Preparation and Characterization of Poly(butylene succinate) Films Modified with Sea Buckthorn (Hippophae rhamnoides L.) Extract for Packaging Applications

Abstract

Food packaging has to preserve food products, not only providing protection against mechanical factors, but also providing antioxidant and antimicrobial protection. This article describes the effects of PBS film modification with two sea buckthorn extracts (chloroform extract and supercritical CO₂ extract) at two different concentrations (1 or 5 g per 100 g of polymer). The films were tested to determine the effect of the active additive on optical properties, mechanical properties, moisture sorption, antioxidant and antimicrobial properties (against Escherichia coli, Staphylococcus aureus, and Candida albicans). The extracts improved free radical scavenging significantly (up to 41.13 ± 1.31% for PBS-CO₂ 0.05) and reduced the number of microorganisms studied (more than a 6000-fold reduction for E. coli, a 1400-fold reduction in S. aureus and a 1200-fold reduction in C. albicans). The ability to block UV radiation was dependent on the concentration of extracts in the polymer matrix. No significant changes were noticed for mechanical properties or FTIR spectra. The films obtained appear to be potential packaging materials for food products with special protective properties.

1. Introduction

Environmental pollution associated with the production and disposal of plastics has become one of the greatest challenges in the modern world [1]. In response to the growing need for sustainable solutions, biodegradable polymers, such as poly(butylene succinate) (PBS), are gaining attention as a potential alternative to the plastics traditionally used in the packaging industry [2]. PBS offers good mechanical strength, adequate biodegradability and processability, making it an attractive material for packaging with functional properties [3,4]. Nevertheless, the properties often need to be modified using active additives [3]. Food packaging plays a key role, not only in protecting products from physical, chemical, and biological damage, but also in extending food product shelf-life and reducing waste. In recent years, there has been growing interest in active packaging, which exhibits additional properties, such as antimicrobial or antioxidant capabilities, in addition to preserving properties [5]. These functionalities are particularly important for perishable products, in which oxidative degradation and microbial contamination are major threats. The addition of natural plant extracts into packaging materials is gaining popularity as a method for introducing these kinds of properties due to their bioactivity and safety of use [6].

Active compounds such as plant extracts, essential oils, flavonoids, phenolic acids, proanthocyanidins, cellulose, stilbenoids, chitosan, and lignans, are often introduced into biopolymers such as PBS film to improve their antioxidant and/or antimicrobial properties [7,8]. Good examples can be found in the work of Petchwattana and Naknaen [9], who incorporated thymol as an active agent into a PBS matrix through a blown film extruder. The authors demonstrated that the processing technique led to the facilitation of the plasticization effect of thymol. Additionally, modified PBS inhibited the growth of Staphylococcus aureus and Escherichia coli cells. On the other hand, Wiburanawong et al. [10] introduced carvacrol essential oil into a PBS matrix using a blowing technique, also showing that modified, active PBS films were active against S. aureus and E. coli strains.

As Nurzia M. et al. [11] confirmed, PBS may also be used as the main polymeric material for preparing an active film containing active compounds, i.e., kesum, thymol, or curry via a solvent-casting method. The authors confirmed that kesum filled PBS film demonstrated a well-integrated film structure; however, PBS films with curry and/or thymol incorporated in the polymer matrix showed poor adhesion features in comparison to neat PBS film. Moreover, the PBS films containing 10% thymol and kesum were confirmed to be effective against S. aureus. Previous studies also confirmed that PBS films modified with natural plant-derived additives can be suitable for potential packaging materials for food products, providing improved antimicrobial and antioxidant protection. Among the compounds tested in the context of bioactive additives in the PBS matrix are quercetin [12], gallic acid [13], thymol [14], roselle calyx and sappan heartwood extracts [15], or curcumin and carvacrol [16].

Sea buckthorn (Hippophae rhamnoides L.) is a plant known for its exceptional wealth of bioactive compounds, such as flavonoids, carotenoids and vitamins, which exhibit strong antioxidant activity [17]. As Fan J. et al. mentioned [18], natural antioxidants are chain-breaking compounds which react with lipid radicals and convert them into more stable products. The authors revealed that the sea buckthorn extracts are rich in proanthocyanidins, which display direct free radical scavenging. In addition, Rajkowska K. et al. [19], who detected phenolic compounds, such as gallic acid, isorhamnetin 3-rhamnosylglucoside, isorhamnetin 3-glucoside, quercetin 3-glucoside-7-rhamnoside, and Isorhamnetin 3-rutinoside in sea buckthorn extract, found that these compounds exhibited an affinity for bacterial cell walls, leading to perturbations in membrane integrity and subsequent efflux of intracellular constituents.

Extracts produced from sea buckthorn can be used as natural additives for packaging materials to increase their functionality. To date, sea buckthorn extracts have been tested for use in the modification of polymers, such as chitosan [20,21], polyvinyl alcohol (PVA) [22], starch [23], and carboxymethyl cellulose (CMC) [24]. The use of sea buckthorn extracts in PBS has yet to be studied, but it is suspected that it may not only impart antioxidant activity, but may also improve the antimicrobial properties of the materials, which are particularly important for packaging foods sensitive to oxidation and microbial contamination. Key aspects, such as the dispersion of bioactive compounds, their stability in a polymer matrix, and their effect on film properties remain the subject of intensive research. Understanding these interactions is crucial for designing materials that effectively combine sustainability with improved food-preservation capabilities.

The differences resulting from the use of supercritical CO₂ and chloroform as solvents for the extraction of active compounds from sea buckthorn are mainly due to their different physicochemical properties, which affect the selectivity and composition of the extracts obtained. Supercritical CO₂ extraction is an ideal process for isolating lipophilic bioactive compounds, like carotenoids, tocopherols, and unsaturated fatty acids. Thanks to controlled temperature and pressure, CO₂ allows pure extracts with high biological activity to be obtained without the risk of the thermal degradation of valuable components [25]. Alternatively, chloroform, as an organic solvent, makes it possible to extract compounds of more diverse composition. In the context of applications in PBS film modification, CO₂ extracts provide compounds with strong antioxidant properties, while chloroform extracts can introduce additional functional groups, potentially affecting interactions with the polymer matrix. The choice of extraction method should therefore take into account the intended application and properties of the target material.

The aim of this study was to investigate the effect of the addition of sea buckthorn extracts on the physicochemical, mechanical, and functional properties of PBS films in the context of their potential use as food packaging. Key characteristics of the films, such as their mechanical properties, as well as their antimicrobial and antioxidant activities, were considered in this study. These results provide new data on the use of natural plant extracts in biodegradable materials, highlighting their potential to create more sustainable and functional solutions for the food industry.

2. Materials and Methods

2.1. Materials and Reagents

Poly(butylene succinate)—PBS (FZ91PM BioPBS™) was purchased from Mitsubishi Chemical (Tokyo, Japan). Dried sea buckthorn (Hippophae rhamnoides L.) fruits were purchased from a local market in Szczecin, Poland. Sea buckthorn (Hippophae rhamnoides L.) fruit supercritical CO₂ (scCO₂) extract was purchased from ECOSPA (Józefosław, Poland). Calcium chloride and 2,2-diphenyl-1-picrylhydrazyl (DPPH), agar—agar, Plate Count Agar, Potato Dextrose Agar, and Tryptic Soy Broth were purchased from Merck (Darmstadt, Germany). Chloroform and methanol were supplied by Chempur (Piekary Śląskie, Poland). All the chemicals were of analytical grade. Escherichia coli ATCC25922, Staphylococcus aureus ATCC43300, and Candida albicans ATCC10231 were procured from ATCC (American Type Culture Collection, Manassas, VA, USA).

2.2. Preparation of Sea Buckthorn Chloroform Extracts

The finely ground sea buckthorn fruits (100 g) were placed in a glass bottle with a magnetic stirrer bar and poured into 250 mL of chloroform. The extraction was carried out at room temperature with continuous stirring (500 rpm) for 48 h. The extract was separated from the solids via vacuum filtration. The extract was concentrated by evaporating the solvent under a fume hood with continuous stirring (250 rpm) until a constant extract volume was reached (final volume of 16 mL). The concentrated extract prepared in this manner was used to modify PBS films.

2.3. Preparation of PBS-Based Films

A mass of 8 g of PBS was introduced into glass bottles containing 100 mL of chloroform. Then, sea buckthorn chloroform extract or sea buckthorn scCO₂ extract were added to glass bottles (separately) at a rate of 0.01 g or 0.05 g per gram of PBS. The mixtures were stirred overnight until the PBS was completely dissolved. Then, 20 mL of each film-forming solution was poured onto a glass Petri dish (175 mm in diameter) and left overnight under a fume hood to evaporate the solvent. Later, the films were removed from the plates and conditioned for 3 days at 25 °C and 50% RH in a climate-controlled room before any tests commenced. The films were labeled as PBS-X Y, where X is Chl or CO₂ (chloroform extract or scCO₂ extract, respectively) and Y is 0.01 or 0.05 (grams of the extract per 1 g of PBS). The exact scheme of sample acronyms is shown in

Table 1. The acronyms of neat and modified PBS films.

Sample Name	Type of Extract Added	g of Extract/1 g of PBS
PBS	-	-
PBS-Chl 0.01	Chloroform extract	0.01
PBS-Chl 0.05	Chloroform extract	0.05
PBS-CO2 0.01	scCO2 extract	0.01
PBS-CO2 0.05	scCO2 extract	0.05

.

2.4. Determination of Moisture Content

A change in the film weight (2 cm × 2 cm) after 24 h of drying at 105 °C was used to determine the moisture content (MC). Each material underwent three tests and average values were calculated. The following formula was used to calculate film moisture content:

$$MC(\%)={\displaystyle \frac{W_1-W_2}{W_1}}\times 100$$

(1)

where W₁ is the initial weight and W₂ is the weight of the dried films.

2.5. Thickness and Mechanical Properties

A digital micrometer (Dial Thickness Gauge 7301, Mitutoyo Corporation, Kanagawa, Japan, accuracy of 0.001 mm) was used to measure film thickness at ten random points on each film sample, with results expressed as the mean ± standard deviation.

A mechanical test was performed using a Zwick/Roell Z2.5 tensile tester (Ulm, Germany) in accordance with the ASTM D822-02 standard [26]. Films were cut into 10 mm wide strips with an initial grip separation of 25 mm and a crosshead speed of 10 mm/min. A minimum of six replicate samples were analyzed. TestXpert II software was used to calculate the elongation at break (EB) and maximum tensile strength (TS) with standard deviations.

2.6. Spectral Analysis of Films

A UV–Vis Thermo Scientific Evolution 220 spectrophotometer (Waltham, MA, USA) was employed to measure the UV–Vis spectra (transmittance and absorbance in the 200–800 nm range) of the film samples.

Transmission infrared spectra of the films were obtained at room temperature using a Perkin Elmer Spectrum 100 FTIR spectrometer (Waltham, MA, USA). Measurements were taken in a range of 4000–650 cm⁻¹ with 32 scans at a resolution of 1 cm⁻¹. The films were positioned in a sample holder for analysis. SPECTRUM software was used to perform baseline correction and normalization of the spectra.

2.7. Film Color Analysis

Color measurements were conducted using a CR-5 colorimeter (Konica Minolta, Tokyo, Japan) employing the CIELab color scale. For each of the films examined, ten measurements were taken at random points. The total color difference (ΔE) and yellowness index (YI) were determined using specific equations [27]:

$$\Delta E={\left[{\left(L_{standard}-L_{sample}\right)}^2+{\left(a_{standard}-a_{sample}\right)}^2+{\left(b_{standard}-b_{sample}\right)}^2\right]}^{0.5}$$

(2)

$$YI=142.86\times b\times L^{-1}$$

(3)

L—lightness; a—value relative to a green–red opponents, with negative values toward green and positive values toward red; b—value relative to blue–yellow opponents, with negative numbers toward blue and positive toward yellow.

The opacity of neat PBS films and PBS films with added Chl or scCO₂ extracts was determined using an Opacimeter EE Model 12 (Diffusion Systems Ltd., London, UK). The opacimeter was pre-calibrated using a standard white plate (value 100 ± 1, Diffusion Systems Ltd.), and measurements were taken from each film on ten occasions and presented as the mean ± standard deviation.

2.8. Antioxidant Potential of Films

The free radical scavenging ability of the films was evaluated using a spectroscopic technique to measure antioxidant activity. This assessment involved placing 100 mg of each film sample in 25 mL of 0.01 mM DPPH solution in methanol and allowing it to incubate for 30 min at room temperature. The absorbance was then measured at a wavelength of 517 nm. The antioxidant capacity was quantified as a percentage relative to the control sample, which consisted of DPPH incubated without any film additions.

2.9. Antimicrobial Activity

Antimicrobial activity tests were conducted following a modified version of ASTM E 2180-01, as detailed in [16]. Briefly, film samples (2.5 cm × 2.5 cm) were cut and sterilized using UV light. Agar slurries were created by mixing 0.15 g agar–agar and 0.45 g NaCl in 50 mL of distilled water, then sterilized. Once cooled, these slurries were combined with microbial suspensions to achieve a concentration equivalent to 0.5 on the McFarland scale. The resulting mixtures (0.5 mL) were then carefully applied to the sample surfaces and incubated for 24 h at 30 °C with 90% relative humidity. Following incubation, the samples were carefully removed from the Petri dishes, placed in 10 mL of sterile 0.9% NaCl solution, and thoroughly vortexed. Serial dilutions were prepared and cultures were grown on Plate Count Agar (for E. coli and S. aureus) and Potato Dextrose Agar (for C. albicans). These cultures were then incubated at 37 °C for 24 h. The findings are presented as mean values with standard deviations.

2.10. Microscopic Analysis

The surface of the PBS and PBS modified with the Chl and scCO₂ sea buckthorn extracts was examined using an SEM (scanning electron microscope). Preliminarily, the films/samples were placed on pin stubs and covered with a thin layer of gold in a sputter coater at room temperature (Quorum Technologies Q150R S, Laughton, East Sussex, UK). Next, SEM micrographs were obtained using a Vega 3 LMU microscope (Tescan, Brno-Kohoutovice, Czech Republic). A microscopic examination was performed using a tungsten filament with an accelerating voltage of 10 kV.

2.11. Statistical Analyses

All analyses were performed at least in triplicate. Statistical analysis was performed using Statistica version 13 software (StatSoft Poland, Krakow, Poland). Differences between means were determined using an analysis of variance (ANOVA) followed by Fisher’s post hoc LSD test at a significance threshold of p < 0.05.

3. Results and Discussion

3.1. Moisture Content

The moisture content (MC) analysis results are presented in

Table 2. Moisture content (MC) of neat and modified PBS films.

Sample	MC (%)
PBS	1.47 ± 0.30 c
PBS-Chl 0.01	1.73 ± 0.61 a,c
PBS-Chl 0.05	2.55 ± 0.45 a,b
PBS-CO2 0.01	2.54 ± 0.47 a,b
PBS-CO2 0.05	3.34 ± 0.58 b

Values are presented as the mean ± standard deviation. Means with different lowercases are significantly different at p < 0.05.

. The results showed that adding sea buckthorn extracts to the PBS polymer led to an increase in the moisture content of the films compared to that of a control sample of neat PBS (1.47 ± 0.30%). Both chloroform extract and scCO₂ extract affected hygroscopicity but differed in efficiency. Samples containing added Chl extract (0.01 g/1 g PBS) exhibited a small increase in moisture content (1.73 ± 0.61%), while for the scCO₂ extract at the same concentration, the increase was much higher (2.54 ± 0.47%), indicating that the scCO₂ extract is more hygroscopic. Higher extract concentrations (0.05 g/1 g PBS) intensified this effect, reaching values of 2.55 ± 0.45% for the Chl extract and values as high as 3.34 ± 0.58% for the scCO₂ extract. These results suggest that hygroscopicity depends on both the type and concentration of the additive, with the scCO₂ extract exhibiting stronger moisture-absorbing properties. These modifications may potentially be suitable for applications where higher material moisture content is desirable, such as biodegradable packaging or medical applications; however, further research into the effects of moisture on the mechanical properties and thermal stability of the films is required.

Earlier studies conducted on films based on poly(vinyl alcohol) modified with citric acid and sea buckthorn leaf extract showed an increase in MC for a film containing 2% plant extract and a decrease in the value of this parameter as the amount of extract increased to 4% and then 6%, respectively [22]. However, it should be noted that PVA is a hot water-soluble polymer, unlike PBS. Therefore, the differences may be due to the properties of the polymer itself and the interaction between the polymer and the active additive rather than the presence of the additive alone.

3.2. The Thickness and Mechanical Properties

Table 3. Thickness and mechanical characteristics (tensile strength (TS) and elongation at break (EB)) of PBS films.

Sample	Thickness (mm)	TS (MPa)	EB (%)
PBS	0.019 ± 0.001 a	6.16 ± 1.70 b,c	14.48 ± 2.54 a
PBS-Chl 0.01	0.018 ± 0.001 a	6.86 ± 2.23 c	13.95 ± 2.59 a
PBS-Chl 0.05	0.020 ± 0.001 a	3.95 ± 1.26 a	14.02 ± 2.99 a
PBS-CO2 0.01	0.021 ± 0.002 a	5.93 ± 1.34 a,b,c	13.17 ± 1.93 a
PBS-CO2 0.05	0.022 ± 0.002 a	4.15 ± 0.61 a,b	13.74 ± 2.64 a

Values are presented as the mean ± standard deviation. Means with different lowercases are significantly different at p < 0.05.

shows the effect of modifying PBS with sea buckthorn extract on the thickness and mechanical properties of the films. The addition of extracts had no statistically significant effect (p > 0.05) on the thickness of the films, but the tensile strength (TS) test of the films with PBS varied depending on the type and concentration of sea buckthorn extract. The control PBS sample achieved a strength of 6.16 ± 1.70 MPa, as a point of reference. The addition of Chl extract (0.01 g/1 g PBS) increased the value to 6.86 ± 2.23 MPa, but this result was not statistically different from neat PBS (p > 0.05). However, increasing the concentration of Chl extract to 0.05 g/1 g of PBS resulted in a significant decrease (p < 0.05) in strength to 3.95 ± 1.26 MPa, which may have been the result of a weakening of the structure in response to the higher amount of additive. In the case of the scCO₂ extract, the TS was 5.93 ± 1.34 MPa at 0.01 g/1 g of PBS concentration and 4.15 ± 0.61 MPa at 0.05 g of extract per 1 g of PBS, suggesting a lesser effect on the change in TS value, as both results are not significantly different from the control (p > 0.05). These results indicate that the addition of sea buckthorn extract can modify the mechanical properties of the films; higher concentrations of both extracts weaken the material. This may be important in material design, where the balance between mechanical strength and other functional properties is important.

In the elongation at break (EB) test, the PBS films showed relatively stable values, regardless of the presence or type of plant extract. The control PBS sample achieved an elongation of 14.48 ± 2.54%, which was the highest value among all samples. The addition of 0.01 and 0.05 Chl extract resulted in a slight decrease in values to 13.95 ± 2.59% and 14.02 ± 2.99%, respectively, but these results were not statistically different from the control (p > 0.05), indicating that this additive had no effect on the material’s ability to deform before breaking. In the case of the scCO₂ extract, the values were 13.17 ± 1.93 and 13.74 ± 2.64% for PBS-CO₂ 0.01 and PBS-CO₂ 0.05, respectively, also indicating that this additive had no effect on the mechanical parameter studied. The results suggest that both the type and concentration of the sea buckthorn extracts did not significantly affect the elongation at break of the PBS films, indicating the stability of this parameter independent of the amount and type of extract. Such properties may be advantageous in applications in which it is necessary to preserve the material elasticity while modifying other functional characteristics. Additionally, the lack of an EB increase shows that the extract does not have a plasticizing effect within the polymer. Similar results were observed by the authors [10], who observed that the tensile strength and the tensile modulus of the PBS film containing carvacrol in the matrix decreased with the addition of carvacrol beyond 4 wt%. Meanwhile, PBS could elongate more in the presence of carvacrol; the maximum elongation at 19% was noted when carvacrol was added at 10 wt%. However, PBS modified with carvacrol was developed by the authors via melt compounding.

3.3. Spectral Analysis

The UV-Vis spectra of the films are shown in Figure 1. Analysis of the UV-Vis spectra of the PBS films showed clear differences in absorbance depending on the type and concentration of the extracts. The control PBS sample (without additives) and the sample modified with 0.01 g of Chl or scCO₂ extract per 1 g of PBS exhibited similar absorbance over the entire visible-light spectral range (300–800 nm), indicating a higher optical density or higher light absorption capacity compared to samples modified with 0.05 g extracts. The addition of 0.05 g Chl extract lowered the absorbance compared to that of the control and modified it with the lower-concentration extract samples. An even greater trend was observed for the PBS containing 0.05 g of scCO₂ extract. Overall, the use of the extracts led to a reduction in the optical permeability of the films, with the scCO₂ extract having a greater effect than the Chl extract. It is interesting to note that extracts at lower concentration led to an increase in absorbance in UV range, while the addition of a higher amount resulted in a decrease in absorbance in this range. These results suggest that modification of PBS with extracts may be useful in applications requiring controlled light transmission, such as UV-protective packaging. Similarly, Ali et al. modified PVA films with sea buckthorn leaf extracts and observed shifts in their UV-Vis spectra depending on the concentration of the plant extract in the polymer matrix [22].

Figure 2 shows the FTIR spectra of the scCO₂ and Chl extracts from the sea buckthorn. The analysis of the FTIR spectra indicated the presence of characteristic functional groups in their chemical structures. The spectra of both extracts show intense bands in the range of 2800–3000 cm⁻¹, corresponding to the stretching vibrations of methyl groups (-CH₂ and -CH₃) present in long-chain hydrocarbons or lipids. The scCO₂ extract exhibited noticeably stronger bands in this range, suggesting that it contained a higher amount of lipid compounds. In addition, an intense band associated with the presence of carbonyl groups (C=O), characteristic of esters or fatty acids, was identified in the range of 1700–1750 cm⁻¹, which was also more prominent in the scCO₂ extract. In the lower ranges (1000–1500 cm⁻¹), bands corresponding to the vibrations of C-O and C-H bonds were present, indicating the presence of compounds, such as alcohols, esters, and phenols. The Chl extract showed more intense bands at lower frequencies (below 1000 cm⁻¹), which may suggest differences in the composition of aromatic or polar compounds compared with the scCO₂ extract. These results indicate that the scCO₂ extract contains more lipid compounds and fatty acids, whereas the Chl extract may be richer in polar compounds. These results are similar to the FTIR spectra obtained for sea buckthorn extracts in other solvents [28].

Figure 3 shows the FTIR spectra of pure and modified PBS films. An analysis of the FTIR spectra of the PBS films and modified PBS films showed subtle differences in peak intensities. Characteristic PBS bands, such as intense vibrations of carbonyl groups (C=O) at 1708 cm⁻¹ and bands corresponding to C-H and C-O vibrations below 1500 cm⁻¹ are visible (C=O stretching of carbonyl group at 1151 cm⁻¹, C-O stretching of ester group at 1332 cm⁻¹). Additionally, a very weak absorption band at 2946 cm⁻¹, assigned to the asymmetric stretching of the −CH− groups, was identified. Moreover, peaks at around 1100 cm⁻¹ and 1250 cm⁻¹ corresponded to the −C–O–C– stretching of the ester bonds, which corresponded to the peaks at 1160 cm⁻¹ and 1210 cm⁻¹, as noted by de Matos Costa A.R. et al. [29]. Similar spectra were observed by Nurzia M. et al. [11], who also obtained neat PBS film via a solvent-casting method.

The addition of the extracts (both Chl and scCO₂) led to small changes in the intensity of these bands, especially around the 1000–1500 cm⁻¹ range, which may be related to the introduction of additive functional groups from the extracts. At a concentration of 0.05 g per 1 g of polymer, the differences in peak intensities were more noticeable, suggesting stronger interactions between the extracts and the polymer matrix, especially for the scCO₂ extract. However, the lack of pronounced shifts in the peak positions indicates that the modifications are more superficial and do not significantly affect the chemical structure of the PBS. The lack of differences in the FTIR spectra of the studied films may be due to the use of lesser amounts of active additives and the fact that they were mixed in a volume of the polymer where the signal from their chemical bonds is imperceptible on the film’s surface. Nurzia M. et al. also observed slight changes between spectra observed for neat PBS and PBS with an active agent incorporated into the biopolymer matrix [11]. The authors determined that a sharp peak at 2969 cm⁻¹ was observed for neat PBS film. The intensities of this absorption peak were reduced for all active PBS films, indicating that the short-chain active agent compounds had assimilated with PBS polymers. Additionally, the appearance of new peaks at 1216 cm⁻¹ and 2128 cm⁻¹ showing some interfacial interactions had formed between the active agent phenolic groups and PBS ester groups confirmed that the active agents were effectively incorporated into the PBS matrix. The results of the study suggest that the extracts may be useful in imparting additional functional properties for the materials while maintaining their basic chemical and structural characteristics, in line with the results obtained by other researchers for PVA films modified with sea buckthorn leaf extracts [22].

3.4. Color Measurement

Table 4. Color (L*, a*, b*), total color difference (∆E), yellowness index (YI), and opacity of unmodified and modified PBS films.

Sample	L*	a*	b*	∆E	YI	Opacity
PBS	64.40 ± 0.33 c	14.49 ± 0.14 b	39.27 ± 0.47 b	0.00	87.12 ± 0.96 a	14.26 ± 0.73 b,c
PBS-Chl 0.01	63.82 ± 0.18 a	14.65 ± 0.13 a	40.16 ± 0.56 a	1.12 ± 0.48 a	89.89 ± 1.32 c	12.04 ± 1.52 a
PBS-Chl 0.05	64.75 ± 0.08 d	14.62 ± 0.06 a	40.17 ± 0.47 a	1.01 ± 0.39 a	88.62 ± 1.11 b	15.70 ± 2.32 c
PBS-CO2 0.01	63.90 ± 0.34 a	14.87 ± 0.02 c	41.22 ± 0.32 d	2.05 ± 0.39 b	92.15 ± 1.18 d	13.73 ± 3.01 a,b
PBS-CO2 0.05	61.94 ± 0.10 b	16.11 ± 0.03 d	46.68 ± 0.26 d	7.97 ± 0.25 c	107.67 ± 0.65 e	18.80 ± 1.56 d

Values are presented as the mean ± standard deviation. Means with different lowercases are significantly different at p < 0.05.

shows the effect of sea buckthorn extract addition on the color and opacity of the films. The brightness (L*) of the films decreased with the addition of 0.01 g and 0.05 g scCO₂ extract per 1 g of PBS (63.90 ± 0.34 and 61.94 ± 0.10, respectively) and increased slightly for Chl extracts, especially at 0.05 g per 1 g of PBS concentration (64.75 ± 0.08). The red (a*) and yellow (b*) tints increased in the extract-modified films, especially for the scCO₂ extract, which may be due to the higher concentration of dyes in these extracts. The total color change (ΔE) was highest for PBS-CO₂ 0.05 (7.97 ± 0.25), indicating the greatest effect on film color. The yellowness index (YI) increased significantly with the scCO₂ extract (up to 107.67 ± 0.65), confirming its coloring properties, while the Chl extract had less effect on this parameter. Opacity also increased, especially for PBS-CO₂ 0.01 (18.80 ± 1.56), indicating greater light scattering in these samples.

Ali et al. investigated PVA films modified with citric acid and sea buckthorn leaf extract and observed an increase in the opacity of films with the addition of the extract; however, at the additive concentrations tested (0%, 2%, 4%, and 6%), only the 4% concentration showed a statistically significant increase (p < 0.05) [22].

3.5. Antioxidant Characteristics

The antioxidant properties of the PBS films modified with the chloroform extract and sea buckthorn scCO₂ extract are shown in

Table 5. Radical scavenging activity of neat and modified PBS films.

Sample	DPPH (%)
PBS	1.74 ± 1.92 b
PBS-Chl 0.01	15.60 ± 2.91 a
PBS-Chl 0.05	26.44 ± 3.01 c
PBS-CO2 0.01	19.55 ± 2.07 a
PBS-CO2 0.05	41.13 ± 1.31 d

Values are presented as the mean ± standard deviation. Means with different lowercases are significantly different at p < 0.05.

. The unmodified PBS showed poor properties for scavenging DPPH free radicals (1.74 ± 1.92%). This finding has already been established for PBS and has been demonstrated previously [12,16]. The addition of chloroform and scCO₂ sea buckthorn extracts increased the antioxidant properties of the films. The addition of 0.01 g extract per 1 g of PBS resulted in similar improvements in these properties, to 19.55 ± 2.07% and 15.60 ± 2.91% for scCO₂ and chloroform extracts, respectively. Increasing the additive amount resulted in even more significant antioxidant properties within the PBS films, which reached 41.13 ± 1.31% for films modified with scCO₂ extract and 26.44 ± 3.01% for films modified with chloroform extract. Thus, in the case of films modified with 0.05 g of extracts per 1 g of PBS, a significant increase in antioxidant properties was achieved, and the sea buckthorn scCO₂ extract showed outstanding effects. These results were confirmed by researchers [30], who reported that sea buckthorn extracts containing a high amount of phenolics (such as glycosides isorhamnetin and quercetin, with small amounts of triterpenes) had strong antioxidant activity, while the activity of the non-polar extract was lower, but still evident. The authors assumed that alterations in the profile of active compounds in the sea buckhorn extract may have contributed to the differences in the case of their antioxidant activity. It is worth mentioning that the activity of the non-polar extract indicates that the triterpenoids that were identified also possess antioxidant potential.

These results are also in line with those obtained for sea buckthorn leaf extracts in combination with chitosan, where a significant improvement in antioxidant properties was observed with an increase in the amount of extract [20], and sea buckthorn leaf extracts were used to modify PVA films; the stronger the antioxidant properties, the greater the proportion of applied extract [22].

Interestingly, in a study of apple slices coated with CMC modified with sea buckthorn leaf extract, it was found that in the tested range (from 0.5% to 2% w/w extract), the strongest antioxidant properties over 10 days of storage were exhibited by a coating with 1.5% extract [24], with a similar effect also being observed for chitosan coatings with sea buckthorn leaf extract [21].

Michel et al. tested extracts based on various parts of the sea buckthorn plant (seed, root, leaves, stem) and the strongest antioxidant properties were identified in extracts from the root and seeds. The aqueous fraction of the ethanol extract had the strongest properties among those tested [31].

3.6. Antimicrobial Characteristics

The antimicrobial properties of PBS films modified with chloroform extract and scCO₂ extract compared to those of neat PBS films are shown in Figure 4. An analysis of the antimicrobial activity of the films showed that the modification significantly affected the growth of the tested microorganisms: E. coli, S. aureus, and C. albicans. The unmodified PBS film did not exhibit antimicrobial activity and was used as a control sample (CFU/mL count in the range of 10⁷–10⁶). Films modified with the extracts, especially at a concentration of 0.05 g per 1 g of PBS, significantly reduced the growth of microorganisms; this was particularly evident for E. coli and S. aureus. The scCO₂ extract reduced a number of colony-forming units (CFU/mL) to a greater extent than the Chl extract, which may indicate that the components present in the CO₂ extract had stronger antimicrobial activity. The activity against C. albicans was lower than that against the bacteria, but there was also a noticeable tendency for the number of CFU/mL to decrease as the concentration of the extracts increased. In conclusion, modifying PBS films with plant extracts, especially CO₂, significantly increases their antimicrobial potential, which may be useful in various applications, such as food packaging and medical materials. Merijs-Meri R. et al. [32] confirmed that PBS and its copolymer (PBS with 20 mol.% of polybutylene adipate) PBSA containing 7 or 10% of fungi chitosan oligosaccharide and crustacean chitosan were utilized as antimicrobial additives and demonstrated high (higher than 4.7 log) levels of activity towards E. coli. However, these biopolymers were developed by melt compounding. Similarly, carvacrol incorporated into the PBS matrix, also via melt compounding, was found to be effective against S. aureus cells starting from the small addition of 4 wt%, but it could be active against E. coli strains when 10 wt% was employed [10]. Similar research to our experiments was performed by Nurzia M. et al. [11], who prepared PBS with active agents, such as thymol, kesum, and curry, incorporated into its matrix via the solvent-casting method. The authors confirmed that the addition of 10% of kesum and thymol led to the creation of films which were active against S. aureus.

Sea buckthorn-based extracts from various parts of this plant (including the leaves, stem, coleus, and seeds) have been repeatedly tested to determine their antimicrobial properties [31]. Brobbey et al., in their study, used an ethanolic extract of sea buckthorn in combination with carboxymethylcellulose to coat the paper. In a test of antimicrobial properties using a method of measuring zones of growth inhibition on the medium (disc diffusion), activity against both S. aureus (as a representative of Gram-positive bacteria) and Pseudomonas aeruginosa (as a representative of Gram-negative bacteria) was observed, but a stronger effect was observed against the latter [33]. Despite the use of different extraction solvents, these observations were in agreement with those achieved in this study. In contrast, Feng et al. observed an inverse relationship between sea buckthorn leaf extract and chitosan and observed a stronger effect for S. aureus than for E. coli [20]. In another study, extracts from Hippophae rhamnoides L. in combination with extracts from Achillea millefolium L. and Hypericum L. were used to obtain active coatings in a hydroxypropyl methyl cellulose carrier. PLA/PHBV films coated with active coatings showed complete inhibition of the growth of S. aureus and E. coli [34]. Nevertheless, in this study, the plant extracts accounted for 10% of the weight of the coating solution and were composed of a combination of three different plant extracts. In addition, despite the use of a higher concentration of active additives than we used in our study and the use of a combination of three types of plant extracts, the test using C. albicans also only resulted in a reduction in the survival of this microorganism, and not its complete inhibition. Guo et al. observed a slower proliferation of microorganisms (including harmful ones) in beef jerky packed in sea buckthorn pomace extract esterified potato starch film, and the higher the concentration of the extract used, the stronger the effect [23].

3.7. Microscopic Examination

The microstructure surface of PBS films and PBS films with additives was examined by SEM, and the micrographs revealed increased surface roughness in all of the examined samples (Figure 5). The neat PBS demonstrated rough surface properties, with a kind of microbead structure, with cracks and holes being noted. A similar film microstructure was noted by Tallawai M. et al. [35]. However, these authors analyzed PBS-DLA (poly (butylene succinate-butylene dilinoleate)), a polymer composed of saturated dilinoleic acid (DLA) as the soft segment and poly(butylene succinate) as the hard segment) films. Ebrahimpour M. et al. [36] and Karakehya N. found contradictory results [36,37], observing that neat PBS had a homogenous, clear, and smooth surface. Meanwhile, a rough surface with a microbead structure was noted by Ebrahimpour [36], though only for PBS blends with poly (dioxanone) (PDO). Similarly, Karakehya [37] noted a rough surface with various forms of holes, but in the case of NCC (nanocrystalline cellulose), reinforced PBS. A SEM analysis performed by Nurzia M. et al. [11] demonstrated that the PBS neat film showed an apparently smooth surface, confirming the toughness of the pure PBS polymer structure. However, an area of clumps appeared on the surface of PBS film containing thymol in a polymer matrix, revealing a poor dispersion of the active agent within the PBS matrix, probably caused by an aggregation of solid thymol leading to the phase separation between the two materials. Meanwhile, PBS film with kesum showed a well-integrated structure, confirming good compatibility between kesum and PBS compounds. A microscopic analysis demonstrated that significant differences between PBS films and PBS films containing added scCO₂ and Chl sea buckthorn extracts were not observed. It might be concluded that both extracts were well distributed in the PBS matrix. However, more holes/cracks were noted in the case of the PBS film containing scCO₂ extract, perhaps influencing a faster release of active substances from the biopolymer matrix. This assumption was confirmed by an examination of the antimicrobial properties of the film, which showed that PBS-CO₂ 5 was the most active against S. aureus and E. coli, and also had the strongest antioxidant properties. In addition, films which contained 0.05 g of this extract per 1 g of PBS were more porous than those containing a lower concentration of the scCO₂ extract, confirming that not only the method of extraction, but also the amount of extract has an influence on the microstructure of PBS films.

4. Conclusions

This article presents the results of the effect of sea buckthorn extract (from chloroform and supercritical CO₂) on the physicochemical and antimicrobial properties of poly(butylene succinate) materials. The modification of PBS with sea buckthorn extracts significantly improved the antioxidant and antimicrobial properties, allowing us to obtain films which were effective towards S. aureus, E. coli and C. albicans. Films modified with scCO₂ extract exhibited a greater impact on antimicrobial and antioxidant properties than chloroform extracts. Despite the use of equal amounts of chloroform or scCO₂ extracts, individual results differed, which indicates the differences in the extraction of the individual compounds from the fruits and the potential applicability in the search for the properties desired. In summary, these films combine three features: antibacterial and antifungal activity, antioxidant properties, and biodegradability. As a result, they can extend the shelf life of food products and, after use, can be composted. It can be underlined that modified PBS films demonstrate their potential for application in the food industry as ecofriendly, active packaging materials. Nevertheless, further research is required to determine the feasibility of using the extracts and films studied here in the food industry.