The Impact of the Antimicrobial Packaging Covered with Coatings Containing Carvacrol or Geraniol with the Addition of Zinc Oxide on the Quality of Sliced Plant-Based Sausages

Abstract

The aim of this work was to estimate the impact of polypropylene (BOPP) films with active coatings applied on their surface on the quality of sliced, plant-based meat analogue (PBMA) sausages. The coatings contained zinc oxide nanoparticles and geraniol (AG) or zinc oxide and carvacrol (AC) as active compounds. The outcomes of the study indicated that the total microbial count of ready-to-eat, sliced PBMAs bought from a local store was high, confirming that the plant-based sausage must have been contaminated during slicing. It was shown that BOPP bags and spacers covered with the AG layer reduced the number of mesophilic bacteria in sliced plant-based sausages stored for 96 h, proving that this packaging material maintained the microbial quality of PBMA samples. It has to be underlined that neither S. aureus, L. monocytogenes, Salmonella sp. nor coliform bacteria were detected in the plant sausage samples after 48 h and 96 h of storage in the BOPP packaging covered with the AG and AC coatings, confirming that these slices were acceptable for consumption. However, the textural analysis showed that bags coated with the AC layer were the best bags for 96 h of storage.

1. Introduction

Plant-based meat alternatives/analogues (PBMAs) have been investigated and developed to replace animal protein-based food with plant-based protein to consumers who intend to reduce their meat consumption to achieve a healthier diet and out of concern for the environment. Meat alternatives are products designed to mimic the texture, appearance, taste, nutritional aspects and cooking process of meat while being composed of plant-based ingredients such as soy, pea, wheat, potatoes, rice or fungi-based mycoprotein as the main protein source, as well as other vegetable-based products such as legumes or quinoa [1,2,3,4,5]. Moreover, meat analogues contain significant amounts of water, fats or oils, flavourings, colouring agents, and binding agents. It is worth mentioning that PBMAs belong to the ultra-processed food (UPF) category [2,3,6,7]. Ultra-processed foods are formulations of substances derived from whole foods, such as starches, fats and protein isolates, often with added flavouring, pigments, emulsifiers and other additives to extend their shelf-life and improve their palatability and visual appearance [2,8,9]. Among these ingredients are extra-virgin olive oils, polyunsaturated-fatty-acid-rich oils and vegetable protein-rich plants such as soya flour and hydrocolloids. Vegetable-sourced gels from soy and wheat protein are used to prepare PBMAs such as meat sausage analogues. Emulsion gels with a gel-type structure and solid-type mechanical properties consist of an emulsion stabilized by proteins with emulsifying agents and the addition of a thickener, such as a hydrocolloid (e.g., alginate), or other gelling ingredients that are able to create an emulsion gel either by aggregating emulsion droplets or by continuous-phase gelation [10]. The PBMA market is expanding, with novel products such as plant-based burgers and sausages becoming available to consumers. The global plant-based meat market was valued at USD 5.3 billion in 2021 and is projected to reach USD 85 billion by 2030 [2,3]. Considering the lack of public awareness regarding the nutritional value of PBMAs [2,6], the WHO in Europe has issued many recommendations regarding PBMAs, emphasizing the need to compare meat and dairy substitutes with their animal-source counterparts when analyzing nutritional content [2,11]. When comparing meat-based sausages with their plant analogues, one might notice that the manufacturing of sausages involves a number of handling steps, making them very susceptible to contamination by microbial strains responsible for food spoilage or microorganisms that can be human pathogens. The high water content and nutritional richness (e.g., proteins) of plant-based or meat-based sausages can enhance microbial growth [10,12,13]. Bacteria, such as Staphylococcus spp., Listeria monocytogenes, Salmonella spp., etc., are common causes of foodborne infections associated with sausages [12,13]. According to Sampson et al. [14], Salmonella spp. may also be detected in plant-based meat analogues. Liu et al. [15] analyzed the microbial quality and safety of plant-based meat analogues in comparison with meat-based products. The authors investigated the changes in native microflora present in pea-based and soy-based meat analogues and compared them with ground beef. Additionally, the authors artificially inoculated both meat- and plant-based products with meat spoilage microorganisms, such as Pseudomonas fluorescens and Brochothrix thermosphacta, as well as with pathogenic microorganisms such as Salmonella spp., Escherichia coli O157: H7 and Listeria monocytogenes. The authors’ results demonstrated that, despite the different initial concentrations of microorganisms, there was no difference among total bacterial counts between the two types of plant-based analogues and meat-based products by the end of the 10-day storage period at 4 °C, with all approaching ca. 7.00 log CFU/g. Artificially inoculated B. thermosphacta increased by 0.76, 1.58 and 0.96 log CFU/g in beef, pea-based and soy-based products, respectively, by the end of the storage period. P. fluorescens increased by 4.92, 3.00 and 0.40 log CFU/g in beef, pea-based and soy-based products, respectively. Moreover, pathogenic bacteria did not change in beef meat and soy-based analogues. L. monocytogenes increased by 0.74 log in a pea-based analogue during the 7-day storage period at 4 °C. All three pathogens grew well, regardless of the type of food product. The authors confirmed that plant-based analogues may support the survival and growth of pathogenic and spoilage-causing microorganisms just as well as meat products can. In conclusion, microorganisms responsible for the spoilage of meat-based sausages, as well as foodborne pathogens, can also contaminate plant-based sausages. Furthermore, contamination during handling and cutting may significantly reduce the shelf-life of sliced sausages (either plant- or meat-based) in addition to the effects caused by the initial, natural contamination of raw food products [13,16,17]. The growth of microbial strains leads to food discolouration and the release of off-odours and off-flavours, resulting in decreased product quality or even decreased shelf life. To avoid this problem, food preservatives such as sodium benzoate, potassium sorbate, lactate acetate or nisin are added by food manufacturers. These additives not only protect food from microbial growth but can also extend the shelf life of protein-based food products [13,17,18]. Unfortunately, they may also lead to health problems in humans. Among the methods aimed at improving the microbial safety of ready-to-eat PBMAs or meat products are gamma radiation, electron beam radiation or pressure and heat processes. These processes can reduce the microbial load. However, they may also change the texture or microstructure of protein-based foods and alter the chemical compounds present on their surfaces [13,17].

In light of this, the use of active packaging covered with antimicrobial coatings for the preservation of sliced PBMAs (e.g., sliced sausages) may prolong the product shelf-life by decreasing the number of microorganisms responsible for meat spoilage or by inhibiting their growth [13,17,18,19,20]. It is worth mentioning that when consumers decide to purchase sliced sausages, ham or plant-based analogues, they have a choice. They can buy products packed in a MAP (modified atmosphere), in a vacuum system, or decide to purchase products which were sliced by a shop assistant. However, slicing may lead to the food becoming contaminated while being handled by the shop assistant or slicing machine operator [20,21]. This consideration is very important for the estimation of applicable secondary shelf life—the time after package opening or after purchasing a plant-based meat analogue sliced in a store by a staff member. During this time, the PBMA should retain an acceptable/required quality level, leading to a reduction in food waste [20,21,22]. The importance of producing healthy and safe PBMAs not based on synthetic preservatives has resulted in the use of active food packaging. Polymer- or biopolymer-based films covered with active coatings are designed to release active antimicrobial agents into the food product to extend its shelf-life and maintain its safety and/or quality [20]. Single-layer or multi-layer films or laminates can be produced through the coating of a surface with different biobased carriers and antibacterial/antifungal agents in order to develop antimicrobial coatings which allow for prolonging the shelf life of food [13,20,23].

Carvacrol is an agent confirmed to be effective against food-borne microorganisms such as Escherichia coli O157:H7 and Salmonella sp. which are often responsible for the spoilage of meat or plant protein-based products. The antimicrobial properties of carvacrol extend to drug-resistant strains such as methicillin-resistant Staphylococcus aureus and S. epidermidis. Furthermore, its effectiveness against bacteria may also be enhanced when combined with different active agents, e.g., antibiotics [23,24]. Similarly, geraniol is a natural substance which was confirmed to exert antimicrobial activity towards S. aureus, Streptococcus pyogenes, S. pneumoniae, Escherichia coli and Salmonella typhimurium [25]. Geraniol also exhibits synergistic interactions when combined with other compounds, such as selected antibiotics [26]. Due to their antimicrobial effectiveness, carvacrol and geraniol can be added to a coating carrier to create an active coating when applied on a surface of a packaging material. However, an important requirement for many applications is to obtain an antibacterial coating which is almost transparent; however, a high concentration of an active compound (e.g., geraniol or carvacrol) may lead to the creation of a non-transparent coating. This is why the amount of the active agent in the coating carrier should be as low as possible [13,20,23]. The results of the previously mentioned work [23] demonstrated that the addition of ZnO nanoparticles to a carrier containing geraniol or carvacrol led to the creation of two antimicrobial coatings with a confirmed synergistic effect between these active agents. The addition of zinc oxide nanoparticles made it possible to decrease the quantity of geraniol and/or carvacrol in the active coating. As a result, the active layers were almost transparent; they inhibited the growth of S. aureus and reduced the number of E. coli.

Drawing inspiration from the previous work’s results, the purpose of the current work was to analyze the influence of polypropylene (BOPP) films covered with active coatings containing geraniol with the addition of ZnO nanoparticles (AG) and/or carvacrol with nano ZnO (AC) as active agents on the microbial purity, microstructure and texture of sliced PBMA sausage.

2. Materials and Methods

Fresh PBMA sausages were purchased from a local store and brought (in commercial, polyethylene bags) to the CBIMO’s (Center of Bioimmobilisation and Innovative Packaging Materials) laboratory.

Polypropylene foil (BOPP, A4, 20 μm) (MarDruk, Andrychów, Poland) was used as the packaging film/material. Hydroxy-propyl-methyl cellulose (HPMC) (Chempur, Piekary Śląskie, Poland) was the coating carrier used in the experiment to cover the BOPP film. Tween 20 and Tween 80 (Sigma-Aldrich, Poznań, Poland) were used as emulsifiers to improve the adhesion of the coatings (active layers) to the BOPP foil. ZnO AA 44899, (particle sizes of ~70 nm), carvacrol (Avitale Pure Liquids, Białystok, Poland) and geraniol (Sigma-Aldrich, Poznań, Poland) were used as antimicrobial agents. To verify the count of coliform microorganisms in the sliced meat analogue (sausage), the VRBG (Violet Red Bile Glucose Agar) was used (Merck KGAA, Darmstadt, Germany). To determine the S. aureus count, the tbc (total bacterial count) and the total psychrotrophic bacteria count of the sliced plant-based sausage, the Baird–Parker medium (Merck KGAA, Darmstadt, Germany), PPS (PPS: 0.1% m/v peptone; 0.85% m/v NaCl) and PCA (BTL, Łódź, Poland) were used. To verify the Salmonella sp. cell count, the MKTTn (Muller–Kauffmann Tetrathionate–Novobiocin), Rappaport Vassiliadis Broth, XLD and BGA (Scharlau, Barcelona, Spain) media were prepared. To verify the Listeria monocytogenes count, half Fraser broth, Fraser broth and Fraser agar were prepared. The media were prepared according to BTL, Merck and Scharlau protocols. All media, except VRBG and XLD agars, were weighed as the manufacturer recommended, then introduced into 1000 mL of distilled water and autoclaved at 121 °C for 15 min. VRBG and XLD agars were weighed as the manufacturer recommended, then introduced into 1000 mL of distilled water and heated to boiling.

2.1. Coating Preparation

The amount of 0.082 g of zinc oxide nanoparticles was added into 100 mL of water. The dispersion was mixed for 60 s using a magnetic stirrer (450 rpm, Ika, Warsaw, Poland). The second step of the experiment was a 30 min sonication process of the dispersion (sonication parameters: cycle: 0.5, amplitude: 20%, power: 400 W and frequency: 24 kHz). At the same time, the coating carriers based on 4% HPMC were prepared as follows: (1) a total of 0.0125 g of carvacrol, 1 g of Tween 20 and 1 g of Tween 80 were mixed with 97.9875 g of HPMC; (2) a total of 0.0125 g of geraniol, 1 g of Tween 20 and 1 g of Tween 80 were mixed with 97.9875 g of HPMC. Following this, 50 mL of the water dispersion of the ZnO nanoparticles was introduced into 50 mL of the coating carrier containing geraniol (AG), and 50 mL of the nano zinc oxide dispersion was introduced into 50 mL of the coating carrier with the addition of carvacrol (AC). The coating carriers containing active agents were sonicated (with sonication parameters as described above).

The AG and AC coatings were applied on the surface of the polypropylene (BOPP) film with a 40 μm diameter roller using Unicoater 409 (Erichsen, Hemer, Germany) at 25 °C. The BOPP foil was coated on one side to obtain active bags. However, both sides were covered to obtain spacers. The neat BOPP film that was not coated with any antimicrobial layers was examined as the control sample (C). The coated and neat BOPP foils were cut and used to prepare square foil spacers and bags. To obtain bags, the neat and active films were joined using a welder (HSE-3, RDM Test Equipment, Hertfordshire, Great Britain) under regular atmospheric conditions. The welding parameters were as follows: temperature—117 °C, pressure—4 kN and time—4 s.

2.2. Coatings’ Optical Property Examination

The UV-Vis spectrophotometer UV-Vis Thermo Scientific Evolution 220 (Waltham, MA, USA) was used to examine the transparency (T, transmittance at 700 nm) and UV-ray absorption capacity in the wavelength range of 190–900 nm.

2.3. Coatings’ Surface Examination

The surface of the neat BOPP film as well as the film coated with the thin AC and AG antimicrobial layers were studied/examined using a scanning electron microscope (SEM) as follows: 1. All samples were placed on pin stubs and sprayed with a thin layer of gold in a sputter coater at 24 °C (Quorum Technologies Q150R S, Laughton, Wealden, East Sussex, UK). 2. SEM micrographs were taken using a Vega 3 LMU microscope (Tescan, Brno-Kohoutovice, Czech Republic). The microscopic evaluation was performed using a tungsten filament with an accelerating voltage of 10 kV.

2.4. Packaging and Storage

The plant-based meat analogue sausage was cut into 5 mm slices (Figure 1a). The PBMA sausage was also cut into 20 mm slices; however, these slices were used only for texture analysis. Each PBMA slice was separated with a square spacer (Figure 1b). The portions were then aseptically introduced into active and neat BOPP bags, and they were welded as follows:

a.

Control samples (C)—neat BOPP bags: PBMA slices separated with BOPP spacers;

b.

AG bags—BOPP bags with the AG coating applied on the internal side of the bag: PBMA slices separated with spacers with the AG coating applied on both sides of the bag;

c.

AC bags—BOPP bags with the AC coating applied on the internal side of the bag: PBMA slices separated with spacers with the AC coating applied on both sides of the bag.

Figure 1. The sliced chicken sausage (a) “0” samples; (b) “0” samples separated with spacers.

Figure 1. The sliced chicken sausage (a) “0” samples; (b) “0” samples separated with spacers.

The joining of all bags was conducted using a welder as described above.

The bags with PBMA sausages were stored for 4 days at 5 °C. The quality of the PBMA portions/slices was examined after 48 h and 96 h of storage.

2.5. The Textural Analysis

The texture examination of the PBMA sausage (20 mm slice) was conducted according to the PN-ISO 11036:1999 standard: “Sensory analysis. Methodology. Texture profiling” [27]. The texture analysis was performed using Zwick/Roell Z 2.5 (Wrocław, Poland).

2.6. The Microbiological Purity Examination

For the microbiological purity examination, 10 ± 0.1 g of individual 5 mm PBMA slices was aseptically introduced into a sterile stomacher bag containing physiological saline peptone solution (PPS: 0.85% m/v NaCl and 0.1% m/v peptone). The plant-based meat analogue was homogenized in a bag mixer (Interscience, Saint-Nom-la-Brèteche, France) for 60 s, and decimal dilutions were prepared in PPS. The total count (mesophilic bacteria) was examined according to PN-EN ISO 4833-2:2013-12 [28]; the total psychrotrophic count (TPC) was evaluated according to the PN-ISO 17410:2004 standard [29]; the S. aureus count was analyzed according to PN-EN ISO 6888-1 [30], and the total coliform bacteria count was determined according to PN-ISO 4832:2007 [31]. To evaluate the Salmonella sp. count, 25 ± 0.1 g of the PBMA sausage was aseptically introduced into 225 mL of sterile PPS. The total Salmonella sp. count was evaluated after 18 h of incubation according to PN-EN ISO 6579-1:2017-04 [32]. To determine the L. monocytogenes count, 25 ± 0.1 g of PBMA slices was aseptically introduced into 225 mL of sterile, half Fraser broth. The total L. monocytogenes count was examined according to the PN EN ISO 11290-1:2017 standard [33].

2.7. The Microstructure Analysis

Before PBMA microtexture analysis, the samples of the plant-based meat analogue were submerged into a medium containing 2% glutaraldehyde in 0.1 M sodium cacodylate with a pH of 7.4 (for 18 h at 4 °C). Then, portions of the slices were washed with 0.1 M sodium cacodylate. Next, they were dehydrated in serial concentrations (20%, 40%, 60%, 80% and 100%) of ice-cold methyl alcohol (−20 °C) for 120 min. The samples of the PBMA sausage were placed in a Petri dish to dry (in atmospheric conditions). After 5 min of drying, they were placed on pin stubs. Then, a thin layer of gold was applied on the surface of the samples using a sputter coater at 25 °C (Quorum Technologies Q150R S, Laughton, Wealden, East Sussex, UK). The PBMA samples were evaluated with a Vega 3 LMU scanning electron microscope (Tescan, Brno-Kohoutovice, Czech Republic).

2.8. Dry Mass Tests

The dry mass of the fresh plant-based meat analogue sausage was examined before storage and after 48 h and 96 h of storage. Dry mass examination was performed (in duplicate) using a weight dryer (Radwag, Warsaw, Poland).

2.9. L* a* b* Tests

PBMA colour analysis was based on the average of 9 evaluations from selected analogue/sausage slice spots and was performed with a colorimeter (NR 20 XE, EnviSense, Lublin Poland), and associated data processing software. The colour was examined through the aperture (with a diameter of 8 mm) using the CIE L* a* b*colour space with a standard observer and Illuminant D65. The evaluated parameters were ∆L (the difference between lightness and darkness) and ∆E_lab (total colour aberration). The parameters were calculated according to the EnviSense protocol.

2.10. Statistical Analysis

The analysis of variance (ANOVA) followed by a one-way ANOVA test was used to evaluate the statistical significance of the PBMA samples. The values were noted to be significantly different when p < 0.05. All tests were carried out with GraphPad Prism 8 (GraphPad Software, Version 9, San Diego, CA, USA).

3. Results and Discussion

3.1. Optical Properties of Coatings

The UV-Vis spectroscopy (transmittance) outcomes are presented in Figure 2. As was emphasized in the figure, all studied samples exhibited low barrier properties toward UV radiation. Upon analyzing the transmittance in visible light, the neat BOPP film did exhibit partial opacity, but its transparency measured at 700 nm clearly exceeded 80%. The opacity of both the AC and AG coatings was characterized by transmittance values of about 80%. These slightly lower values remain comparable with the neat film, despite the fact that carvacrol as the active additive was dark brown. One may suspect that the almost transparent AC and AG coatings which contained very low amounts of ZnO nanoparticles together with geraniol or carvacrol as antibacterial additives should not exhibit any noticeable activity. However, previous work confirmed that these coatings inhibited the growth of Gram-positive bacteria and reduced the number of Gram-negative cells [23].

3.2. Coatings’ Surface Analysis

As was emphasized in Figure 3, the neat BOPP film had a slightly rougher surface compared to the uncovered film (Figure 3a. SEM micrograph under the magnification of 500×). As was showed in Figure 3b,c, the antimicrobial coatings (AC and AG) had no significant effect on BOPP surface morphology. However, a homogenous, thin and smooth appearance of the thoroughly applied active layers was noticed. Additionally, small halls or surface irregularities in the form of indentations were visible on both the AC and AG coatings, while higher halls or indentations were observed on the surface of the AC layer. Similar results were noted in the previous studies [13,20], in which the packaging coated with the active layers based on Uncaria tomentosa and Formitopsis extracts with the addition of zinc oxide nanoparticles [20] or with the mixtures of Glycyrrhiza L. and Scutellaria baicalensis extracts [13] was prepared. The analysis of the cross-section of the AC and AG layers confirmed that both active layers were relatively thin. The range of the AC coating’s thickness was 0.69–0.73 µm (Figure 4a,b), while the range of the AG active layer was found to be 0.61–0.75 µm (Figure 4c,d). The low thickness and colourlessness of both antimicrobial layers contributed to their transparency. An additional advantage of the coatings was the lack of odour despite the presence of carvacrol and geraniol as antimicrobial additives. Based on current and previous observations, one can conclude that homogenous active coatings with convex, spherical irregularities and small halls can have an effect on the release of active/antimicrobial additives/agents on the whole surface of the food product.

3.3. Microbial Analysis

The outcome of the research work indicated that the number of mesophilic bacterial cells (total count) from the sliced plant-based meat sausage analogue kept in neat BOPP bags (C—control sample) increased insignificantly after 48 h at 5 °C in atmospheric conditions. However, after 96 h, a significant (almost 3 log) increase in the number of bacterial cells was noted (compared to the total count detected for the “0” sample—before storage). Figure 5 demonstrated that an AG coating containing geraniol and zinc oxide nanoparticles had an impact on the growth of mesophilic microorganisms. It decreased the number of these bacteria after 48 h of storage. Furthermore, a lower than 1 log increase in the number of bacterial cells was noticed (compared to the “0” sample—before storage) for the slices stored for 96 h. Analyzing the influence of the AG coating on the total count of psychrotrophic bacteria (Figure 6) showed that the AG layer inhibited the growth of these microorganisms after 48 h of storage and reduced their number significantly after 96 h of being kept in active bags (compared to the samples stored for 96 h in neat BOPP bags). It is tempting to suggest that the AG coating was effective towards mesophilic and psychrotrophic bacteria. As was emphasized in Figure 5, the BOPP film covered with the AC layer (containing ZnO nanoparticles and carvacrol) did not influence the total bacterial count of PBMA slices after 48 h of storage. However, it had an impact on the number of these bacteria after 96 h of storage, decreasing their numerosity (compared to the neat BOPP films). Similarly, the effectiveness of AC1 against psychrotrophic bacterial cells was confirmed. As highlighted in Figure 6, complete inhibition of the psychrotrophic bacteria by the AC coating was noted. To summarize, it was concluded that the number of mesophilic and psychrotrophic microorganisms detected from the PBMA samples stored in BOPP bags and in BOPP bags that had been coated with the effective AG and AC layers was higher after 96 h of storage, thereby confirming that the coatings had antimicrobial properties.

Taking into account that there are no guidelines for the microbiological purity of plant-based sausages and the fact that these products are considered to be meat analogues/substitutes, standards defining acceptable limits for meat products can be used to determine the shelf life of PBMAs. It is generally accepted that the acceptable limit for mesophilic bacteria in meat and its products is 6–7 log CFU/g [34]. However, Grzybowski and Reiss [35] underlined that the total bacterial count of sliced meat products should be lower than 10³ CFU/g to be satisfactory/acceptable as a ready-to-eat food. The same authors [35] also mentioned that 10⁴ CFU/g is commonly considered to be the highest acceptable bacterial/microbial load for meat-based sliced food. A number greater than 4 log₁₀ CFU/g is noted as a bacterial load that is unacceptable for sliced meat-based food to be eaten/consumed. This means that after 48 h of storage of the PBMA samples, all slices were acceptable to be consumed regardless of the packaging in which the samples were kept. This also means that after 96 h of storage, in contrast to the plant-based sausage slices kept in neat BOPP bags or in BOPP bags covered with the AC active layer, the shelf life of the sliced PBMA samples kept in bags covered with AG coating can be 96 h, as the total count for these samples was still observed to represent a satisfactory bacterial load for meat product portions. A previous study [20] confirmed that the antibacterial/antimicrobial packaging based on Uncaria tomentosa and/or Formitopsis betulina extracts with zinc oxide nanoparticles inhibited the growth of E. coli and S. aureus. Additionally, both active coatings had an impact on maintaining the microbial quality of the cooked ham. However, the coating containing the U. tomentosa extract and nano ZnO as active agents had a more significant impact on the total count compared to the coating with the F. betulina extract and zinc oxide.

Geraniol, as a monoterpenoid alcohol, was confirmed to exhibit strong antimicrobial activity. Its mechanism of action involves multiple cellular targets, including membrane disruption and metabolic interference. It may also increase the levels of reactive oxygen species (ROS), causing oxidative damage to DNA, proteins and lipids. The outer membrane of Gram-negative microorganisms limits the uptake of hydrophobic substances like geraniol, which explains why this compound is more effective towards Gram-positive strains than Gram-negative bacterial cells. It is worth mentioning that carvacrol was found to be even more effective than geraniol thanks to its hydroxyl group that enables stronger hydrogen bonding and membrane destabilization [36,37,38]. On the other hand, inducing the formation of reactive oxygen species (ROS) is the most common and widely confirmed mechanism for the antimicrobial effectiveness of zinc oxide nanoparticles [39]. The previous work [23] confirmed the existence of a synergistic effect between ZnO nanoparticles and carvacrol or geraniol. The current work’s results determined that a coating containing geraniol and zinc oxide nanoparticles was more effective than the coating based on carvacrol with the addition of nano ZnO. The results may lead to the conclusion that a synergistic effect between geraniol and zinc oxide nanoparticles was stronger than the synergistic effect between ZnO NPs and carvacrol.

Another authors’ work [13] demonstrated that antibacterial bags and antibacterial spacers based on the mixture of Scutelaria baicalensis* and Glycyrrhiza L. extracts (mixed in the ratio of 1*:2) used for the preservation of sliced chicken sausages had a bacteriolytic effect on mesophilic strains after 72 h of storage. These bags decreased the number of these bacteria after 144 h of storage. It is worth mentioning that the total count evaluated for the sliced plant-based sausage on the day of purchasing (“0” sample—before storage) was 1.42 × 10³ CFU/g (more than 3 log₁₀ CFU/g), showing that the slicing of the PBMA by a shop assistant must have contaminated the product. Similar observations were noted in the previous work [13], which indicated that the quantity of mesophilic microorganisms detected in sausage slices purchased from a local store/butcher (“0” sample) was also high (almost 3 log₁₀ CFU/g). In summary, consumers purchase ready-to-eat PBMA products which are often cut/sliced by staff, potentially introducing contamination. This is why polyethylene, polypropylene or paper packaging covered with the AG coating might be developed to preserve the portions/slices of the plant-based sausage during its transportation and short-term storage (96 h). The findings observed in the manuscript were verified by Shiji et al. [12], who developed polythene, antimicrobial pouches to pack chicken sausages which could then be stored for a brief period. PE pouches were used as the control packaging, while a biodegradable PVA–montmorillonite K10 clay nanocomposite blend with the addition of silver nanoparticles as antimicrobial agents was used as the active packaging. The slices were stored for 4 days at 4 °C. The authors determined that the sausage slices in the PE pouches demonstrated higher bacterial growth comparing to the antimicrobial pouches. The results of the current work showed that neat BOPP bags and bags coated with the AC layer were not effective enough to maintain the quality of PBMA slices after 4 days of storage. On the other hand, Shiji et al. [12] confirmed that antimicrobial pouches (based on nanocomposites) strongly reduced the number of bacterial cells in sausage slices after 96 h of storage. Shahrampour et al. [40] prepared circle-shaped, antimicrobial films containing green tea extract as the active agent and put them on sausage slices. The antimicrobial spacers and vacuum system were developed for this product (which was stored for a month at 4 °C). The authors’ results demonstrated that the total count in the control sausage kept in the uncoated package was noticeably higher than the number of these microorganisms in samples which were kept in the packaging with antimicrobial spacers, confirming that antimicrobial packaging had an impact on maintaining the quality of the wrapped food slices.

As Grzybowski and Reiss [35] mentioned, no coliform or L. monocytogenes cells should be detectable in a 1 g portion of the sliced meat sausage. Likewise, Salmonella sp. cells should not be detectable in 25 g of the sliced meat product. Moreover, the quantity of the S. aureus cells in 1 g of ready-to-eat slices or portions of the meat-based food should be less than 100 to be considered appropriate for consumption. A previous paper [23] confirmed that the AC and AG coatings inhibited the growth of S. aureus and reduced the number of E. coli cells. In accordance with these results, it was assumed that the coating may inhibit or limit the growth of pathogenic bacteria on the surface of PBMAs. The examination of a plant-based sausage before and after storage indicated that neither the coliforms, L. monocytogenes, Salmonella sp. nor S. aureus cells were isolated from the “0” sample and from all of the examined sliced plant-based sausage slices stored in neat BOPP bags and in BOPP bags coated with the AC and AG coatings. From this point of view, all samples were acceptable for consumption even after 96 h of storage. The outcomes of the work confirmed our previous assumptions. Similar results were observed in previous studies [13,20], confirming that active packaging may preserve food products rich in protein against pathogenic bacteria such as coliform microorganisms, L. monocytogenes, Salmonella sp. and S. aureus. It is worth mentioning that Van Paepeghem et al. [41] suggested that plant-based meat analogue slices are subjected to similar processing steps as meat-based slices. During the slicing and pre-packing step, the risk of L. monocytogenes contamination is potentially high. This is why it is very difficult for food manufacturers and shops/local butchers to achieve prolonged shelf life while at the same time controlling L. monocytogenes outgrowth.

3.4. The Textural Analysis’ Results

The quality of sliced products such as meat- or plant-based sausages is determined through the examination of their texture. Sausage slices which are not sufficiently cohesive are considered to be too soft, and the consumer can doubt their quality and freshness. Cohesiveness representing the forces which hold the sausage (or another food product) together is one of the crucial parameters for assessing processed food quality. Additionally, stingy or gummy products are not acceptable to consumers as they exhibit very strong resistance during mastication [42]. The outcomes of the work indicated that the springiness of the sliced plant-based sausage did not change after 48 h and 96 h of storage in neat BOPP films. A modification of the packaging with an antimicrobial AC coating containing carvacrol and zinc oxide nanoparticles did exert an influence on the springiness of the sliced meat analogue after 48 h and 96 h of storage. Similar observations were noticed for the bags coated with the layer containing geraniol and nano ZnO after 96 h of sausage storage. It was observed that after 48 h of storage in bags covered with the AG coating, an insignificant decrease in springiness was noted (Figure 7). The results showed that the AC and AG layers did not improve the quality of PBMA slices in comparison with the neat BOPP bags. Similar results were obtained in previous work [20], which showed that coatings based on U. tomentosa and/or F. betulina extracts with the zinc oxide nanoparticles did not significantly influence the springiness of sliced cooked ham. However, coatings based on S. baicalensis and Glycyrrhiza L. [13] decreased the springiness of the sliced chicken sausage.

As emphasized below (Figure 8), the gumminess of the sliced plant-based sausage kept in neat BOPP bags neither increased nor decreased significantly after 48 h and 96 h of storage (when compared to the “0” sample). When analyzing the sausage slices which were stored for 48 h in the packaging material covered with the AC and AG coatings, it was observed that gumminess did not change, confirming that active films did not influence this parameter. After 96 h of storage, the gumminess of the sliced PBMA kept in the packaging based on carvacrol and zinc oxide decreased when compared to the samples stored for 96 h in uncoated bags and compared to the “0” sample. Moreover, the differences between the samples were found to be insignificant (p > 0.05). The results from the previous study [20] determined that only the coatings based on U. tomentosa extract with the addition of zinc oxide nanoparticles decreased the gumminess of sliced cooked ham, while such an effect has not been observed for neat BOPP bags and bags covered with the layer containing F. betulina and ZnO nanoparticles. However, coatings based on S. baicalensis and Glycyrrhiza L. extracts [13] decreased the gumminess of the sliced chicken sausage when compared with uncoated bags.

While analyzing cohesiveness, it was noted that this parameter value for the PBMA slices stored in neat BOPP bags did not change significantly after 48 h and 96 h of storage (Figure 9). The cohesiveness values, evaluated for the same meat analogue slices, but kept/stored for 48 h in bags coated with the AC layer, increased. However, this parameter decreased for the slices kept in bags covered with the AG coating (compared to the “0” sample), though the change was not significant. Opposite results were observed for the samples stored for 96 h in active packaging. The outcomes from the previous work [20] demonstrated that some significant changes in cohesiveness for the sliced cooked ham samples that were stored in bags covered with the coatings based on U. tomentosa and/or F. betulina extracts with the addition of zinc oxide were noticed. Comparing packaging coated with the layers based on S. baicalensis and Glycyrrhiza L. extracts to the neat BOPP bags [13], it was observed that cohesiveness decreased after 72 h of sliced chicken sausage storage and increased after 144 h. However, Patiño et al. [43] indicated that the cohesiveness of meat-based sausages decreased during storage. The authors mentioned that a decrease in cohesiveness might be caused by alterations in the interaction of proteins and fats with the rest of the components, influencing the textural parameters of the sausages.

Comparing the average values of chewiness obtained for the “0” sample and for the slices kept in neat BOPP bags with the values of this parameter observed for the PBMA portions stored in active packaging, it was demonstrated that the decrease in chewiness was noted only for the samples kept for 48 h in bags coated with the AG layer and 96 h for the samples stored in bags with the AC coating applied on the internal surface of the bags. Moreover, the differences between chewiness values were found to be insignificant (Figure 10). The results from the previous study [20] determined that the coatings based on U. tomentosa and F. betulina extracts with zinc oxide nanoparticles had an insignificant impact on the chewiness of sliced cooked ham. Comparing the bags covered with the coatings based on S. baicalensis and Glycyrrhiza L. extracts to the neat BOPP bags [13], it was observed that chewiness decreased after 72 h of sliced chicken sausage storage and increased after 144 h. In summary, the current results showed that, unfortunately, chewiness and gumminess increased after 96 h of storage of PBMA slices kept in neat BOPP bags and in bags covered with the AG coating. This is considered a clear disadvantage. As Carhuancho-Colca et al. mentioned [16], the starch content in plant sausages could generate an increase in hardness and chewiness due to the swelling of the starch granules embedded in the protein matrix. The only package which led to a decrease in gumminess and chewiness values was the bag covered with the AC coating. As was noticed by Zeraatpisheh et al. [17], when the water content decreases, proteins converge due to the formation of new crosslinks; as a result, the gumminess and chewiness of the food samples are elevated. An increase in gumminess and chewiness, as observed in neat BOPP bags and BOPP bags covered with the AG coating, was a clear disadvantage because these parameters are associated with food hardness. Thus, in PBMA sausage slices with increased gumminess and chewiness after 96 h of storage, their hardness and difficulty in swallowing for the consumer could be noticeable. While the plant-based sausage kept in active bags coated with the AC layer was characterized by slightly decreased gumminess and chewiness values, the sausage slices would still be satisfactory for consumption.

3.5. Microstructure Analysis

The plant-based sausage samples (Figure 11a) demonstrated large and small protein particles involved in the gel/hydrocolloid network. It was observed that the general surface of the plant sausage was not homogenous but rather smooth. The porous structure with air holes was noted. While analyzing the microstructure of meat analogues after storage, it was noticed that the slices had a gelatinous appearance (Figure 11b–g). As was seen on Figure 11b,d–g, a significant change in the PBMA samples’ microstructure was not noted. After 96 h of storage in neat BOPP bags and in bags covered with the AG coating, the microstructure of the plant sausage has become more compact (Figure 11c,e). This was confirmed/backed up by results of the texture examination which indicated that the gumminess and chewiness of the PBMA slices kept in bags coated with the AG layer and neat BOPP bags increased. Similar results were observed for sliced ham [20] stored in bags covered with the coating containing F. betulina extract as an active agent. It was noticed that the surface of the samples of the plant sausage introduced into bags with the AC coating for 96 h (Figure 11g) was less compact and dense than the microstructures of the samples stored in neat BOPP bags and in the bags with the AC layer, confirming water migration. Additionally, air bubbles (holes) were noticed along the surface of the slices, as well as small cracks which were present on the surfaces of all samples before and after their storage.

3.6. Dry Mass Examination

The outcomes of the work indicated that the dry mass of the portioned meat analogue was 41.24%. The storage of sausage slices/portions in neat BOPP films led to a decrease in the dry mass to 30.75% after 48 h of sample storage and an increase in the dry mass to 42.76% after 96 h. It was noticed that the dry mass of the portioned PBMA kept in bags covered with the AC coating was lower than the dry mass of the sausage portions kept in uncoated packaging after 48 h and higher after 96 h of storage. The AG coating applied on the internal surface of the BOPP bags led to a decrease in the dry mass of the sliced PBMA sausage after 48 h of sample storage and an increase after 96 h of storage (

Table 1. The dry mass of the sliced plant-based sausage after 48 h and 96 h of storage.

Time [h]	Dry Mass [%]
	C	AC	AG
0	41.24 ± 3.30 *	41.24 ± 3.30 *	41.24 ± 3.30 *
48	30.75 ± 3.82 *	30.58 ± 2.91 *	31.26 ± 3.96 *
96	42.76 ± 1.40 *	44.72 ± 0.60 *	44.84 ± 0.01 *

* SD—standard deviation.

). It is worth mentioning that after 96 h of storage of the PBMA slices, the dry mass was the highest for the samples kept in the bags coated with the AG layer. These findings were confirmed via microbial purity analysis, which demonstrated that the bags coated with this coating were the best packaging for the short-term (96 h) storage of the sliced plant-based sausage. As was mentioned in the previous study [20], the high water loss could have led to the release of antibacterial agents from the antimicrobial layers and thus could improve their antimicrobial/antibacterial effectiveness. However, when investigating the gumminess and chewiness of the PBMA portions kept in bags with the AG layer after 96 h of storage, unfortunately, an increase in these two parameters values was noticed.

3.7. L* a* b* Analysis

It was indicated in this work that ∆E_lab depended significantly on the bags in which the sliced PBMA sausage was kept (

Table 2. The colour changes in the sliced plant-based sausage after 48 h and 96 h of storage.

Time [h]		C	AC	AG
ΔElab	0	0.44 ± 0.15 *	0.44 ± 0.15 *	0.44 ± 0.15 *
ΔL		−0.04 ± 0.17 *	−0.04 ± 0.17 *	−0.04 ± 0.17 *
ΔElab	48	0.56 ± 0.16 *	0.93 ± 0.56 *	0.65 ± 0.27 *
ΔL		−0.46 ± 0.04 *	0.05 ± 0.38 *	0.07 ± 0.34 *
ΔElab	96	0.51 ± 0.06 *	1.14 ± 0.95 *	2.24 ± 0.47 *
ΔL		−0.18 ± 0.41 *	0.43 ± 0.73 *	1.34 ± 0.44 *

* SD—standard deviation.

). The ∆E_lab of the sliced PBMA sausage that was introduced into neat BOPP bags for 48 h was lower than of sausage portions kept in the bags covered with the AC and AG coatings. Additionally, the highest ∆E_lab was observed for samples kept in films coated with the AC layer. Similarly, the ∆E_lab of PBMA slices stored in bags covered with active coatings was higher than the ∆E_lab of PBMA portions kept in neat BOPP bags after 96 h of storage. After 4 days of sample storage, the highest ∆E_lab was noticed for slices introduced into the bags covered with AG coatings. Opposite findings were demonstrated in the previous study, which indicated that the greatest ∆Elab values were observed for cooked ham slices kept in the uncoated bags [20]. The results of the current study showed that the highest values of the ∆L were noted for the sausages kept in active packaging rather than in the neat BOPP bags. The highest ∆L was observed for the portions kept in bags with the AG layer. This indicated that the sausage slices taken from AG bags were the lightest. Moreover, it could be added that ∆L increased for PBMA slices kept in all active bags. The outcomes were proven by Azlin-Hasim et al. [44], who showed that L* values increased in meat portions kept for 6 and 12 days in bags/packaging with Ag nanoparticles as the antibacterial/antimicrobial compound. It may be assumed [45] that the release of water from meat analogues enhanced the light reflection on the sausage surface. Moreover, this effect could also be caused by bacterial strains that exert an influence on pigment degradation processes. Additionally, Patiño et al. [43] observed that in the case of meat-based sausages, colour changes may be caused by chemical reactions between proteins and sodium nitrite or the other additives used in sausage formulation.

4. Conclusions

The results of this study indicated that the total count from the ready-to-eat, sliced plant-based meat analogue bought from a local butcher (“0”—before samples were kept/stored) was high, proving that the plant-based sausage could have been polluted during cutting/slicing. It was shown that BOPP spacers and bags with the AG layer reduced the number of mesophilic strains for portioned plant-based sausages that were kept for 96 h, confirming that this packaging material was the best one to maintain the microbial quality of PBMA samples. It has to be underlined that neither the S. aureus, L. monocytogenes, Salmonella sp. nor coliform bacteria were isolated from the plant sausage portions/samples after 48 h and 96 h of storage in BOPP packaging/bags with the AG and AC coatings, confirming that these slices were satisfactory for consumption. However, the textural analysis showed that bags coated with the AC layer were the best bag for 96 h of storage.

In summary, from a microbiological point of view, the spacers and bags coated with layers containing geraniol and zinc oxide nanoparticles were noticed to be the best BOPP bags for ready-to-eat PBMA sausages that were bought from a local butcher’s shop. However, upon analyzing the textural parameters, the AC coating was noticed to be the most effective, active layer.