3.1. Active properties of the RS extracts.
Table 1 shows the phenolic content, antioxidant parameters, and MIC values of each active extract. A different phenolic content and antioxidant capacity were obtained via the different extraction processes. SWE was more effective than USHT process at phenolic extraction, the efficiency increasing when the temperature rose. The phenolic content of the extracts was linearly correlated with their ABTS radical scavenging capacity (r = 0.996), but a poor correlation was observed between EC50 values and TPC (r =-0.828). The higher the TPC, the lower the EC50 values (a smaller extract amount was required to reduce the DDPH radical activity by 50%), although SWE extracts seem to exhibit a different tendency with respect to TPC. This suggests that phenolic compounds detected from the Folin-Ciocalteu assay in the extracts did not have the same composition profile in USHT extract as in the SWE extracts and exhibited different DPPH scavenging capacity. In this sense, other antioxidant compounds neo-formed through Maillard and caramelisation reactions at the highest SWE temperature could also contribute to the EC50 values, as described by other authors as regards the SWE of different plant matrices (Plaza et al., 2010a). Plaza et al. (2010b) studied the formation of Maillard and caramelisation products during SWE using glycation model systems with amino acids and glucose, as a function of temperature, and found that the extent of non-enzymatic browning reactions was higher when the temperature increased. Therefore, SWE180 extracts could contain a greater ratio of these kinds of brown compounds.
For comparison purposes, the EC50 values of potent antioxidant compounds, such as ascorbic acid or α-tocopherol, are 0.12 and 0.26 mg compound /mg DPPH, respectively (Brand-Williams et al., 1995), which indicates that the SWE extracts, especially SWE180, exhibited a high DPPH radical scavenging capacity, nearer to that of the strong antioxidants.
As concerns the antimicrobial capacity of the extracts, the MIC values of the extracts for both Gram + and Gram- bacteria also suggested greater antibacterial activity of the SWE extracts, since lower MIC values were obtained for these extracts. Although in no case were the MIC values of USHT extracts reached at the maximum concentration (200 mg.mL− 1) used, SWE extracts reached the MIC value of L. innocua at 30 and 50 mg/mL− 1 for SWE160 and SWE180 extracts, respectively. E coli was less sensitive to these extracts, exhibiting higher MIC values (over 200 mg.mL− 1, for SWE160 extract). Previous studies with phenolic acids also pointed to the differences as regards the effectiveness against each bacterium (Ordoñez et al., 2021), which are the main phenolic compounds in RS aqueous extracts (Menzel et al. 2020). Ferulic acid, one of the main constituents of rice straw aqueous extract (Menzel et al. 2020) was also more effective against L. innocua than against E. coli, as observed by other authors (Takahashi et al. 2013; Andrade et al. 2022). Gram-negative bacteria, such as E. coli, are expected to be more resistant to antimicrobials, as their outer membrane limits the crossing of small molecules to a greater degree (Liu et a. 2020).
Table 1
Total solid yield (TSY), total phenolic content (TPC), antioxidant capacity (EC50 and Trolox equivalent antioxidant capacity (TEAC)), and minimum inhibitory concentration (MIC) for L. innocua and E. coli of the bioactive extracts obtained with different extraction methods.
|
USHT*
|
SWE160*
|
SWE180*
|
TSY (g dry extract. 100 g− 1 RS)
|
13.95 ± 0.13b
|
23.1 ± 0.3a
|
23.4 ± 1.1a
|
TPC (mg GAE. g− 1 dry extract)**
|
37.1 ± 0.4c
|
51.1 ± 2.4b
|
82.5 ± 3.2a
|
EC50 (mg dry extract. mg− 1 DPPH)
|
6.3 ± 0.3a
|
2.0 ± 0.1b
|
1.2 ± 0.1c
|
TEAC (µmol Trolox.mg− 1 dry extract)
|
0.59 ± 0.02c
|
0.67 ± 0.01b
|
0.94 ± 0.02a
|
MIC L. innocua (mg.mL− 1)
|
> 200
|
50 ± 3a
|
30 ± 3b
|
MIC E. coli (mg.mL− 1)
|
> 200
|
> 200
|
182 ± 3
|
*Different letters in the same column indicate significant differences between films by the Tukey test (α = 0.05).
**GAE: gallic acid equivalent.
3.2. Properties of the films
3.2.1 Microstructure and optical properties of the films
Figure 1 shows the cross-sections of PLA films with and without RS extracts obtained by different extraction methods (USHT, SWE160 and SWE180). The control PLA films exhibited a plastic cryofracture, with brittle and rubbery domains, characteristic of this amorphous polymer, as observed by other authors (Muller et al., 2017; Sanyang et al., 2016). The incorporation of RS extracts promoted appreciable differences in the microstructure of the films, according to the new interactions established between the extract components and PLA chains. The phenolic-rich composition of the extracts, including ferulic acid, p-coumaric acid, protocatechuic acid, and caffeic acid (Menzel et al., 2020), may imply the extensive formation of hydrogen bonds between the phenol OH groups and carbonyl of the PLA chains or with the end chain hydroxyl, which could promote a new interchain entanglement. Likewise, the presence of small aggregates, or small holes, in the polymer matrix, depending on the extract incorporated, suggested the lack of total compatibility of the extract compounds and polymer, giving rise to phase separation and a more heterogenous structure. The PLA-USHT film showed a high proportion of small aggregates that were well adhered to the polymer matrix, whereas films with SWE extracts showed small holes that, in some cases, contained loose particles. This suggests a different affinity of non-miscible compounds of the extracts with the polymer, giving rise to differing adhesion forces of dispersed particles with the matrix, depending on their composition. A rough surface pattern of the cross-section of PLA films was also reported by Díaz-Galindo et al. (2020) when the a greater concentration of grapevine cane extract was incorporated (5–15% wt.). The SWE promotes the extraction of less polar compounds, such as phenolic acids, (Ong et al., 2006; Plaza et al., 2010) but also favours the extraction of hemicellulose (Requena et al. 2019), which could be less compatible with the PLA chains, given their polymeric, hydrophilic nature. Therefore, interchain interactions with miscible compounds of the extracts and dispersion of the non-compatible fractions provoked a structural modification in the PLA matrix that can affect its functional properties as a packaging material, such as its optical, barrier or mechanical properties.
As concerns the optical characteristics of the films, their visual appearance, colour coordinates and UV-vis transmittance spectra are shown in Fig. 2a. The incorporation of extracts promoted marked changes in the film colour that becomes reddish, more vivid, and darker than the control PLA film. Active films exhibited a notable decrease in lightness (L*: from 90.7 to 61.0), with a pronounced increase in colour saturation (Cab*: from 2.7 to 49.2), and a significant change in the hue angle (hab*: from 99.0 to 75.2), in which the extract SWE180 promoted the highest changes. The total colour difference (∆E*) of the active films with respect to the control PLA film was higher for PLA-SWE180 films (55.7) than PLA-SWE160 (45.1), while the lowest value was obtained for PLA-USHT films (36.7). This is coherent with a higher ratio of coloured compounds in the extracts, since many chemicals present in the lignin fraction are responsible for the dark colour in plant matrices (Do et al., 2020). Likewise, the high temperatures and pressures applied in the SWE process could promote browning reactions, such as Maillard and caramelisation reactions, giving rise to more coloured/more intensely coloured extracts, mainly at 180°C, as previously reported (Plaza et al. 2010b).
As concerns the UV-vis transmittance spectra, the net PLA film was highly transparent, with 70–80% transmittance in the visible wave-length range, with a slight light-blocking effect in the UV region (400 − 250 nm). In contrast, the active films exhibited a marked decrease in light transmission in both the visible and UV ranges, due to the presence of the extract compounds in the polymer matrix with the ability to absorb UV light. In fact, phenolic compounds are considered as photo-protective compounds due to their molecular structure, with conjugated double bonds and aromatic rings (Woo et al., 2011). The most intense UV-vis light blocking effect was achieved for the films with SWE extracts (SWE160 and SWE180). These transmitted approximately 20% in the visible light range and exhibited a total blocking effect between 400–450 nm. The PLA-USHT film was slightly more transparent than the other active films, especially in the UV region. Wen et al. (2020) also reported an excellent light-blocking effect of poly (vinyl alcohol) films with green tea extract. Thus, the presence of active RS extracts, especially those obtained by SWE, prevented the UV light transmission, which is responsible for promoting the photo-oxidation of the components present in foods, such as vitamins, fatty acids, or pigments.
3.2.2 Thermal, mechanical and barrier properties of the films.
Thermal analysis of the films obtained by DSC (second heating to delete the polymer thermal history) (Fig. 3b and c) showed the typical glass transition of the amorphous PLA at 55.8°C, in the previously reported range by other authors (Ordoñez et al., 2022). The incorporation of RS extracts provoked a plasticizing effect, reducing the Tg values by about 5°C, regardless of the kind of extract (Table 2).
Table 2
Thermal parameters of the PLA films obtained from DSC (glass transition temperature (Tg)) and TGA (initial temperature (To), peak temperature (Tp), and residue).
|
TGA*
|
DSC*
|
Formulation
|
To (°C)
|
Tp (°C)
|
Residue (%)
|
Tg (°C)
|
PLA
|
269 ± 6a
|
357 ± 1a
|
1.1 ± 0.5a
|
55.8 ± 0.4a
|
PLA-USHT
|
218 ± 4b
|
342 ± 1b
|
1.7 ± 0.4a
|
49.6 ± 2.5b
|
PLA-SWE160
|
214 ± 2b
|
344 ± 1b
|
1.4 ± 0.5a
|
47.7 ± 2.2b
|
PLA-SWE180
|
216 ± 3b
|
345 ± 1b
|
1.8 ± 0.4a
|
48.5 ± 1.5b
|
* Different letters in the same column indicate significant differences between films by the Tukey test (α = 0.05).
Likewise, TGA also revealed the effect of RS extracts on thermal stability of PLA (Fig. 3a and b). The typical degradation curve of amorphous PLA was obtained, with a single thermal event starting at 269°C (To) and maximum degradation rate at 357°C (Tp), as reported by other authors (Ordoñez et al. 2022). The incorporation of RS extracts provoked a decrease in both To and Tp of about 50 and 15°C, respectively, regardless of the extract incorporated, as shown in Table 2. These effects reflected the structural changes in the PLA matrix due to action of some extract components, such as phenolic acids or bonded water. These compounds could promote the partial hydrolyses of PLA during thermal processing, with the subsequent formation of shorter chains. The low molecular weight chains would contribute to a reduction in the Tg and thermal stability of the matrix, whose degradation started at a lower temperature. Similar effects on Tg of PLA matrices have been observed when plant extracts were incorporated (Khakestani et al. 2017).
Figure 4. (a) Stress–strain curves and tensile properties (inserted table) (TS, E, and EM) (different subscript letters in the same column indicate significant differences between samples by Tukey test), and (b) oxygen permeability (OP), and water vapour permeability (WVP) of the PLA films with different RS extracts (for each property, different letters indicate significant differences).
The control PLA films showed TS, E, and EM values in the range found by other authors for PLA films of similar characteristics (Muller et al. 2017; Cvek et al., 2022). Nevertheless, the incorporation of active extracts markedly affected the tensile behaviour of the films, making them less extensible and reducing their resistance to break, as shown in Fig. 2a. Therefore, the active films exhibited a slightly worsened mechanical performance with respect to the neat PLA film, with reductions of about 18 and 36%, respectively, in the TS and E values. This behaviour reflected the weakening effect provoked by the extract compounds in the polymer matrix resulting from the compound interactions with the PLA chains that globally reduced the interchain forces and matrix cohesiveness, as previously observed in PLA films when molecular compounds of different characteristics, such as phenolic acids (Ordoñez et al. 2022) or other antioxidant compounds (Bassani et al. 2019; Freitas et al., 2023) were incorporated. Additionally, the phenolic acids present in the extracts could promote the hydrolysis of the PLA chains during the film thermo-manufacturing steps to a certain degree, as deduced from the thermal analysis. This would also contribute to the weakening of the PLA network. Nevertheless, the described interactions did not significantly affect the film stiffness since no notable changes were observed in the elastic modulus of the films with extracts.
Figure 4b shows the WVP and OP values of the control PLA film and those containing different RS extracts. The extract-free film exhibited a WVP value of 0.077 g.mm.kPa− 1.h− 1.m− 2, similar to that reported by other authors for PLA films (Díaz-Galindo et al., 2020; Jamshidian et al., 2012). Nevertheless, the incorporation of the active extract slightly worsened the water vapour barrier capacity of the films, leading to an increase in the WVP values of about 30% (p < 0.05). This could be associated with the presence of hydrophilic components from the RS extracts in the matrix, which would enhance the solubility of water molecules through the polymer network, and with the weakening effect of the compound extracts in the polymer network cohesion forces that enhances molecular mobility and diffusion.
The OP values of the films were also greatly influenced by the incorporation of the active extracts (p < 0.05). All of the active films showed a decrease in the OP values with respect to the control PLA film, which could be attributed to several reasons: 1) the promotion of the hydrophilic nature of the polymer matrix when RS extracts were present that enhances the oxygen solubility in the matrix, and 2) the oxygen scavenging capacity of the extract compounds that limits the oxygen transfer in the active films (Bonilla et al., 2013). The PLA-USHT, PLA-SWE160, PLA-SWE180 films exhibited reductions in the OP values of approximately 16, 20, and 24%, respectively, which correlate with the respective antioxidant capacity of the present extracts. The greater the radical scavenging capacity of the extracts, the more significant the OP reduction in the films. This points to a greater impact of the antioxidant power on the oxygen transfer through the films.
Therefore, despite the slightly worse mechanical properties and water vapour permeability of the films with RS extracts, their oxygen and light barrier capacity as well as their potential active properties could represent a significant improvement in terms of their food packaging potential, as a means of preventing food oxidation reactions during storage.
3.2.3 Pork meat preservation capacity of the films
The active and control PLA films were used to evaluate their ability to package and preserve fresh pork meat during storage. For this purpose, the quality parameters of packaged meat, namely weight loss, pH, TBARS index, microbial count, and colour were analysed throughout 16 days of cold storage. Figure 5a shows the weight loss of the meat fillets packaged in the active and control film bags. The samples packaged with the control PLA films exhibited the greatest weight loss, reaching values of about 10% after 16 storage days, in the range reported by other authors (Hernández-García et al., 2022). The active film bags were more effective at preventing the sample weight loss (~ 4% after 6 storage days), with no significant differences between active bags. The meat water retention capacity may be affected by different processing factors, such as cutting, temperature, salt addition or grinding, and by the intrinsic characteristics of the meat, such as the pH value that directly affects the water retention capacity of meat proteins (Haque et al., 2016).
Figure 5b shows the pH values of the meat as a function of storage time, where the expected increase can be observed in every sample. The pH increase is associated with the deterioration of proteins through enzymatic reactions or the metabolism of microorganisms that leads to the production of alkaline compounds, such as biogenic amines. The initial pH value of the meat was 5.59, consistent with the values reported by other studies for fresh pork meat (Yang et al., 2019). The meat packaged in the control film bag exhibited a greater increase in pH, (pH final value of 5.70) than the samples packaged in active bags, which also coincides with the higher weight loss value of this sample. Meat samples packaged with PLA-SWE160 and PLA-SWE180 films showed the smallest changes in the pH values throughout time, which could be attributed to the active properties of the films, as discussed below for the other quality parameters.
As concerns meat oxidation, the development of malondialdehyde (MDA) levels, one of the end-products originating from hydroperoxide decomposition, as a function of storage time is shown in Fig. 4c for the different treatments. Initially, the meat exhibited a TBARS index of 0.1 mg MDA.kg− 1 sample, typical of fresh pork meat (Kaczmarek et al., 2017). The storage time and the type of packaging significantly affected the TBARS values. All of the meat samples showed a progressive increase in the TBARS index, but the samples packaged with the control film had a much higher oxidation rate. This indicates that the extracts incorporated into PLA films were efficient at retarding meat oxidation during storage. The ability to slow down the oxidative processes in meat could be associated with the release of antioxidant compounds from the films to the meat surface. Likewise, the lower OP values and the significant UV-light barrier of the active films could produce additional antioxidant effects, since the lower oxygen and UV light exposure of the meat samples prevent meat photo-oxidation. The antioxidant effects were mainly appreciated after 16 storage days and were aligned with the different antioxidant activity of the extracts incorporated into the films (PLA-SWE180 > PLA-SWE160 > PLA-USHT). Therefore, the films with a SWE180 extract with the lowest oxygen permeability, more intense UV light blocking effect and the highest antioxidant capacity of the extract were the most effective at preserving the meat samples from oxidative deterioration.
Figure 6 shows the microbial counts in the packaged meat, in terms of the TV, LA, TC, and PB bacteria, at the different storage times. As reported by other authors (Kim et al., 2016), all the microorganisms tested in the meat samples exhibited progressive microbial growth. The microbial counts in the control sample (packaged in an extract-free PLA bag) were higher than those of the samples packaged in active films, which indicates the capacity of these films to inhibit microbial growth. Of the active films, the PLA-USHT was the least efficient at reducing microbial counts, especially in the case of TV and PB bacteria, with the samples exhibiting similar counts to the control sample. In fact, the samples packaged in PLA and PLA-USHT bags exceeded the acceptability limit of the total viable count in pork meat (6 log CFU.g− 1, Commission Regulation No 2073/2005) on day 16 of cold storage, whereas the samples packaged in SWE160 and SWE180 films remained below this limit throughout the entire period tested. The PLA-SWE180 film was the most effective at inhibiting microbial growth, exhibiting a log reduction of TV, LA, TC, and PB bacteria of about 1.9, 0.9, 1.1, and 1.4, respectively, after 16 storage days with respect to the control sample. This is coherent with the different antibacterial activity of the extracts deduced from the MIC values obtained for Gram + and Gram- bacteria. The antibacterial effect of the released phenolic compounds is associated with their ability to inhibit bacterial virulence factors, such as enzymes and toxins, interact with the cytoplasmic membrane, and suppress the formation of bacterial biofilms (Miklasińska-Majdanik et al., 2018).
The colour changes of packaged meat, in terms of the L*, Cab*, hab* coordinates, and the total colour difference (∆E*) with respect to the initial coordinates are shown in Fig. 7, as a function of storage time at 4°C. All of the meat samples became darker (L* decrease) and less vivid in colour (Cab*) decrease), with small changes in hue, throughout storage (Fig. 7). However, the samples packaged in the extract-free film exhibited the most significant changes in the colour coordinates, which was reflected in the higher values of ∆E* at each storage time. The colour changes are associated with water loss and pigment oxidation during storage. Water loss modifies the sample surface reflectance and pigment concentration, which affect sample colour (Hernández-García et al., 2022). Myoglobin is the heme protein responsible for meat colour, and the oxidation of the central iron atom within the heme group is responsible for discoloration, producing changes from red OxyMb to brownish MetMb. This oxidation has been linked to lipid oxidation since the oxidation of one of these compounds produces chemical species that can promote the oxidation of the others. Therefore, previous studies reported that meat colour was preserved by the incorporation of antioxidant compounds (Faustman et al., 2010). In fact, active films with antioxidant extracts better preserved the colour of the meat samples, leading to smaller colour differences throughout storage. The colour development was coherent with the sample oxidation pattern reflected in the TBARS analysis and indicates the key role played by the antioxidant extracts in delaying the oxidative decay of the samples that produces both rancidity and colour degradation.
Active PLA films with RS extracts exhibited good preservation capacity to package fresh pork meat, compared to extract free PLA films, maintaining the quality paremeters throughout longer times and extending the product shelf life. SWE (especilly a 180°C) produced extracts with greater antioxidant and antibacterial activity, which were reflected in a greater efectiveness of the films containing these extracts to preserve meat from oxidative and microbial spoilage.