Antibacterial Activity of Chitosan–Polylactate Fabricated Plastic Film and Its Application on the Preservation of Fish Fillet

shows both PLA and chitosan–PLA films’ water vapor transmission rate, moisture content, and solubility. With the increasing concentration of chitosan, the PLA films’ water vapor transmission rate and moisture content increase, with 2% chitosan–PLA film being the best one. This result may be due to chitosan’s hydrophilic properties [ 26 ]. In contrast, due to the PLA’s hydrophobicity, Suyatma et al. [ 29 ] proved that PLA addition reduced the chitosan–based films’ water vapor transmission rate. The solubility of test films was quite low and there was no significant difference among them.

The test groups’ tensile strengths of machine direction (MD) and transverse direction (TD) were 448–155 and 271–79 kgf/cm 2 , respectively. The chitosan concentration’s increase in PLA decreased the tensile strength, and. therefore, 0.5% chitosan–PLA film had the best tensile strength among all chitosan–PLA composite films. The addition of chitosan particle may result in irregularities and discontinuities in the oriented PLA matrix [ 23 ], which caused the decrease of chitosan–PLA film’s tensile strength. However, in this study, when the chitosan concentration was 0.5%, the PLA’s MD elongation at break increased, although its value slightly decreased when the chitosan concentration was further increased. Besides, adding chitosan to PLA significantly increased both the MD and TD tearing strength (gf). Although we are currently unable to provide the correct reason, the helical configuration [ 28 ] and the size of chitosan molecule may help increase the elongation and tear strength of PLA film containing appropriate amount of chitosan.

The visual appearance of pure PLA films and chitosan–PLA composites films are shown in . The PLA film is more transparent than the chitosan–PLA composite films, and this is consistent with the observations previously reported in [ 23 ]. The chitosan–PLA films are yellowish, which may be due to chitosan’s partial miscibility affecting the continuous matrix’s color. The film’s tensile strength, the elongation at break, and the tearing strength are shown in .

shows the PLA and 0.5%, 1.0%, and 2.0% chitosan–PLA films’ antimicrobial activities against E. coli, S. aureus, P. fluorescens, and V. parahaemolyticus. Except for V. parahaemolyticus, 0.5% chitosan–PLA film showed the highest activity. This plastic film’s inhibition efficiency against E. coli, P. fluorescens, and S. aureus was over 95%. In addition, among the four target bacteria, P. fluorescens seems to be more susceptible to chitosan–PLA films. Except for V. parahaemolyticus, the activity of higher chitosan concentrations (1.0% and 2.0%) in PLA was significantly lower than that of lower chitosan concentrations (0.5%). Several reports have shown that the presence of more -NH 3 + residues in chitosan is conducive to binding to bacterial cells, resulting in bacterial structural instability [ 12 , 15 , 30 , 31 , 32 ]. The pKa value for chitosan is approximately 6.3–6.5, dependent on chitosan’s MW [ 33 ]. We speculate that, when the diluted bacterial culture is added to the film surface, some of the amine residues protruding from the film surface are protonated, thereby having antibacterial activity. However, when the content of chitosan in the film increases, it may cause the film structure to become tighter, hinder the exposure of amine groups, or generate electrostatic attraction within or between chitosan molecules [ 28 ], thereby reducing its antibacterial activity. In addition, our previous study demonstrated that the chitosan’s antimicrobial activity varies considerably with the DD, MW, and reaction pH [ 15 ]. Since we only used one chitosan sample with 93% DD and 220 kDa in this study, the comprehensive effects of chitosan DD, MW, and concentration on the chitosan–PLA film’s structure and antibacterial activity merit future investigation.

3.3. Chitosan–PLA Film’s Application for Preservation Fish Fillet

Due to the fresh fish products’ high perishable characteristics, chitosan has been used to extend the salmon fish fillet’s shelf life [14]. Our study evaluated the PLA and the 0.5% chitosan–PLA films application’s preservation effects on grouper fish fillets stored at 25 °C and 4 °C. shows the differences among fish fillets uncovered with film (control) or covered with PLA or 0.5% chitosan–PLA (0.5% CH–PLA) films during storage at 25 °C. Specifically, we measured the samples’ changes in the mesophilic count, psychrotrophic count, TVBN content, and pH value. The mesophilic counts of the control, PLA, and chitosan–PLA groups were similar, although the mesophilic counts of fish fillets covered with a 0.5% chitosan–PLA film after 24 h incubation were slightly lower than other fish fillets ( A). After 24 h of incubation, the psychrotrophic count in the 0.5% chitosan–PLA group dropped to 3.51 Log CFU/g, which was significantly lower than the control (5.91 Log CFU/g) and PLA (5.56 Log CFU/g) groups. After 48 h incubation, the psychrotrophic count of the 0.5% chitosan–PLA group remained at about 4.0 Log CFU/g, which was significantly lower than that of the control ( B). The TVBN is a quantitative parameter to determine the content of ammonium and type I, II, and III amines in fish. An increase in TVBN indicates fish and bacterial enzyme actions increase, leading to fish spoilage [34]. C shows the TVBN value changes of uncovered control fish fillets and fish samples covered with PLA and 0.5% chitosan–PLA film. The TVBN of the 0.5% chitosan–PLA group was significantly lower than that of the control and PLA group after 48 h of incubation ( C). There was no difference in pH between the test groups ( D).

shows the cell count changes of various microbiomes in fish fillet covered with PLA or 0.5% chitosan–PLA film during the 48 h of storage at 25 °C. After 12 and 48 h of incubation, the coliform counts ( A) and Aeromonas counts ( B) for the 0.5% chitosan–PLA film group were significantly lower than those of the uncovered control group. After 12 h of incubation, the Pseudomonas counts for PLA and 0.5% chitosan–PLA film groups were 4.9 and 5.2 Log CFU/g, respectively, which were significantly lower than that of the control group (over 6 Log CFU/g) ( C). The sample groups’ Vibrio counts were comparably lower than the control group ( D).

shows the changes in the mesophilic count, psychrotrophic count, TVBN, and pH value of fish fillet uncovered with film (control) or covered with PLA or 0.5% chitosan–PLA films (0.5%CH–PLA) during storage at 4 °C. At least three days after storage, covering fish fillets with PLA or 0.5% chitosan–PLA film effectively inhibited the increase in mesophilic bacteria count. After seven days of storage, the control’s mesophilic count exceeded 6 Log CFU/g (control limit), while the count for the 0.5% chitosan–PLA group was 3.16 Log CFU/g, which was significantly lower than that of control and PLA groups. The PLA and 0.5% chitosan–PLA groups’ counts after nine days were still below the control limit ( A). In all groups, there was no psychrotrophic growth during the first three days of storage. After nine days of storage, the 0.5% chitosan–PLA group’s psychrotrophic counts were significantly lower than those of the control and PLA groups ( B). After nine days of storage at 4 °C, the TVBN content of all tested fish samples was below 10 mg/100 g, which is far below the control limit for raw fish (25 mg/100 g). By the ninth day, the TVBN content of fish fillets covered with a 0.5% chitosan–PLA film was 7.42 mg/100 g, which was significantly lower than the control and PLA groups ( C). There was no significant difference in all groups’ pH values after nine storage days ( D).

When stored at 4 °C for nine days, the PLA and 0.5% chitosan–PLA films effectively retarded the increase in the counts of coliform, Aeromonas, and Pseudomonas in the fish fillets. Especially when stored for seven days, these bacterial counts of fish fillets covered with 0.5% chitosan–PLA film were all lower than 4Log CFU/g ( A–C). The Vibrio counts in PLA and 0.5% chitosan–PLA film covered fish fillets during storage at 4 °C were not detectable, while the Vibrio count in control was about 2 Log CFU/g ( D) during storage at 4 °C.

Chitosan has antibacterial activity and non-toxic properties and has been demonstrated to be an ideal antibacterial packaging material. Remya et al. [19] prepared chitosan–ginger essential oil film (CH–GEO film) and demonstrated that covering it with CH–GEO film effectively reduced the fish fillet’s (Sphyraena jello) TVBN content and the mesophilic count. This process effectively prolonged the fish fillets’ shelf life at 2 °C up to 20 days. Chitosan–polyethylene film was shown to have good antibacterial activity against E. coli, Listeria monocytogenes, and Salmonella enteritidis, thereby effectively extending the refrigeration beef’s shelf life [25]. Fathima et al. [35] used ethylene glycol and vinyl alcohol as crosslinkers and plasticizers to prepare nano-chitosan–PLA film. They further demonstrated that covering this film could effectively prolong Indian white prawn’s (Fenneropenaeus indicus) shelf life. In this study, 0.5% chitosan–PLA film led to significant reduction of microbes in fish fillets stored at 4 and 25 °C; especially when compared with 25 °C, the reduction in fish fillets observed at 4 °C was greater. In addition, the PLA film also showed a slight decrease in microbial numbers in the fish fillet, which may be related to the oxygen impermeability of the PLA film [23]. In summary, the overall effects of the chitosan in the PLA film, the low temperature environment, and the low oxygen permeability of the film led to the chitosan–PLA film greatly reducing the bacteria in the fish fillet and extending its shelf life at 4 °C.