Active food packaging: safe, biodegradable, bactericidal

• Scientists utilized the bactericidal properties of eugenol and phenazine subjected to co-crystallization.
• Nine variants of polylactide foams were also studied using supercritical fluid technology.
• The foams were tested for salmonella and listeria.
• The tested materials completely inhibited bacterial deposition.
• Many active substances are used in modern packaging.

 – One of the serious problems faced by the food industry is food spoilage and contamination with pathogens. Synthetic preservatives, pasteurization, or heat treatment are not the only solutions. Alternatives can be active materials that not only serve to package food but also protect it from harmful microorganisms – says Prof. Dušan Mišić from the Department of Development of Functional Food Products at UPWr. Along with his team, he published an article in the prestigious "Chemical Engineering Journal" titled "Supercritical fluid and co-crystallization technologies for designing nanocomposite packaging foams with antibacterial properties saturated with eugenol co-crystals with extended-release". The topic of active packaging was addressed jointly by scientists from the University of Santiago in Chile, the Wrocław University of Environmental and Life Sciences, and the Wrocław University of Science and Technology.

Eugenol and phenazine, a perfect duo

Aleksandra Rajewska retrieves string bags with small pieces of light plastic from the laboratory refrigerator. They are the size of a fingernail, looking unassuming, yet capable of much. They somewhat resemble Styrofoam and surprisingly emit a scent, evoking the atmosphere of Christmas. They flew to Wrocław from Chile, from the laboratory of chemist Dr. Adrián Rojas, who is the originator and leader of the research published in the "Chemical Engineering Journal." At the University of Santiago, he primarily deals with the application of supercritical fluids in experimental nanomaterials for food packaging. Meanwhile, microbiologist Dr. Dušan Mišić, a professor at the Department of Development of Functional Food Products at UPWr, studies the antibacterial action of experimental materials and natural molecules. Both scientists have been collaborating for over two years, seeking solutions not only in the world of synthetic chemistry but also delving into natural resources.

This time they utilized eugenol, which is a basic component of clove oil, also found in cinnamon—hence the aromatic Christmas association. It not only adds flavor to gingerbread but also destroys bacteria and fungi, hence its use in medicine. Dentists have long used its antiseptic properties. Dr. Rojas saturated biodegradable polylactide (PLA) foams with eugenol but subjected it to co-crystallization. This process involves combining molecules of different active substances into a new molecule with altered, more desirable physicochemical properties. This time, eugenol was combined with phenazine, an organic compound with antibacterial properties, and these co-crystals were impregnated into the foams. Nanoclay Cloisite was also included in them, allowing the eugenol-phenazine co-crystals to release slowly over time.

– Essential oils are highly volatile compounds. The challenge was to design foams with extended release of eugenol. So that the active ingredients would work during food packaging, not before or after – says Aleksandra Rajewska from the Doctoral School of UPWr, a food technologist and co-author of the article.

Supercritical carbon dioxide

In year-and-a-half-long studies initiated in the autumn of 2022, nine foam variants were tested, differing in concentrations of ingredients and structure. In addition to co-crystallization, supercritical fluid technology was also utilized. A fluid in a supercritical state is formed when temperature and pressure exceed the so-called critical values, above which the substance changes its physical state. Supercritical fluids have high penetrability, making them excellent solvents. They are used in the pharmaceutical, chemical, textile, wood, material engineering industries, and for extracting biologically active components. In the food industry, they are used for extracting natural dyes (e.g., capsanthin or capsorubin from peppers), which are used in soups and juices.

Capsaicin, the hot component of chili peppers, is also used as an insect repellent, which does not exhibit toxic properties. The most commonly used fluid in a supercritical state is supercritical carbon dioxide (scCO2)—nontoxic, readily available, and environmentally friendly because no waste or effluents remain after its use. It is used as a safe solvent for the active substance to impregnate solid bodies. It is also a foaming agent, so it is used for foaming polymers, i.e., creating foams. The authors of the publication used scCO2 to produce foams and embed eugenol-phenazine co-crystals within their pores. For comparison, foams were also saturated with pure eugenol. Then, the compounds impregnated in the foams were examined, among other things, through X-ray analysis and electron microscopy, to determine the rate at which they were released.

The foams were designed at the University of Santiago. They were also examined by Prof. Irena Zizovic from the Faculty of Chemistry at Wrocław University of Technology, an expert in supercritical extraction and experimental active materials. She verified whether the structure of the eugenol-phenazine co-crystal changed under high pressure and scCO2. In the final phase of the project at the University of Environmental and Life Sciences, the antibacterial properties of all nine foam types were tested against two bacteria: Listeria monocytogenes and Salmonella enteritidis.

Salmonella and listeria under scrutiny

– The research was very time-consuming, so we narrowed it down to these two microorganisms, which are exceptionally widespread and dangerous – says Prof. Mišić.

Listeria monocytogenes is one of the most virulent foodborne pathogens, transmitted through meat, cheese, butter, raw vegetables, and fruits. Leafy green vegetables and melons are particularly risky. "It can be present in raw milk, but also pasteurized milk if the pasteurization process was not properly carried out," notes the professor. "It is commonly found in soil and animal organisms. It is resistant to high temperatures, so hygiene and thorough washing of vegetables and fruits are important," he adds.

Salmonella enteritidis is not as widespread as Listeria but is the most common cause of bacterial food poisoning. According to Sanepid, in 2023, there were 250 cases of listeriosis and 10,000 cases of salmonellosis registered.

Salmonella can even be found in peanut butter, tea, chocolate, or chips. It thrives in the excreta of human and animal carriers; infection occurs through the consumption of contaminated fruits, and vegetables, especially undercooked meat, and raw eggs.

– The World Health Organization estimates that one in 12-15 thousand egg yolks is certainly contaminated with salmonella and has banned the use of raw eggs as a source of vitamins for children. Yet, 35 years ago, beaten egg yolks with sugar were the best source of vitamins for children and adults – says the microbiologist.

Patience pays off

– First, we checked whether both bacteria could attach to the foams – says Aleksandra Rajewska. Pathogens often form a biofilm, a type of coating, a biological membrane that protects them from external factors and is difficult to remove. This is a problem not only for the food industry but also in medicine and wherever harmful bacteria reside. When they settle, for example, on implants or drains, they can cause hospital-acquired infections. Due to its compact and complex structure, the biofilm is more resistant to antibiotics or other bactericidal agents.

– The biofilm poses a serious microbiological threat on the surface of animal and human organisms, on contaminated meat, cheese, vegetables, and fruits, as well as on materials such as glass, plastic, metal, rubber, ceramics, wood – emphasizes Aleksandra. Therefore, packaging material should be anti-adhesive, i.e., prevent pathogens from adhering. It's not easy because each bacterium has a different mechanism of attaching to the substrate.

Foams tested for this purpose were immersed in a bacterial broth for 24 and 48 hours.

– These different time variants are a novelty because in similar studies, usually only the effects after a day are checked – adds Prof. Mišić. Initially, the results seemed unpromising: after the first day, bacteria effectively attached to the surface of some materials. However, after 48 hours, the material was "activated," and the attached bacteria fell off. In the next stage of Wrocław research, it was examined whether foams immersed in a bacterial broth released beneficial compounds with which they were saturated, i.e., eugenol-phenazine co-crystals. For 96 hours, the number of bacteria was determined using a spectrophotometer until the compounds were completely released.

– The results surprised us because every day the foams worked differently. Their properties changed: under the influence of increasing moisture from the liquid broth, they began to activate more and were more effective. We didn't expect them to be active for so long – says Prof. Mišić – Patience and curiosity, however, paid off.

Thanks to the addition of nanoclay and co-crystals, the foams completely inhibited the deposition of Listeria and Salmonella and provided extended antibacterial activity against both pathogens. Therefore, they can work perfectly in creating active food packaging, innovative, and environmentally friendly. The results are so interesting that UPWr scientists want to continue them, including using an electron microscope.

–We plan to check how other active substances, such as usnic acid from the common beard lichen, a lichen with antibacterial and antifungal activity, affect food pathogens – says Aleksandra Rajewska, who, along with Prof. Mišić, in previous work, examined the effect of some natural molecules under different temperature conditions on pathogens such as Listeria monocytogenes, Campylobacter, Bacillus cereus, Staphylococcus aureus.

Packaging for special tasks

The Polish-Chilean team, through their research, has joined the increasingly present trend in global science: designing packaging materials that perform more tasks than simple storage. They are not only used for transportation, advertising (as information carriers), and isolation from harmful external factors—temperature, light, water, dust, and pollutants. The perfect packaging is safe, resistant to fats, and serves as a barrier to aromas. It is designed from recyclable materials, making it biodegradable or compostable, thereby reducing the carbon and water footprint. It is a way to extend the shelf life of food products without the use of synthetic preservatives.

As a graduate student, Aleksandra Rajewska participated in classes on innovative solutions used in food packaging at Miguel Hernandez University of Elche (UMH), in the food technology field.

– We used, among others, modified atmosphere packaging (MAP), which involves changing the composition of the atmosphere inside the packaging, as well as edible coatings based on aloe and alginate, which were applied to fruits and vegetables. Then we tested them organoleptically, microbiologically, or physicochemically – she says.

Modern packaging can be divided into intelligent and active. The former, still rare in our stores, monitors the state of stored food through temperature, time, humidity, and gas content indicators. Discoloring labels provide information about conditions inside the packaging. Thus, there is no need to open the box or bottle to find out whether the product was transported and stored in proper conditions, whether bacteria have multiplied in it, or what the content of oxygen or carbon dioxide in it due to packaging leakage.

On the other hand, active packaging acts on the product in two ways: either absorbing certain components or releasing them. They may contain moisture-absorbing sachets, oxygen (e.g., to prevent oxidation of vitamins and fat rancidity), and ethylene (to slow down the ripening of fruits and vegetables). Active substances, such as essential oils like eugenol or thymol, with strong antimicrobial and antioxidant properties, are impregnated through packaging.

Other active substances of plant origin are also used, e.g., resveratrol from grapes, gallic acid from tea, and coumarin from cinnamon, which inhibit the growth of pathogenic microorganisms.

This reduces the risk of illness due to contaminated or spoiled food and allows maintaining the highest quality of food products for as long as possible and extending their shelf life.

– This can also help reduce food waste, which is common. It is estimated that we waste one-third of the world's production. Discarding food also means wasting money, labor, energy, and water. We hope that our projects will also contribute to reducing this problem – say the scientists.

Aneta Augustyn