# Hake Fish Preservation Using Plant-Based Impregnated Polylactic Acid Food Films as Active Packaging
## 3 Results and Discussion
### Preservation of Hake Fillets
#### 3.2.3 Effects of Storage on the Microbial Count  
Fish is excellent for microbial growth due to its high-water content, relatively high pH, and abundance of nutrients such as proteins and non-protein nitrogen compounds  
Figure 5: Evolution of the (**a**) total volatile base (TVB-N) and (**b**) trimethylamine (TMA-N) during preservation at 4 \({}^{\circ}\)C at each sampling point. Data are expressed as the mean \(\pm\) standard deviation (\(t=3\)). Lowercase letters indicate differences between the impregnated and control samples at the same storage time. Capital letters indicate differences along storage days in the same treatment (\(p<0.05\)). typically found in fish tissues [70]. Microbial proliferation depends on the initial microflora, conditioned largely by the aquatic environment from which it originates and the handling it has undergone. This proliferation generates the metabolization of non-protein nitrogenous compounds present in fish. These compounds contribute to the characteristic off-flavors and odors of spoiled fish as well as biogenic amines, which are a health and food safety concern [70; 71; 72]. Microbial spoilage and pathogenic bacteria will overgrow on chilled fish and cause the final deterioration. After this, the fish is no longer organoleptically, visually, and microbiologically acceptable for consumption. The recommended microbiological limit for seafood, particularly for fresh fish, is 10\({}^{7}\) CFU g\({}^{-1}\), according to the International Commission on Microbiological Specifications for Foods [73]. Figure 6 shows the microbial count of the packaged hake fillets during the storage time. In this work, the initial microbial count values were very high, as were the pH values, volatile bases, and TMA. As seen in previous studies of fresh hake [74; 75], fresh samples of excellent quality showed TMA-N levels below 1 mg 100 g\({}^{-1}\), maintained for 5-6 days. However, in this study, the initial value was 3-4 mg 100 g\({}^{-1}\) and immediately increased exponentially. This phenomenon suggests that the hake may have been caught several days before purchase. Nonetheless, the results showed an identical evolution in both cases, obtaining statistically lower values for the hake packaged into impregnated films from day 5. Between days 2 and 5, the impregnated films seemed to exert a bacteriostatic effect, delaying the microbial growth by 3 days. In this sense, the microbial count remained in the range of 10\({}^{7}\) CFU g\({}^{-1}\) for three days longer, which could potentially increase the commercial shelf life of the fish. The bacteriostatic effect of active packaging has previously been reported in the application of fresh food. For instance, [PERSON] et al. [76] reported a delay in the growth of mesophilic, lactic acid, and psychotrophic bacteria, molds, and yeasts of dry-cured shoulder sliced from Iberian pigs when packaged into polyamide polyethylene films enriched with olive leaf extract. The experiments were compared with the application alone and in combination with high-pressure processing. The authors concluded that the physical treatment exerted a bactericidal action instead of the bacteriostatic  
Figure 6: Evolution of the microbial count (log UFC g\({}^{-1}\) of sample) during preservation at 4 \({}^{\circ}\)C at each sampling point. Data are expressed as the mean \(\pm\) standard deviation (\(n=2\)). Lowercase letters indicate differences between the impregnated and control samples at the same storage time. Capital letters indicate differences along storage days in the same treatment (\(p<0.05\)). action of the AP tested. [PERSON] et al. [77] analyzed the application of carvacrol-loaded curdlan hydrogels for the preservation of seabass. Although the antimicrobial effect of carvacrol was demonstrated in vitro against _Shewanella putrefaciens, Vibrio parahaemolyticus_, and _Vibrio harveyi_, the effectiveness in the inactivation of the pathogens when inoculated over the fish was not that evident, showing, in the best cases, a bacteriostatic effect.