directed microreactors [11]. Qu's group reported a strong calcium alginate microfiber achieved by microfluidic spinning [12]. Our previous work had constructed ordered microfiber arrays and sustained drug release hybrid microfibers via MST [13,14]. In addition, the obtained microfilms fabricated by MST would have large specific surface area and consist of continuous, oriented, controllable length and spacing microfibers. However, it is still challengeable for MST to construct micro-films for food packaging as it has high requirements on the mechanical properties of the material. Considering most natural biopolymers including KGM are not available to form micro-films ascribe to their poor mechanical properties [15,16], polylactic acid (PLA) which has strong

W. Lin et al. / International Journal of Biological Macromolecules 137 (2019) 982–991

mechanical tensile strength was used to prepare the micro-films. It is a commercially available bio-based and biodegradable thermoplastic polyesters [17]. The incorporation of PLA could confer the micro-films with good mechanical strength, which meets not only the requirements of food packaging materials but also the technology itself. Besides, the methods of preparing PLA-based materials are mainly electrospinning and solvent casting [18,19]. And there are no reports on fabricating PLA films via microfluidic spinning for food packaging applications to the best of our knowledge. Therefore, we employed KGM and PLA to prepare the micro-films via MST. Then, we loaded trans-cinnamic acid (t-CA) with antibacterial activities, a naturally occurring phenolic compound found in various plant sources to construct the active micro-films [20]. Last but not least, inspired by the hydrophilicity and hydrophobicity theory, we artfully use KGM to drive the release of active substances. The antibacterial activities and mechanisms were further explored. 2. Materials and methods 2.1. Materials Konjac glucomannan (KGM) (Mw = 1.0 × 106 Da, purity ≥ 95%, viscosity: 1% solution, ≥35,000 MPa·s at 30 °C) was supplied by San Ai Konjac Food Co. Ltd. (Shaotong, Yunnan, China). Polylactic acid (PLA) (Ingeo 3052D, Mn ≈ 12 × 104), containing 4% D-lactic acid and 96% Llactic acid, was supplied by Nature Works Co. Ltd. (USA). Transcinnamic acid (t-CA) (purity ≥99.5%) was purchased from Sigma Chemical Reagent Co., Ltd. (St. Louis, MO, USA). Other analytical grade chemical reagents were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Food borne pathogenic microorganisms, including the Gram-negative bacteria Escherichia coli (ATCC 25922) and the Gram-positive bacteria Staphylococcus aureus (ATCC 25923), were provided by Hope Bio-Technology Co. Ltd. (Qingdao, Shandong, China). 2.2. Preparation of KGM/PLA solutions and exploration of microfluidic spinning feasibility A 20 wt% PLA solution was prepared in 100 mL of CH2Cl2/CH₃CH₂OH (2:1, V/V) with stirring rate at 600 rad/min for 10 h. The KGM/PLA solutions were prepared by mixing KGM and 20 wt% PLA at weight ratio of 1: 5. The microfluidic spinning feasibility of KGM/PLA solutions was investigated by rheology. The apparent viscosities of KGM/PLA solutions were determined using an Anton Paar MCR 301 rheometer (Austria) equipped with a parallel-plate geometry (diameter: 50 mm, lift distance is 0.1–50 mm) at a shear rate of 0.1–500 s−1 (25 °C). Images were captured using an Apple mobile phone (iPhone 6s) and the relevant distance was recorded.

2.3. Preparation of KGM/PLA/t-CA micro-films (KPTMF) KPTMF were prepared through a microfluidic spinning machine (JNS-MS-04, Nanjing, China) in accordance with previously described method with some modifications [14]. In brief, t-CA (50% of KGM (w/ w)) was added to the KGM/PLA solutions and the solutions were stirred at the rate of 500 rad/min for 5 h. And the solutions were then centrifuged at 5000 rad/min for 10 min to remove air bubbles. Finally, the solutions were loaded into a syringe (20 mL) with 18 G stainless steel needles. KGM/PLA/t-CA spinning solutions were ejected by a syringe pump, then was stretched and twined by a frame receiver. The instrument parameters of the microfluidic spinning process were as follows: 0.3 mL/h (push injection pump speeds), 500 rad/min (motor rotation speeds), and 250 μm/s (forward speeds). Finally, micro-films were immobilized under the condition of relatively humidity of 48 ± 2% and temperature of 25 ± 2 °C for 4 h in the microfluidic spinning machine. The formation process was illustrated in Fig. 1 and Video 1.

983

2.4. Morphologies of KPTMF The morphologies were observed by scanning electron microscopy (SEM, Hitachi S-4800, Japan). KPTMF were fixed on a cylindrical microscope stub covered with a carbon strip and coated with a 100–200 Åthick layer of gold. The sputtered time was about 60 s and the accelerating voltage was 5 kV. 2.5. Fourier-transform infrared (FT-IR) spectra The purified samples were ground into powder with KBr and squashed into a wafer. FT-IR spectra were measured using a Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientific, MA, USA) at ambient temperature. The wavelength range is 4000–400 cm−1. In all the cases, 32 scans at a resolution of 4 cm−1 were used to record the spectra. 2.6. Infrared imaging (IR imaging) IR images were obtained using a Thermo Scientific Nicolet iN10 infrared microscope (Thermo Electron Corporation, USA). An analysis of the IR microscopy data was performed using OMNIC picta software (Thermo Electron Corporation, USA). 2.7. X-ray diffraction (XRD) XRD patterns were obtained by a Bruker AXS, D8 Advance X-ray diffractometer (Bruker Inc., Germany) equipped with Ni-filtered Cu Kα radiation source (λ = 0.1542 nm). The data were collected at a scanning speed of 5 min−1 with 2θ range from 5° to 80°. 2.8. Thermal properties The thermal properties were analyzed by differential scanning calorimeter (DSC) and thermogravimetric analysis (TGA). DSC was measured through a DSC200F3 (Netzsch, Germany) with the temperature increasing from 25 °C to 300 °C at a heating rate of 10 °C/min under nitrogen flow (25 mL/min). TGA was analyzed by a synchronous thermal analyzer (STA409-PC, Netzsch, Germany) at the heat rate of 15 °C/min from 25 °C to 600 °C. All samples were placed in a dry environment prior to testing. 2.9. Water vapor permeability (WVP) The WVP of the micro-films was determined according to the Chinese National Standard GB/T 1037-70 with some modifications [21]. In brief, the micro-films were sealed by tying with a rubber band to a weighing bottle (25 mm × 40 mm, containing 3.0 ± 0.1 g anhydrous CaCl2). Saturated NaCl solution was used to keep the relative humidity at 75 ± 5% in the desiccators. And the weighing bottles were put into a desiccator. The desiccator's temperature was kept at 25 ± 5 °C. The weighing bottle was taken out and weighed until the weight change was stable. Water vapor permeance (g/(m·h·kPa)) was calculated by the following equation: WVP ¼

Δm  d S  Δt  Δp

ð1Þ

where Δm (g) is the weight difference between initial and final, d (mm) is the thickness of micro-films, S (m2) is the area of micro-films, Δt (h) is the time to reach the equilibrium of the micro-films, Δp (kPa) is the pressure difference on both sides of the micro-films. 2.10. Water contact angle (WCA) Static contact angles with water were tested by a sessile drop method at room temperature (Optical Contact Angle Meter, OCA 20,

984

W. Lin et al. / International Journal of Biological Macromolecules 137 (2019) 982–991

Fig. 1. The formation process of preparing KPTMF via MST.

Germany). In brief, samples were tested by carefully depositing equal volume of distilled water (4 μL) onto the surface of samples using motor driven syringe. Angles were measured at two different regions of each surface (n = 3–5). 2.11. Swelling degree (SD) and water solubility (WS) Firstly, all micro-films (squares of 2 cm × 2 cm) were dried by an oven at 60 °C for 24 h to obtained an initial constant weight (Wi) before immersing into 50 mL of distilled water. Films were soaked in the liquid condition at ambient temperature for a period of time. Then, the final wet weight (Ww) of films was determined. After 72 h, the micro-films were dried at 60 °C to a final constant weight (Wc). The SD and WS were calculated according to the following formulas: SDð%Þ ¼

W w −W i  100 Wi

ð2Þ

W i −W c  100 Wi

ð3Þ

WSð%Þ ¼

where Wi, Ww and Wc represent the weights of initial constant, final wet and final constant micro-films. 2.12. Antibacterial activity test 2.12.1. Inhibition zone measurement The antibacterial activities of KPTMF were tested by the inhibitionzone method with some suitable modifications [22]. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) were used as the representative Gram-positive and Gram-negative bacteria, respectively. All tested films were sterilized by UV for 20 min. The bacteria were cultivated in nutrient broth (3 g beef extract, 10 g peptone and 5 g sodium chloride per 1000 mL water). S. aureus and E. coli (approximately 107– 108 CFU/mL) were then evenly spread over LB agar plates. The KPTMF (circular sheet, 10 mm diameter) were placed on the LB agar plates. KGM/PLA and PLA/t-CA micro-films prepared in the same condition were used as comparison. Then, the plates were incubated at 37 °C for 24 h and the inhibition zone diameters were measured with calipers. Images were captured by an Apple mobile phone (iPhone 6s). 2.12.2. Bacterial viability assessment The plate count method reported by Shankar’ group was used for evaluating the antibacterial activities of micro-films, with some modifications [23]. Two common food-borne pathogens S. aureus and E. coli were tested. In brief, the two bacteria were aseptically inoculated in the TSB at 37 °C for

24 h before diluted in 0.9% of sterile saline to obtained approximately 107–108 CFU/mL. All tested micro-films were sterilized prior to the tests by UV for 20 min. Then, 100 μL of the diluted bacterial suspension was aseptically transferred to 10 mL of TSB medium in tubes containing 200 mg of micro-films for incubation at 37 °C for 24 h with shaking rate of 200 rpm. The micro-films were taken out and the bacterium suspension was diluted by sterile saline and then 150 μL of each dilution suspension was plated on the TSA for colony counting. The incubation condition for the TSA plates was set at 37 °C for 24 h. 2.13. Mechanical properties In order to evaluate the mechanical properties of KPTMF, the tensile strength (TS) and elongation at break (E) were measured according to GB 13022-91. A texture analyzer (EZ-SX, Shimadzu, Japan) with a 500 N load cell was employed. The samples were cut with a dimension of 10 mm × 40 mm. The crosshead speed and initial grip separation were set at 10 mm/min and 20 mm, respectively. The TS and E were calculated by the following equations: TS ¼

F S

ð4Þ

where TS (Pa) is the tensile strength, F (N) is the maximum stress of stretching, S (m2) is the initial sectional area of sample. E ð%Þ ¼

ðL−L0 Þ  100 L0

ð5Þ

where E is the elongation at break, L0 (mm) is the initial length of sample, L (mm) is the length of the sample at the time of break. 2.14. Statistical analysis Data were presented as the mean ± standard deviation of each treatment. Analysis of variance (ANOVA) was performed using a SPSS software statistical analysis system (SPSS 20.0 for windows, SPSS Inc., Chicago, IL). 3. Results and discussion 3.1. Apparent viscosity We firstly try to qualitatively explore the microfluidic spinning feasibility of KGM/PLA solutions by apparent viscosity. As shown in Fig. 2, the apparent viscosity (ηap) of KGM solutions or KGM/PLA solutions decreased as the shear rate increased, which indicated the pseudoplastic properties or the shear thinning region of these solutions. These

W. Lin et al. / International Journal of Biological Macromolecules 137 (2019) 982–991

985

Fig. 2. Apparent viscosity behavior: (A) 2% (w/w) KGM solutions and (B) KGM/PLA solutions.

findings were consistent with the polysaccharide-based film-forming solutions [24]. However, the ηap of KGM/PLA solutions (initial apparent viscosity is 2750 Pa·s, WKGM:WPLA = 1:5, 20 wt% PLA) was bigger than that of the neat KGM solutions (initial apparent viscosity is 45 Pa·s, 2% KGM), which may be attributed to the excellent mechanical strength of PLA or the intermolecular interactions (KGM-PLA) [25]. Moreover, KGM solutions were stretched to break at 26 mm, which suggested that their solutions could not form microfibers via MST due to poor inherent viscosity. This results were consistent with our previous reports [13]. By contrast, KGM/PLA solutions could form a wire and keep still continuous at 50 mm when the rotor lifted, which indicated their potential microfluidic spinning ability.

measures, which is beneficial for maintaining the bioactivities of t-CA. In addition, with the help of curing devices, we could achieve mass production of micro-films at a short time. Just 60 min, we calculated the theoretical length of microfibers to be reached at about 4710 m according to the equation: L = πdvt (d: frame length is 5 cm, v: rotational speed is 500 rad/min, t: time is 60 min). The preparation process of the limitless length microfibers is flexible, controllable, simple, versatile, highly efficient and environmental-friendly. In summary, the KPTMF are completely green, non-toxic, biodegradable, and low-cost, which could be a promising candidate for active food packaging materials.

3.2. Morphologies of KPTMF

We investigated the compatibility of the materials in KPTMF by FTIR spectra. As shown in Fig. 4, the FT-IR spectra of KGM showed the following main absorption bands: the bands at 3491 cm−1 (O\\H stretching), 2925 cm−1 (C\\H stretching), 895 cm−1 (mannose unit stretching). These findings were consistent with previous reports [27]. The main characteristic peaks for PLA were presented at 3495 cm−1 (O\\H stretching), 2998 cm−1 (C\\H stretching), 1750 cm−1 (C_O stretching), 1480 cm−1 (CH(CH3) stretching) [28]. In addition, the peaks at 1646 cm−1 (COOH stretching) and 1070 cm−1 (C\\O stretching) were observed in t-CA [29]. Compared with KGM and PLA, the stretching vibration of O\\H in KGM/PLA shifted to 3441 cm−1, which indicated the formation of hydrogen bonds between KGM and PLA chains [30]. Similar changes were also observed by Pang's group

The whole morphology of the films is shown in Fig. 3A and the microscopic appearance is shown in Fig. 3B. It is clear that the films were composed of ordered, uniform and compact microfibers. The average diameter of a single microfiber was 5 ± 0.2 μm, which could be easily adjusted by pushing injection pump, motor rotation and forward speeds. It is worth noting that the special microstructure of the film is conducive to enhance the release of t-CA therefore could protect the quality of food [26]. Compared with the films prepared through the conventional method, the KPTMF have large specific surface areas, which was directly correlated with the antibacterial effectiveness. Moreover, the green and mild preparation process does not need high voltage or other auxiliary

3.3. FT-IR spectra and IR imaging

Fig. 3. (A) Digital image and (B) SEM image of KPTMF.

986

W. Lin et al. / International Journal of Biological Macromolecules 137 (2019) 982–991

Fig. 4. FT-IR spectra and IR images of KGM, PLA, t-CA, KGM/PLA and KPTMF.

in FT-IR spectra when evaluating the compatibility of KGM and PVDF [14]. Furthermore, the bands of O\\H stretching vibration of KPTMF also shifted, which suggested the formation of hydrogen bonds in the films. Meanwhile, compared with KGM/PLA and t-CA, the stretching vibration of COOH (1646 cm−1) in KPTMF significantly reduced. This might be attributed to the new formation of hydrogen bonds by COOH from t-CA, which resulted in the enhanced physicochemical properties of KPTMF. We further employed IR images to obtain insight into the compatible mechanism of KPTMF. IR images exhibit chemical changes, directly illustrating the distribution of the groups through color variation. Red color represents high intensity, green color indicates uniform dispersion and blue color represents low intensity [31]. At 3491 cm−1 and 1646 cm−1, KPTMF images are green (ignoring the influence of the edge effect), which revealed that hydroxyl groups or carboxyl groups became relatively uniform [14]. This results further suggested the formation of hydrogen bonds (OH-OH or OH-COOH). 3.4. XRD analysis XRD was performed to verify the compatibility of KGM, PLA and t-CA in the micro-films (Fig. 5). A distinguished peak at 2θ ≈ 22° was observed in the KGM, indicating that KGM is an amorphous material, which is consistent with previous findings [32]. The XRD pattern of PLA showed one distinguished diffraction peak at 2θ ≈ 17° and one small characteristic peak at 2θ ≈ 19° [33]. The XRD patterns of KGM/ PLA are similar to those of PLA. And the intensity of the two diffraction peaks (2θ ≈ 17° or 19°) arising from PLA slightly increased, which suggested that there existed intermolecular interactions between KGM and PLA in the micro-films. This is similar to previous research [4]. Interestingly, similar characteristic peaks were also observed at 2θ ≈ 17° or 2θ ≈ 19° in the XRD pattern of KPTMF. This result suggested the good compatibility of KGM, PLA and t-CA in the KPTMF, which supported the results of FT-IR analysis.

Obviously, the KGM and t-CA is thermal degraded earlier than PLA, which signified the good thermal performances of PLA. It's worth noting that the KPTMF and KGM/PLA had relatively higher endothermic temperatures than that of PLA, which may ascribe to the hydrogen bonds formed in the micro-films. This is consistent with the FT-IR analysis. As shown in Fig. 6B, the TGA profiles of KGM companioned by three weight loss stages. The first stage occurred between 40 °C and 50 °C, which was due to the water evaporation in the KGM. The next stage of weight loss started at around 127 °C with a slow weight loss rate. This was caused by the degradation of oxygen-containing functional groups in KGM [37]. The last stage that started at about 245 °C should be resulted from the thermal decomposition of KGM and the structure damages of its molecular chains [37]. As for the rest four samples, there are not any significant weight losses before reaching up to certain temperatures (130 °C for t-CA and 300 °C for PLA-contained micro-films). This might owe to little moisture contained in those samples because both tCA and PLA are highly hydrophobic. The four samples began to decompose at different temperatures with various amounts of residue of 0.4% (t-CA), 3.26% (PLA), 2.18% (KGM/PLA) and 1.2% (KPTMF). And the degradation behaviour of PLA is consistent with our previous work [25]. The

3.5. Thermal properties analysis Thermal stability is an important parameter for active packaging films [34]. Hence, we investigated the thermal properties of KPTMF by DSC and TGA measurements. The results are depicted in Fig. 6. As shown in Fig. 6A, the endothermic peak appeared at 124.6 °C (KGM), 164.8 °C (PLA), 134.7 °C (t-CA), 165 °C (KGM/PLA) and 165.2 °C (KPTMF), which was due to the degradation of tested samples [35,36].

Fig. 5. XRD patterns of KGM, PLA, t-CA, KGM/PLA and KPTMF.

W. Lin et al. / International Journal of Biological Macromolecules 137 (2019) 982–991

987

Fig. 6. (A) DSC and (B)TGA curves of KGM, PLA, t-CA, KGM/PLA and KPTMF.

postponed temperatures for weight loss of KPTMF and KGM/PLA indicated the improved thermal stabilities of KGM-based micro-films. This may be attributed to the formation of hydrogen bonds which could endow with a more stable thermal performance. This is also in the line with the FT-IR analysis. In summary, KPTMF had an excellent thermal degradation temperature of 326 °C, which could meet the common requirements of food packaging based on natural biopolymers.

3.6. WVP and WCA of the micro-films As previously mentioned, water sensitiveness of KGM-based films is their main drawback in food packaging applications. For this reason, the water barrier properties of KPTMF were studied by WVP and WCA measurements. Generally, a low WVP value of packaging films is desirable for extending food shelf life. As shown in Fig. 7A, the WVP of KPTMF

Fig. 7. (A) WVP and (B) WCA of KGM/PLA micro-films, PLA/t-CA micro-films and KPTMF.

988

W. Lin et al. / International Journal of Biological Macromolecules 137 (2019) 982–991

(4.81 × 10−6 g/(m·h·kPa)) was significantly lower than the common KGM matrix films, which indicated its potential applications in food packaging [38]. Because most natural and biodegradable biopolymers such as KGM-based films are highly hydrophilic [39]. The decreased WVP value of KPTMF could be explained by the formation of hydrogen bonds in the micro-films, which reduced the number of available polar groups in the system. According to previous reports, the WVP value is dependable on the quantity of available polar groups contained in the polymers [40]. What's more, the formation of hydrogen bonds could confer the micro-films with a more compact structure, which could decrease the WVP value as well. Compared with KPTMF, the WVP value of KGM/PLA was relatively higher and PLA/t-CA was relatively lower. There may be two reasons why the WVP value of KGM/ PLA is higher than that of KPTMF. One is the formation of hydrogen bonds between PLA and t-CA. In our FT-IR analysis, we had inferred the hydrogen bond connections between KGM and PLA. Based on this, we may further deduce that hydrogen bonds could be formed between PLA and t-CA hence a more compact structure was obtained in the KPTMF. Another is the hydrophobic nature of both PLA and t-CA, which could also lead to the decreased WVP value. As for the reason why the WVP value of PLA/t-CA is lower than that of KPTMF, the primary cause may be due to the hydrophilic nature of KGM, which could result in the formation of repulsive forces between PLA or t-CA hence had negative effects on the compact structures of KPTMF. Moreover, the hydrophilicity of KGM could affect the hydrophobicity of KPTMF so that may reduce the WVP value. In addition, we estimated the hydrophobicity of micro-films by WCA. Generally, a higher WCA value means a more hydrophobic performance of the films [7]. As shown in Fig. 7B, the WCA of KPTMF is 99.2°, indicating that it had good water resistivity, which might be the contribution of hydrogen bonds. The high WCA value of KPTMF is favorable for eliminating water condensation inside fresh produce packaging and improving printability [41]. Hence, such hydrophobic micro-films could guarantee food quality by resisting the reproduction of microorganisms [42]. The WCA of PLA/t-CA micro-films is the highest (102.6°) among the three kinds of micro-film, which resulted from the good hydrophobicity of PLA and t-CA. This is in accordance with previous reports [20]. Moreover, the hydrogen bonds formed in PLA/t-CA might be another reason. KGM is highly sensitive to moisture ascribe to its hydrophilic nature, which should be the cause of relatively lower WCA value of the KGM/ PLA (84.7°) [43]. These results agreed with the WVP analysis. Overall, the hydrophobicity of KPTMF was significantly improved, which indicated it has potential applications in food packaging. 3.7. SD and WS of the micro-films A good SD could reflect the release performance of active compounds in the micro-films. Thus, the swelling properties of the films were evaluated at room temperature. As presented in Table 1, the KPTMF showed excellent water holding capacity, which was ascribe to the incorporation of good water-absorbent KGM. The high SD of the micro-films could promote the release of t-CA [15]. Besides, KGM could form a large number of hydrogen bonds with PLA, which could lead to the decrease the quantity of hydrogen bonds formed between PLA and t-CA, indirectly causing the release of t-CA. Meanwhile, the repulsive forces existed between hydrophilic KGM and hydrophobic t-CA Table 1 Swelling degree and solubility of KPTMF, KGM/PLA micro-films and PLA/t-CA micro-films. Sample code KPTMF KGM/PLA PLA/t-CA

Swelling degree (%) a

81.36 ± 5.79 72.12 ± 14.34a 3.31 ± 0.26b

Solubility (%)

could directly contribute to the release of t-CA. The swelling characteristics of KGM/PLA was similar to KPTMF. But the lowest SD was clearly seen in the PLA/t-CA micro-films, which was due to the hydrophobic PLA and t-CA. The WS has the similar trend with SD in each film. Overall, the good SD of KPTMF is beneficial for the release of t-CA. 3.8. Test of antibacterial activities 3.8.1. Inhibition zone The antibacterial performances of KGM/PLA micro-films, PLA/t-CA micro-films and KPTMF against S. aureus (Gram-positive) and E. coli (Gram-negative) were investigated. As shown in Fig. 8A, the KGM/PLA micro-films do not have any inhibition effect on both S. aureus and E. coli, which suggested both KGM and PLA do not have antibacterial activities. These results are in the line with previous report [44]. In our present work, t-CA, a natural antimicrobial compound, was used as the antibacterial agent [20]. It is interesting to remark the inhibition effect of bacterial growth not occurred in the PLA/t-CA group in Fig. 8A. However, the inhibition zones in the KPTMF are clearly observed. This phenomenon suggested that the addition of KGM into the KPTMF could improve the antibacterial activities. There were mainly two reasons for why PLA/t-CA did not exhibit antibacterial efficiency. One is the formation of hydrogen bonds and the other is the attractive forces. According to the FT-IR and WVP analysis, hydrogen bonds were formed between PLA and t-CA. Hence, the network of PLA/t-CA was compact and the connection inside was tight, which could impede the release of t-CA from the micro-films. Moreover, the attractive forces between PLA and t-CA could also prevent the release of t-CA. As for the remarkable antibacterial activities of KPTMF, it could be explained in terms of hydrogen bonds and the hydrophilic/hydrophobic forces. The formation of hydrogen bonds between KGM and PLA could reduce the number of hydrogen bonds formed between PLA and t-CA therefore further affect the connections between PLA and t-CA to some extent. This could lead to the release of t-CA indirectly. More importantly, there are repulsive forces between KGM and t-CA ascribe to the hydrophilicity of KGM and the hydrophobicity of t-CA, which could promote the release of tCA directly. Thus, the KPTMF exhibited good antibacterial effects. The antibacterial mechanism is shown in Fig. 8C. Similar conclusions were obtained by our previous report [14]. In addition, the diameter of inhibition zones of KPTMF against S. aureus (9.50 ± 2.12 mm) is larger than that of E. coli (2.25 ± 0.35 mm) (Fig. 8B). Because the t-CA has stronger antibacterial activities against Gram-positive bacteria than that of the Gram-negative ones [20]. In summary, the hydrophilic KGM improved the antibacterial activities of KPTMF by driving the release of hydrophobic t-CA. Meanwhile, the limitations of KGM-based films including relatively poor thermal stability and water resistance were solved by incorporating PLA. Hence, the KPTMF could act as potential efficient antimicrobial films in active food packaging. 3.8.2. Bacterial viability In addition to the inhibition-zone method, the total colony count method was applied to quantify the antibacterial efficiency of microfilms. As shown in Fig. 9, the KPTMF displayed remarkable antibacterial activity, which could be ascribe to their good SD confirmed by the swelling experiment. By contrast, the antibacterial effects of PLA/t-CA were far from desired. Moreover, the micro-films had stronger antibacterial activity on Gram-positive bacteria than that on Gram-negative bacteria, which is in the line with the inhibition zone test. 3.9. Measurement of mechanical properties

a

17.27 ± 6.43 12.15 ± 4.91a 2.57 ± 0.32b

Date are expressed by the form of mean ± SD (n = 3). Column with different superscript letters means that values are significantly different according to Duncan's Multiple Range Test (p b 0.05).

Finally, the mechanical properties of KPTMF were evaluated by using PLA as the control since it has good mechanical properties [17]. As shown in Table 2, both TS and E of the KPTMF is closer to that of PLA, which indicated the well mechanical properties of KPTMF. This may result from the formation of hydrogen bonds in the micro-films, which is

W. Lin et al. / International Journal of Biological Macromolecules 137 (2019) 982–991

989

Fig. 8. Antibacterial activities evaluated by the assay of inhibition zone against S. aureus and E. coli: (A) photos of inhibition zone (the red circles represent the inhibition zone, the black and red arrows represent the diameter of sample and inhibition zone respectively), (B) diameters of inhibition zone, (C) schematic diagram of antibacterial mechanism of KPTMF.

consistent with the FT-IR analysis. Meanwhile, the addition of PLA could also enhance the TS and E of KPTMF. Therefore, the KPTMF could be a suitable candidate for present food packaging. 4. Conclusion A novel method of preparing micro-films was provided. The obtained KPTMF had microstructures and large specific surface area. And

the activities of compounds in the micro-films were remained under the mild preparation process, MST. Besides, konjac glucomannan (KGM) was used artfully to drive the release of active substance. And the good swelling degree had promoted the release of functional compound. Hence, the KPTMF exhibited excellent antibacterial activities, which suggested its potentials in food packaging. The hydrophilicity of KGM played a significant role in enhancing the antibacterial activities of micro-films, which is a breakthrough in KGM-based films. Also, the

990

W. Lin et al. / International Journal of Biological Macromolecules 137 (2019) 982–991

Fig. 9. Log CFU/mL of KGM/PLA micro-films, PLA/t-CA micro-films, KPTMF and t-CA against S. aureus and E. coli.

Table 2 Mechanical properties of PLA and KPTMF. Sample code

TS (MPa)

E (%)

PLA KPTMF

14.48 ± 0.41a 14.09 ± 2.97a

4.02 ± 1.72a 3.12 ± 0.57a

Date are expressed by the form of mean ± SD (n = 3). Column with different superscript letters means that values are significantly different according to Duncan's Multiple Range Test (p b 0.05).

problems of poor mechanical strength, thermal stabilities and hydrophobicity of KGM-based films were solved. Overall, this work offered a new and advanced method to construct active food packaging films. More importantly, it offered a new idea for producing active food packaging materials with high performance by using hydrophilic polysaccharides represented by KGM. Supplementary data to this article can be found online at https://doi. org/10.1016/j.ijbiomac.2019.07.045.

Acknowledgement The authors are grateful to the National Natural Science Foundation of China (grant number: 31772045, 31471704). References [1] T. Liang, G. Sun, L. Cao, J. Li, L. Wang, Rheological behavior of film-forming solutions and film properties from Artemisia sphaerocephala Krasch. gum and purple onion peel extract, Food Hydrocoll. 82 (2018) 124–134. [2] Z. Aytac, S. Ipek, E. Durgun, T. Tekinay, T. Uyar, Antibacterial electrospun zein nanofibrous web encapsulating thymol/cyclodextrin-inclusion complex for food packaging, Food Chem. 233 (2017) 117–124. [3] Y. Ni, R.J. Mu, X.D. Tan, R.X. Huang, Y. Yuan, H.B. Cheng, J. Pang, Stability of the konjac glucomannan topological chain based on quantum spin model, Chinese J. Struc. Chem. 36 (2017) 1043–1048. [4] Y. Ni, W. Lin, R. Mu, C. Wu, Z. Lin, S. Chen, J. Pang, Facile fabrication of novel konjac glucomannan films with antibacterial properties via microfluidic spinning strategy, Carbohydr. Polym. 208 (2019) 469–476. [5] W. Lin, Y. Ni, L. Wang, D. Liu, C. Wu, J. Pang, Physicochemical properties of degraded konjac glucomannan prepared by laser assisted with hydrogen peroxide, Int. J. Biol. Macromol. 129 (2019) 78–83. [6] R. Zhang, X. Wang, L. Li, M. Cheng, L. Zhang, Optimization of konjac glucomannan/ carrageenan/nano-SiO2 coatings for extending the shelf-life of Agaricus bisporus, Int. J. Biol. Macromol. 122 (2019) 857–865. [7] K. Wu, Q. Zhu, H. Qian, M. Xiao, H. Corke, K. Nishinari, F. Jiang, Controllable hydrophilicity-hydrophobicity and related properties of konjac glucomannan and ethyl cellulose composite films, Food Hydrocoll. 79 (2018) 301–309.

[8] Y. Lei, H. Wu, C. Jiao, Y. Jiang, R. Liu, D. Xiao, J. Lu, Z. Zhang, G. Shen, S. Li, Investigation of the structural and physical properties, antioxidant and antimicrobial activity of pectin-konjac glucomannan composite edible films incorporated with tea polyphenol, Food Hydrocoll. 94 (2019) 128–135. [9] A. Aydogdu, G. Sumnu, S. Sahin, Fabrication of gallic acid loaded Hydroxypropyl methylcellulose nanofibers by electrospinning technique as active packaging material, Carbohydr. Polym. 208 (2019) 241–250. [10] T. Cui, Z. Zhu, R. Cheng, Y.L. Tong, G. Peng, C.F. Wang, S. Chen, Facile access to wearable device via microfluidic spinning of robust and aligned fluorescent microfibers, ACS Appl. Mater. Inter. 10 (2018) 30785–30793. [11] Y. Zhang, C.F. Wang, L. Chen, S. Chen, A.J. Ryan, Microfluidic-spinning-directed microreactors toward generation of multiple nanocrystals loaded anisotropic fluorescent microfibers, Adv. Funct. Mater. 25 (2015) 7253–7262. [12] X. Hu, M. Tian, B. Sun, L. Qu, S. Zhu, X. Zhang, Hydrodynamic alignment and microfluidic spinning of strength-reinforced calcium alginate microfibers, Mater. Lett. 230 (2018) 148–151. [13] R.J. Mu, Y. Ni, L. Wang, Y. Yuan, Z. Yan, J. Pang, S. Chen, Fabrication of ordered konjac glucomannan microfiber arrays via facile microfluidic spinning method, Mater. Lett. 196 (2017) 410–413. [14] Y. Ni, W. Lin, R.J. Mu, C. Wu, L. Wang, D. Wu, S. Chen, J. Pang, Robust microfluidic construction of hybrid microfibers based on konjac glucomannan and their drug release performance, RSC Adv. 8 (2018) 26432–26439. [15] W. Lin, Y. Ni, J. Pang, Microfluidic spinning of poly (methyl methacrylate)/konjac glucomannan active food packaging films based on hydrophilic/hydrophobic strategy, Carbohydr. Polym. 222 (2019), 114986. [16] Y. Ni, W. Lin, R.J. Mu, L. Wang, X. Zhang, C. Wu, J. Pang, Microfluidic fabrication of robust konjac glucomannan-based microfiber scaffolds with high antioxidant performance, J. Sol-gel Sci Tech. 90 (2019) 214–220. [17] K. Fernandez, J. Aburto, C. von Plessing, M. Rockel, E. Aspe, Factorial design optimization and characterization of poly-lactic acid (PLA) nanoparticle formation for the delivery of grape extracts, Food Chem. 207 (2016) 75–85. [18] Y. Li, C.T. Lim, M. Kotaki, Study on structural and mechanical properties of porous PLA nanofibers electrospun by channel-based electrospinning system, Polymer 56 (2015) 572–580. [19] S. Shankar, L.F. Wang, J.W. Rhim, Incorporation of zinc oxide nanoparticles improved the mechanical, water vapor barrier, UV-light barrier, and antibacterial properties of PLA-based nanocomposite films, Mater. Sci. Eng. C 93 (2018) 289–298. [20] K.S. Letsididi, Z. Lou, R. Letsididi, K. Mohammed, B.L. Maguy, Antimicrobial and antibiofilm effects of trans-cinnamic acid nanoemulsion and its potential application on lettuce, Lwt-Food Sci. Technol. 94 (2018) 25–32. [21] L. Lin, X. Liao, D. Surendhiran, H. Cui, Preparation of ε-polylysine/chitosan nanofibers for food packaging against Salmonella on chicken, Food Packaging Shelf 17 (2018) 134–141. [22] F. Kayaci, O.C. Umu, T. Tekinay, T. Uyar, Antibacterial electrospun poly(lactic acid) (PLA) nanofibrous webs incorporating triclosan/cyclodextrin inclusion complexes, J. Agric. Food Chem. 61 (2013) 3901–3908. [23] S. Shankar, J.W. Rhim, Preparation and characterization of agar/lignin/silver nanoparticles composite films with ultraviolet light barrier and antibacterial properties, Food Hydrocoll. 71 (2017) 76–84. [24] F. Ma, Y. Zhang, N. Liu, J. Zhang, G. Tan, B. Kannan, X. Liu, A.E. Bell, Rheological properties of polysaccharides from Dioscorea opposita Thunb, Food Chem. 227 (2017) 64–72. [25] L. Wang, Y. Yuan, R.J. Mu, J. Gong, Y. Ni, X. Hong, J. Pang, C. Wu, Mussel-inspired fabrication of konjac glucomannan/poly (lactic acid) cryogels with enhanced thermal and mechanical properties, Int. J. Mol. Sci. 18 (2017) 2714. [26] Z. Wu, X. Huang, Y.C. Li, H. Xiao, X. Wang, Novel chitosan films with laponite immobilized Ag nanoparticles for active food packaging, Carbohydr. Polym. 199 (2018) 210–218. [27] R.J. Mu, Y. Yuan, L. Wang, Y. Ni, M. Li, H. Chen, J. Pang, Microencapsulation of Lactobacillus acidophilus with konjac glucomannan hydrogel, Food Hydrocoll. 76 (2018) 42–48. [28] J. Ye, S. Wang, W. Lan, W. Qin, Y. Liu, Preparation and properties of polylactic acidtea polyphenol-chitosan composite membranes, Int. J. Biol. Macromol. 117 (2018) 632–639. [29] Y. Sert, H. Dogan, A. Navarrete, R. Somanathan, G. Aguirre, C. Cirak, Experimental FTIR, Laser-Raman and DFT spectroscopic analysis of 2,3,4,5,6-Pentafluoro-transcinnamic acid, Spectrochim. Acta A 128 (2014) 119–126. [30] H. Jia, X.R. Song, S. Liu, X. Xu, X.C. Zheng, Z.K. Peng, P. Liu, Assembly and superior performance of palladium nano-catalysts anchored to a magnetic konjac glucomannan-graphene oxide hybrid for H2 generation from ammonia borane, J. Taiwan Inst. Chem. E. 100 (2019) 137–143. [31] C. Xu, Q. Li, X.T. Hu, C.F. Wang, S. Chen, Dually crosslinked self-healing hydrogels originating from cell-enhanced effect, J. Mater. Chem. B 5 (2017) 3816–3822. [32] Y. Yuan, L. Wang, R.J. Mu, J. Gong, Y. Wang, Y. Li, J. Ma, J. Pang, C. Wu, Effects of konjac glucomannan on the structure, properties, and drug release characteristics of agarose hydrogels, Carbohydr. Polym. 190 (2018) 196–203. [33] C. Xu, X. Luo, X. Lin, X. Zhuo, L. Liang, Preparation and characterization of polylactide/thermoplastic konjac glucomannan blends, Polymer 50 (2009) 3698–3705. [34] M.L. Picchio, Y.G. Linck, G.A. Monti, L.M. Gugliotta, R.J. Minari, C.I. Alvarez Igarzabal, Casein films crosslinked by tannic acid for food packaging applications, Food Hydrocoll. 84 (2018) 424–434. [35] L. Peponi, V. Sessini, M.P. Arrieta, I. Navarro-Baena, A. Sonseca, F. Dominici, E. Gimenez, L. Torre, A. Tercjak, D. López, J.M. Kenny, Thermally-activated shape memory effect on biodegradable nanocomposites based on PLA/PCL blend reinforced with hydroxyapatite, Polym. Degrad. Stabil. 151 (2018) 36–51.

W. Lin et al. / International Journal of Biological Macromolecules 137 (2019) 982–991 [36] L. Costes, M. Aguedo, L. Brison, S. Brohez, A. Richel, F. Laoutid, Lignin fractionation as an efficient route for enhancing Polylactide thermal stability and flame retardancy, Flame Retardancy and Thermal Stability of Materials 1 (2018) 14–24. [37] T. Chen, P. Shi, J. Zhang, Y. Li, T. Duan, L. Dai, L. Wang, X. Yu, W. Zhu, Natural polymer konjac glucomannan mediated assembly of graphene oxide as versatile sponges for water pollution control, Carbohydr. Polym. 202 (2018) 425–433. [38] L.H. Cheng, A. Abd Karim, C.C. Seow, Characterisation of composite films made of konjac glucomannan (KGM), carboxymethyl cellulose (CMC) and lipid, Food Chem. 107 (2008) 411–418. [39] F. Garavand, M. Rouhi, S.H. Razavi, I. Cacciotti, R. Mohammadi, Improving the integrity of natural biopolymer films used in food packaging by crosslinking approach: a review, Int. J. Biol. Macromol. 104 (2017) 687–707. [40] R. Suriyatem, R.A. Auras, P. Rachtanapun, Improvement of mechanical properties and thermal stability of biodegradable rice starch-based films blended with carboxymethyl chitosan, Ind. Crop. Prod. 122 (2018) 37–48.

991

[41] D. Briassoulis, A. Giannoulis, Evaluation of the functionality of bio-based food packaging films, Polym.Test. 69 (2018) 39–51. [42] A. Mousavi Khaneghah, S.M.B. Hashemi, S. Limbo, Antimicrobial agents and packaging systems in antimicrobial active food packaging: an overview of approaches and interactions, Food Bioprod. Proc. 111 (2018) 1–19. [43] X. Li, F. Jiang, X. Ni, W. Yan, Y. Fang, H. Corke, M. Xiao, Preparation and characterization of konjac glucomannan and ethyl cellulose blend films, Food Hydrocoll. 44 (2015) 229–236. [44] C. He, Q. Chen, M.A. Yarmolenko, A.A. Rogachev, D.G. Piliptsou, X. Jiang, A.V. Rogachev, Structure and antibacterial activity of PLA-based biodegradable nanocomposite coatings by electron beam deposition from active gas phase, Prog. Org. Coat. 123 (2018) 282–291.