**About the Editors**

**Matteo Alessandro Del Nobile** received his Ph.D. Degree in Material Science from the University of Naples "Federico II". Since 2001, he has worked at the University of Foggia where he is full professor. Most of his early research efforts were put towards determining the relationships that exist between the physical and chemical structure of polymers and their mass transport properties. This topic has been approached both from a theoretical and from an experimental point of view. The knowledge he gained studying polymers has more recently been applied to food science. He has published over 250 papers on topics related to food science with particular attention on food packaging, food preservation and functional food. He has been Principal Investigator of many national projects and is the coordinator of the Ph.D. in "Translational Medicine and Food: Innovation, Safety and Management" at the University of Foggia.

**Amalia Conte** She graduated at the University of Foggia in Food Science and Technology and since 2008 she has been a researcher. Her research interests include food packaging, functional food, shelf-life prolongation and novel technologies. In the last 10 years, she has been involved in successful national research projects. To date, she has co-authored more than 200 scientific publications in international peer-reviewed journals, 20 book chapters, 1 book and 1 edited book, as well as serving as guest editor for four Special Issues. She is a peer-reviewer for various scientific journals in the food science sector. She is the CEO of Spin Off and is co-inventor of five patents.

### *Editorial* **Introduction to the Special Issue: Advanced Strategies to Preserve Quality and Extend Shelf Life of Foods**

**Amalia Conte \* and Matteo A. Del Nobile \***

Department of Agricultural Sciences, Food and Environment, University of Foggia, Via Napoli 25, 71122 Foggia, Italy

**\*** Correspondence: amalia.conte@unifg.it (A.C.); matteo.delnobile@unifg.it (M.A.D.N.)

We are pleased to present this Special Issue, which includes 13 papers that highlight the most important research activities in the field of food quality assurance and shelflife extension. The goal of this Special Issue was to broaden the current knowledge of advanced approaches to guarantee the maintenance of the properties of packaged products during storage. The most consolidated strategies in the literature concern the use of heat and modified atmospheres. However, knowledge gained in the sector has broadened the perspective and found valid and effective alternative in the use of bioactive compounds, industrial food by-products, adoption of active packaging solutions or the application of novel mild treatments, such as pulsed light, ultrasounds, high-pressure processing and cold plasma.

The 11 research articles/communication/ and 2 reviews that comprise this Special Issue highlight the most recent research and investigations into this exciting area, covering the following topics: (i) vacuum packaging; (ii) cork closures; (iii) innovative active packaging; (iv) emerging technologies; (v) the reuse of by-products; and (vi) secondary shelf life.

Interesting results that bring to light the issues concerning the effects of vacuum packaging on surface color and lipid oxidation of beef steaks were presented by Reyes et al. [1]. The results from this study suggest that the use of vacuum packaging for beef steaks is plausible for maintaining quality characteristics during extended display periods.

The study of Amaro et al. [2] aimed at investigating the impact of different technical cork stoppers on the quality preservation and shelf life of sparkling wines. The volatile compositions of two Italian sparkling wines sealed with a sparkling cork with two natural cork discs (2D) and a micro-agglomerated (MA) cork were determined during bottle aging (12 to 42 months) after disgorging. The results unveiled that the type of closure has a greater impact on the volatile composition of sparkling wines at longer post-bottling periods, and 2D stoppers preserve the fruity and sweet aromas of sparkling wines better after 42 months of bottle storage.

The next four papers dealt specifically with the effects of active packaging on food shelf life. In particular, Ambrosio et al. [3] proved the positive effects of an active polypropylenebased packaging functionalized with the antimicrobial peptide 1018K6 on microbial growth, physicochemical properties and sensory attributes of raw salmon fillets and hamburgers of Sarda sarda during storage. Roy et al. [4] developed a pullulan/chitosan-based multifunctional edible composite film by reinforcing mushroom-mediated zinc oxide nanoparticles (ZnONPs) and propolis. The system was advantageously used for wrapping pork belly. Gutiérrez-Jara et al. [5] coated sweet cherries by electro-spraying with an edible nanoemulsion of alginate and soybean oil, with or without a CaCl2 cross-linker to reduce fruit cracking. It was interesting to observe that the use of the nano-emulsion + CaCl2 coating on sweet cherries helps to reduce cracking and maintain fruit quality at 4 ◦C for about 1 month. Finally, Socaciu et al. [6] studied the effects of a whey-protein-isolate-based film incorporated with tarragon essential oil on the quality and shelf-life of refrigerated brook

**Citation:** Conte, A.; Del Nobile, M.A. Introduction to the Special Issue: Advanced Strategies to Preserve Quality and Extend Shelf Life of Foods. *Foods* **2022**, *11*, 1052. https:// doi.org/10.3390/foods11071052

Received: 15 March 2022 Accepted: 2 April 2022 Published: 6 April 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

trout. The selected essential oil conferred antioxidant and antimicrobial properties to the film. Thus, the developed active packaging system could be a promising material for fresh fish packaging.

As regards the adoption of active compounds of natural origin, two papers have been published, one dealing with shelf-life extension of chilled pork by optimal ultrasonicated ceylon spinach (*Basella alba*) extracts [7] and another one on the potential of algae extracts for extending the shelf life of rainbow trout (*Oncorhynchus mykiss*) fillets [8]. In the first study, Phimolsiripol et al. [7] found that fresh pork treated with the ultrasonicated extracts at 100 and 120 mg/mL had lower values of thiobarbituric acid reactive substances (TBARS) than the control (without dipping). From the food safety standpoint, as measured by the total microbial count, the fresh pork dipped with 100–120 mg/mL spinach extract could be kept at 0 ◦C for 7 days, 2 to 3 days longer than control meat at 0 and 4 ◦C, respectively. The results of Saez et al. [8] on the shelf life of rainbow trout demonstrated that algae extracts are also naturally effective agents for preserving fish.

In the context of natural compounds used for shelf-life extension, another two studies have been also published in the current Special Issue. This is the case of one article and one review dealing with fruit and vegetable by-products, whose valorization is considered a hot topic. In the article of Panza et al. [9], olive paste, a by-product from olive oil production, was valorized as breading for fresh fish sticks stored for 15 days at 4 ◦C. The results proved that the enrichment with olive paste increased the total phenols, the flavonoids and the antioxidant activity of the breaded fish samples compared to the control, without compromising the sensory parameters. The overview of Nardella et al. [10] collects the recent applications of fruit and vegetable by-products as valid components to prolong food shelf life. This review provides a detailed picture of the state-of-art of the literature on the topic in the last 10 years. The review highlights the potential of by-products and the clear advances in terms of food sustainability, even though the current situation still limits by-product diffusion. The authors also underlined that for future perspectives of by-products recycling, multidisciplinary research is of striking importance, as it is able to promote the scale-up of by-products and encourage their adoption at the industrial level.

As regards the emerging technologies and food shelf life, one article and one review were published in the current Special Issue. In particular, the article deals with the effects of gaseous ozone on microbiological quality of Andean blackberries (*Rubus glaucus* benth) [11]. Andean blackberries are highly perishable. Ozone was applied prior to storage at 0.4, 0.5, 0.6 and 0.7 ppm for 3 min, and this treatment was found effective in maintaining the quality of blackberries throughout refrigerated storage. The authors suggest that higher doses could be advisable to enhance its antimicrobial activity. The review of Tavares et al. [12] deals with emergent preservation techniques (chilling and super-chilling) as a complement, or even replacement of conventional preservation methodologies (refrigeration and freezing), to assure fish safety and extend shelf life without compromising food safety. In addition, the use of novel food packaging methodologies (edible films and coatings) was also presented and discussed, along with a new storage methodology, hyperbaric storage, that uses storage pressure control as a hurdle microbial development and slows down organoleptic decay at subzero, refrigeration and room temperatures.

One paper dealing with secondary shelf life (SSL) is also included in this Special Issue. SSL represents the time after package opening during which the food product retains a required level of quality. The study of Nicosia et al. [13] suggests the possibility to significantly extend or even omit the SSL indications for industrial pesto sauces because the product remained acceptable for a time longer than that reported on the label. This study could have practical outcomes at the domestic level in terms of food waste reduction and at industrial level in terms of reduced household stock turnover and consequent cost savings.

Taken together, these studies are clear evidence of how the achievement of food shelflife extension is still a complex and multifaceted process. Food manufacturers have to meet consumer demands for freshness and convenience without compromising the safety of foods, and the food industry is thus continuously challenged and seeking sustainable and

practical methods to ensure the safety of products and guarantee the maximum level of security for consumers. The innovative and exciting research included in this Special Issue highlights the interest and potential of this emerging area, addressing some of the most pressing global issues.

In summary, all the papers published in this Special Issue highlighted a large portion of the research activities in the field of advanced application of novel processing, antimicrobial/antioxidant substances as well as from by-products and active packaging. The development of these topics and the exploration of their combined use will remain a very active research area in the coming decades.

We sincerely hope that the readers will find this Special Issue interesting and informative.

**Author Contributions:** A.C. and M.A.D.N. equally contributed to organizing the Special Issue, to editorial work and to writing this editorial. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We thank and congratulate all authors for submitting the manuscripts of high quality. We are very grateful to the authors who have shared their scientific knowledge. We also thank the reviewers' willingness for their careful evaluations to improve the papers.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **Vacuum Packaging Can Extend Fresh Color Characteristics of Beef Steaks during Simulated Display Conditions**

**Tristan M. Reyes 1, Madison P. Wagoner 1, Virginia E. Zorn 1, Madison M. Coursen 1, Barney S. Wilborn 1, Tom Bonner 2, Terry D. Brandebourg 1, Soren P. Rodning <sup>1</sup> and Jason T. Sawyer 1,\***


**Abstract:** Packaging technology is evolving, and the objectives of this study were to evaluate instrumental surface color, expert color evaluation, and lipid oxidation (TBARS) on beef *longissimus lumborum* steaks packaged in vacuum-ready packaging (VRF) or polyvinyl chloride (PVC) overwrap packaging. Paired strip loins (Institutional Meat Purchasing Specifications # 180) were cut into 2.54-cm-thick steaks and assigned randomly to one of two packaging treatments, VRF or PVC. Steaks packaged in VRF were lighter in color (*p* < 0.05) as the display period increased, whereas steaks packaged in PVC became darker (*p* < 0.05). Redness (a\*) values were greater (*p* < 0.05) for PVC steaks until day 5, whereas VRF steaks had a greater (*p* < 0.05) surface redness from day 10 to 35 of the display period. Calculated spectral values of red to brown were greater (*p* < 0.05) for steaks in VRF than PVC. In addition, expert color evaluators confirmed VRF steaks were less brown and less discolored (*p* < 0.05) from day 5 to 35 of the display. Nonetheless, lipid oxidation was greater (*p* < 0.05) for PVC steaks from day 10 through day 35 of the display. Results from this study suggest that the use of vacuum packaging for beef steaks is plausible for maintaining surface color characteristics during extended display periods.

**Keywords:** instrumental color; overwrapped packaging; simulated retail display; TBARS; vacuum packaging

#### **1. Introduction**

Vacuum packaging using form-and-fill technology is a packaging method that is becoming one of the most prominent packaging systems in use within the retail meat industry [1]. Unfortunately, previous research focused on form-and-fill vacuum packaging for use with fresh meat storage in a retail setting is limited. Previous efforts in vacuum packaging uses for fresh meat have focused on using bag or skin technologies [2]. Formand-fill packaging systems use one film to construct a pouch with time, pressure, and heat. After forming the pouch, meat products are placed into the pouch and a second film is overlayed and sealed within the vacuum chamber. Furthermore, vacuum packaging has accounted for 40% of packaging types within meat cases, with most products packaged using a roll-stock machine [1]. It has been noted that PVC overwrapped packaged beef has decreased in use by 46% from 2018 to 2021 [1].

While the meat surface color is still regarded as one of the greatest determining factors consumers utilize when purchasing fresh beef in the retail setting [3,4], packaging technologies are pivotal in maintaining the surface color of fresh meat. PVC is a packaging method used with fresh meat that allows oxygen and other gasses to permeate through the film in large quantities allowing oxygen to bind with myoglobin. The oxymyoglobin state of beef is often correlated with a fresher and more wholesome product by consumers due to a bright cherry red color [5]. Creating a shift from the current industry's primary

**Citation:** Reyes, T.M.; Wagoner, M.P.; Zorn, V.E.; Coursen, M.M.; Wilborn, B.S.; Bonner, T.; Brandebourg, T.D.; Rodning, S.P.; Sawyer, J.T. Vacuum Packaging Can Extend Fresh Color Characteristics of Beef Steaks during Simulated Display Conditions. *Foods* **2022**, *11*, 520. https://doi.org/ 10.3390/foods11040520

Academic Editors: Matteo Alessandro Del Nobile and Amalia Conte

Received: 22 December 2021 Accepted: 9 February 2022 Published: 11 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

packaging methods of PVC to vacuum packaging is unclear; however, many advantages such as the extension of shelf life and color stability may exist with the use of vacuum packaging in fresh meat applications. Vacuum packaging allows meat products to remain more color stable over extended periods of time within retail coolers [6]. Reportedly, vacuum packaging has been known to extend the storage period of fresh meat products by reducing the amount of residual oxygen within the package [7,8].

With the ability to extend fresh meat storage through the use of vacuum packaging, it is a packaging system that is quickly becoming an essential part of the solution for meeting sustainability programs and reducing food waste for the meat industry. Food waste has been characterized as edible food that is not consumed and often discarded by consumers or retailers [9]. It has also been reported that meat, poultry, and fish were the top food groups contributing to an estimated food loss approaching \$48 billion in 2010 [9]. In addition, approximately 43 billion pounds of food at the retail level and 90 billion pounds at the consumer level have not been consumed [9]. Aside from food loss and food waste issues that still reside within the meat and food industry, there exist excessive food packaging materials entering the waste management system destined for landfills. In 2017, there were approximately 26.3 billion pounds of beef, 25.6 billion pounds of pork, and 42.2 billion pounds of chicken that American meat companies processed [10].

The packaging of fresh meat products is a necessity for the purpose of maintaining a fresh and wholesome product during retail display for consumer purchases. With the volume of packaging necessary to address the meat industry's demand of packaged meat products, it is essential that a packaging option be investigated for extending storage times of meat products. New packaging technologies could assist in reducing the volume of markdowns and throwaways that occur at the retail counter. A large percentage of fresh meat has been packaged with a form-and-fill roll stock machine, which utilizes multilayered packaging films [11]. Multi-layered vacuum packaging is constructed with a wide variety of materials that can include amorphous polyethelene terephthalate, polyolefines, ethylene vinyle alcohol, polyvinylidene di-chloride, and nylon [10–13]. Currently, the ability to recycle multi-layered films lacks economic viability due to the nature of the film layering [14].

Nonetheless, multi-layered vacuum packaging films are growing in popularity for vacuum packaging platforms; unfortunately, these packaging films are often constructed without sustainable or recycle-ready materials. Limitations in recycle-ready packaging materials can create difficulties downstream from the consumer with sustainable meat packaging due to challenges in the delamination process of multi-layered films [10,15]. Nevertheless, an investigation into using multi-layered films is a necessity to extend the fresh-meat shelf life. With a need for greater storage periods of fresh meat by retailers, customers, and consumers, the agriculture industry could focus its efforts on becoming more sustainable through innovative developments of packaging materials for meat and meat products. Therefore, the objectives of the current study were to investigate the feasibility of using VRF vacuum packaging film in place of PVC overwrapping on beef strip loin steaks and the subsequent impacts on surface color characteristics during a simulated retail display period.

#### **2. Materials and Methods**

#### *2.1. Raw Materials*

Cattle (*n* = 7) were harvested under simulated commercial conditions according to USDA humane slaughter standards at the Auburn University Lambert Powell Meat Laboratory after a 12 h rest period. After harvest, carcasses were chilled for 48 h at 2 ◦C. Following carcass chilling, beef carcasses were subsequently fabricated into left- and rightside paired (IMPS # 180) boneless beef strip loins, vacuum packaged (3 mil, Clarity Vacuum Pouches, Kansas City, MO, USA), and stored in the absence of light for 10 days to simulate boxed beef fabrication and logistics. After aging, beef strip loins were cut into 2.54-cm-thick steaks (N = 112 steaks/packaging treatment) using a BIRO bandsaw (Model 3334, BIRO

Manufacturing Company, Marblehead, Ohio, USA). At the time of steak cutting, steaks from each loin were allocated randomly to one of two packaging treatments, VRF or PVC. The allocated steaks were placed onto a plastic tray and allowed to bloom for 30 min prior to packaging.

#### *2.2. Packaging and Simulated Display Conditions*

After steak portioning, steaks allocated to vacuum packaging (VRF) were packaged using a Reiser form-and-fill vacuum packaging machine (Optimus OL0924, Variovac, Zarrentin, Germany) and sealed. Steaks were packaged in VRF packaging films (O2 transmission rate = 0.8 cc/sq. m2/24 h/atm). Steaks allocated to traditional overwrapping (PVC) were placed onto a foam tray (2s, Genpak, Charlotte, NC, USA) with an absorbent moisture pad (DRI-LOC AC-50, Novipax, Oak Brook, IL, USA) and wrapped by hand with a polyvinyl chloride film (O2 transmission rate = 14,000 cc O2/m/24 h/atm).

Packaged steaks were placed onto lighted shelves within a refrigerated retail display case (Model TOM- labels 60DXB-N, Turbo Air Inc., Long Beach, CA, USA). Packages of steaks were displayed for 35 days at 3 ◦C ± 1.2 ◦C, and the case temperature throughout the display period was monitored with temperature data recorders (Model-TD2F, ThermoWorks, American Fork, UT, USA) placed on the center of each display shelf. Packages of steaks were displayed on shelves under continuous LED lighting with an intensity of 2297 lux for each shelf. Lighting intensity was measured (ILT10C, International Light Technologies, Peabody, MA, USA) throughout the duration of the simulated display period. Additionally, packages of steaks were distributed evenly across all shelves and rotated daily from side to side and front to back to simulate consumer movement. Fresh meat characteristics of instrumental color, surface color, lipid oxidation, purge loss, and pH were measured on days 0, 5, 10, 15, 20, 25, 30, and 35 throughout the simulated display period.

#### *2.3. Instrumental Color*

Throughout the 35-day simulated retail display period, the instrumental surface color was measured on packaged steaks (*n* = 28) with a HunterLab MiniScan EZ colorimeter, Model 45/0 LAV (Hunter Associates Laboratory Inc., Reston, WV, USA). Prior to surface color readings, the colorimeter was standardized using a black and white tile. Instrumental color values were determined from the mean of three readings through the surface of each unopened package using illuminant A, an aperture of 31.8 mm, and a 10◦ observer. Packages of steaks were evaluated for lightness (L\*), redness (a\*), and yellowness (b\*) using the Commission Internationale de l' Eclairage guidelines for surface color [16]. In addition, the hue angle was calculated as tan−<sup>1</sup> (b\*/a\*), with a greater value indicative of the surface color shifting from red to yellow. Chroma (C\*) was calculated as √a\*<sup>2</sup> + b\*2, where a larger value indicates a more vivid color. Lastly, reflectance values within the spectral range of 400 to 700 nm were used to capture the surface color changes from red to brown by calculating the reflectance ratio of 630 nm:580 nm and the relative values of deoxymyoglobin (DMb = {[1.395 − ({A572 − A700}/{A525 − A700})]} × 100), metmyoglobin (MMb = {2.375 × [1 − ({A473 − A700}/{A525 − A700})]} × 100), and oxymyoglobin (OMb = DMb − MMb) according to color guidelines previously described [17].

#### *2.4. Expert Color Evaluation*

A five-member, expert color panel was used to evaluate the surface color of packaged beef boneless strip steaks during the simulated retail display period. Color measuring experts used anchors for scoring surface color discoloration previously described and modified from meat color guidelines [12]. At 16:00 h on the day of simulated display, experts rated surface color changes for steaks (*n* = 28) every 5 days for 35 days of refrigerated storage. Surface color ratings were created for steaks packaged under vacuum (VRF) for the initial beef color (1 = extremely bright purple-red, 2 = bright purple-red, 3 = moderately bright purple-red, 4 = slightly purple-red, 5 = slightly dark purple, 6 = moderately dark purple, 7 = dark purple, 8 = extremely dark purple), whereas packages of PVC overwrapped steaks

were rated for the initial beef color (1 = extremely bright cherry-red, 2 = bright cherryred, 3 = moderately bright, 4 = slightly bright cherry-red, 5 = slightly dark cherry-red, 6 = moderately dark red, 7 = dark red, 8 = extremely dark red). Both VRF- and PVCpackaged steaks were rated for the amount of browning (1 = no evidence of browning, 2 = dull, 3 = grayish, 4 = brownish gray, 5 = brown, and 6 = dark brown) and percent (%) discoloration (1 = no discoloration [0%], 2 = slight discoloration [1–20%], 3 = small discoloration [21–40%], 4 = modest discoloration [41–60%], 5 = moderate discoloration [61–80%], 6 = extensive discoloration [81–100%]).

#### *2.5. Purge Loss and Fresh Muscle pH*

Prior to conducting lipid oxidation analysis, steaks were removed from their respective packaging materials, blotted dry, and weighed on an analytic balance (PB3002-S, Mettler Toledo, Columbus, OH, USA). Purge loss was calculated as [(packaged steak weight − steak weight) ÷ packaged steak weight × 100)]. After capturing the purge loss for each steak, fresh muscle pH was measured in duplicate with a glass electrode inserted into two random locations within the steak and attached to a pH meter (Model-HI99163, Hanna Instruments, Woonsocket, RI, USA). Prior to measuring, the pH probe was calibrated (pH 4.0 and 7.0) using 2-point standard buffers (Thermo Fisher Scientific, Chelmsford, MA, USA) and again after 10 readings.

#### *2.6. Lipid Oxidation*

Packaged steaks (*n* = 56) were removed from their packaging material and sampled for 2-thiobarbituric acid reactive substances (TBARS) using a previously described method [18]. Steaks were trimmed of all external fat and connective tissue then minced together to form a uniform sample of the entire steak. Approximately 2 g of minced muscle was homogenized with 8 mL of cold (1 ◦C) 50 mM phosphate buffer (pH of 7.0 at 4 ◦C) containing 0.1% EDTA, 0.1% n-propyl gallate, and 2 mL trichloroacetic acid (Sigma-Aldrich, Saint Louis, MO, USA). Homogenized samples were subsequently filtered through Whatmann No. 4 filter paper, and duplicate 2-mL aliquots of the clear filtrate were transferred into 10-mL borosilicate tubes, mixed with 2 mL of 0.02 M 2-thiobarbituric acid reagent (Sigma-Aldrich, Saint Louis, MO, USA) then boiled for 20 min. After boiling, tubes were placed into an ice bath for 15 min. Absorbance was measured at 533 nm with a spectrophotometer (Turner Model–SM110245, Barnstead International, Dubuque, IA, USA) and multiplied using a factor of 12.21 to obtain the TBARS value (mg malonaldehyde/kg of meat).

#### *2.7. Statistical Analysis*

Data were analyzed with the GLIMMIX procedures of SAS (ver. 9.4; SAS Institute Inc. Cary, NC, USA) with treatment serving as the lone fixed effect and replication serving as the random effect for instrumental color, expert color, lipid oxidation, purge loss, and pH. All data were analyzed in a modified randomized design with steak serving as the experimental unit. For expert surface color rating data, the expert color panelist was included as a random factor, and panelist × day of display was included as a random, repeated factor (with a first-order autoregressive covariance structure). Least-squares means were generated, and when significant (*p* ≤ 0.05) F-values were observed, least-squares means were separated using a pair-wise *t*-test (PDIFF option).

#### **3. Results and Discussion**

#### *3.1. Instrumental Beef Color*

The instrumental surface color of packaged steaks was measured throughout a 35-day simulated retail period. An interaction of the packaging method × day of display on steak surface lightness (L\*) occurred (Table 1). Steaks packaged in PVC were lightest (*p* < 0.05) on day 0 and became darker as the length of display period increased (Table 1). However, from day 20 through day 35 of the display, steaks packaged using VRF were lighter (*p* < 0.05) than steaks packaged using PVC methods (Table 1). Additionally, surface redness (a\*) for beef steaks packaged in PVC were redder (*p* < 0.05) from day 0 through day 15 of the display period (Table 1), whereas steaks packaged in VRF became significantly redder (*p* < 0.05) until the conclusion of the study on day 35 (Table 1). Greater a\* values are indicative of a redder fresher color and have a greater consumer appeal at the time of the consumers' purchasing decision. PVC-packaged steaks maintained greater (*p* < 0.05) values for yellowness (b\*) throughout the duration of simulated retail display than steaks packaged in VRF (Table 1). The changes in surface color for steaks packaged using VRF indicated surface lightness and redness were more stable throughout the entire simulated retail period than steaks packaged in PVC. As expected during a simulated retail period, fresh steaks packaged in an oxygen-rich permeable method such as PVC will have a brighter surface color initially. Similar findings have reported that ground beef packaged using PVC methods resulted in greater L\* values on day 0, along with greater a\* and b\* through only 50% of the display period [5] when displayed up to 35 days. Moreover, ground beef patties when packaged with PVC materials have recorded similar results, indicating a\* values will decline within the first 5 days of the display period [19]. However, a\* values for ground beef patties packaged using a vacuum packaging platform have been reported to increase throughout a display period [19]. Furthermore, a study evaluating the surface color of beef steaks indicated a\* values were greater for vacuum packaging rather than other packaging types at the conclusion of a 35-day study [20]. It appears the results for b\* values of ground beef and steaks are consistent with the current study, resulting in a decline during a 5-day retail storage period when packaged in PVC. Regardless of the fluctuation of yellowness, the current and previous results suggest b\* values are less stable regardless of the packaging method [5,19–21].

**Table 1.** The interactive impact of packaging method × day of display for instrumental surface color values on fresh beef strip loin steaks during a simulated retail display.


<sup>1</sup> L\* Values are a measure of darkness to lightness (larger value indicates a lighter color); a\* values are a measure of redness (larger value indicates a redder color); and b\* values are a measure of yellowness (larger value indicates a more yellow color). <sup>2</sup> C\* (Chroma) is a measure of total color (larger number indicates a more vivid color). <sup>3</sup> Hue ( ◦) angle represents the change from the true red axis (larger number indicates a greater shift from red to yellow). <sup>4</sup> RTB calculated as 630 nm ÷ 580 nm, which represents a change in the color of red to brown (larger value indicates a redder color). <sup>5</sup> Calculated percentages of deoxymyoglobin (DMb), metmyoglobin (MMb), and oxymyoglobin (OMb) using relative spectral values. a—h Mean values within a row and a packaging method lacking common superscripts differ (*p* ≤ 0.05). \* SEM, Standard error of the mean. Bold font, the packaging methods investigated.

There was a packaging method × day of display interaction for surface color chroma (C\*) and hue angles (Table 1). The instrumental surface color of steaks packaged in PVC was more vivid (*p* < 0.05) on day 0 but C\* values declined as the duration of display increased. However, steaks packaged with VRF became more vivid (*p* < 0.05) from day 25 through 35 of the simulated retail display period (Table 1). In addition, steaks packaged with PVC had greater (*p* < 0.05) hue angles indicative of a surface color shift from red to yellow from day 5 through 35. It appears that the reduction in oxygen exposure for steaks in VRF packages protected the surface color of steaks by sustaining the vividness and reducing the shift from red to yellow. Similar results for fresh packaged beef C\* and hue angle values have been reported to decline during the initial 10 days of a simulated display period when using an oxygen-rich packaging method such as PVC [22]. Changes in surface color values for the hue angle and C\* can be used as a great indicator for observing meat discoloration in retail display settings [19–24]. Interestingly, C\* (vividness) for steaks packaged in VFR in the current study differ from previous C\* results that did not differ throughout a 35-day display period [23]. It should be noted that as the percentage of oxygen exposure to the steak surface increases a reduction in the hue angle and C\* will likely occur during retail display periods as the surface color shifts from red to brown with the formation of metmyoglobin [22–24].

The interactive influence for packaging method × day of display remained for calculated spectral values of red to brown (630:580 nm) and relative forms of myoglobin (Table 1). Red to brown values were greater (*p* < 0.05) for steaks packaged in PVC until day 5 of the simulated display period. However, from day 10 through 35, PVC-packaged steaks' surface color showed a greater shift from red to brown. Steaks packaged in VRF had less (*p* < 0.05) discoloration from red to brown after day 5 through day 35 (Table 1). Previous studies have [25] reported similar findings indicating a decline in calculated red to brown values throughout 7 days of simulated display for beef packaged in PVC [25]. It is expected that calculated spectral values for the surface color of fresh beef will shift from a brighter red to brown as the duration of a simulated retail display increases. Steaks packaged in VRF had the greatest (*p* < 0.05) amount of calculated metmyoglobin (MMb) on day 0 (Table 1). However, as expected from days 5 to 35, steaks packaged using PVC had greater (*p* < 0.05) calculated relative values for MMb. As expected, steaks packaged in VRF had greater (*p* < 0.05) calculated deoxymyoglobin (DMb) values throughout the entire simulated retail display period (Table 1) because of limited oxygen exposure. Interestingly, calculated relative values of oxymyoglobin (OMb) were greater (*p* < 0.05) for steaks packaged using PVC packaging materials throughout the entire simulated retail display period (Table 1). The results for calculated spectral values reported are likely due to the oxygen permeability of the PVC package resulting in greater exposure of the steak surface to an oxygen-rich atmosphere. Greater formations of MMb in PVC have been associated with greater amounts of lipid oxidation [26,27] and the relationship of oxidation during the transition of myoglobin pigment from OMb to MMb [26–28].

#### *3.2. Expert Color Evaluation*

Fresh steaks were evaluated by experts for visual surface color variations during a simulated retail display for up to 35 days. However, the evaluation of steaks packaged in aerobic PVC packaging materials was discontinued after day 20 due to total surface color deterioration. An interaction of the packaging method × day of display occurred for the surface color evaluation (Table 2). Trained expert evaluators noted that values for the initial beef color, amount of browning, and surface discoloration deteriorated (*p* < 0.05) for steaks packaged in PVC from day 5 through day 20 (Table 2). The surface color of steaks packaged in PVC materials became darker, with a greater amount of browning, and a greater percentage of discoloration as the duration of display increased. As a result of significant surface discoloration, PVC-packaged steaks used for expert color evaluation were discarded on day 20 of the display period. The changes in visual surface color are influential in driving consumer purchasing intent and the lack of storage for PVC steaks

may contribute to greater throwaway by the retailer. Steaks packaged in VRF had initial beef colors that decreased (*p* < 0.05), and the amount of browning and surface discoloration were less (*p* < 0.05) than steaks packaged in PVC throughout the duration of the study (Table 2). Interestingly, steaks packaged in VRF were darker at day 0, but the visual steak color turned brighter purple red with less browning and surface discoloration throughout a 35-day simulated retail period. Results from the current study agree with previous findings when using vacuum packaging. Beef's surface color tends to remain visually stable throughout the duration of the study, whereas high-oxygen packaging of fresh beef can show rapid color deterioration [29]. The color stability of fresh beef is dependent on controlling countless factors such as pH, temperature, light, lipid oxidation, residual oxygen, MMb-reducing systems, reducing equivalents, and the oxygen consumption rate [30,31]. It is plausible that the transformation from OMb to MMb in PVC-packaged steaks was due to greater amounts of lipid oxidation. Furthermore, limited surface color variation of steaks packaged in VRF may be attributed to a lack of residual oxygen within the packaging, influencing and reducing the amount of oxidation occurring in vacuum-packaged fresh beef products.

**Table 2.** Interactive influence of packaging method × day of display for expert surface color evaluation on fresh beef strip loin steaks during a simulated retail display.


<sup>1</sup> PVC color anchors: Initial Beef Color (1 = Extremely bright cherry-red to 8 = Extremely dark red); Amount of Browning (1 = No Evidence of Browning to 6 = Dark Brown); Surface Discoloration (1 = No discoloration (0%) to 6 = Extensive discoloration (81–100%). <sup>2</sup> VRF color anchors: Initial Beef Color (1 = Extremely bright purple red to 8 = extremely dark purple red); Amount of Browning (1 = No Evidence of Browning to 6 = Dark Brown); Surface Discoloration (1 = No discoloration (0%) to 6 = Extensive discoloration (81–100%). a–h Mean values within a row and packaging method lacking common superscripts differ (*p* ≤ 0.05). \* SEM, Standard error of the mean. Bold font, the packaging methods investigated.

#### *3.3. Lipid Oxidation*

There was an interactive effect of the packaging method × day of display for lipid oxidation on fresh beef steaks (Figure 1). The packaging method did not alter (*p* > 0.05) lipid oxidation through day 5 of the simulated retail display period. However, from days 10 through 35 of the storage period, lipid oxidation was greater (*p* < 0.05) for steaks packaged using PVC methods. Lipid oxidation of fresh steaks using PVC packaging from the current study agrees with previous simulated retail storage studies measuring an expected storage period in a retail setting of 3 to 7 days [32]. The exposure to greater amounts of oxygen across the packaging material can result in increased catalysis of lipid oxidation [33,34]. Moreover, greater lipid oxidation can be correlated to reduced consumer palatability due to the deterioration of the surface color and accumulation of off flavors [35]. Unfortunately, the evaluation of sensory taste characteristics was not completed during the current study, but future studies on the extended storage of fresh beef influencing lipid oxidation and sensory characteristics would be warranted.

**Figure 1.** Interactive influence of packaging method × day of display for 2-Thiobarbituric acid reactive substances (TBARS) on beef strip loin steaks during a simulated retail display. Bars lacking common letters differ (*p* ≤ 0.05).

#### *3.4. Purge Loss*

A packaging method × day of display interaction occurred for the purge loss of fresh beef steaks (Figure 2). The purge loss was greatest (*p* < 0.05) for steaks packaged in PVC materials on day 25 of the simulated display period and the lowest on day 0. The packaging method influenced the purge loss on day 0, with steaks packaged in VRF having a greater (*p* < 0.05) percentage of moisture loss. It is plausible that the method of vacuum packaging using the form-and-fill machine caused more moisture to be pressed out of the steak at the time of package sealing. However, the purge loss in vacuum-packaged meat products can result in an unappealing visual appearance for consumers due to the accumulation of purge in the packaging [36,37]. The results from the current study differ from previous results where values for purge loss using vacuum-packaging platforms were greater than PVC or alternative packaging such as modified atmosphere packaging platforms [38].

#### *3.5. pH*

The interactive influence of the packaging method × day of display for fresh muscle pH values is presented in Figure 3. Fresh muscle pH values were recorded within muscle pH values (5.1 to 5.8) throughout the duration of the simulated display period. Values for fresh muscle pH were greatest (*p* < 0.05) on day 10 in steaks packaged using PVC methods. At the time of harvest and before chilling, carcasses were rinsed with an FDA-GRAS (U.S. Food and Drug Administration-Generally Recognized as Safe) organic acid (lactic acid). The combination of vacuum packaging and the organic carcass wash may have contributed to the decline in fresh muscle pH of VRF-packaged steaks, causing a shift in the visual and instrumental surface color variations reported within the current study. Furthermore, it is plausible that pH values for VRF declined due to an increase in lactic acid bacteria that can be present in vacuum-packaged fresh meats. With limited residual oxygen within the

vacuum package, favorable conditions for anerobic lactic acid bacteria may have caused fresh muscle pH to decline as lactic acid bacteria populations increased [5,39]. In addition, lactic acid bacteria can be associated with low-pH (<5.8) vacuum-packaged meats due to a lower residual oxygen environment [40].

**Figure 2.** Interactive influence of packaging method × day of display for purge loss (%) on beef strip loin steaks during a simulated retail display. Bars lacking common letters differ (*p* ≤ 0.05).

**Figure 3.** The interactive influence of packaging method × day of display for fresh muscle pH on beef strip loin steaks. Bars lacking common letters differ (*p* ≤ 0.05).

#### **4. Conclusions**

It is feasible that the storage of beef strip loin steaks using vacuum packaging, VRF, can provide a longer, fresh, refrigerated storage period than steaks packaged in traditional PVC packaging. It is evident that VRF displayed a more color-stable product throughout the duration of simulated retail display. Additionally, VRF maintained less oxidation throughout the display period, whereas steaks packaged in PVC tended to have greater oxidation leading to greater amounts of surface discoloration in beef products. The current results suggest that the vacuum-packaged film used within the current study is an acceptable replacement to traditional packaging methods of PVC for packaging whole-muscle beef steaks for up to 35 days of refrigerated retail storage. However, additional research should be considered to evaluate the sensory taste profiles of vacuum packaging used for extended storage periods and the implications for flavor characteristics of beef steaks.

**Author Contributions:** Conceptualization, T.M.R., T.B. and J.T.S.; methodology, T.M.R.; validation, M.P.W., V.E.Z. and M.M.C.; formal analysis, J.T.S.; investigation, T.M.R., M.P.W., V.E.Z. and M.M.C.; resources, T.B.; data curation, T.M.R.; writing—original draft preparation, T.M.R.; writing—review and editing, T.M.R., M.P.W., V.E.Z., M.M.C., T.D.B., S.P.R. and J.T.S.; supervision, J.T.S. and B.S.W.; project administration, J.T.S. and B.S.W.; funding acquisition, J.T.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not Applicable.

**Informed Consent Statement:** Not Applicable.

**Data Availability Statement:** Not Applicable.

**Acknowledgments:** The authors would like to extend their upmost appreciation to WINPAK for providing the Variovac thermoforming machine and supplying thermoforming films to complete the study. Additionally, the authors would like to thank the staff of the Lambert Powell Meats Laboratory.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


### *Article* **An Active Peptide-Based Packaging System to Improve the Freshness and Safety of Fish Products: A Case Study**

**Rosa Luisa Ambrosio 1,†, Marta Gogliettino 2,†, Bruna Agrillo 2,3,4, Yolande T. R. Proroga 5, Marco Balestrieri 2, Lorena Gratino 2, Daniela Cristiano 5, Gianna Palmieri 2,3,\* and Aniello Anastasio <sup>1</sup>**


**Abstract:** Fresh fish are highly perishable, owing mainly to their moisture content, high amount of free amino acids and polyunsaturated fatty acids. Microorganisms and chemical reactions cause the spoilage, leading to loss in quality, human health risks and a market value reduction. Therefore, the fishing industry has always been willing to explore new technologies to increase quality and safety of fish products through a decrease of the microbiological and biochemical damage. In this context, antimicrobial active packaging is one such promising solution to meet consumer demands. The main objective of this study was to evaluate the effects of an active polypropylene-based packaging functionalized with the antimicrobial peptide 1018K6 on microbial growth, physicochemical properties and the sensory attributes of raw salmon fillets. The results showed that application of 1018K6-polypropylene strongly inhibited the microbial growth of both pathogenic and specific spoilage organisms (SSOs) on fish fillets after 7 days. Moreover, salmon also kept its freshness as per volatile chemical spoilage indices (CSIs) during storage. Similar results were obtained on hamburgers of *Sarda sarda* performing the same analyses. This work provides further evidence that 1018K6-polymers have good potential as antimicrobial packaging for application in the food market to enhance quality and preserve the sensorial properties of fish products.

**Keywords:** antimicrobial polymers; antimicrobial peptides; fresh fish; spoilage; fish quality; food safety; food packaging

#### **1. Introduction**

Today, health, nutrition and convenience are the major drivers in the global food industry. In this context, fish products have attracted considerable attention as a source of important nutritional components, such as high-quality protein, essential vitamins, minerals and polyunsaturated fatty acids (PUFA) [1,2]. Indeed, fish is considered of key importance for human nutrition all over the world, providing about 17% of the global intake of animal proteins [3]. However, its consumption in many parts of the world is far below the recommended level. As such, high-quality food with an extended shelflife is essential for both producers and consumers. However, fish is a highly perishable product due to its relevant water activity, nearly neutral pH and specific composition that make it vulnerable to various biochemical, physical and microbial forms of deterioration throughout the production chain, thus causing rejection by consumers. Indeed, spoilage

**Citation:** Ambrosio, R.L.; Gogliettino, M.; Agrillo, B.; Proroga, Y.T.R.; Balestrieri, M.; Gratino, L.; Cristiano, D.; Palmieri, G.; Anastasio, A. An Active Peptide-Based Packaging System to Improve the Freshness and Safety of Fish Products: A Case Study. *Foods* **2022**, *11*, 338. https://doi.org/ 10.3390/foods11030338

Academic Editors: Matteo Alessandro Del Nobile and Amalia Conte

Received: 21 December 2021 Accepted: 21 January 2022 Published: 25 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

starts quickly after fish are caught, and rigor mortis is responsible for changes in fish after death.

Specifically, the degradation of various components and the formation of new products are accountable for the alterations in odour, flavour, colour and texture that happen during the spoilage process, so that deterioration occurs very rapidly due to mechanisms triggered by the microbial community, endogenous enzymatic activity (autolysis), and the chemical oxidation of lipids [4–8]. Due to all these changes, the shelf-life and quality of fresh fish are very limited, resulting in health risks as well as in enormous economic loss. Therefore, the fish industry is focused on preventing and controlling foodborne illness and microbial growth, which can lead to food spoilage, the major cause of fish loss, spoil estimated at millions of tons per year and accounting for 10% of the total production from capture fisheries and aquaculture [9]. These phenomena are even more evident in lower-income economies, in which spoilage and the quality downgrading of fish products occur due to high ambient temperatures, lack of infrastructure, basic technology and lack of cooling (cold chain) facilities.

Salmon (*Salmo salar*) is one of the most consumed seafood in the world, either fresh or frozen, and a main product of aquaculture, accounting for 93% of total production [10]. Recently, its consumption has increased substantially because it offers several health benefits, mostly due to the presence of essential and vital nutrients for human body, such as omega-3 long-chain fatty acids. As this marine species has become a luxury product in the fishery market, any new strategy developed to enhance its safety and preserve its quality is considered essential for this economic sector. A considerable support in the fight against microbial spoilage may derive from food packaging, which can serve as a carrier of active substances, such as antimicrobials, playing an active role in food quality and shelf life, besides acting as a barrier against moisture, water vapor, gases and solutes. Specifically, antimicrobial packaging is considered a promising form of active packaging based on the immobilization of antimicrobial agents on the surfaces of polymers, thus providing antimicrobial properties to specific materials.

In this context, polypropylene (PP) is an ideal food-safe thermoplastic material for packaging applications because of its low cost and its physical and chemical parameters. Therefore, it would be advantageous to impart antibacterial activity to these polymers, given the growing consumer interest in foods with fewer preservatives. In a previous study, the in-silico-designed antimicrobial peptide 1018K6, extensively characterized both from functional and structural points of view, was covalently bonded to commercial PET (polyethylene terephthalate) films and the ability of the developed antimicrobial packaging to improve the microbial quality and safety of dairy products was clearly demonstrated [11–14]. The applied procedure fulfilled the criteria of an efficient immobilization reaction, such as high yields and remarkable stability of the activated polymers, with no peptide release under different environmental conditions of use even after prolonged incubation times. Surprisingly, the AMP (antimicrobial peptide) was still active and preserved its excellent antimicrobial and antibiofilm abilities against a panel of Gram+ and Gram- bacterial pathogens upon polymer surface functionalization, along with potent activity against moulds and fungal species, without exhibiting cytotoxic effects on human cells. Specifically, 1018K6 was able to explicate its bactericidal activity against both fungi, such as *Aspergillus brasiliensis,* the Gram+ pathogens *Listeria monocytogenes* and *Staphylococcus aureus* and the Gram− *Salmonella* Typhimurium and *Escherichia coli* [15]. Furthermore, the optimized technology revealed the possibility of re-using the peptide polymers at least six times, while preserving its antimicrobial properties.

In this paper, 1018K6 was covalently immobilized onto PP surfaces, previously activated by plasma treatment, in order to extend the application field of our AMP-based system. Therefore, the impacts of prepared films on the physicochemical, microbial and sensory properties of fresh salmon fillets throughout storage at 4 ◦C for 7 days were investigated. A challenge test with *L. monocytogenes* was also performed. In order to validate the antimicrobial effectiveness of 1018K6-PP packaging in controlling the quality decay of fresh

fish, the same analyses were performed on a different typology of fish-based foods, burgers of the bonito fish (*Sarda sarda*). Indeed, this typology of product has a softer texture with a lower shear force than other meat products reported in the literature, and it represents a valid solution for meeting consumer preferences for foods that have high nutritional value and are very convenient, being ready-to-cook. Moreover, it is well-known that the production phases of burgers are responsible for higher microbial concentrations than fillets due to the handling and the increased superficial area of the matrix for grinding. All these aspects outline the insidious profile of fish burgers and the challenge of testing the innovative packaging on these products.

#### **2. Materials and Methods**

#### *2.1. Atmospheric Plasma Treatments*

Openair-Plasma® Technology was used as plasma treatment. The surfaces of commercial PP were cut into square-shaped pieces (4.0 × 4.0 cm dimension), which were cleaned with ethanol prior to use and then were placed on the plate at a distance of 3 cm to the nozzle. In all the treatments, air was used as the processing gas with a power of 440 watts and a speed of 10 mm/s. The effect of plasma on the polymer surfaces was evaluated by using the test Ink (Plasmatreat) in order to assess the wettability of the material.

#### *2.2. Production of 1018K6*

The peptide 1018K6 (VRLIVKVRIWRR-NH2) used in this work was purchased from GenScript Biotech (Leiden, Netherlands). It was stored as a lyophilized powder at −20 ◦C. Analysis by mass spectrometry confirmed the identity of peptide.

#### *2.3. 1018K6 Immobilization on Polymer Surfaces and Release Test*

Polymer surfaces pre-activated by atmospheric plasma treatment were incubated into solutions (3 mL) of 1018K6 prepared in distilled water at three concentrations (50, 100 and 200 μM) for about 4 h at 70 ◦C to fully remove the water. After drying, the functionalized PPs were immersed in a volume of distilled water equal to that evaporated for 16 h at room temperature in agitation, then they were sonicated for 20 min and the recovered solutions analysed by reverse-phase high-performance liquid chromatography (RP-HPLC) to indirectly estimate the immobilization yield. For these analyses, 200 μL of the samples were injected over a μBondapak C18 reverse-phase column (3.9 mm × 300 mm, Waters Corp., Milford, MA, USA) connected to a HPLC system (Shimadzu, Milan, Italy), using a linear gradient of 5–95% 0.1% TFA (Trifluoroacetic acid) in acetonitrile, at a flow rate of 1 mL/min. A reference solution was prepared with the initial peptide concentration used in the coupling reaction and was run in parallel. Therefore, by knowing the added peptide concentration (reference solution), the amount of peptide not covalently attached on the polymer surface was calculated by comparing the peak area and expressed as a percentage. A calibration curve of the C18 column using different 1018K6 concentrations was constructed. All measurements were performed in triplicate on three different preparations.

To determine the stability of 1018K6 on the functionalized polymers, a release assay was performed by RP-HPLC using a linear gradient of 5–95% acetonitrile in 0.1% TFA, at a flow rate of 1 mL/min. A volume of 1 mL of pure water or NaCl 1 M was poured onto the functionalized polymers, which were incubated for 7 days at 4 ◦C, sonicated for 20 min and then the recovered solutions were loaded on RP-HPLC column. The solutions in contact with the functionalized polymers at time t = 0 were used as control samples and were run in parallel. All measurements were performed in triplicate on three different preparations.

#### *2.4. Sample Preparation*

Raw salmon (*Salmo salar*, Linnaeus 1758) from different batches were freshly bought from a local fishery industry (Naples, Italy). To evaluate the antimicrobial effects of the functionalized 1018K6-PPs polymers, two samples were prepared under aseptic conditions from each fillet that was sliced into pieces of approximatively 50 g. As a whole, the samples were separated into two groups: the control group (CTR-PP), including salmon fillets packaged in pre-activated PPs films not-functionalized with 1018K6 and the treated group, including salmon fillets packaged in PPs films functionalized with 1018K6 (1018K6-PP). Both 1018K6-PPs and non-functionalized PPs squares (4 × 4 cm) were placed on Petri dishes (both lid and base) in order to ensure constant contact between the pieces of salmon and the PPs. Subsequently, the packaged samples were refrigerated at 4 ± 1 ◦C for 7 days. The samples' microbiological, physicochemical properties and quality aspects were analysed at days 0, 4 and 7.

Fish burgers of Atlantic bonito (*Sarda sarda*, Bloch 1793) were purchased from a fishing industry in Naples (Italy). A total of 21 burgers (200 g) were included in the experimental design. The samples were randomly divided into CTR-PP and 1018K6-PP groups and prepared as described above for the salmon samples. Once the fish burgers were packaged, the samples were stored at refrigeration temperature (4 ± 1◦C) and sampled at days 0, 3, 5 and 7 to carry out the same analyses as were conducted on the salmon fillets.

The same analyses were performed also on fish samples packaged in PP films that were not subjected to any surface modification.

#### *2.5. pH and aw Measurements*

The pH measurements were carried out with a digital pH meter (Crison-Micro TT 2022, Crison Instruments, Barcelona, Spain). Water activity (aw) was measured with Aqualab 4 TE (Decagon Devices Inc., Northeast Hopkins Court Pullman, Pullman, WA, USA).

#### *2.6. Microbiological Analyses*

Ten grams of each sample were added to 90 mL (1:10 *w*/*v*) of sterilized Peptone Water (PW, Oxoid, Madrid, Spain) in a sterile stomacher bag to be homogenized for three minutes at 230 rpm using a peristaltic homogenizer (BagMixer®400 P, Interscience, Saint Nom, France). Ten-fold serial dilutions of each homogenate were prepared. In order to better describe the microbial profile of samples and follow the growth trend of each bacterium responsible for the food alteration, the viable counts of various microorganisms were carried out. Total aerobic bacterial counts (TAB), both mesophilic and psychrophilic, were performed on plate count agar (PCA, Oxoid, Madrid, Spain) incubated at 30 ◦C for 48/72 h and 7 ◦C for 10 days, respectively (ISO 4833-1:2013 and ISO 17410:2019); total coliforms on violet red bile lactose agar (VRBL, Oxoid, Madrid, Spain) incubated at 37 ◦C for 48 h (ISO 4831:2006); Enterobacteriaceae on violet red bile glucose agar (VRBG, Oxoid, Madrid, Spain) incubated at 37 ◦C for 48 h (ISO 21528-2:2017); lactic acid bacteria (LAB) on MRS agar with Tween 80 (Oxoid, Madrid, Spain), incubated at 30 ◦C for 72 h (ISO 15214:2015); *Pseudomonas* spp. on pseudomonas agar base with CFC supplement (Oxoid, Madrid, Spain) incubated at 25 ◦C for 48 h (ISO 13720:2010); β-glucuronidase-positive *Escherichia coli* (ISO 16649-1:2018) on Triptone Bile X-glucoronide Agar (TBX, Oxoid, Madrid, Spain) at 44 ◦C for 24 h; *Brochothrix thermosphacta* on STAA (streptomycin thallous acetate actidione agar, Oxoid, Madrid, Spain) at 37 ◦C for 48 h; *Enterococcus faecalis* on KAA (kanamycin aesculin azide, Oxoid, Madrid, Spain) at 37 ◦C for 48 h; coagulase positive *staphylococci* on Baird-Parker agar (Oxoid, Madrid, Spain) at 37 ◦C for 24/48 h (ISO 6888-1:1999). After counting, the data were expressed as logarithms of the number of colony-forming units (CFU/g) and means and standard error were calculated.

#### *2.7. Challenge Test*

Four fillets of approximately 150 g and from different batches were used to evaluate the inter-batch variability. All of them were tested in agreement with the AFNOR-BRD 07/10-04/05-Real Time PCR method in order to evaluate the absence of *L. monocytogenes* contamination. Three strains of *L. monocytogenes* isolated from fish samples were selected, following ISO 11290-1, to perform these analyses and stored in the Zooprophilactic Experimental Institute of Mezzogiorno biobank. All strains were re-suspended in diluent at 0.5 Mcfarland concentration, then a series of 10 times gradient dilution of *L. monocytogenes*

was performed until to reach a concentration of 150 CFU/mL. All fillets were contaminated at surface to mimic contamination during the slicing, using one fillet not contaminated as a control. After artificial contamination, the samples were packed between two functionalized films and stored at 5 ◦C until 96 h. The enumeration of *L. monocytogenes* was performed according to Annex 1 of Reg (CE) 2073/2005 [16] at 24 h, 48 h, 72 h, and 96 h, in agreement with the reference methods EN ISO 11290-2.

#### *2.8. Colour*

Colourimetric measurements of the surface appearance of salmon fillets and bonito fish burgers were performed using a Konica Minolta CR 300 colourimeter (Minolta, Osaka, Japan). The data were analysed in the CIELAB colour space, organizing in three orthogonal axes in a Cartesian coordinate system: lightness (*L\**), redness (*a\**) and yellowness (*b\**). Additionally, the angular coordinates of Hue angle [hab = ArcTan(*b\**/*a\**)], and chroma [Cab = (*a\**<sup>2</sup> + *b\**2) 1/2] were calculated. Total colour difference (Δ*E*), variation in *a\** (Δ*a*\*) and in *b\** (Δ*b*\*) were calculated as:

$$
\Delta E = \sqrt{\left(L^\* \, \_1 - L^\* \, \_2\right)^2 + \left(a^\* \, \_1 - a^\* \, \_2\right)^2 + \left(b^\* \, \_1 - b^\* \, \_2\right)^2}
$$

$$
\Delta a^\* = a^\* \, \_2 - a^\* \, \_1
$$

$$
\Delta b^\* = b^\* \, \_2 - b^\* \, \_1
$$

where *L*\*2, *a*\*2, and *b*\*2 are the values recorded at a specific day during the storage; instead, *L*\*1, *a*\*1, and *b*\*1 are values collected at day 0.

Δ*E* represents the result of changes in lightness (Δ*L*\*), redness (Δ*a\**) and yellowness (Δ*b\**), which do not always change in parallel. For this reason, Δ*a\** and Δ*b\** were taken into account. Since the colour may not be homogeneous over the entire surface of fillets and burgers, five superficial measurements were performed for each sample to obtain representative results.

#### *2.9. TBARS, Total Volatile Basic Nitrogen (TVB-N) and Trimethylamine (TMA) Analyses*

Lipid oxidation was monitored by determining the thiobarbituric acid (C4H4N2O2S) substances expressed as malondialdehyde (CH2(CHO)2) concentration (mg/Kg), which represent secondary oxidation products. Measurements were performed according to the method proposed by Ambrosio et al. [17].

The TVB-N and TMA values for all salmon and fish burger samples were quantified according to Conway's micro-diffusion method [18]. The results were expressed in mg of nitrogen per 100 g of sample.

#### *2.10. Sensory Testing*

Sensory testing of salmon fillets and bonito fish burgers was undertaken by a panel consisting of five trained panellists. The judge's acceptability study was assessed through a sensory evaluation, taking into account odour, colour and general acceptability. Appropriate attributes have been fixed in order to minimize individual differences and ensure the results' repeatability. Sensory assessments were performed under controlled humidity, light and temperature. A Likert scale (9-point) was used to assess each attribute; in the scale, 9 corresponded to excellent, 8 to very good, 7 to good, 6 to reasonable, 5 to not good (acceptable limit), 4 to disliked, 3 to bad, 2 to very bad, and 1 to completely unacceptable [19]. Coded samples were randomly and simultaneously distributed to each panellist.

#### *2.11. Statistical Analyses*

Physicochemical and microbiological data were statistically analysed with generalized linear mixed model of SPSS version 26 (IBM Analytics, Armonk, NY, USA). Analysis of variance was performed to study parameters of salmon fillets and bonito fish burgers at each sampling time, including the fixed effect of packaging used and storage times. An a posteriori contrast was carried out using the Tukey test, considering a *p* value of <0.05 as statistically significant.

#### **3. Results**

#### *3.1. 1018K6 Immobilization on PP Surface*

Following confirmation of the excellent antimicrobial properties preserved by 1018K6, even after bonding on different materials, such as PET and nanoparticles [13,20], the peptide was further immobilized on another plastic polymer commonly used in food packaging, polypropylene (PP).

To this aim, commercial PP slides were exposed to plasma treatment to activate the inert polymeric surfaces with reactive -COOH\* functional groups that are available to interact with the amine moieties of 1018K6, forming amide bonds [21].

In order to develop an antimicrobial packaging more adequate for food application, the conditions applied in our previous studies to functionalize the polymeric materials were modified. Specifically, the covalent attachment of 1018K6 on the pre-activated PP polymers was executed by a one-step immobilization process involving the immersion of the polymeric surfaces in a water solution containing the peptide at different concentrations. Thereafter, the slides were kept at 70 ◦C for about 4 h to completely remove the water and to drive the coupling reaction. To validate the success of our immobilization procedure, the test ink was applied, confirming the increase in the surface hydrophilicity of the AMP-functionalized PP slides following the immobilization procedure, due to the introduction of polar groups on the hydrophobic polymer. Moreover, reverse-phase high-precision liquid chromatography (RP-HPLC) analysis was performed to quantify the amount of 1018K6 immobilized on PP surfaces. For this investigation, 1018K6-PP slides, after the coupling reactions, were immersed in distilled water and incubated for 16 h at room temperature under agitation. Then, the polymers were subjected to sonication for 20 min at room temperature and the recovered solutions loaded on an RP-C18 column. By knowing the initial peptide concentrations that were used in the conjugation reaction, the amount of the peptide attached to PP slides was indirectly determined by comparing the peak area in the RP-chromatograms. The data obtained from these analyses showed that the immobilization yield varied from 23%, starting from a peptide concentration of 50 μM, to 5%, when 200 μM was used. The maximum peptide binding (31%) was obtained at 100 μM, which corresponded to a surface coverage of approximately 5.8 nmol/cm2, confirming that the initial amount of 1018K6 strongly influenced its binding to synthetic slides (Figure 1). Therefore, 100 μM was selected as the peptide concentration for performing all further experiments.

**Figure 1.** Immobilization yield (%) of 1018K6 on PP surfaces determined by RP-HPLC chromatography on a C18 column after the coupling reaction. PP surfaces pre-activated by plasma treatment, were incubated with a water solution of 1018K6 (100 μM). After the coupling reactions, the supernatants were recovered and analysed by RP-HPLC. The peptide solution (100 μM) at time 0 (t = 0) was used as control. The reported chromatograms are representative of three independent experiments.

Concerning the low binding capacity associated with the highest peptide concentration used, it could be attributed to a steric hindrance effect, which limits polymer–peptide interactions and a phenomenon producing water-soluble microaggregates, which can strongly reduce the availability of bioactive molecules for the immobilization reaction.

One of the most important requirements in applying an antimicrobial packaging in the food industries is the stability of the peptide immobilized on the polymers in the conditions of use, because, in this way, it does not require approval as food additive by EFSA (European Food Safety Authority). For this purpose, the slides functionalized with 100 μM 1018K6 were incubated in pure water or in NaCl 1 M at 4 ◦C for 7 days and the potential release of the peptide from the polymeric support was monitored by RP-HPLC, using the free peptide as a control. Following these analyses, no peptide-release was observed during 7 days of incubation, confirming the strong attachment via the covalent coupling of the bioactive compound, preventing its release from the surface and highlighting the high stability of the system.

It is worth noting that the projected packaging was reused at least six times in all the subsequent analyses, after washing with EtOH 70% for 1 min, rinsing with water and exposition to UV radiations for 1 h. Surely, this represents an important advantage from the industrial point of view, allowing a substantial decrease in environmental impact based on the concept that "reuse is better than recycling".

#### *3.2. Effects of 1018K6-PP on the Physical Properties and on the Microbiological Quality of Salmon Fillets*

It is known that the physicochemical characteristics of raw salmon fillets, such as pH (close to 6) and a high water activity (aw), make them highly susceptible to microbial growth, which affects the storability of these products [22]. Therefore, the fish industry is actively seeking methods of preservation to improve quality and marketability of this luxury marine food while economizing on costs.

To this aim, the antimicrobial effects of 1018K6-PP on the spoilage microbiota and the intrinsic properties of fresh salmon fillets during refrigerated storage were assessed, using the pre-activated and not-functionalized PP slides as control (CTR). In Figure 2, a representative scheme of the different steps applied for the preparation of salmon fillets employed in the microbiological and physicochemical analyses, is shown.

**Figure 2.** Representative scheme of the experimental preparation of salmon fillets.

As reported in Table 1, the initial pH values (pH > 6) for both samples (CTR and 1018K6-PP) were similar to those reported by other authors [23]. Throughout storage, the pH of salmon fillets in contact with the not-functionalized PP slides (CTR) and 1018K6-PP slightly decreased, recording a significant difference between the two groups on the 4th day. This result could be justified by an increase of acid production due to the homogenous proliferation of lactic acid bacteria occurring in these samples during the experimental analysis [24], although Gonzalez-Rodriguez et al. [25] registered an increase of alkalinity in prepacked salmon slices as a result of the ammonia and amines production by bacteria. As far as the water activity is concerned, no significant differences were observed among the experimental groups, with only a minor increase on the fourth day (Table 1). From the microbiological point of view, the initial concentrations of TAB (total aerobic bacteria) at

both 30 ◦C and 7 ◦C in raw salmons were somewhat higher compared with values reported in previous works [26,27], probably due to poor handling practices during the processing of fish fillets. However, similar data were reported by Wiernasz et al. [28], which referred to a concentration of 4.3 ± 0.2 Log (CFU/g) for total mesophilic bacteria. Indeed, the performed analyses showed that 1018K6-PP samples did not display significant differences (*p* > 0.05) in the growth kinetics of TAB at 30 ◦C and 7 ◦C compared with the control samples at the end of the storage period, indicating that the antimicrobial packaging did not have any effect, either positive or negative, on the microbiota of salmon fillets (Table 1). Albeit the total bacterial count represents a key factor in assessing the microbiological quality and safety of foods, it is well known that *Pseudomonas* spp., Enterobacteriaceae and *Brochothrix thermosphacta* are the main microbial family and genera responsible for the off-flavours and the unpleasant odours typical of deteriorating fish and fish products [29]. As far as the evolution of these bacteria is concerned, the samples stored in 1018K6-PP packaging revealed a significant slowdown in the replication of these microorganisms at the 4th day of conservation. Specifically, the sensitivity of bacteria belonging to the Enterobacteriaceae family to antimicrobial activity of the active packaging could make this product interesting for the food industry and promote its applicability as a potential "controller tool" for *Escherichia coli*. Indeed, the inhibitory effect of the innovative packaging was also evident towards beta-glucuronidase-positive *E. coli*, whose levels in treated samples were always below 1.0 Log (CFU/g) in contrast to the CTR [>2.0 Log (CFU/g)]. This finding becomes more relevant when the microbiological limits recommended for *E. coli* (1.0 and 2.7 Log (CFU/g) for minimum and maximum limit, respectively) by the International Commission on Microbiological Specifications for Foods (ICMSF) for the commercialization of fish and fish products [30], are taking into account. Actually, the innovative packaging makes the salmon fillets hygienically suitable throughout the storage period.

Regarding total coliforms and *Enterococcus faecalis*, the growth curves were very similar for the control and treated groups, but a significant antimicrobial effect of the 1018K6-PP was observed only on the 4th day of storage. Finally, the microbiological results pointed out the ability of the bound peptide to affect the growth of bacteria belonging to *Staphylococcus* genera. Therefore, the antimicrobial coating appears to successfully act on the survival and replicative capacity of this class of microorganisms, showing a significant (*p* < 0.01) difference between the control groups and the treat one on 4th and 7th days. All these findings confirm the results previously obtained with the peptide 1018K6 in a free status [31]. It is worth noting that the same microbiological analyses were performed on salmon fillets packaged in PP films that were not subjected to any surface modification. Interestingly, the obtained results were comparable to those achieved with the pre-activated and not-funzionalized PP films, thus excluding the occurrence of a potential antimicrobial effect determined by the polymeric surfaces activated by plasma alone. It should be pointed out that the microbiological results were not obvious on the basis of two main considerations: (i) the antimicrobial packaging could be unable to kill microbes under conditions of intended use due to the complexity of the fish matrix, which can inactivate the bioactive compound; (ii) 1018K6 could not retain its antimicrobial activity when bound to PP polymers, because the immobilization process could restrict its conformational freedom and influence its orientation, both of which are important features for the peptide activity.

As far as the potential antimicrobial mechanism, two factors could play a dominant role of the bound 1018K6 with respect to its soluble form:


To sum up, 1018K6-PPs can be considered a promising instrument to positively affect the quality of perishable products such as fresh salmon based on the microbial effects observed. This suggestion is supported by the important antimicrobial data that the new package exerts against specific spoilage microorganisms responsible for spoilage processes in fish and fish products. However, a potential role of 1018K6-PP in the food safety cannot be excluded, taking into account its action against Enterobacteriaceae and *Staphylococcus* spp. In this scenario, the introduction of 1018K6-PP into the food marketplace could guarantee the availability of safe and natural tools capable of limiting damages of bacterial origin.


**Table 1.** Evaluation of microbiological counts [Log (CFU/g)] in salmon fillets packaged in active PP films functionalized with 1018K6 by storage time.

*ni*: not isolated. In each storage day, three samples by experimental group were analysed. Statistical analysis was performed comparing experimental groups at each sampling time and within each experimental group along the ripening period. All data were presented as mean (m) ± standard error (sem). Different superscript uppercase letters indicate a significant difference at *p* < 0.01. Different superscript lowercase letters indicate a significant difference at *p* < 0.05. a–c In the same row mean values (same group in different days) followed by different letters show significant differences. x,y In the same column mean values (different groups on the same sampling time) followed by different letters show significant differences.

#### *3.3. Instrumental Colour Analysis of Salmon Fillets*

The colour in fish foods is one of the most important qualities influencing consumer decisions to purchase. Therefore, the impact of 1018K6-PPs on the colour of the packaged salmon fillets was investigated for various storage periods. As reported in Table 2, the lightness (*L*\*) was the only parameter to be influenced significantly by the use of the active packaging, although this phenomenon did not visibly affect the general appearance of the product. Indeed, chroma values are similar in all the samples during the whole experimental period. Our findings are in agreement with Merlo et al. [32], who reported that the use of chitosan film reduces the change in structure of proteins, conferring a darker aspect to treated salmon fillets. This result was justified considering the strong connection between the change in light scattering of the muscle and the variation in lightness. Furthermore, samples packed in 1018K6-PP were found to be slightly more reddish and yellowish (higher values of *a*\* and *b*\*, respectively) than the control ones. According to several authors [32–34] the main value taken into account for this fish family is the redness, which is associated to the consumer's preference and acceptability. Changes in *a\** value in salmon are due to the addition of carotenoids, such as astaxanthin and cataxanthines, and related to reddish colour of salmonid fishes. However, the scientific community disagrees, and different opinions are reported in literature. Ye¸silayer et al. [35] demonstrated that fillets of farmed Atlantic salmons fed with feed containing carotenoids showed high values of yellowness, demonstrating that the typical red-orange colour is represented by both redness and yellowness values. It is worth underling that *a*\* and *b*\* did not differ significantly among all samples by storage time, exhibiting similar ΔE (total colour differences), Δ*a\**, and Δ*b\** values. Therefore, 1018K6-PPs did not produce negative effects on colours parameters, potentially preserving this aspect of salmon samples.

**Table 2.** Changes in colour indices of the salmon fillets packaged in active PP films functionalized with 1018K6 by storage time.


On each sampling day, three samples by experimental group were analysed. Statistical analysis was performed comparing experimental groups at each sampling time and within each experimental group along the ripening period. All data were presented as mean (m) ± standard error (sem). Different superscript uppercase letters indicate a significant difference at *p* < 0.01. Different superscript lowercase letters indicate a significant difference at *p* < 0.05. a,b In the same row mean values (same group in different days) followed by different letters show significant differences. x,y In the same column mean values (different groups on the same sampling time) followed by different letters show significant differences.

#### *3.4. Effect of 1018K6-PP on Chemical Parameters of Salmon Fillets*

It is common to evaluate the "age" of the food through the study of the microbiological community in order to evaluate the presence and the concentration of specific spoilage microorganisms (SSOs). However, the spoilage of fish and fish products is associated with the occurrence of off-odours due to the production of volatile substances as a result of the bacterial metabolism. Changes in the odour affect the acceptability to consumers, who associate the freshness of fish products to typical organoleptic features. Due to perishable foods being sensitive to variations in appearance, some of the characteristic volatile organic compounds (VOCs) produced by bacteria can be used as potential chemical spoilage indices (CSIs) in fish and fish products [36]. In this study, two chemical quality indicators were used to assess the ability of 1018K6-PP to preserve the quality and sensorial properties, such as the total volatile basic-nitrogen (TVB-N) and trimethylamine-nitrogen (TMA-N) (Figure 3). The choice to detect these two VOCs is dictated by the key role that these chemicals play in the freshness of salmon, being the final products of protein degradation [37].

TVB-N includes the measurement of volatile basic nitrogenous compounds, such as trimethylamine (TMA), dimethylamine (DMA) and other nitrogenic substances, which are produced by bacterial or tissue enzymes from the deamination of amino acids. In the current study, the initial amount of TVB-N in all salmon fillets analysed was 7.89 ± 0.21 mg/100 g (Figure 3A). Significant differences (*p* < 0.01) between salmon samples packaged with CTR-PP and 1018K6-PP slides were observed after 4 days of storage. Specifically, 1018K6-PP appeared, indirectly, to slow down the protein degradation in salmon fillets through the control of microbial growth. Indeed, the great amount of the free amino acids in fish [38,39] are used as substrate by bacteria in their metabolism, with the final production of organic acids, sulphur compounds, ammonia and biogenic amines (BAs) [40,41]. Overall, though 1018K6-PP demonstrated to be efficient in reducing the protein degradation, throughout the entire storage period TVB-N values never reached and overcame the legislative limit of 35 mg/100 g specified by the EU 2019/627 for this fish typology [42].

The TMA-N origins by decomposition of trimethylamine N-oxide (TMAO), used from bacteria as a donor of oxygen molecules in their respiratory metabolism in fish and fish products stored at refrigeration temperature [43–45]. Due to the importance of the initial amount of TMAO in the muscle, the concentration of TMA-N is strongly related to the species of fish, and *Salmo salar* is naturally rich in trimethylamine N-oxide [46]. In Figure 3B, the trends of TMA-N over time are displayed. Specifically, the samples packed with 1018K6-PPs showed the lowest TMA-N values (*p* < 0.01) at both 4 and 7 days of storage, in agreement with the above reported values of TVB-N, thus reinforcing the hypothesis that active slides affect the spoilage microbial communities. In fact, it is well-known that the TMA production is mainly operated by bacteria belonging to the Enterobacteriaceae family, which, the results showed, proved sensitive to the antimicrobial activity of the bound peptide, as already reported in Table 1 [47,48]. Although TMA is considered a good indicator of the deterioration progress, no maximum legislative limits for TMA concentrations were defined and different values were proposed. However, according to Shumilina et al. [49], who reported 4.2 mg/100 g as the acceptability limit for fish, the freshness was preserved only in salmon fillets put in contact with 1018K6-PP (TMA < 5 mg/100 g).

Finally, measurements of thiobarbituric acid reactive substances (TBARS) expressed as malonyldialdehyde (MDA) levels, were performed in order to investigate lipid oxidation, which is a very important event determining the quality of foods, especially of those containing highly unsaturated fats, such as fish [50,51]. As shown in Figure 3C, the TBARS values in control fillets increased significantly during refrigerated storage in contrast to that observed in the packaged fillets with 1018K6-PP slides. Therefore, 1018K6-PP is able to exert antioxidant properties, but this finding is not surprising given the well-known correlation between lipid oxidation and bacterial contamination [52].

Indeed, MDA is the main aldehyde produced as a result of the decomposition of unsaturated fatty acids—also a bacterial operation—thus, remarking on the antimicrobial efficacy of the active films. The chemical analyses performed on the salmon fillets packaged in unmodified PP films demonstrated that the plasma activation by itself was not able to allow the polymers to affect the quality of these fish products.

Overall, the comprehensive analyses of microbiological and chemical parameters pointed out two main aspects: the key roles of TVB-N, TMA and MDA as chemical spoilage indices in perishable food and the effectiveness of 1018K6-PP in preserving salmon fillets. As reported by Prabhakar et al. [53], the assumption of the interconnection among bacterial concentrations and chemical metabolites production is already consolidated, as is the link between TVB-N/TMA levels and quality. Therefore, our findings confirmed this strong link, and the candidate 1018K6-PP as a valuable packaging technology capable of guaranteeing longer durability for highly perishable foods, such as raw salmon.

**Figure 3.** Effects of 1018k6-PP surfaces on the chemical quality of salmon fillets. (**A**) Changes in TVB-N (A), TMA-N (**B**), and TBARS (**C**) of *Salmon salar* fillets packaged in active 1018K6-PP films during storage at 4 ◦C. CTR (blue lines)—PP films without 1018K6; 1018K6-PP (green lines)—PP films functionalized with 1018K6. Results are means of three independent experiments and error bars represent the standard error (sem). Different letters at each sampling time are used for significantly different samples, according to Tukey test (uppercase letters: *p* < 0.01).

#### *3.5. Panelists' Sensory Evaluation*

Sensory perception is the tool through which the consumers choose foods at a store, trusting in their senses and adopting an immediate and easy system for evaluating freshness and quality [54]. In this study, the organoleptic features appeared to be partially influenced by the packaging technology used. As reported in Figure 4, the representation of observed sensory characteristics highlighted an important consequence of the use of antimicrobial slides on the production of off-odours. Despite the initial good quality of all samples, the salmon fillets belonging to the control groups showed signs of spoilage as early as the 4th day of storage at refrigeration temperature. The judges rated the control samples as "poor freshness quality" products, due to the score of odour and of the general appearance obtained by the end of the trial. Contrarily, the treated samples maintained good sensory characteristics over time. In agreement with those reported above for VOCs, the demonstrated antimicrobial activity (Table 1) of 1018K6-PP seems to indirectly control the negative changes in the chemical structure and metabolites production of salmon fillets occurring during storage [55,56]. Furthermore, the scores of overall appearances of treated samples pointed out the absence of negative influences of the novel active packaging on the sensory features, due to the colourless and odourless nature of the 1018K6 molecules.

#### *3.6. Microbial Challenge Testing of L. monocytogenes on Salmon Fillets Packaged with 1018K6-PP*

Foodborne diseases are a reality affecting thousands of people in industrialized countries every year. Amongst the bacterial pathogens responsible of severe human toxiinfections*, Listeria monocytogenes* is considered one of the most dangerous. Due to their origin and the way in which they are processed, fish products show an increased incidence rate of listeriosis, and then they represent typical food vehicles of high levels of microbiological contamination, taking into account that this bacterium is able to grow also at refrigeration temperatures. Therefore, challenge testing of the food products with

*L. monocytogenes* is recommended to assess the potential for growth, both qualitatively and quantitatively, in the foods at risk.

**Figure 4.** Changes in colour (**A**), odour (**B**), and overall appearance (**C**) of salmon fillets during storage period. CTR (blue lines)—PP films without 1018K6; 1018K6-PP (green lines)—PP films functionalized with 1018K6.

In this context, the anti-listerial efficacy of 1018K6-PP was evaluated in salmon fillets stored at 5 ◦C for 96 h (Figure 5). In order to confer greater confidence in the assessment of the likelihood of a particular strain to compromise food safety, mixed cultures of three *L. monocytogenes* strains isolated from fish were used at a concentration of ca. 150 CFU/mL. This value of inoculum is representative for the natural contamination of *L. monocytogenes* commonly encountered in fresh foods, taking into account that 100 CFU/mL is the threshold limit considered as low risk for causing listeriosis. In addition, the use of food isolates is recommended because it is likely to represent better the behaviour of naturally contaminating strains.

**Figure 5.** Bactericidal activity of polymer functionalized with 1018K6 against *L. monocytogenes* on salmon fillets. Negative control—untreated salmon fillets; positive control—salmon fillets treated with not-functionalized PP; treated control—salmon fillets treated with 1018K6-PP.

The results of the challenge test performed on salmon fillets indicated that the antimicrobial packaging was effective in inhibiting the growth and survival of the pathogen on the surface of the fresh food during storage in contrast to the untreated control. Indeed, a complete inhibition of *L. monocytogenes* was observed after 72 h incubation, with a slight

decrease (95%) at the end of the assay (96 h), thus suggesting that our system could be used to preserve the safety of fish products during storage.

Overall, our results show the positive impact of the effectiveness of 1018K6-PP packaging on food safety when the target microorganism is a foodborne pathogen of great present concern, such as *L*. *monocytogenes*.

#### *3.7. Evaluation of 1018K6-PPs Slides on the Physicochemical, Microbial and Sensorial Properties of Sarda Sarda Burgers*

In order to evaluate the versatility of our active packaging, a different typology of food matrices was included in the experimental design. To this aim, microbiological, physicochemical and sensorial analyses were performed on fish burgers of bonito (*Sarda sarda)* packaged with 1018K6-PP slides. This analysis was aimed at also verifying the effectiveness of 1018K6-PP against minced fish meat, which is notoriously characterized by higher level of microorganisms than fillets because of the shredding process underlying their manufacture [57]. The scheme used to set up the *Sarda sarda* hamburger employed in the experimental trials, is shown in Figure 6.

**Figure 6.** Representative scheme of preparation of *Sarda sarda* burgers employed in the microbiological and physico-chemical analyses. (**A**) Burgers of *Sarda sarda* treated with PPs films not-functionalized with 1018K6 (CTR); (**B**) burgers of *Sarda sarda* treated with PPs films functionalized with 1018K6 (1018K6-PP).

As reported in Table 3, the initial amounts of TAB were significantly different between salmon fillets (Table 1) and fish burgers (Table 3), in which more than 1 Log (CFU/g) of mesophilic bacteria were enumerated. For this reason, fish burgers represent a difficult challenge. Regarding antimicrobial activity, 1018K6-PPs negatively affected the growth of specific microorganisms, including the total bacterial count. During the storage, the mesophilic TAB increased significantly in control samples, until reaching a concentration greater than 8 Log (CFU/g) by the 7th day, in contrast to that observed in the samples packaged with the antimicrobial slides, in which the maximum acceptable limit set by ICMSF for TAB [7 Log (CFU/g)] was never exceeded during 7 days of storage. Furthermore, due to the key role of mesophilic bacteria in the production of metabolites and off-odours, the antimicrobial activity of 1018K6-PP produced a beneficial effect on the overall appearance of fish burgers and their chemical profile. Therefore, our findings not only confirmed the effectiveness of the new package in slowing the growth of the same bacterial communities described for salmon fillets but also the obtained results enhanced the potential role of 1018K6-PP as a tool for monitoring microbiologic contaminations.


**Table 3.** Evaluation of microbiological counts (Log CFU/g) in *Sarda sarda* burgers packaged in active PP films functionalized with 1018K6 by storage time.

*ni*—not isolated. In each sampling day, three samples were analysed by experimental group. Statistical analysis was performed comparing experimental groups at each sampling time and within each experimental group along the ripening period. All data were presented as mean (m) ± standard error (sem). Different superscript uppercase letters indicate a significant difference at *p* < 0.01. Different superscript lowercase letters indicate a significant difference at *p* < 0.05. a–d In the same row mean values (same group in different days) followed by different letters show significant differences. x,y In the same column mean values (different groups on the same sampling time) followed by different letters show significant differences.

It is worth noting that the same analyses were performed on bonito burgers packaged in PP films and not subjected to any surface modification and no discrepancy in the results was observed with respect to those obtained with the pre-activated PP films alone.

As far as the determination of colour values, the changes in this parameter in fish burgers over time overlapped the data collected for salmon fillets (Table 4). Specifically, the samples belonging to the control group appeared less dark than the others, affirming the hypothesis of an increase in proteolysis. Moreover, no differences were highlighted among samples in *a*\* and *b*\* values and, consequently, in total colour differences (ΔE), variations in *a*\* (Δ*a\**), and in *b*\* (Δ*b\**).

The graph reported in Figure 7 showed the positive effect of the active packaging on the redness on 3rd and 7th day, although slightly.


**Table 4.** Changes in colour indices of *Sarda sarda* burgers packaged in active PP films functionalized with 1018K6 by storage time.

In each sampling day, three samples were analysed by experimental group. Statistical analysis was performed comparing experimental groups at each sampling time and within each experimental group along the ripening period. All data were presented as mean (m) ± standard error (sem). Different superscript uppercase letters indicate a significant difference at *p* < 0.01. Different superscript lowercase letters indicate a significant difference at *p* < 0.05. a,b In the same row mean values (same group in different days) followed by different letters show significant differences. x,y In the same column mean values (different groups on the same sampling time) followed by different letters show significant differences.

**Figure 7.** (**A**) Analysis of *a\** variation (Δ*a\**) in bonito fish burgers during the storage period. Results are means of three independent experiments and error bars represent the standard error (sem). CTR (blue)—PP films without 1018K6; 1018K6-PP (green)—PP films functionalized with 1018K6. (**B**) Photos of *Sarda sarda* burgers packaged with the control and functionalized slides at each sampling time.

Moreover, the experimentation on fish burgers marked the important contribution of the antimicrobial molecule in slowing down the protein degradation. Indeed, significant differences were found among samples packed in active films and control ones and the gap recorded between the corresponding TMA-N and TVB-N values proved the concrete beneficial effect of the 1018K6-PP (Figure 8).

**Figure 8.** Effects of 1018K6-PP slides on the chemical quality of *Sarda sarda* burgers. Changes in TVB-N (**A**), TMA-N (**B**) and TBARS (**C**) of *Sarda sarda* burgers packaged in active 1018K6-PP films during storage at 4 ◦C. CTR (blue lines)—PP films without 1018K6; 1018K6-PP (green lines)—PP films functionalized with 1018K6. Results are means of three independent experiments and error bars represent the standard error (sem). Different letters at each sampling time are used for significantly different samples, according to Tukey test (uppercase letters: *p* < 0.01; lowercase letters: *p* < 0.05).

Finally, the off-odours drastically affected the judgments (Figure 9), by which the control samples were labelled as unpleasing foods, probably due to their content in TVB-N and TMA-N. This result was expected considering that the odour weight on the panellists' choices is the most critical sensory characteristic for fish products [58].

**Figure 9.** Changes in colour (**A**), odour (**B**), and overall appearance (**C**) of bonito fish burgers during storage period. CTR (blue lines)—PP films without 1018K6; 1018K6-PP (green lines)—PP films functionalized with 1018K6.

Finally, our findings allowed also supposing a positive effect of 1018K6-PP on the quality parameters of bonito burgers, considering the significant differences between the two experimental groups in microorganisms concentrations and CSIs levels, which strongly affected on the sensory appearance of samples. The off-odours and the changes in lightness were demonstrated to be the main visible properties associated to the spoilage processes, so that they could be considered alarm bells for the consumers.

#### **4. Conclusions**

As stated, fish is a highly perishable food characterized by a short shelf-life. Refrigeration is probably one of the most used methods for fish preservation, but several deteriorative quality changes occur during storage, particularly in texture, colour and flavour, limiting shelf-life. Therefore, there is an important and urgent need to find alternative strategies to overcome existing challenges that are associated with fish spoilage, which will ultimately benefit both the producers and consumers. In the present study, two different kinds of fish foods, *Salmon salar* fillets and *Sarda sarda* burgers, were used to obtain information about the feasibility of the potential application of 1018K6-PP packaging in the food industry. The results showed that 1018K6-PP helped to maintain the chemical and microbial quality of this kind of product without inducing sensory alterations during refrigerated storage. Therefore, the antimicrobial packaging used in the present study represents an excellent and promising option for the preservation of fish foods due to its antimicrobial, non-toxic and re-usability properties, and thereby reduce the occurrence of foodborne illness.

**Author Contributions:** Conceptualization, A.A., Y.T.R.P. and G.P.; methodology, R.L.A., L.G., B.A. and D.C.; software, R.L.A., M.B. and D.C.; validation, M.G., B.A. and G.P.; formal analysis, Y.T.R.P., R.L.A., B.A., L.G. and M.B.; investigation, M.G., R.L.A., B.A. and D.C.; resources, A.A., Y.T.R.P. and G.P.; data curation, M.G., R.L.A. and Y.T.R.P.; writing—original draft preparation, M.G., R.L.A. and G.P.; writing—review and editing, M.G. and G.P.; visualization, M.G., G.P. and A.A.; supervision, M.G. and G.P.; project administration, A.A. and G.P.; funding acquisition, G.P., Y.T.R.P. and A.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by: Ministero della Salute-"Dalle superfici agli alimenti: nuove soluzioni per alimenti più sicuri (NewSan)"-grant number IZSME 06/20 RC, Ricerca Corrente 2020 project; Ministero dello Sviluppo Economico "Sviluppo di piattaforme molecolari e cellulari per l'identificazione di prodotti innovativi ad attività NUTRAceutica da Biotrasformazioni mediante organismi ESTremofili (NUTRABEST)"- grant number F/200050/01-03/X45, Fondo per la Crescita Sostenibile-Sportello "AGRIFOOD" PON I&C 2014-2020 project.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data is contained within the article.

**Acknowledgments:** The authors would like to thank Francesca Segreti for technical support and Valentina Brasiello for administrative assistance.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**


### *Communication* **Impact of Cork Closures on the Volatile Profile of Sparkling Wines during Bottle Aging**

**Filipa Amaro 1,2, Joana Almeida 1,2, Ana Sofia Oliveira 2, Isabel Furtado 2, Maria de Lourdes Bastos 1,2, Paula Guedes de Pinho 1,2 and Joana Pinto 1,2,\***


**Abstract:** This study aimed at investigating the impact of different technical cork stoppers on the quality preservation and shelf life of sparkling wines. The volatile compositions of two Italian sparkling wines sealed with a sparkling cork with two natural cork discs (2D) and a microagglomerated (MA) cork were determined during bottle aging (12 to 42 months) after disgorging, by headspace solidphase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GC/MS). The volatile profile of the sparkling wine #1 sealed with 2D stoppers showed the presence of camphor from 12 to 42 months, along with significant alterations in the levels of several alcohols, ketones, and ethyl esters at 24 and 42 months. The impact of closure type was less pronounced for sparkling wine #2 which also showed the presence of camphor from 12 to 42 months in 2D samples, and significantly higher levels of esters at 24 and 42 months for 2D compared with MA. These results unveiled that the type of closure has a greater impact on the volatile composition of sparkling wines at longer post-bottling periods and 2D stoppers preserve the fruity and sweety aromas of sparkling wines better after 42 months of bottle storage.

**Keywords:** volatile organic compounds; HS-SPME-GC/MS; Italian sparkling wines; cork stoppers; bottle aging

#### **1. Introduction**

Over the last two decades, the global wine market has experienced an increase in the demand for sparkling wines due to changes in consumers' preferences [1]. The aroma composition of these wines is a key attribute to consumers' acceptance and an important indicator of quality [2]. Sparkling wines produced by the traditional method (or *Champenoise*) are relatively complex in terms of aroma composition since they undergo a secondary fermentation in the bottle, followed by different contact periods with lees, when yeasts suffer autolysis releasing nitrogen compounds, polysaccharides, volatile compounds (e.g., ethyl esters), and phenolic compounds, among others (e.g., lipids and nucleic acids) [3], which contribute to the aroma complexity of the greatest sparkling wines.

The impact of several winemaking processes in the aroma composition of sparkling wines has been studied, such as grape variety and maturity [4,5], production methods [6], yeast selection [7], and aging period in contact with lees [7,8]. However, it is well known that the type of bottle closure influences the aroma composition of wines during aging, as has been reported for still wines [9,10], but the aroma comparison between sparkling wines bottled with different types of closures has not been reported so far.

Cork stoppers play a pivotal role in preserving the effervescence (carbon dioxide levels) and the aroma attributes of sparkling wines, making them almost irreplaceable

**Citation:** Amaro, F.; Almeida, J.; Oliveira, A.S.; Furtado, I.; Bastos, M.d.L.; Guedes de Pinho, P.; Pinto, J. Impact of Cork Closures on the Volatile Profile of Sparkling Wines during Bottle Aging. *Foods* **2022**, *11*, 293. https://doi.org/10.3390/ foods11030293

Academic Editors: Matteo Alessandro Del Nobile and Amalia Conte

Received: 29 December 2021 Accepted: 20 January 2022 Published: 22 January 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

for this type of wine. Nowadays, there are several types of sparkling wine cork stoppers available in the market, from microagglomerated to agglomerated corks, and corks made with agglomerated body plus one, two, or three natural cork discs attached to one of the ends [11]. Importantly, the natural cork discs are obtained from high-quality cork planks, allowing a higher contact of the sparkling wines with this type of material. Hence, this study aimed to investigate, for the first time to our knowledge, the impact of two different technical cork stoppers on the volatile composition of two Italian sparkling wines from 12 to 42 months of aging in bottle after disgorging.

#### **2. Materials and Methods**

#### *2.1. Chemicals*

1,4-Cineole (98%), 1-decanol (99.9%), 1-hexanol (99.9%), 1-octanol (≥99%), 2-heptanone (99%), 2-nonanone (97%), 2-undecanone (97%), 3-hexen-1-ol (98%), 5-methyl-2-furfural, benzaldehyde (≥99.5%), camphor (99%), decanal (95%), diethyl succinate (≥99%), ethyl 2-methylbutanoate (99%), ethyl butanoate (99%), ethyl decanoate (≥98%), ethyl heptanoate (≥98%), ethyl hexanoate (99%), ethyl isobutanoate (≥98%), ethyl isovalerate (98%), ethyl nonanoate (≥98%), ethyl octanoate (99%), eucalyptol (99%), furfural (99%), hexyl acetate (98%), isoamyl acetate (≥99%), isoamyl alcohol (98%), limonene (99%), linalool oxide (97%), nonanal (≥95%), octanal (≥98%), phenylacetaldehyde (90%), phenylethyl acetate (99%), phenylethyl alcohol (99%), tartaric acid (≥99.5%), *α*-pinene (99%)), *β*-cyclocitral (90%), *β*-damascenone (≥98%), *α*-ionone (85%), and *β*-linalool (80%) were supplied by Sigma-Aldrich (Madrid, Spain). Ethanol (99.9%) was purchased from ERBA Reagents (Val de Reuil, France).

#### *2.2. Sparkling Wine Samples*

The sparkling wines used in this study were a 2011 Classic Brut Vintage (sparkling wine #1) and a 2005 Reserve Brut Vintage (sparkling wine #2), from different producers in the Piemonte region in Italy. The sparkling wines were produced from the Chardonnay and Pinot Noir grape varieties, using the traditional method with secondary fermentation in the bottle, and were both disgorged in 2017, corresponding to approximately 5 years of aging in contact with lees for sparkling wine #1 and 11 years for sparkling wine #2. Two types of commercially available stoppers were used for bottling of the two sparkling wines, namely one sparkling cork (3–7 mm diameter granules) with two natural cork discs glued at one end, termed as 2D throughout the article, and one microagglomerated cork (1–3 mm diameter granules), termed as MA. After bottling, all samples were kept under controlled temperature conditions in the producers' cellars. Samples were then collected at 12, 24, and 42 months (*n* = 3–5 bottles per sampling point) for analysis of the volatile fraction.

#### *2.3. Analysis of Volatile Composition by HS-SPME-GC/MS*

The analyses of volatile compounds in sparkling wine samples were performed in 2018 (12 months), 2019 (24 months) and 2020 (42 months) using a HS-SPME-GC/MS method adapted from Barros et al. [12]. Briefly, each sparkling wine sample (250 μL) was placed in a 20 mL glass vial which was incubated for 5 min at 45 ◦C, using a Combi-PAL autosampler (Varian Pal Autosampler, Zwingen, Switzerland). The volatile compounds were then extracted by a 50/30 μm divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) fiber (Supelco Inc., Bellefonte, PA, USA) for 30 min at 45 ◦C with a stirring speed of 250 rpm. After extraction, the compounds were thermally desorbed into the GC system for 6 min at 250 ◦C. All samples were randomly injected.

A 436-GC model (Bruker Daltonics, Bremen, Germany) coupled to a SCION single quadrupole (SQ) mass spectrometer (Bruker Daltonics, Bremen, Germany) and a Bruker Daltonics MS workstation (version 8.2.1, Bruker Daltonics, Bremen, Germany) were used for volatile analysis and quantification. The GC system was equipped with a fused silica capillary column (Rxi-5Sil MS, 30 m × 0.25 mm internal diameter × 0.25 μm; Restek Corporation, Bellefonte, PA, USA) and high-purity helium C-60 (Gasin, Leça da Palmeira, Portugal) was used as carrier gas at a constant flow rate of 1.0 mL/min. The oven temperature was programmed at 40 ◦C for 1 min, followed by an increase of 5.0 ◦C/min to 250 ◦C, where it was held for 5 min, and then increased at 5.0 ◦C/min to 300 ◦C. SQ-MS was conducted in the electron ionization (EI) mode at 70 eV and the transfer line, ion source, and manifold temperatures were maintained at 250, 250, 260, and 41 ◦C, respectively. Data acquisition was performed in full scan mode with a mass range of 40 to 400 *m/z* and a 500 ms scan time.

For the quantification of volatile compounds, standard compounds were dissolved in a wine model solution (12% ethanol, 5 g/L of tartaric acid, pH 3.2) and analyzed under the same conditions by HS-SPME/GC-MS. The calibration curves were achieved by injecting a range of known concentrations of each compound and computed by the respective area of the peak versus concentration.

#### *2.4. Statistical Analyses*

Multiple unpaired *t*-tests were applied to evaluate the differences in the levels of volatile compounds in sparkling wines sealed with 2D compared with MA at each postbottling time. In addition, ordinary one-way analysis of variance (ANOVA) was computed to assess the differences in volatile concentrations between different post-bottling times. The concentration levels were considered significantly different for *p*-values < 0.05. All statistical analyses were performed using the software GraphPad Prism 9 (version 9.3.0, San Diego, CA, USA).

#### **3. Results**

Bottle closures can affect the aroma composition of wines during aging by three main factors: (1) the oxygen ingress through the bottle which can lead to wine oxidation and the development of oxidized aromas; (2) the desorption of volatile compounds from closures into wine which can lead to pleasant (e.g., terpenes) or unpleasant (e.g., pyrazines) aromas; and (3) the scalping of volatile compounds present in wine by closures [9].

From the 39 volatile compounds quantified, significant alterations were found for 8 compounds in sparkling wine #1 sealed with 2D compared with MA (Figure 1, Table 1), and 3 compounds for sparkling wine #2 (Figure 2, Table 2), during bottle storage from 12–42 months. Interestingly, a lower number of altered volatile compounds was found for sparkling wine #2 which aged longer in contact with lees (11 years) in contrast with sparkling wine #1 (5 years). The levels of the remaining quantified compounds in both sparkling wines are present in Tables S1 and S2.

**Figure 1.** Bar graphs representing the levels of volatile compounds significantly changing in sparkling wine #1 sealed with a sparkling cork with two natural cork discs (2D in blue) and a microagglomerated cork (MA in red) during bottle aging (12 to 42 months). \*—*p* ≤ 0.05, ND—not detected.

The volatile composition of sparkling wines #1 and #2 was more affected by the type of closure at 24- and 42-months post-bottling, while only a qualitative change in camphor (only present in samples sealed with 2D, Figure 1 and Table 1) was detected in both sparkling wines at 12 months. Camphor is responsible for pleasant aromas—such as herbal, minty, and woody [13]—but the olfactory perception threshold in wine or wine model solution has not been reported so far. At 24 months post-bottling, sparkling wine #1 showed significantly higher levels of 3-hexen-1-ol and *β*-damascenone in samples sealed with 2D compared with MA and the presence of camphor only in 2D samples (Figure 1, Table 1). 3-hexen-1-ol is characterized by green and leafy odors and was present in concentrations below the olfactory perception threshold (<400 μg/L) reported for wine model solution [14], while *β*-damascenone is characterized by woody, floral, and herbal odors [14,15], and was present above its olfactory perception threshold (0.05 μg/L) [14]. At 42 months, this sparkling wine showed significantly higher levels of ethyl isobutanoate, ethyl butanoate, and ethyl isovalerate in samples sealed with 2D, as well as significantly lower levels of 2-undecanone and the presence of camphor (Figure 1, Table 1). From these compounds, the three ethyl esters (ethyl isobutanoate, ethyl butanoate, and ethyl isovalerate), and 1-hexanol were present above their olfactory perception thresholds (Table 1) [14], and can contribute with fruity notes [13,15] to the sparkling wine aroma. Interestingly, the levels of 1-hexanol, ethyl butanoate, ethyl isovalerate, 2-undecanone, *β*-damascenone, and camphor changed significantly with bottle aging, while the levels of 3-hexen-1-ol and ethyl isobutanoate were relatively constant over time (Table 1).

**Table 1.** Levels of volatile compounds significantly changing in sparkling wine #1 sealed with a sparkling cork with two natural cork discs (2D) and a microagglomerated cork (MA) during bottle aging (12 to 42 months).


<sup>1</sup> Average concentration and standard deviation of sparkling wine #1 sealed with 2D and MA corks. A *n* = 3 per closure was considered at 12 and 42 months, and a *n* = 4 at 24 months. <sup>2</sup> Descriptors reported in references [13,15]. <sup>3</sup> Olfactory perception thresholds determined in wine model solution as reported in reference [14]. ns—*p* > 0.05, \*—*p* ≤ 0.05, \*\*\*\*–*p* ≤ 0.0001, BLOQ–below limit of quantification, ND—not detected, NR–not reported, Q—qualitative alteration.

**Figure 2.** Bar graphs representing the levels of volatile compounds significantly changing in sparkling wine #2 sealed with a sparkling cork with two natural cork discs (2D in green) and a microagglomerated cork (MA in orange) during bottle aging (12 to 42 months). \*—*p* ≤ 0.05, ND—not detected.

**Table 2.** Levels of volatile compounds significantly changing in sparkling wine #2 sealed with a sparkling cork with two natural cork discs (2D) and a microagglomerated cork (MA) during bottle aging (12 to 42 months).


<sup>1</sup> Average concentration and standard deviation of sparkling wine #2 sealed with 2D and MA corks. A *n* = 5 per closure was considered at 12 months, a *n* = 4 at 24 months, and a *n* = 3 at 42 months. <sup>2</sup> Descriptors reported in references [13,15]. <sup>3</sup> Olfactory perception thresholds determined in wine model solution as reported in reference [14]. ns—*p* > 0.05, \*—*p* ≤ 0.05, \*\*\*\*—*p* ≤ 0.0001, ND–not detected, NR—not reported, Q—qualitative alteration.

In contrast, at 24 months post-bottling, sparkling wine #2 showed the consistent presence of camphor in samples sealed with 2D, as well as significantly higher levels of ethyl decanoate (Figure 2, Table 2). At 42 months, significantly higher levels of ethyl octanoate and a tendency for higher levels of ethyl decanoate were observed in samples sealed with 2D, along with the presence of camphor (Figure 2, Table 2). The presence of ethyl octanoate in levels above the olfactory perception threshold (>0.6 mg/L) [14] may contribute to the fruity aroma [13,15] of this sparkling wine, while ethyl decanoate may have a lower impact due to its low concentration (<200 μg/L) [14]. Regarding the behavior of these compounds during bottle aging, ethyl octanoate increased significantly, whereas ethyl decanoate showed a significant decrease and camphor levels were constant over time (Table 2).

#### **4. Discussion**

Ethyl esters are the main class of aroma compounds released by the autolysis of yeasts in sparkling wines produced by the traditional method and they contribute to the fruity and floral-like aromas of these wines [16]. In our study, the levels of several ethyl esters were significantly higher in both sparkling wines sealed with 2D corks. The preservation of ethyl esters during bottle storage has been a challenge for winemakers as ethyl esters tend to hydrolyze over time due mostly to the low pH of wines [17]. Hence, a stopper able to preserve better the ethyl ester composition of sparkling wines can improve their shelf life and the sensory attributes expected by consumers. Notably, most ethyl esters present in both sparkling wines (Tables 1, 2, S1 and S2)—with exception of ethyl decanoate, hexyl acetate, and phenylethyl acetate—showed a significant increase over time. Despite the behavior of these compounds has been studied during the aging period in contact with lees [7,8], the information about their evolution trends after disgorging is limited [18].

Camphor has been previously identified by our group as only present in wines sealed with natural cork [19], which agrees with the results observed for both sparkling wines sealed with 2D stoppers. The most probable hypothesis is the desorption of camphor from natural cork to wines, which is also corroborated by the detection of this compound in wine model solution extracts of natural cork granules [20]. Based on these facts, camphor seems to be a good marker to discriminate wines bottled with natural cork discs versus other closures.

The levels of two alcohols (3-hexen-1-ol and 1-hexanol), one ketone (2-undecanone) and one isoprenoid (*β*-damascenone) were also significantly influenced by the type of closure in sparkling wine #1. Alcohols can be substrates for wine oxidation originating their correspondent aldehydes [21]. Thus, the lower levels of 3-hexen-1-ol and 1-hexanol in sparkling wine #1 sealed with MA stoppers may be due to their oxidation in hexanal and 3 hexenal, respectively. Though these aldehydes were not detected in the volatile composition of sparkling wine #1 under our experimental conditions. However, 2-undecanone, a ketone that may be also formed by oxidation [21], was found in higher levels in samples sealed with MA. Finally, *β*-damascenone is mainly produced from direct degradation of carotenoid molecules during fermentation [22]. Higher levels of this isoprenoid were previously found in a dry white wine sealed with natural cork compared with microagglomerated cork [23], in agreement with the results obtained for sparkling wine #1 at 24 months.

#### **5. Conclusions**

These results showed that the type of closure has a greater impact on volatile composition of sparkling wines at longer post-bottling periods (42 months). For both sparkling wines, the sparkling cork with natural cork discs better preserved the fruity and sweety aromas after 42 months of bottle aging, due to the presence of higher amounts of ethyl esters. In addition, the presence of camphor in sparkling wines sealed with a sparkling stopper with natural cork discs seems to be a good marker to discriminate this type of closure versus microagglomerated corks. In general, this work emphasizes the importance of the choice of cork closure for the preservation of the aromatic characteristics of sparkling wines, increasing their shelf life.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/foods11030293/s1, Table S1: Concentrations of other volatile organic compounds determined in sparkling wine #1, during bottle aging (12 to 42 months), for which no significant change was observed according to the type of closure.; Table S2: Concentrations of other volatile organic compounds determined in sparkling wine #2, during bottle aging (12 to 42 months), for which no significant change was observed according to the type of closure.

**Author Contributions:** Conceptualization, P.G.d.P. and J.P.; Methodology, P.G.d.P. and J.P.; Software, J.P.; Validation, F.A. and J.A.; Formal analysis, F.A., J.A., A.S.O. and I.F.; Investigation, F.A., J.A., A.S.O. and I.F.; Resources, P.G.d.P. and M.d.L.B.; Data curation, F.A. and J.A.; Writing—original draft preparation, F.A.; Writing—review and editing, J.A., A.S.O., I.F., M.d.L.B., P.G.d.P. and J.P.; Visualization, J.A., A.S.O., I.F., M.d.L.B., P.G.d.P. and J.P.; Supervision, M.d.L.B., P.G.d.P. and J.P.; Project administration, P.G.d.P. and J.P.; Funding acquisition, M.d.L.B. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by national funds from FCT-*Fundação para a Ciência e a Tecnologia*, I.P., in the scope of the project UIDP/04378/2020 and UIDB/04378/2020 of the Research Unit on Applied Molecular Biosciences—UCIBIO, and the project LA/P/0140/2020 of the Associate Laboratory Institute for Health and Bioeconomy—i4HB.

**Data Availability Statement:** Data available upon request.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

#### **References**

