High relative humidity (RH) in-package of fresh-cut fruits or vegetables, which usually causes microbial spoilage, can be used as an advantageous trigger to deliver hydrophobic antimicrobial compounds from package systems using cyclodextrins (CDs) as a matrix.
Premise I. The rich source of nutrients and high relative humidity are the causes of major microbiological problems in fresh-cut produce
Fresh-cut produce is one of the fastest growing convenience food industries in history. Fresh-cut fruits and vegetables offer a number of advantages over whole produce, including cost control, waste reduction, variety selection, consistent quality and safety, and less in-store labor (Luo 2007). However, the fresh-cut industry and the evolving processes used to sustain freshness face considerable challenges.
The main problem that makes fresh-cut fruit a highly perishable product is the microbial growth caused by the accumulation of leaked juice rich in nutrients in the bottom of containers (Brecht 2006). Operations such as peeling, cutting, or slicing greatly increase tissue damage of fresh-cut fruits, causing the release of intracellular liquids, and consequently increase microbial growth, which is the principal deteriorative and safety problem involved in the fresh-cut industry (Brecht 2006). Additionally, as a response/consequence to wounding during processing and microbial growth during storage, there is an increase in off-flavor compounds, loss of firmness and respiration, reduced fresh-cut shelf life and leads to senescence processes (Artés and others 2007). Therefore, water loss is one of the major deleterious factors that affect fresh-cut produce quality, which in turn influences firmness and enhances microbial growth.
Whole and fresh-cut produce are unique among the food products; they remain metabolically active and their shelf life and storage stability are shortened as consequences of these processes. Whole fruit, contrary to fresh-cut, are covered by specialized skin tissues, which serve as barrier against insect and pathogen attack, avoiding excessive water loss. Therefore whole fruit had less water losses and gas exchange due the to natural barrier that offers the cuticle, which can be affected by storage conditions. If the epidermis or periderm is damaged or removed, which is the case with fresh-cut products, water loss can be massively increased. For this reason, fresh-cut produce must be protected by a packaged system in order to decrease water loss; however, water released from fruit can accumulate inside the package and create a high RH atmosphere.
The rate of water loss is dependent primarily on the external vapor pressure deficit; however, other factors may affect it. Products with a large surface:volume ratio such as leaf vegetables are prone to lose greater percentages of water faster than large spherical fruits. The percentage of water that can be lost without compromising edible quality varies depending on each product but, in general, fruit appearance deteriorates steadily with increasing water loss.
Film packaging reduces water loss of fruit and vegetables. Scott and others (1982) found that fruit kept in unperforated polyethylene bags at 20 °C for 10 d lost less than 2% of their fresh weight, while control (unpacked) fruit lost between 18% and 30%. Edible surface coatings are another possibility. Zhang and Quantick (1997) found that an edible coating of chitosan and L-glutamic acid on litchi fruit reduced water loss at 4 °C by about 20% and significantly slowed browning when compared with untreated fruit. However, this technology has not been adopted commercially yet. Jiang and Fu (1999) observed that litchi fruit stored at 90% RH under a controlled atmosphere of 3% to 5% O2 and 3% to 5% CO2 for 30 d at 1 °C avoided browning and kept fruit quality, while browning of fruit stored at 60% and 70% RHs significantly increased, while pericarp desiccation occurred. In-package RH can be reduced by using films with a high water vapor transmission rate but it is difficult to control to a specific level using only this approach. Since in-package RH is affected by out-package RH in the surrounding environment, variations can be observed during handling and storage in function of the temperature used (Deell and others 2006).
Water activity (aw) is directly related to equilibrium RH of a food, and describes the degree to which water is “bound” in the system, controlling its availability to act as a solvent and participate in chemical/biochemical reactions and growth of microorganisms. The complete concept of aw is the ratio of the vapor pressure of water in equilibrium with a food to the saturation vapor pressure of water at the same temperature. The aw is equal to the equilibrium RH, expressed as a fraction. This important property can be used to predict the stability and safety of food with respect to microbial growth, rates of deteriorative reactions, and chemical/physical properties. The aw principle has been incorporated by various regulatory agencies (FDA CFR Title 21) in definitions of safety regulations regarding the growth and proliferation of undesirable microorganisms, standards of several preserved foods, and packaging requirements.
Food biological safety is still the top priority of the fresh-cut fruit industry. Fresh-cut produce are normally eaten raw, and for this reason they have to be considered potentially hazardous for consumers. Most of the microorganisms affecting shelf life of fresh-cut produce need an optimal environment with an RH higher than 80% for their growth (Frazier and Westhoff 1993). A reduction of humidity to levels below to the critical equilibrium RH for microorganisms could be an effective approach to limit their growth. The limiting factor of most fresh-cut fruit and vegetables shelf life is fungi and bacteria growth, which is associated with high RH within packages (Ben-Yehoshua and others 1998). Fresh-cut produce normally reach rapidly an equilibrium RH close to saturation (100%) (Figure 1). RH in a plastic package of fresh commodities is usually high (> 95%); and fluctuations in the storage temperature may result in condensation, which would greatly increase the proliferation and spread of spoilage microorganisms (Grierson and Wardowski 1978).
Fungi are the most important microorganisms causing postharvest wastage of fresh produce (Eckert and Ogawa 1988). This is particularly true for fruits, where the relatively acid conditions tend to suppress bacterial growth (Frazier and Westhoff 1993). However, in vegetables, bacterial infections are more common, due to their high pH. Nevertheless, bacteria are generally the fastest growing microorganisms; and under favorable conditions bacteria usually outgrow fungi (Frazier and Westhoff 1993). Bacteria are also important as agents of both spoilage and foodborne diseases.
Attempts to control bacterial proliferation through decreasing the relative humidity within the package of fresh produce have been reported (Shirazi and Cameron 1992; Anantheswaran and others 1996; Ben-Yehoshua and others 1998; Deell and others 2006), but change in environmental conditions and wilting/shrinkage due to water loss from the produce make these systems unsuccessfully.
Shirazi and Cameron (1992) introduced the concept “modified humidity packaging.” They also studied the feasibility of controlling RH in MAP of fresh tomatoes using several desiccants. Mold growth was suppressed by low RH; however, fruit stored under dry conditions showed higher water loss, affecting their visual quality. Sorbitol and sodium chloride were used to modify the in-package RH of fresh mushrooms stored in a modified atmosphere package (MAP) at 12 °C. Moisture absorbers used did not improve quality; however, a small amount of sorbitol helped to avoid condensation (Anantheswaran and others 1996). Bell peppers packaged in perforated film lost less weight and maintained higher quality than fruit stored in open carton boxes and, at the same time, had lower decay levels than peppers kept in nonperforated packages (Ben-Yehoshua and others 1998). Broccoli heads stored in MAP plus sorbitol maintained better appearance, firmness, and odor ratings after 29 d of storage at 0 °C compared to controls. Addition of sorbitol significantly reduced water loss (≤ 1.3%) of broccoli (Deell and others 2006).
Therefore, most of the above-explained mechanisms to control in-packaged RH could not be effective in fresh-cut fruits and vegetables, due to the high RH recommended levels for storage of fresh-cut produce (approximately 95%) (Sargent and others 2000). The reductions of RH represent a compromise to prevent excessive weight loss while providing some control of microbial spoilage.
In this context, we propose that alternative systems can be created to take advantage of the high RH in sealed packages of fresh-cut fruit to deliver antimicrobial compounds. These systems are the CDs, which are host-type molecules that can form molecular complexes. Using CDs delivering systems would be valuable for improving safety and quality of fresh-cut produce.
Premise II. CDs are host-type molecules that can form molecular complexes with several antimicrobial compounds (guest molecules)
CDs are nonreducing cyclic glucose oligosaccharides (Figure 2). There are 3 common CDs: α-, β-, and γ-CD, with 6, 7, or 8 D-glucopyranonsyl residues, respectively, linked by α-1,4 glycosidic bonds (Del Valle 2003). CDs present a bottomless bowl-shaped (truncated cone) molecule stiffened by hydrogen bonding between the 3-OH and 2-OH groups around the outer rim. Due to their α-(1-4)-glycosidic linkages, all primary hydroxyl groups (C-6) are orientated toward one of the edges of the truncate cone, while the secondary hydroxyl groups (C-2 and C-3) are placed on the other edge. Since hydrogens of carbons 3, 5, and 6 and the nonbonding electron pairs of the glycosidic oxygen bridges are oriented toward the inside of the cavity, it has a hydrophobic environment with a high electron density (Szejtli 1989). These double characteristics of CDs, (1) the existence of a hydrophobic cavity and (2) the presence of both hydrophilic hydroxyl rims, give them the property to form inclusion complexes in water with a variety of organic molecules. β-CD is the most accessible, the cheapest, and generally the most used in the pharmaceutical and food industries, since it has been approved by the FDA (Del Valle 2003) (Table 1).
|Units of glucose||6||7||8|
|Solubility H2O (g/100 mL)||14.5||1.85||23.2|
|Inner diameter (nm)||0.45||0.62||0.79|
|to 0.57||to 0.78||to 0.95|
|Outer diameter (nm)||1.37||1.53||1.69|
|Cavity volume (nm3)||0.174||0.262||0.472|
CDs possess a unique ability to act as molecular containers by entrapping guest molecules in their internal cavity (Figure 3). During this process, the less polar guest molecule replaces the energetically unfavored water molecules that occupy the CD cavity, as shown in Figure 3. This process is regulated by noncovalent interactions between host and guest: Van der Waals forces, hydrogen bonding, and electrostatic interactions. During formation of the inclusion complex, the guest molecule enters, totally or partially, inside of the slightly apolar cavity of the CD. The simplest, and most frequent complex formed has a 1:1 stoichiometry (CD:guest), in which the guest is totally or partially included inside of a single CD. This ability of a CD to form inclusion complexes with guest molecules is the function of 2 key factors. The 1st factor is steric hindrance and depends on the relative size of both guest molecule and CD cavity. If the guest is the wrong size, it will not fit properly into the CD cavity, and consequently, the stability of the complex will decrease. The 2nd critical factor is the thermodynamic interactions between the different components of the system (CD, guest, and solvent), which has been exhaustively described by Rekharsky and Inoue (1998). These researchers suggest that the most important contributors to the complexation process are (1) penetration of the hydrophobic part of the guest inside of the CD cavity, (2) dehydration of the organic molecule, (3) hydrogen interactions, (4) release of the water molecules from the CD cavity to the bulk (approximately 7 water molecules for β-CD), and (5) conformational changes or strain release of the CD molecule upon complexation. In fact, these researchers have compared the complexation process with typical hydrophobic interaction processes, such as the transfer of an organic molecule from an aqueous to organic phase such as hexane. Therefore, CDs have been recognized as some of the most important host materials for organic molecules in aqueous media where certain hydrophobic chemicals can form stable host–guest complexes within the hydrophobic CDs cavity (Muñoz-Botella and others 1995).
Efficiency of the microencapsulation process depends on the guest and host properties; however, when the inclusion is accomplished, CDs offer protection to the guest. α-CDs can typically complex better low molecular weight molecules or compounds with aliphatic side chains, β-CD will complex aromatics and heterocycles, and γ-CD can accommodate larger molecules such as macrocycles and steroids. When microencapsulation takes place, the guest molecule can suffer advantageous changes in its chemical and physical properties such as protection to light or oxygen; modification of the chemical reactivity; fixation of very volatile substances; improvement of water solubility and handling of hydrophobic, liquids, or gaseous substances to powders; protection against microbial degradation; masking off-odors and taste; masking color; and catalytic activity, as well as control release (Madene and others 2006).
These characteristics of CDs to form stable inclusion complexes make them suitable for applications in food, pharmaceutical, cosmetic, environment protection, bioconversion, packing, and textile industries (Hedges 1998; Dufosse and others 1999; Holland and others 1999; Bhardwaj and others 2000; Fujishima and others 2001; Lezcano and others 2002). CDs have been successfully used to encapsulate essential oils to improve their physicochemical properties (Ayala-Zavala and others 2007; Karathanos and others 2007; Martins and others 2007).
Recently, the food industry has expressed considerable interest in extracts and essential oils from aromatic plants with antimicrobial activities for controlling pathogens and toxin-producing microorganisms (Soliman and Badeea 2002; Tepe and others 2005). The active constituents of these oils are normally volatile, hydrophobic, and very labile compounds, which can be trapped and protected by CD inclusion.
Several essential oils such as oregano, thyme, sage, rosemary, clove, coriander, garlic, and onion oils have been studied as antimicrobial natural products against both bacteria and molds (Nychas 1995). Some spices contain essential oils with antimicrobial activity, such as sulfur compounds in garlic, cinnamaldehyde, and eugenol from cinnamon essential oils (Ayala-Zavala and others 2005). The growth of different microorganisms responsible for quality loss of fruit and vegetables can be diminished using these essential oils (Ponce and others 2004).
Many pathogenic molds, such as Fusarium spp., Alternaria spp., Aspergillus spp., Penicillium spp, and Rhizopus spp., which have been reported as the causal agents of foodborne diseases and/or food spoilage, can be inhibited using essential oils (Tripathi and Dubey 2004). Sokmen and others (2004) reported the capacity of thyme essential oil to inhibit the growth of several molds such as Alternaria ssp., A. flavus, Fusarium spp., and Penicillium spp. According to Soliman and Badeea (2002), the antifungal effect of thyme essential oil might be caused by the presence of β-pinene. In another study, López-Carballo and others (2005) determined that antifungal properties of oregano essential oil are attributed to carvacrol, a major component of oregano (Veres and others 2003). Clove essential oil showed a high antifungal capacity on molds, attributed to eugenol, the major component of this essential oil (Chami and others 2005).
Davidson (2001) reported that the exact cause–effect relation of the mode of action of antimicrobial compounds such as thymol, eugenol, and carvacrol has not been well determined, although it seems that they may inactivate essential enzymes, react with the cell membrane, or disturb genetic material functionality. At low concentrations, these compounds affect enzyme activity, especially those enzymes associated with energy production; and at higher concentrations, they could cause protein denaturalization (Prindle and Wright 1977). They may also alter microbial cell permeability, allowing the loss of macromolecules from the interior and interact with membrane proteins, causing an opposite flow of protons and a deformation of membrane structure and functionality (Nychas 1995).
Some vegetables and herbs also contain substances capable of inhibiting microbial growth (Zaika 1988; Delaquis and Mazza 1995), and their microencapsulation could be an approach to prolong the shelf life of fresh-cut fruits and vegetables (Ayala-Zavala and others 2005).
Bhandari and others (1999) studied the microencapsulation of lemon oil flavor volatiles (α-pinene, sabinene, β-pinene, β-myrcene, limonene, γ-terpinene, terpinolene, linalool, neral, and geranial) with β-CD. It was found that a lemon oil powder can be successfully produced by a microencapsulation technique using β-CD, preserving the constituents of the free oil. In other study, cinnamon leaf (CLO) and garlic oil (GO) microcapsules (major oil constituents were eugenol and allyl disulfide, respectively) presented good antifungal activity against A. alternata (Ayala-Zavala and others 2007). In the generated antifungal CLO:β-CD and GO:β-CD microcapsules, the adsorption of water was observed, showing that at high RHs (63% to 100%) the water adsorption was higher compared to low RHs (18% to 33%).
The main reason for promoting the application of natural products in fresh-cut fruits is the consumer demands for natural and/or organic methods to preserve foods. There is an increasing portion of consumers choosing convenient and fresh-cut fruits and vegetables with a fresh-like quality containing only natural ingredients (Ponce and others 2004). However, these researchers reported that the treatment with essential oils affects the sensory acceptability, due to the strong odor-flavor that can be transmitted from the oil to the vegetable product. Microencapsulation can be a solution to solve this problem, because during the microencapsulation process, the active antimicrobial compounds will be trapped, masking odor and flavor until release to the atmosphere in constant low doses. This can protect the product from microbial growth without affecting its sensory acceptability. Therefore, the above-mentioned natural compounds are good candidates to be encapsulated in CDs matrixes and form controlled delivering systems, as is explained in the following section.
Premise III. CDs are delivery systems that can be controlled throughout high humidity levels in the atmosphere
When a CD–antimicrobial compound molecular complex is exposed to water molecules (high RH), their interaction is weakened and the antimicrobial is passively released to the environment. These mechanisms could be used to generate an antimicrobial active packaging (AP) and therefore protect fresh-cut produce against bacterial and fungal growth, as shown in Figure 4.
To give experimental support to this hypothesis, an experiment to demonstrate the controlled release of a microencapsulated essential oil compound (eugenol) in β-CD was performed. Figure 5 shows the effect of relative humidity on the release of eugenol (expressed as %) from de β-CD:eugenol complex. It can be observed that the stability of the complex is lost at high RH, and consequently the complexed eugenol is released to the headspace. This process can be explained considering that as the water availability increases around the microcapsules, water starts to interact with the polar groups of the CD–antimicrobial complex, causing a displacement of the antimicrobial compound from the interior of the CD cavity. Exchange experiments carried out with D or 18O marked water showed a complete H/D exchange, hence extending also to sterically inaccessible OH groups. In these studies, researchers concluded that the long-range transport of hydrogen takes place by diffusion of water molecules throughout the CDs molecule (Cunha-Silva and Texeira-Dias 2004). When this hydration process occurs with a CD complex, the interaction between the host and guest is weakening, causing a structural distortion and favoring guest release to the environment.
Controlled release packaging is an emerging technology using packaging materials as delivery systems to release active compounds (such as antimicrobials, antioxidants, enzymes, flavors, nutraceuticals) to protect against microbial spoilage and enhance food quality (LaCoste and others 2005). Most attention in this area has been paid to the use of natural antimicrobial packaging instead of synthetic additives (Appendini and Hotchkiss 2002). For this reason, in response to the dynamic changes in current consumer demand and market trends, the area of active packaging (AP) is becoming increasingly significant (LaCoste and others 2005), and CD complexes appear to have great potential to create this kind of packaging material.
The effect of RH on the release of volatile materials indicates that high retention is maintained at low RH levels, where individual structures of the capsule (of several materials) are intact (Rosenberg and others 1990). In maltodextrin/gum arabic or soy matrices spray dry microcapsules of ethyl butyrate, water uptake at high relative humidity destroys the capsule structure, causing the release of microencapsulated ethyl butyrate (Yoshii and others 2001). The rate of release and oxidation of spray dried encapsulated D-limonene are related to the RH level and structural changes of the capsule matrix (Soottitantawat and others 2004). Soottitantawat and others (2004) studied the effect of spray drying on the microencapsulation of l-menthol using gum arabic and modified starch as wall materials, observing a direct correlation between RH levels and l-menthol releasing rates. The main releasing trigger is the hydrophobicity of the guest compound. As water penetrates the encapsulating matrix, the system turns energetically unfavorable for the hydrophobic antimicrobial guest that migrates to a lower water content medium, the atmosphere.
Several active substances can now be incorporated into the packaging material to improve its functionality; CD–antimicrobial complexes can be used for this purpose. For the inclusion of CD complexes in plastic films, a matrix film can comprise a thermoplastic polymer to locate the complex, which can be a dispersed substituted CD forming a molecular complex with an active compound (Wood and Beaverson 1997). The thermoplastic/CD film obtains substantial barrier properties from the interaction between the substituted CD in the film material, with a permeant. The substituents on the CD molecule cause the CD to be dispersible and stable in the film material, resulting in an extrudable thermoplastic. Such materials can be used as a single layer film material or a multilayer film material which can be coated or uncoated and can be used in structural materials (United States Patent 5603974). The cooperation between the CD and the thermoplastic polymer provides barrier properties to the film, and makes possible that active molecules can be complexed, entrapped, or released by the CD within the film. The trapped molecules can comprise a variety of the above-mentioned antimicrobials. However, it has to be highlighted that when a more complex film is created the recycling process would be more difficult.
Most AP systems represent a package/headspace/food system such as sachet release systems. Normally, sachets have been used to control the gas composition inside a package; for example, an ethanol-vapor-generating sachet can inhibit mold growth on bakery products (Smith and others 1987). Evaporation or equilibrated distribution of a substance among the headspace, packaging material, and/or food has to be considered as a part of main migration mechanisms to estimate the interfacial distribution of the substance. Compared to a nonvolatile substance, which can only migrate through the contact area between the package and the food, a volatile substance can migrate through the headspace and air gaps between the package and the food (Han 2000). Lee and others (2006) developed a 1-methylcyclopropene (1-MCP)-CD sachet release system controlled by high RH levels. This ethylene inhibitor action has been widely studied in different whole and fresh-cut produces.
Considering the fresh-cut decay problems and the CDs' capacity to include and release natural antimicrobial compounds, it could be assumed that the integration of this information supports the possibility of developing antimicrobial active packages, which can be widely used to preserve quality of different fresh-cut fruit and vegetables. The proposed essential oil–CD microcapsule has 2 advantages. First of all, stability: to release the essential oil from the complex, a high RH is necessary (Figure 5). This is important because the microcapsule can be easily manipulated, without loosing activity. The 2nd advantage of these microcapsules is that release of active compounds can be achieved controlling the RH of the system, avoiding that high concentrations of the essential oil interact with the food at once (the traditional method to apply essential oils to food matrix), and consequently no significant odor/flavor modifications of the products may occur.