Concentration of phenolic compounds in the OEO and TEO.
The development of active food packaging is addressed using polyolefins such as LDPE and PVOH, as well as biopolymers from flour (sorghum and corn) and by-products of the food industry. Bacteriocins (nisin, natamycin), plant extracts such as oregano and thyme, as well as native plants of the northeast region of Mexico (Larrea tridentata, Schinus molle, Cordia boissieri, Leucophyllum frutescens), and essential oils of oregano and thyme as antimicrobial agents have been studied. The effect exerted by the process of incorporation of the antimicrobial agent (casting, extrusion) on the barrier and mechanical properties of the package as well as the antimicrobial activity of the containers (broad spectrum or selective activity) has been observed and the establishment of methods for their traceability.
- Larrea tridentata
- Schinus molle
- Cordia boissieri
- Leucophyllum frutescens
- Listeria monocytogenes
- Staphylococcus aureus
Since the last decade and a half (2000 to date), the main forces that have unleashed the greatest developments in the packaging of food are the great concern of society for the care of their integral health including its nutritional status through foods with less or no presence of additives but in convenient presentations that facilitate their preparation, heating, and intake as well as foods with therapeutic action. A consumer who is very concerned about the safety of food, where food packaging and storage systems do not represent or have physical, biological, or even toxicological risks, nor for the protection of the environment.
All of the previous demand constantly forces the change on the nature of the food packaging and consequently on the materials of which it is composed . Therefore, new materials are being developed to comply with the above. First, packages that contain in their formulation substances that migrate from the container to the food exert a positive action avoiding deterioration reactions likewise increase the sensory quality through the positive migration of substances or have a therapeutic effect. In this category are the so-called active packaging . Second, in relation to the protection of the environment: the development of biodegradable packaging using, for example, biomaterials obtained from agri-food sources .
An active packaging is defined as the one that produces a change in the state of the packaged food to prolong its shelf life, improve its safety and quality, and provide a barrier between the food and its environment . The mechanisms of action in active packages can be acting as emitting systems or as sequestering systems for substances. In the emitting systems, compounds or additives generally recognized as safe (GRAS), such as antioxidants and antimicrobial agents, are released into the food through the walls of the package. Sequestering systems remove undesirable compounds such as oxygen, H2O, ethylene, CO2, and impurities, among others . Great diversity of active packaging is being developed in order to control the emission or absorption of substances and thus modify the environment of the product or directly the product. Thus, active packaging has substances or systems that absorb oxygen, ethylene, CO2 and humidity; others absorb or release desired aromas [2, 3, 4]. Other active packages contain active enzyme systems and antimicrobial substances or systems. All these active containers seek the elimination of microbial growth, the extension of the useful life, and/or the increase of organoleptic qualities of the product .
The proposal of our line of research is based on obtaining a series of products (active antimicrobial and active biodegradable packages according to a defined food or conservation need), as well as the processes for their elaboration. Most developments use materials obtained only from starches or proteins. With our development, a single product has a biopolymer matrix that includes both biopolymers (starches and proteins) in a single stage. By starting from a matrix that includes both biopolymers (starches and proteins and sometimes antimicrobials or antioxidants), unit operations are eliminated, which reduces costs of equipment and energy, consequently operating costs. The technological impact of the developments of the research line will be reflected in the conservation of food (fresh or dehydrated) through the use of antimicrobial/antioxidant active packaging that contribute to preserve the environment when they are discarded since they are potentially biodegradable.
From the scientific point of view, this solves a couple of problems at the same time, the first concerning the toxicological risk of the abuse of additives in the formulation and conservation of food and the second discarding the ecological and environmental problems generated by food packaging. Our developments will have, on the one hand, low environmental impact due to the development of biodegradable products from nature-friendly processes. On the other hand, they will have a high economic impact since currently in the country there are no companies dedicated to the development of biopolymer containers, creation of own technologies, and high added value to products of low commercial value.
2. Antimicrobial active packaging developed at the Tecnológico de Monterrey
2.1 Biopolymer active packaging
Our first works focused on the use of starches from several varieties of sorghum (high-production cereal in northeast of Mexico) whose different proportion in amylose and amylopectin plays an important role in the water vapor barrier of the containers reinforcing them with prolamines (kafirin and zein) to increase their impermeability  and use of antifungal agents such as the sorbates and benzoates of Na and K. The inclusion of broad-spectrum antimicrobial additives in plastic polymers and/or biopolymers through the proprietary technology generated at the Tecnológico de Monterrey, for example, enabled active packaging to be obtained on a laboratory scale that reduced biological risks by manual or semi-manual packaging (risks of contamination with pathogens such as
Subsequently, Ríos-Licea conducted a search of natural substances of broad spectrum, so he analyzed the antimicrobial activity of aqueous extracts of known plants. Ríos-Licea also succeeded in developing antimicrobial films by incorporating natural extracts of garlic and oregano into the same biopolymer matrix of sorghum flour using the method established by Schause . However, it was necessary to incorporate high concentrations of natural extracts, due to the low potency of the antimicrobial activity of the commercial product tested.
Tinoco-Pérez studied a variety of corn rich in anthocyanins (blue corn) by applying the process of dry milling and establishing the process to obtain active films in antioxidants (anthocyanins) from flour of this cereal . Two biopolymers present in corn with a filmogenic capacity are starch and zein, the first being the most abundant in this grain . There are a significant amount of reports published on films made from corn starch and zein; the effect of different additives, copolymers, and processes on the performance of films for different applications has been evaluated. In 2009, Mexico produced 29.4 million tons of corn using 38.5% of its total cultivated area. The production of this grain has shown an increase in its average annual growth rate of 2.1% in the period from 1994 to 2008. Of total corn production in 2008, 92% was white corn, 7% was blue corn, and 1% was of other varieties. Basically, white corn is destined for national consumption, yellow for export, and the rest of the varieties are commonly produced for self-consumption of rural populations. Among the 1% of the varieties not defined is the blue corn (
Among the processes studied to obtain films from corn fractions are casting, different types of extrusion (double screw/flat die, single screw/flat die, and extrusion/calendering, among others), stretching of zein resins, and pressing by heat [10, 20, 21, 22]. The effects of various additives and chemical treatments, for example, plasticizers, hydrophobic agents, copolymers, and the use of chemically modified starches on the structural, molecular, thermal, mechanical, and barrier performance characteristics, have been studied extensively [22, 23, 24, 25].
In the case of sorghum, the cultivation of this cereal is less demanding in agronomic terms than corn (water and nutrients) [26, 27]. Sorghum is the fifth most important grain in the world, being the United States the country with the highest production in the world, followed by India and Nigeria. For the year 2010, Mexico contributed with 10.5% of the total world production, equivalent to 6,250,000 metric tons [15, 16]. In Mexico, sorghum is the second most important grain in production after corn; during the period 1996–2006, sorghum production contributed with 22% of the total production of cereals .
In Mexico, this cereal is destined mainly for livestock feed and secondarily for human food and obtaining inputs such as starch, alcohol, glucose, acetone, and butanol. One of the great advantages of sorghum is that it has the capacity to adapt to arid and semiarid climatic conditions and to be resistant to drought for long periods . In previous works, it was able to demonstrate that antimicrobial active films can be obtained from corn and sorghum flour [8, 15, 16].
The biopolymers obtained in this way through a technique and process patented by Tecnológico de Monterrey as PCT  have the advantage of being biodegradable because their chemical structure is primarily based on proteins and starches. Additionally, they have the possibility of forming films with plasticity (custom flexibility) and of being formulated also tailored to the requirements of the product to be packaged. Additionally, they can be heat sealed to form bags of different dimensions or not to be sealed and act as “active” pads or pads in combination with other packaging. In addition to the advantages in terms of sustainability, the interest in using these sources to produce biopolymers lies in adding value to agricultural products [8, 29].
It is important to note that for any application of the said technology, it will be necessary to make an adaptation of the formulations and the process to satisfy the specific protection requirements for each food to be packaged. For what it is proposed to demonstrate in this work, the film-like packages obtained by adapting the formulations and process of the said published patent work to preserve and keep refrigerated for 30–45 days a commercial presentation in slices of semi-matured cheese .
The biopolymeric antimicrobial films described in WO2010/024657 A1 from cereals are limited to the packaging of dry foods or as pads for adsorption of exudates and emission of antimicrobial agents for fresh meat and cheese products . Because of its sensitivity to water and low mechanical resistance to contain products with intermediate moisture, the biopolymeric matrix was reformulated to improve both parameters . The results of refrigerated shelf stability of the cheese in terms of the control capacity exercised by the antimicrobials used in this study (nisin and natamycin) through the active packaging against fungi and yeast were effective throughout storage compared to vacuum packaging (control). The results of the microbial kinetics throughout the refrigerated storage for the fungi and yeast count showed the effectiveness of the active packaging. The development of fungi and yeasts remained controlled, showing the effectiveness of this emerging food preservation technology .
The plasticizing effects of two different polyols (glycerol and sorbitol) on the mechanical, thermal, and microstructural properties of flour films were studied by Valderrama and Rojas, and the results showed that films plasticized with sorbitol had better mechanical properties and less affinity for water than those plasticized with glycerol. The attenuated total reflectance-Fourier-transform infrared (ATR-FTIR) spectra of blue corn flour plasticizer with sorbitol showed the presence of the additional band at 1745 cm−1 characteristic of the carbonyl peak, which confirms the chemical linkages between sorbitol and a polymeric matrix. The effect of the plasticizer on the glass transition temperature (Tg) showed that Tg decreased as the plasticizer content increased. Plasticized glycerol films showed lower Tg values than those with sorbitol. Observations by scanning electron microscopy (SEM) showed that it was necessary to add plasticizer to maintain film integrity. The sorbitol-plasticized flour films revealed better adhesion between phases, and these films showed a compact structure .
Finally, bioplastics were produced through thermoplastic processing using different cereals derived raw materials, namely, blue maize flour (BM), white sorghum flour (WS), maize starch, and the maize prolamin (zein). The overall performance of the bioplastics was investigated emphasizing on the study of the effect of different process strategies on the compatibilization of the starch and prolamin using mixtures of urea and formamide (UF) and maleated starch (MS) as compatibilizing agents [32, 33]. Results suggest that two competing phenomena, thermoplasticization and degradation, occurred simultaneously during the thermoplastic process. Fourier-transform infrared (FTIR) spectroscopy analysis evidenced the chemical changes induced by these phenomena. Moreover, chemical modification had also a major effect on the properties of the produced materials. WS films made with chemically modified flour increased their tensile strength in 29%, as compared to their native counterparts. Thermogravimetric analysis and FTIR analysis showed that the chemical interaction between starch and zein occurred more extensively in films made with formamide than those made with maleated starch [32, 33].
2.2 Plastic active packaging
In Valderrama’s work, natural aqueous extracts are exchanged for essential oils because they have a higher concentration of antimicrobial active substances. It analyzed essential oils of oregano, thyme, tea tree, and mint, which have greater antimicrobial activity than the natural extracts used by Ríos-Licea . In particular, the effect of incorporating two essential oils such as oregano (
The mechanical, barrier, and antimicrobial properties of the packaging were evaluated against
A simple and rapid Fourier-transform infrared (FTIR) spectroscopy method was developed by Valderrama and Rojas to determine the main essential oil components (carvacrol, thymol, and p-cymene) in the antimicrobial LDPE films incorporated with oregano (
The work of Rocha is described below, who worked with the same essential oils of oregano and thyme that Valderrama used to obtain active plastic containers. This was due to the fact that they presented greater antimicrobial activity than the aqueous extracts of oregano and garlic from previous studies in our research group [9, 15]. It also proposed the use of a polymeric film for the preparation of the active container with essential oils, in order to present an alternative to vacuum cheese packaging. For this project, polyvinyl alcohol (PVOH) has been chosen for the preparation of the packaging due to its unique characteristics: permeability, biodegradability, and its facility to form films by the casting method. The purpose of this work is to propose an alternative, a packaging that is not dependent on complex plastic structures that requires vacuum packaging for provide the high barrier. The main challenge of the present project is the incorporation of essential oils that are lipophilic to a hydrophilic PVOH matrix, which is why it was suggested encapsulating them in cyclodextrins.
3. Control of the development of
Listeria monocytogenesin fresh cheese during shelf life at refrigeration by means of an antimicrobial PVOH film (pad) with microcapsules of active compounds of oregano and thyme
The presence of
3.1.1 Selection of substances, materials, microorganisms, and cheese
The selected essential oils were oregano (OEO) and thyme (TEO) (Primavera Life) for their potent antimicrobial activity and their availability in the national market. The antimicrobial activity of these inclusion complexes such as films using
3.1.2 Process for developing PVOH films with active microcapsules of essential oils
The PVOH films were elaborated adapting the method used by Schause  and Ríos-Licea , previously studying three methods of incorporation of the essential oils in it: dispersion, emulsification, and formation of inclusion complexes with α and β cyclodextrins (CD) and the following variables: amount of PVOH, type and amount of cyclodextrin, coprecipitation strategy, solvent (water, ethanol), and concentration of EO. The encapsulation with β-CD is being the one selected for the incorporation of EO in the PVOH film. A PVOH film without inclusion complex was made as a control. With the resulting films, pads of 8 × 4 cm were made to be used in the active packaging system.
3.1.3 Thermal stability of essential oils and confirmation of inclusion complex formation by differential thermal analysis (DTA)
In order to determine the degradation temperature of the essential oils and establish if these would be affected during the film making process, the thermal stability of the same and their active compounds were evaluated. Firstly, a thermal evaluation of the essential oils of oregano and thyme was carried out, as well as its main active components with carvacrol, thymol, and p-cymene standards. Next, the β-CD and the inclusion complexes of OEO and TEO to confirm the formation of such complexes and not only a physical mixture. Finally, a thermal evaluation of the PVOH films with the inclusion complexes of CD:EO of oregano and thyme was made to determine the optimal storage temperature of the active films. The thermal evaluation was performed with a home DTA validated by Martínez and collaborators . For each substance, at least two runs of food matrix were performed to verify the repeatability of the analysis.
3.1.4 Microbiological evaluation and disk diffusion method
To determine the antimicrobial activity of PVOH films, the disk diffusion method was applied (Kirby-Bauer method). After preparing and inoculating the agar with 106 CFU of each microorganism, samples of the films were cut in the form of 6 mm diameter disks and deposited on the agar, evaluating both the rough and smooth side of the films [7, 38, 39]. After 24 hours of incubation at 37 ± 1°C in inverted position, the inhibition halo was measured with a digital micrometer (Mitutoyo Digimatic 2,931,051 m, 0.001 mm sensitivity).
3.1.5 Control study of
L. monocytogenesin fresh goat cheese using an active packaging system
The packaging system consisted of a pad of PVOH with EO microcapsules of oregano and thyme in a LDPE bag. First, 7 × 7 cm bags with LDPE film of 0.023 ± 0.003 mm thickness obtained by extrusion by Valderrama  were made, which were obtained by sealing two films on three sides with a vacuum packing machine Torrey brand. In the same way, bags were obtained with the multilayer film (Zublon® 5CR from Zubex Industrial). Second, in aseptic conditions, portions of cheese of 3 cm × 3 cm and 10 ± 0.5 g of weight were cut and exposed to UV treatment for 15 min on each side, a methodology adapted from Suppakul  for the purpose to reduce the interference of microorganisms typical of cheese in the study. Then, the samples were packed in the bags of the four treatments to be analyzed and inoculated with 100 mL of
The four treatments evaluated were (1) multilayer bag for vacuum packaging as control, (2) LDPE bag with PVOH “pad” without essential oils, (3) LDPE bag with PVOH “pad” with inclusion complex of β-CD:OEO, essential oil at a concentration of 25% in the film, and (4) LDPE bag with PVOH “pad” with inclusion complex of β-CD:TEO, essential oil at a concentration of 25% in the film.
The inhibition kinetics of
3.2.1 PVOH films process with microcapsules of essential oils of oregano and thyme
It was possible to produce PVOH films with the inclusion complexes of oregano and thyme in all the experimental conditions. Films with 1, 4, and 15% EO were prepared with molar ratio 1:10, which they are shown in Figure 1. Films made with 1% EO were those most similar to the PVOH control film. Continuous, elastic, and transparent films were obtained. The higher the concentration of the essential oil in the film, the more presence of the inclusion complex affects the transparency of the film, with the PVOH matrix being observed as white, as can be seen in the film at 15% EO (Figure 1). It should be noted, however, that although the inclusion complex is observed in the film, no migration of this or the essential oil to the touch is perceived, which is why it has been well incorporated into the PVOH matrix. The films also presented less transparency when approaching the β-CD:EO ratio at 1:1 molar proportions; this is because a greater amount of inclusion complex tends to saturate the film. The films whose inclusion complex was dissolved in 30% ethanol also showed greater transparency than those in which it was prepared in water; this is because the inclusion complex in the 30% ethanol solution was better solubilized.
3.2.2 Thermal stability of essential oils and confirmation of inclusion complex formation by DTA
Firstly, the thermal stability of β-cyclodextrin and the essential oils of oregano and thyme, as well as its components (carvacrol, thymol, and p-cymene), was evaluated to confirm the formation of inclusion complexes. The thermograms of the carvacrol, thymol, and p-cymene standards are shown in Figure 2 and 3. Here it is shown that these three components are stable up to a temperature of 182°C, which refers to the boiling temperature of the p-cymene. The melting point of thymol shown in Figure 2 is in agreement with that obtained by Ponce, which reports the melting point of thymol at 50°C . The boiling point of p-cymene matches with the one reported by the supplier (178–180°C Sigma-Aldrich). Carvacrol was analyzed by broadening the study temperatures, as shown in Figure 3. This compound has an interesting behavior, since it has a crystallization temperature of −20°C followed by a melting point of 2°C and a point of boiling of 240° C. Sigma-Aldrich reports its melting point at 3–4°C and its boiling point at 236–237°C, which also coincides with that reported by Dahmane, which reports the boiling point of carvacrol at 237.7°C . The closeness of the crystallization and fusion transitions does not allow the existence of a solid state of this intermediate substance at the reported temperatures. A similar behavior is reported by Ponce for cinnamaldehyde . According to the obtained in Figure 5, it was successful to form an inclusion complex, since otherwise the signal of the boiling point would have been shown at 214°C of the essential oils (Figure 4).
The thermogram obtained by DTA of the β-CD:EO inclusion complexes of oregano and thyme (Figure 5) shows that they are stable at temperatures below 115°C, so there is no risk of degradation of the active compounds if the inclusion complex dissolves PVOH in situ in a solution with inclusion complex at 8°C (process B of PVOH film making with inclusion complexes). Figure 6 shows the thermogram of the PVOH films with the inclusion complexes of oregano and thyme. The film made with essential oil of thyme had a little moisture on its surface, so we can see a couple of peaks at 0 and 100°C corresponding to the melting and boiling point of water, respectively. PVOH control films and those with inclusion complexes of essential oil of oregano and thyme are stable up to a temperature of 110°C, which indicates that they can be stored in shelves at room temperature without problem. The PVOH control film has a peak at 150°C, which refers to the point of fusion of unplasticized PVOH. The last peak corresponds to the degradation of PVOH at 230°C, which is supported by Holland and Hay .
3.2.3 Antimicrobial activity of the active films in vitro against L.
monocytogenes, S. typhimurium, and E. coliO157: H7
The films made with a concentration of 25% essential oil of oregano and thyme presented broad-spectrum antimicrobial activity by inhibiting the growth against the Gram-positive and Gram-negative microorganisms evaluated. The antimicrobial activity and the inhibition halo against
The antimicrobial activity of the films is mainly due to the phenol group of carvacrol and p-cymene. The concentration of these compounds in the essential oils of oregano and thyme used for the production of films is shown in Table 1. The phenol group is essential for bacterial inhibition, since it destabilizes the cytoplasmic membrane and also functions as a proton exchanger which reduces the pH gradient in the membrane and causes cell collapse and death [46, 47, 48]. The destabilization of the membrane occurs because carvacrol and thymol have affinity for lipids and accumulates in the bilayer between fatty acid chains, which causes changes in the conformation of the membrane. This mechanism of action does not present p-cymene; however, it has been found to have a synergy with phenols, expanding the membrane and destabilizing it . The position of the hydroxyl group in the phenolic compounds does not seem to influence the degree of antimicrobial activity so that the activity of carvacrol and thymol is similar.
3.2.4 Analysis of the control of
L. monocytogenesin fresh cheese packed during storage in a refrigerated rack
The antimicrobial activity of the PVOH films with inclusion complexes of oregano and thyme in a model with fresh cheese inoculated with
Active PVOH films were obtained with essential oils of oregano and thyme, which showed broad-spectrum antibacterial activity by inhibiting pathogenic Gram-positive and Gram-negative bacteria specifically against
4. Antimicrobial agents from plants of the northeast of Mexico
As Mexico is a country that stands out for its floristic richness and taking into account the extensive knowledge of medicinal plants that since the pre-Columbian Era conserve Mexicans, mainly those of rural communities, it was natural that we were interested in the study of incorporation of some of them as active substances to be included in polymer matrices for food packaging.
Larrea tridentataplant as an alternative source for obtaining antimicrobial extracts
The flora of arid zones represents a great potential for the wealth based on its biological specialization, since it is the product of thousands of years of physiological adaptation for its survival . The governor plant (
The governor plant has a wide range of adaptation in elevation since it is located in the Valley of Death in California located 86 m below sea level, to more than 2500 m in the sierras of northern Mexico. Its growth is good in dry plains and plateaus, also around hills and slopes, and in several types of soils except clayey, saline, or granitic . The lifetime of this plant is negatively correlated with disturbance and soil compaction, being intolerant to soils with high phosphorus content . In Mexico, the distribution of the governor plant is in part of the Sonoran Desert, which includes the states of Baja California, Baja California Sur and Sonora, and in the Chihuahuan Desert, which includes the states of Chihuahua, Durango, Coahuila, Nuevo León, Zacatecas, and San Luis Potosí . The infusion of the whole
Among the uses that the governor plant has traditionally had, it stands out in its use as an antioxidant that was given to it in the United States since 1943; although in the decade of the 1990s, it was suspended by the US Food and Drug Administration (FDA) due to the strong interaction of nordihydroguaiaretic acid (NDGA) comment on results that were not found in the extracts studied, with several enzymatic processes. NDGA inhibits enzymatic activity, in addition to inhibiting the signaling pathway of lipoxygenase in which arachidonic acid generates leukotrienes and other oxygenated products [53, 54, 55].
Cordia boissieriplant as an alternative source for obtaining antimicrobial extracts
The anacahuita plant (
Leucophyllum frutescensplant as an alternative source for obtaining antimicrobial extracts
The ash plant (
Schinus molleplant as an alternative source for obtaining antimicrobial extracts
The plant pirul (
4.5 Inhibition of
Staphylococcus aureuswith extracts of anacahuita ( Cordia boissieri), governor ( Larrea tridentata), ash ( Leucophyllum frutescens), and pirul ( Schinus molle) with potential application in active packaging
The objective of the present investigation was to evaluate if the alcoholic extracts of the plants of anacahuita (
4.5.2 Materials and methods
According to the obtained results in Table 2, we can observe that all alcoholic extracts of the plants
Of the extracts of
Table 3 shows the minimum inhibitory concentration of the extract with the highest inhibition of each of the plants studied. The extract of the plant
From GC/MS of
For the four plants evaluated, there is greater inhibition in the extracts formulated with the harvested plants than with the purchased plants, since in the purchased plants, the storage time and the management that has been given are not known. In the extracts of the plants
The presence of 9, 12-octadecanoic acid and 3, 4 ‘, 5, 6, 7-pentahydroxyflavone was identified in the ethanolic extract of
In a previous work, Sáenz-Collins demonstrated that it was possible to obtain active antimicrobial PVOH biofilms against
In the present work, it was demonstrated that the alcoholic extracts (ethanolic and methanolic) of the plants