Preparation of coaxial polylactic acidâpropyl gallate electrospun fibers

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Functional Nanostructured Materials (including low-D carbon)

Preparation of coaxial polylactic acid–propyl gallate electrospun fibers and the effect of their coating on salmon slices during chilled storage Ting Ding, Tingting Li, and Jianrong Li ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b00461 • Publication Date (Web): 22 Jan 2019 Downloaded from http://pubs.acs.org on January 28, 2019

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ACS Applied Materials & Interfaces

Preparation of Coaxial Polylactic Acid–propyl Gallate Electrospun Fibers and the Effect of Their Coating on Salmon Slices During Chilled Storage Ting Dinga, Tingting Lib, Jianrong Lia*, c a

School of Food Science and Technology, Jiangnan University, Wuxi 214122, China

b

Key Laboratory of Biotechnology and Bioresources Utilization (Dalian Minzu

University), Ministry of Education, Dalian, Liaoning, 116600. China c College

of Food Science and Technology, Bohai University; Food Safety Key Lab of

Liaoning Province; National & Local Joint Engineering Research Center of Storage, Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products; Jinzhou, Liaoning, 121013, China *Correspondence: Professor Jianrong Li, College of Food Science and Technology, Bohai University, Jinzhou, Liaoning, China. Tel: +86-416-3400008; Email: [email protected]

Key words: Electrospinning; quorum sensing; P. fluorescens; propyl gallate; yield factor

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ABSTRACT Pseudomonas fluorescens bacteria can grow well in cold-storage conditions and cause food spoilage. Quorum sensing (QS) is a biological pathway existing in a large number of microorganisms, through which bacteria regulate several of their physiological activities. A number of substances have been identified as quorum sensing inhibitors (QSIs); they can interfere with the QS system and control bacterial spoilage characteristics and production of virulence factors. In our previous study, propyl gallate at sub-minimum inhibitory concentration (sub-MIC) levels showed a potent anti-QS activity. Thus, in this study, coaxial polylactic acid (PLA)–propyl gallate electrospun fibers were fabricated, and their physicochemical properties were characterized. Salmon slices were coated with these electrospun fibers and the effect of coating on the salmon slices during chilled storage was evaluated. The results showed that the electrospun fibers had a small diameter and smooth surface with no beads or other defects. The thermal stability, tensile strength, and other properties of the fibers were suitable for refrigerated storage conditions. Without inhibiting the bacterial growth in the salmon slices, the QSI-containing electrospun fibers exerted a significant inhibitory effect on the production of total volatile base nitrogen (TVB-N) and trimethylamine (TMA). Furthermore, the deterioration of muscle tissue in the salmon slices was significantly delayed during cold storage. A quantitative analysis indicated that the electrospun fibers had a significant inhibitory effect on the bacterial spoilage ability. The results suggested that the electrospun fibers loaded with QSIs might be an effective strategy to control food spoilage and enhance the quality of aquatic food products. 1. INTRODUCTION Aquatic foods are highly appreciated by consumers worldwide owing to their low fat and high protein and vitamin content, among other properties. However, these very qualities of aquatic food products render them highly susceptible to spoilage during harvesting, transportation, processing, and storage; in addition, the presence of moisture, easy oxidation of unsaturated fatty acids, loose muscle tissue, neutral pH 2


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value, and the presence of lysozymes result in huge economic losses 1-2. Pseudomonas fluorescens (P. fluorescens) are specific spoilage organisms occurring in aerobic environments, especially in refrigerated aquatic food products. They can not only secrete extracellular proteases to break down proteins in food, but also produce a large number of biofilms on the food material. Biofilms are extracellular polymers that can protect bacteria and induce drug resistance, which makes food harmful if consumed. Quorum sensing (QS) is a cell-to-cell communication pathway through which bacteria stay connected with their neighbors. Bacteria can supervise their population density by secreting low molecular weight signals called autoinducers (AIs)

3-5,

such

as N-acyl-homoserine lactones (AHLs), oligopeptides, diffusible signal factor (DSF) 6, 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) 7, diketopiperazines (DKPs) 8, furanosyl borate diester (AI-2) 9, 3-hydroxypalmitic acid methyl ester 10, and hydroxyl-palmitic acid methyl ester (PAME)

11.

Most Gram-negative and Gram-positive bacteria use

AHLs and oligopeptides, respectively, as their signal molecules

12-14. 15-16.

Studies

have shown that a number of cellular functions, such as the production of extracellular protease, biofilm formation, and swimming and swarming motility, are regulated by the QS system. Therefore, finding quorum sensing inhibitors (QSIs) to interfere with the QS might be a possible way to inhibit bacterial resistance, biofilm formation, and prolong the shelf life of food products 17-19. Propyl gallate is an antioxidant widely used in foods to inhibit the oxidation and rancidity of oils and fats. We have found that propyl gallate shows potent anti-QS activity under sub-minimum inhibitory concentration (sub-MIC) levels. Unfortunately, its instability to light and easy decomposition limit its bioavailability. However, this problem may be solved using the coaxial electrospinning technology. Through coaxial electrospinning, fibers with a small diameter, high porosity, and large specific surface area can be fabricated. More importantly, coaxial electrospinning results in the formation of fibers with a core–shell structure. Various difficult-to-electrospin-alone chemicals, such as drugs, proteins, magnetic particles, activators, and DNA, can be loaded in the core of the fibers and degradable polymers, such as polycaprolactone (PCL) and polylactic acid (PLA), can be used as shell materials 3


20-22 23.

Coaxial


electrospun fibers can act as a barrier to drugs and form a drug-release system; thus, they can prevent changes in the biological activities of the core materials. Further, the drug release rate can be controlled by adjusting the decomposition rate of the shell fibers

24.

Therefore, the coaxial electrospinning technology has broad application

prospects. In this study, we prepared PLA–propyl gallate coaxial electrospun fibers and analyzed their properties. Later, they were used to coat salmon slices during chilled storage and the effects of coating were evaluated. 2. EXPERIMENTAL SECTION 2.1 Preparation of coaxial electrospun-fiber membranes PLA (molecular weight = 100,000) was dissolved in a mixed solvent (dimethylformamide: dichloromethane = 1:1) at a concentration of 9% (w/v) and this solution was used as the shell electrospinning solution. Propyl gallate was dissolved in 70% methanol at a concentration of 1.25 mg/mL, and this solution was used as the core electrospinning solution. The shell electrospinning solution was stirred using a magnetic stirrer at 25 °C for 12 h, after which it was subjected to ultrasonic treatment for 2 h to remove air bubbles. The shell and core electrospinning solutions were then loaded into two syringes (10 mL) and placed in an electrospinning equipment (Elite electrospinning equipment, Beijing Yongkang Le Ye Technology Development Co., Ltd.). The parameters of the coaxial electrospinning process were as follows: core and shell electrospinning solution velocities of 0.005 mm/min and 0.2 mm/min, respectively, positive voltage of 25.74 kV, negative voltage of 1.78 kV, receiving distance of 20 cm, room temperature, relative humidity of 50%, coaxial needle: 18G/25G, and drum speed of 20 r/min. PLA uniaxial electrospun fibers were used as the blank control. The parameters of uniaxial electrospinning were as follows: positive voltage of 7.5 kV, negative voltage of 1.77 kV, receiving distance of 14 cm, and 21 uniaxial needles. A silver paper was used as the receiver. The injection time was approximately 1 h, after which the receiver was found to be paved evenly with

4


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the fibers. After electrospinning, the fibers were dried in a vacuum oven for 24 h and their properties were characterized. 2.2 Characterization of the electrospun fibers 2.2.1 Morphology characterization The prepared electrospun fibers were cut into 3 mm × 3 mm pieces and their morphological features were observed by scanning electron microscopy (SEM) (S4800, Hitachi, Japan) after gold-spraying treatment. The electrospun fibers were also analyzed by transmission electron microscopy (TEM) (FEI Tecnai TM G2 Spirit BioTWIN, USA) to observe their core–shell structure. 2.2.2 Chemical structure analysis The fibers were cut into debris, ground with KBr, and pressed into tablets. Later, these sample tablets were analyzed by Fourier-transform infrared spectroscopy (FTIR) (Alpha-Centauri 560; Nicolet Company, America) in the wavenumber range of 400–4000 cm–1. 2.2.3 Tensile strength testing The electrospun fiber mats were separated from the silver paper slowly and cut into 70 mm × 25 mm samples. The tensile strength of the electrospun fibers was measured using a tensile probe on the texture analyzer (TA. XT-plus, Stable Micro Systems Company, England). The test parameters were as follows: test length of 40 mm, tensile rate of 5 mm/min, and the room temperature was set at the test temperature. From these tests, the tensile strength (σt) and elongation at break (εt) values of the electrospun fibers were calculated using formula (1) and formula (2), respectively. The experiments were repeated thrice to obtain the average values. σt = P/(b × d)

(1)

where σt is the tensile strength (MPa), P is the tensile force at break (N), b is the sample width (mm), and d is the sample thickness (mm). Elongation at break εt = (L–L0)/L0 × 100 (%)

(2)

In this formula, L0 represents the original length of the sample (mm) and L is the length at break (mm). 2.2.4 Hydrophobicity analysis 5



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The electrospun fiber membranes were cut into 2 cm × 2 cm pieces and tested on a contact angle measuring instrument (OCA20, Dataphysics Company, Germany) to evaluate the hydrophobicity at room temperature. Water droplets (4 μL) were used as the test liquid. The samples were measured at six different locations and the results were averaged. 2.2.5 Thermal property analysis Fiber samples weighing approximately 3 mg were cut into debris, ground evenly in a mortar, and then tested using a thermogravimetry-differential scanning calorimetry (TG-DSC) thermal analyzer (Pyris DIAMOND TG-DTA, PerkinElmer, America) in a nitrogen atmosphere in the temperature range of 40–600 °C; the heating rate was 10 °C/min. 2.2.6 Thickness analysis The samples were cut into 3 cm × 3 cm pieces and folded repeatedly, after which the total thickness of the sample was measured using a Vernier caliper. Each sample was measured three times and the results were averaged. Thickness of the film = Total thickness/number of layers

(3)

2.2.7 X ray diffraction (XRD) analysis The crystalline properties of the electrospun fibers were determined by XRD (Rigaku ultima IV, Neo Confucianism Co., Ltd., Japan) using Cu Kα radiation (λ = 0.15418 Å) at an operating voltage and current of 40 kV and 40 mA, respectively; the scanning speed was set at 6°/min and measurements were carried out in the 2θ range of 5° to 90°. 2.2.8 In vitro release behavior Propyl gallate was dissolved in 70% methanol and diluted by PBS, and different concentrations of the propyl gallate solution were prepared. Then, the absorbance of propyl gallate was determined using an ultraviolet spectrophotometer. A linear regression analysis was conducted with respect to the concentration and absorbance of propyl gallate and fitting equations were obtained. Ten milliliters of PBS phosphate buffer (pH = 7.4) were added to an Erlenmeyer flask containing 10 mg of the electrospun fibers and the flask was then transferred to a constant-speed oscillator (4 6


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°C). Quantitative liquid samples were taken out periodically to determine the absorbance of the released propyl gallate. At the same time, an equal amount of the release medium was added to the flask. The drug concentration was calculated from the standard curve, using which the cumulative drug release was calculated. Each test was repeated three times and the average values were obtained. 2.3 Determination of the MIC and QS inhibitory activity of propyl gallate The MIC of propyl gallate and its QS inhibitory activity were determined according to the protocols described in the literature 25. Chromobacterium violaceum CV026 and Agrobacterium tumefaciens A136 were used as the indicator strains to identify whether the compound possessed a QS inhibitory activity. C. violaceum CV026 produces violacein in the presence of exogenous short-chain AHLs, but it cannot synthesize AHLs endogenously β-galactosidase,

26.

A. tumefaciens A136 produces

which

breaks

5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside

(X-Gal)

down when it

encounters

exogenous long-chain AHLs, leading to a blue color in the culture 27. Methanol (70%) served as control. 2.4 Coating effects of the electrospun fibers on the quality of salmon slices during cold storage 2.4.1 Preparation of the P. fluorescens P07 bacterial suspension P. fluorescens P07 was stored at −80 °C in a sterile refrigerator in our laboratory. The bacteria were cultured for 12–18 h until the concentration reached a value of 108 CFU/mL (160 rpm, 28 °C). The bacterial liquid was centrifuged (at 4000 r/min for 10 min), and subsequently, the supernatant was removed, and the cells were re-suspended in sterile normal saline (0.85%, w/v) at a concentration of 106 CFU/mL. The bacterial suspension thus prepared was stored at 4 °C until further use. 2.4.2 Preparation of aseptic salmon slices and determination of the spoilage ability of P. fluorescens P07 Ice-fresh Norwegian salmon (it takes about 3 days to transport salmon from Norway to China) was purchased from the JinZhou LinXi aquatic product market. The salmon was transported to the laboratory under temperature-controlled conditions 7



by layering with flaked ice. Later, the salmon was peeled and the surfaces were wiped with 75% alcohol to reduce the number of colonies to less than 102 CFU/mL. Subsequently, the salmon slices were immersed in the prepared bacterial suspension for 10 s and drip dried. The experiment was divided into four groups. The first group was coated with a UV-sterilized (15 min) PLA–propyl gallate electrospun fiber membrane. Non-coated salmon slices were considered as the second group (control group). The salmon slices of the third group were immersed in 1.25 mg/mL of propyl gallate for 10 s and drip dried. The salmon slices of the fourth group were coated with UV-sterilized (15 min) PLA uniaxial electrostatic electrospun fibers. The salmon slices of all groups were placed on sterilized glass slides, wrapped with a sterilized preservative film, and stored at 4 °C. The number of P. fluorescens P07, muscle tissue status, total volatile basic nitrogen (TVB-N), and trimethylamine (TMA) content were determined regularly, and the spoilage ability of P. fluorescens P07 was quantitatively analyzed. 2.4.3 Determination of TVB-N Salmon fillets (10 g), MgO (1 g), dH2O (50 mL), and 3 drops of n-octanol were placed in a distillation tube and distilled. The distillate was titrated, and the TVB-N content was calculated using a FOSS Kjeldahl apparatus. 2.4.4 Determination of TMA The TMA content was determined according to the GB/T 5009.179-2016 standard 28. Briefly, 10 g (accurate to 0.001 g) of shredded salmon slices and 20 mL of 5% trichloroacetic acid were added to a 50-mL centrifuge tube. The sample was homogenized for 1 min, centrifuged for 5 min (4000 r/min), and filtered. The residue was extracted twice with 15 mL and 10 mL of 5% trichloroacetic acid sequentially. The filtrates were combined and fixed to 50 mL with 5% trichloroacetic acid. Five milliliters of 50% NaOH were added to 2 mL of the extraction solution, after which the TMA content was determined by gas chromatography-mass spectrometry (GC-MS). The chromatographic conditions were as follows. Quartz capillary column: 30 m × 0.25 mm × 0.25 μm; stationary phase: polyethylene glycol; carrier gas: high-purity 8


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helium; flow rate: 1.0 mL/min; inlet temperature: 220 °C. Split ratio: 10:1; temperature program: 40 °C for 3 min, followed by heating at 30 °C/min to 220 °C and equilibrating for 1 min. Mass spectrum conditions: EI source; 220 °C; ionizing energy: 70 eV; transmission line temperature: 230 °C; solvent delay: 1.5 min; scanning mode: selective ion scanning (SIM). 2.4.5 Growth curve of P. fluorescens P07 The growth curve of P. fluorescens P07 was determined according to a previously reported method

29

with slight modifications. Approximately 25 g of the

fish samples were homogenized in 225 mL of normal saline (0.85%, w/v) for 1 min and then decimally diluted with sterile NaCl. Three dilutions were selected, and 1-mL aliquots of each solution were transferred to Petri dishes (in triplicate). Approximately 20 mL of plate count agar (PCA, AoBoXing Bio-Tech, Beijing, China) was dumped into the dishes. One branch of a CFC-selective medium additive was added to the medium when the temperature dropped to 50 °C. The number of colony-forming units was calculated after incubation at 30 °C for 72 h. 2.4.6 Quantitative analysis of the spoilage ability of P. fluorescens P07 The spoilage metabolites produced by unit spoilage bacteria are used as quantitative indicators of spoilage ability. The yield factors of TVB-N and TMA, i.e., YTVB-N/CFU and YTMA/CFU, respectively, were used as the quantitative indices of the spoilage ability of P. fluorescens P07 in chilled salmon slices. The formulae corresponding to the yield factors are as follows:

YTVB  N / CFU 

YTMA / CFU 

(TVB  N ) s  (TVB  N ) 0 Ns  N0

(4)

(TMA )s (TMA )0 N s N 0

(5)

YTVB-N/CFU and YTMA/CFU represent the yield factors of TVB-N and TMA (mg TVB-N/CFU)/(mg TMA/CFU), respectively; N0 and Ns represent the number of spoilage bacteria at the beginning and end of the food shelf life, respectively (cfu/g); (TVB-N)0 and (TMA)0 indicate the contents of TVB-N and TMA, respectively, at the

9



beginning point (mg/100g); (TVB-N)s and (TMA)s refer to the contents of TVB-N and TMA, respectively, at the end point (mg/100g). 2.4.7 Muscle tissue observation The salmon muscles were cut into 3 cm × 3 cm × 3 cm pieces and stored at −20 °C for 30 min; later, the pieces were cut into 20-μm slices using a freezing slicer and stained according to the instructions provided with the HE (hematoxylin violet-eosin) staining kit. The slices were placed on glass slides for microscopic observation (Ti-S Inverted microscope Nikon, Japan) and analyzed. 2.5 Statistical analysis The results were expressed in the form of mean ± standard deviation (SD) and analyzed by one-way ANOVA of SPSS Statistics 16.0 (SPSS, Inc.). A significance level of p < 0.05 was considered. All the tests were repeated thrice.

3. RESULTS AND DISCUSSION 3.1 MIC of propyl gallate and violacein inhibition and β-galactosidase assay results

Fig. 1 (A) Violacein inhibition assay and (B) β-galactosidase assay of propyl gallate at sub-MIC levels The MIC of propyl gallate was determined on indicator strains (C. violaceum CV026 and A. tumefaciens A136) and P. fluorescens P07 to ensure that its QS inhibitory activity was due to interference with the signaling system of bacteria rather than the antibacterial activity. The results showed that the MIC levels of propyl gallate on C. violaceum CV026, A. tumefaciens A136, and P. fluorescens P07 were 2.25, 2.25, and 2.5 mg/mL, respectively. Thus, further assays were carried out on propyl gallate at sub-MIC levels. As shown in Fig. 1, the plates were purple, 10


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indicating C. violaceum CV026, and blue, indicating A. tumefaciens A136. A yellow and opaque halo appeared in the plates when injected with propyl gallate below sub-MIC levels, suggesting that propyl gallate showed anti-QS activity; on the contrary, 70% methanol, which was used as control, did not exhibit any anti-QS activity. The results also showed that propyl gallate could inhibit the production of both short-chain and long-chain AHLs. 3.2 Preparation and characterization of electrospun fibers 3.2.1 Morphology of the electrospun fibers

Fig. 2 (A) Photograph of a PLA–gallate coaxial electrospun fiber mat (30 cm × 30 cm), (B) SEM image of PLA uniaxial electrostatic electrospun fibers, (C) SEM image of PLA–propyl gallate coaxial electrospun fibers, and (D) TEM image of PLA–propyl gallate coaxial electrospun fibers Fig. 2A shows a photograph of the coaxial PLA–propyl gallate electrospun fiber mat. The surfaces of the fibers were even and smooth and the membrane could be easily separated from the silver paper. Fig. 2B and Fig. 2C depict the SEM images of uniaxial PLA electrospun fibers and coaxial PLA–propyl gallate electrospun fibers, respectively. The diameters of the uniaxial and coaxial electrospun fibers were uniform and small; the direction of the fibers was random, and they interlaced to form a network structure. The surfaces of the fibers were smooth and there were no beads or defects. As shown in Fig. 2D, the coaxial PLA–propyl gallate electrospun fibers 11



exhibited an obvious core–shell structure. 3.2.2 FTIR and XRD analysis of the electrospun fibers

Fig. 3 FTIR spectra of (A) PLA uniaxial electrostatic electrospun fibers and (B) PLA–propyl gallate coaxial electrospun fibers. XRD patterns of (C) PLA uniaxial electrostatic electrospun fibers and (D) PLA–propyl gallate coaxial electrospun fibers Fig. 3A illustrates the infrared spectrum of PLA uniaxial electrospun fibers. In the spectrum, a strong absorption peak was observed at 1761.0 cm–1, which was caused by C=O stretching vibrations; the peaks at 1056.99, 1089.78, and 1228.66 cm–1 were characteristic of ether bond vibrations (-C-O-stretching). The peak at 1435.04 cm–1 corresponded to the bending vibrations of -CH3, while that at 1373.32 cm–1 corresponded to the bending vibrations of -CH. In the infrared spectrum of coaxial PLA–propyl gallate electrospun fibers (Fig. 3B), the peak at 3626.17 cm–1 could be attributed to hydroxyl (-OH) stretching vibrations; the peak at 2862.36 cm–1 was ascribed to C-H stretching vibrations, and the peak at 1764.87 cm–1 represented the stretching vibrations of carbonyl (C=O) groups. Meanwhile, the peaks at 1450.47, 1496.76, and 1597.06 cm–1 represented the stretching vibrations of the double bonds 12


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of benzene rings (C=C); further, the peak at 1228.66 cm–1 was characteristic of ether bonds (-C-O-C- stretching vibrations). The FTIR results indicated that the PLA–propyl gallate fibers contained all the functional groups of propyl gallate, including the benzene ring, carbonyl groups, and hydroxyl groups. In addition, the characteristic peaks of PLA appeared at 1759.08 cm–1 (C=O stretching vibrations), while the peaks at 1055.06, 1090.71, and 1228.65 cm–1 were characteristic absorption peaks of PLA (-C-O- stretching vibrations). The bending vibration absorption peak at 1435.04 cm–1 was ascribed to the -CH3 groups and the bending vibration peak at 1373.32 cm–1 was attributed to -CH groups. The FTIR data indicated that PLA used as the shell material could encapsulate the core material (propyl gallate) well without affecting its structure. Similarly, the core materials did not affect the structure and properties of the shell materials; the two materials mainly underwent physical mixing. An XRD analysis can be used to identify the different phases present in a polymer along with their features, including the crystalline and amorphous states, crystal type, crystallinity, and grain size. PLA is a polycrystalline polymer with four crystalline forms – α, α’, β, and γ. Of these, α is the most common and stable crystalline form. In the XRD pattern of PLA, the main characteristic peaks of α crystals appeared at 2θ = 15°, 17°, 19°, 29°, 31°, and 32°, which corresponded to the (010), (110)/(200), (203), (216), (0010), and (1010) growth surfaces, respectively. Meanwhile, the diffraction peaks of β crystals mainly occurred at 2θ = 25°, 26.5°, 27.9°, 29.8°, and 31°. Fig. 3C shows the XRD pattern of PLA uniaxial electrospun fibers. It showed a wide diffraction peak at 2θ = 21.60°. This is a characteristic diffraction peak of PLA, which showed crystalline characteristics. The crystalline peaks of PLA electrospun fibers experienced a significant deviation, suggesting that the high-voltage current employed affected the crystalline structure of PLA electrospun fibers. The diffraction peaks of PLA electrospun fibers were wide, indicating that the crystallinity of PLA electrospun fibers reduced. Fig. 3D shows the XRD pattern of coaxial PLA–propyl gallate electrospun fibers, which exhibited diffraction peaks at 2θ = 28.71° and 32.56°. The two peaks corresponded to the characteristic diffraction peaks of α crystals. However, the width and sharpness of the 13



diffraction peaks decreased, indicating that the crystallinity of PLA electrospun fibers decreased when propyl gallate was used as the core material; this was also evidence of the successful loading of propyl gallate into PLA electrospun fibers. 3.2.3 Thermal properties, tensile strength, thickness, and hydrophobicity of the electrospun fibers

Fig. 4 TG-DSC of (A) PLA uniaxial electrostatic electrospun fibers and (B) PLA–propyl gallate coaxial electrospun fibers (Exo: exothermic; Endo: endothermic). Tensile strength analysis of (C) PLA uniaxial electrostatic electrospun fibers and (D) PLA–propyl gallate coaxial electrospun fibers The thermal properties of the electrospun fibers were analyzed by TG-DSC testing, as shown in Fig. 4A and 4B. The changes in the mass fraction of the electrospun fibers with respect to the temperature in a N2 atmosphere are illustrated in Fig. 4. It can be seen in Fig. 4A that the initial decomposing temperature of PLA fibers was approximately 270 °C and the half decomposing temperature (T50%) was approximately 390 °C. The pyrolysis of PLA electrospun fibers was divided into two parts. The first part occurred in the range of 270 to 430 °C, in which the mass decreased sharply. This was the first weight-loss platform and the weight-loss rate was 87.8%. Subsequently, there occurred the platform area of the TG curve, which 14


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was also representative of the thermal stability of the sample. The second part occurred in the range of 460 to 520 °C; in this range, the TG curve descended gently; this area was referred to as the second weight-loss platform (the weight-loss rate was 7.3%). The TG curve of the sample did not change when the temperature rose to 520 °C, indicating that the samples tended to be stable and did not decompose. Fig. 4B shows the TG-DSC result of coaxial PLA–propyl gallate electrospun fibers. The thermal decomposition behavior of the fibers wrapped around propyl gallate was similar to that of PLA uniaxial electrospun fibers. The first weight-loss platform occurred at approximately 270 to 420 °C and the weight loss rate was 89.9%. The second weight-loss platform occurred in the range of 460 to 520 °C; in this area, the TG curve decreased more smoothly, and the weight loss rate was approximately 5%. The TG curve no longer changed and the sample no longer decomposed when the temperature was greater than 520 °C. Compared to the PLA electrospun fibers, the weight-loss rate of the PLA–propyl gallate electrospun fibers was higher in the first weight-loss platform, but it was lower in the second weight-loss platform, indicating that the thermal stability of the electrospun fibers changed after the incorporation of propyl gallate. The above results showed that the core material exerted an influence on the thermal stability of the shell material. The results of the DSC analysis of the electrospun fibers are shown in Fig. 4A and 4B. There was an endothermic peak at approximately 65 °C, which corresponded to the glass-transition temperature (Tg) of PLA. A gentle endothermic peak appeared at approximately 110 °C, but the corresponding TG curve showed no weight loss; therefore, it was concluded that this temperature represented the melting temperature (Tm) of PLA electrospun fibers. There were two exothermic peaks at 390 °C and 410 °C, indicating that the sample decomposed at high temperatures following an exothermic reaction mechanism. During this period, the sample underwent a large weight loss, which corresponded to the first weight-loss platform in the TG curve. There was an obvious exothermic peak at 460 °C, which corresponded to the second weight-loss platform in the TG curve, indicating that the sample decomposed further according to an exothermic mechanism. In the DSC curve of the coaxial PLA–propyl 15



gallate electrospun fibers (Fig. 4B), it can be seen that an endothermic peak appeared at 60 °C, which corresponded to the Tg of the electrospun fibers. The melting temperature (Tm) of the PLA–propyl gallate electrospun fibers was 105 °C. The reduction in Tg and Tm indicated that the thermal properties of the PLA electrospun fibers were affected by the encapsulation of propyl gallate. An obvious exothermic peak was observed at 395 °C, indicating that the sample decomposed at this temperature according to an exothermic mechanism, which corresponded to the first weight-loss platform of the TG curve. Further, there was an obvious exothermic peak at 570 °C, which corresponded to the second weight-loss platform of the TG curve, indicating further decomposition of the sample. The tensile strengths of the electrospun fibers were analyzed, as shown in Fig. 4C and 4D. The tensile strength (σt) and elongation at break (εt) of the fibers were calculated and the results showed that the tensile strengths of PLA uniaxial and coaxial PLA–propyl gallate electrospun fibers were (1.1337 ± 0.0058) MPa and (1.6680 ± 0.0056) MPa, respectively. The elongations at break values were (24.21% ± 0.393%) and (30.57% ± 0.476%), respectively. It was also observed that the electrospun fibers were elongated and deformed with an increase in the tensile force, and the fibers broke when the tensile force increased to a certain extent. The tensile strength and elongation at break of the coaxial PLA–propyl gallate electrospun fibers were obviously higher than those of the PLA uniaxial electrospun fibers. The thickness of the fiber mats was determined, and the results showed that the thicknesses of the PLA uniaxial and coaxial PLA–propyl gallate electrospun fibers were (0.0178 ± 0.00046) mm and (0.0184 ± 0.00025) mm, respectively. The hydrophobicity of the electrospun fibers was tested and the results showed that the contact angles of the PLA uniaxial and coaxial PLA–propyl gallate electrospun fibers were (132.82° ± 8.83°) and (103.72° ± 3.52°), respectively. A static water contact angle of 90° is the critical point at which a material is classified as either hydrophilic or hydrophobic. The contact angles of the electrospun fibers were all greater than 90°, indicating their strong hydrophobicity. The hydrophobicity of the coaxial electrospun fibers decreased when they were used as shell materials to 16


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encapsulate propyl gallate. 3.2.4 Slow-release behavior of the coaxial electrospun fibers and its effect on salmon slices during chilled storage The slow-release behavior of coaxial electrospun fibers was analyzed; further, the growth curve of P. fluorescens P07 and variation in the TVB-N and TMA content in salmon slices coated with electrospun fibers were monitored, as illustrated in Fig. 5.

Fig. 5 Slow-release behavior of PLA– propyl gallate coaxial electrospun fibers and the effect of coating these fibers on refrigerated salmon slices. (A) Slow-release behavior of PLA–propyl gallate electrospun fibers. (B) Growth curve of P. fluorescens P07. Variations in (C) TVB-N and (D) TMA content Fig. 5A illustrates the cumulative release behavior of the coaxial PLA–propyl gallate electrospun fibers. The results showed that free propyl gallate was released very rapidly in the initial stages and the release rate was as high as 82.28% at 12 h. However, the release rate slowed in the later stages. On the contrary, the release of propyl gallate encapsulated in the electrospun fibers was relatively slow and there was no sudden release. The release curve indicated a slow release behavior. The drug release rate reached a value of 49.87% after 48 h and 86.46% after 192 h. Even after 17



192 h, release occurred, indicating a slow and lasting release behavior. Therefore, it could be concluded that the coaxial electrospun fibers played an effective role in drug delivery applications. Fig. 5B depicts the growth curve of P. fluorescens P07 in chilled salmon slices of the four groups. The number of P. fluorescens P07 colonies in the four groups of salmon slices increased with time; the growth trend was similar in the four cases. The number of colonies of P. fluorescens P07 in the salmon slices of the four groups were 3.89 lgcfu/g, 3.83 lgcfu/g, 4.09 lgcfu/g, and 3.74 lgcfu/g at 48 h. Subsequently, the bacteria began to multiply rapidly. The number of colonies in the four groups of salmon slices reached 6.44 lgcfu/g, 6.36 lgcfu/g, 6.64 lgcfu/g, and 6.61 lgcfu/g at 120 h, which exceeded the acceptable limit of the total number of fish colonies (5 × 105 cfu/g) and approached the maximum safety limit (107 cfu/g) as stipulated by the International Committee on Microbial Specifications (ICMSF). Fig. 5C depicts the variations occurring in the TVB-N content in refrigerated salmon slices of the four groups. It can be seen in the figure that the TVB-N content increased in the four groups with an increase in the storage time, but the TVB-N content in the salmon slices of the first group (coated with PLA–propyl gallate electrospun fibers) increased much more slowly when compared to those of the second (control) and fourth groups (coated with PLA uniaxial electrostatic electrospun fibers). The TVB-N content in the salmon slices of the third group was lower than that of the first group in the first 48 h, while it was higher than that of the first group after 72 h. In the control group, the TVB-N content increased slowly in the initial stages (48 h), after which it increased rapidly. The growth trend of the TVB-N in the salmon slices of the fourth group was similar to that of the control group. In the control group, the TVB-N content reached a value of 29.98 mg/100 g at 144 h, which was close to the hygienic standard of fresh and frozen animal aquatic products; the GB 2707-2016 standard stipulates that the content of TVB-N in seawater fish should be less than 30 mg/100 g. Compared to the control group, the TVB-N content of the salmon slices coated with PLA–propyl gallate electrospun fibers increased slowly. The TVB-N content reached a value of 11.20 mg/100 g at 96 h, which exceeded the 18


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first-grade freshness standard (TVB-N ≤ 10 mg/100 g). The TVB-N value was 16.02 mg/100 g at 144 h, which was much lower than that of the control group; it decreased by 46.56% when compared to the control group. Fig. 5D illustrates the variations in the TMA content in refrigerated salmon slices of the four groups; it can be seen that while the TMA content in the fresh salmon slices was low, it increased with an increase in the storage time. The TMA content in the salmon slices increased more rapidly in the second (control group) and fourth groups (coated with PLA uniaxial electrostatic electrospun fibers) than in the first group; the TMA content of the control group was 13.82 mg/100 g, while that in the first group was just 6.73 mg/100 g at 144 h. The TMA content in the salmon slices of the third group (treated with propyl gallate under sub-MIC) was lower than that of the first group before 72 h, but it was higher than that of the first group after 72 h during cold storage. 3.2.5 Quantitative analysis of the spoilage ability of P. fluorescens P07 Table 1 lists the values of YTVB-N/CFU and YTMA/CFU of P. fluorescens P07 in salmon slices. Table 1 Yield factors of P. fluorescens P07 in the salmon slices Group PLA–propyl gallate electrospun fibers Control Propyl gallate PLA uniaxial electrostatic electrospun fiber

YTVB-N/CFU (mgTVB-N/CFU) 3.225 × 10–8 8.818 × 10–8 4.755 × 10–8 8.228 × 10–8

YTMA/CFU (mgTMA/CFU) 1.392 × 10–8 4.677 × 10–8 2.037 × 10–8 4.343 × 10–8

As listed in Table 1, the YTVB-N/CFU and YTMA/CFU of the control group were 8.818 × 10–8 mgTVB-N/CFU and 4.677 × 10–8 mgTMA/CFU, respectively. The yield factors, YTVB-N/CFU and YTMA/CFU, of P. fluorescens P07 in salmon slices coated with PLA–propyl gallate electrospun fibers were lower than those in the other groups, indicating that the PLA–propyl gallate electrospun fiber inhibited the spoilage ability of P. fluorescens P07. 3.2.6 Muscle tissue observation of salmon slices coated and uncoated with PLA–propyl gallate electrospun fibers 19



Fig. 6 Muscle tissue changes in refrigerated salmon slices (A) Salmon slices coated with PLA–propyl gallate electrospun fibers; (B) Control group; (C) Salmon slices treated with propyl gallate; (D) Salmon slices coated with PLA uniaxial electrostatic electrospun fibers. Pictures from left to right were taken at 0, 48, 96, 144, and 192 h. Fig. 6 shows the changes occurred in salmon muscle tissues of the four groups during cold storage. The muscle tissues were arranged tightly and neatly; further, the muscle fibers were connected in a network structure with a few pores, indicating that the salmon slices were fresh in the early stages. Large pores appeared in the muscle fibers in the control group over time. Fragmentation started to occur and the fibers became loose at 96 h. Subsequently, the muscle tissues distorted and fractured, and irregular fragments were observed in the control group in the later stages of cold storage. Besides, the muscle tissue became soft and its hardness reduced. The breakdown rate of the muscle tissue structure of salmon slices coated with PLA uniaxial electrostatic electrospun fibers was similar to that of the control group. Meanwhile, the breakdown rate of the muscle tissue structure of salmon slices treated with propyl gallate was slower than that of the control group. Among the four groups, the breakdown rate of the muscle tissue structure of salmon slices coated with PLA–propyl gallate electrospun fibers was the slowest. Large pores appeared in the muscle tissue at 96 h and the muscle fibers appeared aggregated and broke down at 20


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192 h. The results suggested that the PLA–propyl gallate electrospun fibers could effectively delay the rate of deterioration of the muscle tissue of salmon slices. Discussion Turbot (Scophthalmus maximus) is noted for its delicious and nutritious meat, which is favored by consumers worldwide. It is also an important marine economic fish in China. In this study, P. fluorescens P07 was isolated from deteriorated turbot during cold storage. Therefore, interfering with the bacterial QS system may be a promising approach for the preservation of refrigerated aquatic products. P. fluorescens are major spoilage bacteria often found in refrigerated aquatic food products. They can produce heat-resistant proteases and esterases to decompose proteins in food, resulting in the spoilage of aquatic products during refrigeration and sensory unacceptability. Because some types of P. fluorescens have developed resistance to ampicillin and streptomycin

30,

new methods are urgently needed to

control food spoilage and the virulence factor of P. fluorescens. Controlling the QS system may be a feasible strategy to solve this problem without causing bacterial resistance. A large number of compounds with QS inhibitory activity have been discovered. However, some of them have various shortcomings, such as water insolubility, easy decomposition, and oxidation, which limit their application in aquatic food-product preservation. Using the electrospinning technology, fibers with a core–shell structure can be prepared, which can compensate the application limitations of QSIs. PLA is an environmentally friendly material with good biodegradability; it can be degraded into carbon dioxide and water by microorganisms in nature. In addition, PLA also has good tensile strength, air permeability, and oxygen permeability. Most importantly, the human body also contains lactic acid in the form of monomers and no serious acute tissue reaction or toxicological reaction was found in clinical trials on PLA

31.

Therefore, PLA is deemed to be highly safe. Its nontoxic and non-irritating

properties make PLA a great biomaterial. Wen et al. prepared PLA electrospun fibers containing a cinnamon essential oil/β-cyclodextrin inclusion complex (CEO/β-CD-IC). They found that the fibers exhibited good antimicrobial activity against both 21



Gram-positive and Gram-negative bacteria. Besides, the fibers could prolong the shelf-life of pork 32. In this study, we prepared PLA–propyl gallate electrospun fibers and explored their preservation effects on salmon slices. The compounds in these materials were analyzed qualitatively and quantitatively by FTIR. The FTIR results showed that PLA as the shell material did not affect the activity and structure of propyl gallate, which is generally easy to oxidize. Thus, the shell material could protect the bioactivity of the core material. This may be because the shell and core solutions came into contact only transitorily in the coaxial needle, after which the core material was immediately wrapped in the shell material under the action of a high electrostatic voltage during the electrospinning process. This reduced the effect of the external environment on the structure and biological activity of the core material. Crystallization plays a key role in controlling the properties of polymers. An XRD analysis showed that the crystallization peak of the PLA electrospun fibers experienced a shift, possibly due to the electric field applied during the electrospinning process. In the electrospinning process, curing of the fibers occurred rapidly and there was no time for crystalline structure formation, which led to imperfections in the lattice of the electrospun fibers. Therefore, they exhibited a low degree of crystallization. The positions of the crystalline peaks of the fibers remained unchanged after propyl gallate was encapsulated, indicating that the addition of propyl gallate did not change the crystalline nature of the shell material. However, the width and sharpness of the diffraction peaks decreased, indicating that the crystallinity of the fibers decreased after coating with propyl gallate. The crystallinity of the coaxial electrospun fibers was lower than that of uniaxial electrospinning fibers, which may be because the core material interacted with the shell material. These interactions destroyed the hydrogen bonds and van der Waals forces between the molecules of the shell material, disturbing its original crystallization behavior, leading to gradual changes in the shell material from a crystalline state to an amorphous state. TG and DSC tests were conducted to characterize the thermal properties of the electrospun fibers. The rate of weight loss in the electrospun fibers was high; a large 22


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mass loss was observed in the first weight-loss platform, which was caused by the oxidative decomposition of organic matter. Compared to PLA uniaxial electrospun fibers, the rate of weight loss in the coaxial PLA–propyl gallate electrospun fibers in the first weight-loss platform was higher, indicating that the thermal stability of the fibers changed after propyl gallate encapsulation. This may be because during the process of thermal decomposition, the surface materials first decomposed and produced volatile small molecules. The rate of volatilization and diffusion of these substances is positively correlated with the pyrolysis rate of polymer materials 33. The interactions between small molecules and polymer chains can accelerate the pyrolysis rate of the polymer 33. The thermal movement in the polymer increased, accelerating the escape of small molecular products after QSI encapsulation. This in turn accelerated the heat transfer and pyrolysis rate, resulting in an increase in the weight-loss rate in the first weight-loss platform. The first weight loss of the electrospun fibers was attributed to the decomposition of organic matter, while the second weight-loss platform was due to further oxidation of the electrospun fibers, decomposition of organic matter, and a breakdown of the macromolecular chains. After reaching a certain temperature, the TG curve remained unchanged, indicating that the water and organic matter in the fibers decomposed completely and volatilized; at this point, the remaining products were inorganic. The DSC results showed that the Tm of PLA electrospun fibers was lower than that of PLA, which may be due to the large specific surface area of the electrospun fibers and fast heat transfer during the heating process. The Tg and Tm values of coaxial PLA–propyl gallate electrospun fibers were lower than those of PLA uniaxial electrospun fibers, which may be because the molecular chain space structure of the polymer changed after QSI encapsulation, resulting in a reduction in the degree of crystallinity and an increase in the amorphous content. The mechanical properties of the electrospun fibers were characterized in terms of their tensile strength and elongation at break. PLA exhibited good tensile strength and high brittleness. The tensile strength and elongation at break of coaxial PLA–propyl gallate electrospun fibers were higher than those of uniaxial PLA 23



electrospun fibers; the original brittle fracture property of PLA changed after wrapping small molecular QSIs. In addition, PLA had good tensile strength and the thickness of coaxial electrospun fibers was larger than that of uniaxial PLA electrospun fibers. Thus, coaxial electrospun fibers could not be easily broken, resulting in an increase in the tensile strength and elongation at break. The thickness of the electrospun fibers was measured and the results showed that the coaxial electrospun fibers were thicker than uniaxial electrospun fibers. During the preparation of coaxial electrospun fibers, the addition of the core material led to an increase in the amount of material ejected from the needle per unit time, resulting in an increase in the diameter of the coaxial electrospun fibers. Contact-angle measurement is the most commonly used method to characterize the hydrophobicity of a material. The larger the contact angle is, the stronger the hydrophobicity of the material. If the contact angle is less than 90°, the material is considered hydrophilic; if the contact angle is greater than 90°, the material is hydrophobic. The results showed that the contact angles of the electrospun fibers were greater than 90°, indicating that they were hydrophobic. The hydrophobicity of coaxial electrospun fibers decreased to some extent after wrapping different core materials, which may be because water was more likely to enter the hydrophobic electrospun fibers due to the small QSIs, thus leading to a decrease in the hydrophobicity. From the cumulative release rate curve of the electrospun fibers, it was found that the release rate of the QSI-coated fibers was slower than that of free QSI and there was no obvious sudden release. This was because the QSI was wrapped in the core layer of the hydrophobic coaxial electrospun fibers, which were not very soluble in the buffer solution. Water molecules need to penetrate into the fibers before dissolving the QSI and the core material needs to pass through the shell material to be released. Thus, it was difficult to release the QSI quickly in the fibers. In addition, the electrospun fibers degraded under the action of microorganisms and enzymes, resulting in a slow release of QSI, which continued with an increase in time. The number of P. fluorescens P07 colonies increased with storage time, 24


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indicating that P. fluorescens P07 grew well under cold-storage conditions. The growth curves of bacteria in the salmon slices in the four groups exhibited a similar trend, indicating that the QSI released from the electrospun fibers had no antibacterial effect on P. fluorescens P07. TVB-N is an important index to evaluate the freshness of meat according to the Chinese food hygiene standards. Proteins in food are decomposed by enzymes and microorganisms, and volatile basic nitrogen-containing substances, such as ammonia, DMA (dimethylamine), and TMA (trimethylamine) 34-35 are produced. The higher the TVB-N value is, the lower the freshness of the food. Fish is considered to have deteriorated when the TVB-N content reaches a value of 30 mg/100 g. The TVB-N value of salmon slices coated with PLA–propyl gallate electrospun fibers increased slowly, much lower than that of the control group, indicating that the QSI released from the coaxial electrospun fibers inhibited the spoilage ability of P. fluorescens P07. This may be because the QSI interfered with the QS system of bacteria, resulting in a decrease in the production of QS-regulated extracellular protease and eventually leading to a decrease in the TVB-N content. The TVB-N content of salmon slices treated with propyl gallate was less than that of PLA–propyl gallate electrospun fibers at the beginning of cold storage (0–48 h), probably because the amount of propyl gallate released from PLA–propyl gallate electrospun fibers was small in the early stage of cold storage. Trimethylamine oxide (TMAO) is a natural chemical responsible for the characteristic flavor of aquatic food products. However, TMAO is unstable and can be easily reduced to trimethylamine under the combined action of bacteria and enzymes 36.

TMA is the main component of amines, which constitute TVB-N. The strong fishy

smell of TMA is often used to evaluate the degree of decomposition of aquatic food products during chilled storage. The TMA content in fresh salmon slices in our study was low and it increased with an increase in the storage time. The rate of increase in TMA in the salmon slices coated with the PLA–propyl gallate electrospun fibers was slower than that in the control group. This result was consistent with the TVB-N analysis. The results indicated that the QSI released from the PLA–propyl gallate 25



electrospun fibers had a significant retarding effect on the spoilage rate of salmon slices. Quantitative analysis of the spoilage ability of P. fluorescens P07 showed that the yield factors YTVB-N/CFU and YTMA/CFU in the first group were significantly lower than those of other groups, indicating that the QSI released from the electrospun fibers significantly inhibited the spoilage ability of P. fluorescens P07. Muscle tissue observation of salmon slices of the four groups showed that the deterioration rate of muscle tissue in the first group was slower than that in other groups, indicating that the PLA–propyl gallate electrospun fibers significantly improved the quality of salmon slices during chilled storage. 4. CONCLUSIONS In this study, PLA–propyl gallate electrospun fibers were prepared by coaxial electrospinning. The electrospun fibers were characterized and it could be seen that they exhibited good thermal stability, tensile strength, hydrophobicity, and other properties, which could meet the refrigeration requirements. The fibers were efficient at encapsulating substances with a strong QS inhibitory activity and exhibited a slow-release behavior. Further, the PLA–propyl gallate electrospun fibers could inhibit the spoilage ability of P. fluorescens P07 and enhance the quality of refrigerated salmon slices. Therefore, electrospinning technology combined with the QSI technology provides a new and efficient method for the preservation of aquatic food products. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ORCID Ting Ding: 0000-0003-3768-0751 Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS 26


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This research was financially supported by the National Natural Science Foundation of China (No. 31471639; 31301572) and the National Key Research and Development Programme of China (2017YFD0400106).

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Table of Contents Graphic

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Fig. 1 (A) Violacein inhibition assay and (B) β-galactosidase assay of propyl gallate at sub-MIC levels


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Fig. 2 (A) Photograph of a PLA–gallate coaxial electrospun fiber mat (30 cm × 30 cm), (B) SEM image of PLA uniaxial electrostatic electrospun fibers, (C) SEM image of PLA–propyl gallate coaxial electrospun fibers, and (D) TEM image of PLA–propyl gallate coaxial electrospun fibers



Fig. 3 FTIR spectra of (A) PLA uniaxial electrostatic electrospun fibers and (B) PLA–propyl gallate coaxial electrospun fibers. XRD patterns of (C) PLA uniaxial electrostatic electrospun fibers and (D) PLA–propyl gallate coaxial electrospun fibers


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Fig. 4 TG-DSC of (A) PLA uniaxial electrostatic electrospun fibers and (B) PLA–propyl gallate coaxial electrospun fibers (Exo: exothermic; Endo: endothermic). Tensile strength analysis of (C) PLA uniaxial electrostatic electrospun fibers and (D) PLA–propyl gallate coaxial electrospun fibers



Fig. 5 Slow-release behavior of PLA– propyl gallate coaxial electrospun fibers and the effect of coating these fibers on refrigerated salmon slices. (A) Slow-release behavior of PLA–propyl gallate electrospun fibers. (B) Growth curve of P. fluorescens P07. Variations in (C) TVB-N and (D) TMA content 1305x873mm (96 x 96 DPI)


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Fig. 6 Muscle tissue changes in refrigerated salmon slices (A) Salmon slices coated with PLA–propyl gallate electrospun fibers; (B) Control group; (C) Salmon slices treated with propyl gallate; (D) Salmon slices coated with PLA uniaxial electrostatic electrospun fibers. Pictures from left to right were taken at 0, 48, 96, 144, and 192 h. 1801x1117mm (96 x 96 DPI)


Preparation of coaxial polylactic acidâpropyl gallate electrospun fibers

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Preparation of coaxial polylactic acidâpropyl gallate electrospun fibers