*3.3. Potential Applications of High O2*

It is known that low O2 modified atmosphere packaging is universally accepted to prolong the shelf-life of minimally-processed fruits and vegetables; however, the growth of some anaerobic pathogen strains might be allowed or even stimulated. In addition, when O2 levels are too low, this induces anaerobic respiration, leading to fermentation processes and to the production of undesirable metabolites [89].

The application of high O2 concentrations (>70% O2) could overcome the disadvantages of low O2 modified atmosphere packaging for some ready-to-eat fruits and vegetables. High O2 was found to be particularly effective at inhibiting enzymatic discoloration, preventing anaerobic fermentation reactions and inhibiting microbial growth [90]. It is hypothesized that high O2 levels may cause substrate inhibition of the enzyme polyphenol oxidase (PPO), which is the enzyme responsible for initiating discoloration on the cut surfaces of processed products [91].

High O2-modified atmosphere packaging has been used in some vegetables and fruits, such as minimally-processed carrots, strawberry, minimally-processed baby spinach, fresh-cut mango, apple slices, and so on [89].

Amanatidou *et al.* [92] screened numerous microorganisms (Pseudomonas fluorescens, Enterobacter agglomerans, Aureobacterium strains 27, Candida guilliermondii, Candida sake, *Salmonella* Typhimurium, *Salmonella* Enteritidis, *E. coli*, *L. monocytogenes*, *Leuconostoc mesenteroides* var. *mesenteroides*, *Lactobacillus plantarum* and *Lactococcus lactis*) associated with the spoilage and safety of minimally-processed vegetables. High oxygen levels (80%–90%) caused a reduction in the growth rate of microbial targets at 8 ◦C, whilst the lag phase was prolonged (it was more evident at 90% O2 concentrations). Amanatidou *et al.* [92] and Kader and Ben Yehoshua [93] hypothesized that high O2 concentrations lead to intracellular generation of reactive oxygen radical species (ROS, O2 −, H2O2, OH−), causing damage to the vital cellular macromolecules, thus reducing cell viability when oxidative stresses overwhelm cellular protection systems.

Jacxsens *et al.* [90] studied the effect of high oxygen-modified atmosphere packaging (*i.e.*, >70% O2) on microbial growth and the sensorial qualities of mushroom slices, grated celeriac and shredded chicory endive and observed that high O2 atmospheres were effective at inhibiting enzymatic browning of the tested vegetables. In addition, an improvement of the microbial quality (as a reduction in yeast growth) was obtained.

In an interesting paper, Allende *et al.* [94] studied the effect of superatmospheric oxygen packaging on sensorial quality, spoilage and *L. monocytogenes* and *Aeromonas caviae* growth in fresh processed mixed salads. An initial O2 concentration of 95 kPa was combined with two plastic films (low and high barrier impermeability for O2). Packaged salads were stored up to eight days at 4 ◦C and at temperatures simulating the chilled distribution chain.

The authors reported that superatmospheric O2 does not affect all microorganisms in the same way: high oxygen levels affected lactic acid bacteria and *Enterobacteriaceae*, which were inhibited in both plastic films (with low and high permeability). On the contrary, the growth of yeast and *A. caviae* seemed to be stimulated, whereas the growth of psychrotrophic bacteria and *L. monocytogenes* was not affected. The general appearance was maintained for longer, and the shelf-life of the mixed salads was prolonged by using O2 concentrations higher than 60 kPa throughout the storage period.

Chunyang *et al.* [89] studied the effect of high oxygen-modified atmosphere packaging on fresh-cut onion quality at room temperature. Onion slices were packaged in a high-barrier film package of 70 μm in thickness and stored in five different modified atmospheres (100% O2; 95% O2/5% CO2; 80% O2/20% CO2, 75% O2/25% CO2; and air). Results showed that the fresh-cut onions packaged in air had a short shelf-life, because the respiration rate was quickened due to mechanical damage, and microorganisms were not inhibited. High O2 reduced the weight loss, the respiration rate, the total reducing sugar loss and the total titrable acidity increase of the fresh-cut onions. The best modified atmosphere seemed to be 80% O2/20% CO2, where total bacteria counts increased slowly. High oxygen affected also the sensory characteristics: sensory quality was acceptable up to five days, while air-packaged fresh-cut onions were not acceptable after two days of storage at room temperature.

Day [91] also confirmed that the most effective high O2 gas mixtures were found to be 80%–85% O2/15%–20% CO2. This had the most noticeable sensory quality and antimicrobial benefits on a range of freshly-prepared produce items.

In fact, recommended optimal headspace gas levels immediately after freshly-prepared produce package sealing are: 80%–95% O2 and 5%–20% N2. After package sealing, headspace O2 levels will decline, whereas CO2 levels will increase during storage, due to the intrinsic respiratory nature of fresh product.

The levels of O2 and CO2 within packages are influenced by numerous variables:


It would be preferable to maintain headspace levels of O2 > 40% and CO2 between 10% and 25% to maximize the benefits of high O2 MAP. This can be achieved by:


A further measure to maintain a gas mixture O2 > 40%/CO2 10%–25% could be obtained by introducing the highest level of O2 (balanced with N2) possible just prior to prepared produce package sealing. Generally, it is not necessary to have any levels of CO2 in the initial gas mixture, since it will increase rapidly during chilled storage.

Numerous products (iceberg lettuce, sliced mushrooms, broccoli florets, lettuce, baby-leaf spinach, Lollo Rossa lettuce, flat-leaf parsley, cubed swede, coriander, raspberries, strawberries, grapes and

oranges) processed with high O2 MAP reported beneficial effects on sensory quality if compared to industry-standard air packing and low O2 MAP [91].

Finally, the recommended packaging material for high O2-modified atmosphere is 30-μm orientated polypropylene (OPP), which has sufficient O2 barrier properties (to maintain high in-pack O2 levels >40%), and it is sufficiently permeable to ensure that CO2 did not rise above 25% after 7–10 days of storage at 5–8 ◦C.

Other packaging materials suitable for high O2 MAP of fresh prepared produce are:


or other medium to very high O2 permeability films.

#### **4. Conditioning Solutions with Antimicrobials or Natural Compounds for Fruit Salad**

Coatings, dipping, spraying, *etc.*, are the main applications of antimicrobial compounds, as natural alternatives to chemical additives, whilst little is known about the use of antimicrobials as filling liquids.

An initial work by Senesi *et al.* [95] on the use of rectified apple juice as a filling liquid to increase the quality and shelf-life of fresh-cut apples was followed, more recently, by D'Amato *et al.* [96]. The authors evaluated the possibility of using a chitosan, honey and pineapple juice solution as filling liquids to prolong the microbiological shelf-life of a fruit-based salad.

"Granny Smith" apples, "Gialla" first crop cactus pear fruits and "Regina" table grapes were washed; then, apples and cactus pears were peeled and sliced, while grapes were cut without seeds. Mixed fruits were placed in conical frustum-shaped cups, filled with four different solutions (sterile distilled water as the control; 30% of acacia honey and 70% of distilled water; 50% of pineapple juice and 50% of distilled water; 50% of low molecular weight chitosan solution and 50% of sterile distilled) and stored at 4, 8 and 12 ◦C.

The authors observed that the use of the natural antimicrobial compounds, as a filling liquid, affected the microbiological shelf-life of salad.

At 4 ◦C, mesophilic and psychrotrophic bacteria did not reach the established limit (1 × 106 CFU/g) during the whole storage period (14 days), in samples with honey and chitosan solutions, whereas it ranged from about 6.34–7.81 days and from 9.38 to 11.49 for the control and samples with pineapple juice, respectively.

At 8 ◦C, all of the samples were stored for 10 days. For the samples treated with honey and chitosan solutions, psychrotrophic bacteria did not reach the established limit during the storage, while in control and pineapple juice, it was of 5–6 days. Concerning mesophilic bacteria, microbiological shelf-life was strongly reduced in the control samples and in pineapple juice (about 3.7–4.3 days), whilst in chitosan and honey, it was over six days. In addition, in the samples with chitosan solution, the microbiological shelf-life, calculated by using the growth of yeasts, was over seven days and, by using lactic acid bacteria, did not reach the established limit.

At 12 ◦C, the samples were stored for six days. The microbiological shelf-life of fruit salad without antimicrobial compounds and with pineapple juice was always very low (1.5 and 4 days, respectively).

In the samples honey added, mesophilic and psychrotrophic bacteria did not reach the limit of <sup>1</sup> × <sup>10</sup><sup>6</sup> CFU/g. For lactic acid bacteria and yeasts, microbiological shelf-life reached 3.82 and 1.81 days, respectively.

Chitosan exhibited its high antimicrobial activity towards psychrotrophic bacteria, which did not reach the established limit, while no advantage resulted for mesophilic bacteria and lactic acid bacteria and yeasts.

The authors concluded that:


Further investigations are needed to improve the antimicrobial efficacy of honey and chitosan for potential commercial applications. The literature is poor in articles addressing this topic; thus, it would be advisable to explore the use of antimicrobial compounds as filling liquids.

### **5. Biopreservation and the Use of Probiotic Coatings**

In recent years, biological preservation has emerged as a promising strategy to extend the shelf-life and to improve the microbiological safety of foods [97], since it fits well with the diffuse desire to preserve foods by natural means. Several bacteria and yeasts have been already identified as bioprotective agents [98], and different studies have been carried out on their application to fresh-cut fruits and vegetables [99]. Table 3 summarizes the most recent studies performed on this topic.

In general, lactic acid bacteria (LAB) have shown the greatest potential as biocontrol agents of several minimally-processed foods, because they are widely used in fermented foods and have a long history of safe use [100]. However, several other bacteria and yeasts, often selected among the naturally-occurring microflora, including strains of *Pseudomonas syringae*, *Pseudomonas graminis*, *Gluconobacter asaii*, *Candida* spp., *Dicosphaerina fagi*, *Metschnikowia pulcherrima* and *C. sake*, have been proposed as biocontrol agents in fresh-cut fruits and vegetables [101–104].


**Table 3.** Overview of the most recent studies performed on the use of biocontrol microorganisms to biopreserve minimally-processed products.

The success of LAB in preventing the growth and activity of undesirable microorganisms is due to a large diversity of mechanisms of action related to the production of antimicrobial compounds, organic acids, hydrogen peroxide, bacteriocins and diacetyl [103,104,115]. The combination of low pH values and antibacterial activities of organic molecules produced by LAB remains the main mechanism for biopreservation [97]. Several bacteriocin-producing LAB have been shown to be effective against spoilage and pathogenic microorganisms in minimally-processed fruits and vegetables [116,117]. However, the direct application of bacteriocins to fresh-cut products did not provide completely satisfactory results probably due to the adsorption or inactivation of the added compound into the food product [116]; on the other hand, the direct use of living bacteriocinogenic bioprotective strains could lead to a more effective protection of the food product by circumventing the mentioned problems because of the localized and constant delivery of the antibacterial compound, which will add to other advantages, like space colonization by the strain [116,117]. The inhibitory properties against contaminating foodborne pathogens and spoilage microorganisms could also consist of a mere competition for nutrients (vitamins, minerals, trace elements and peptides), and therefore, via competition or antibiosis, LAB are able to function as a hurdle to pathogen growth and survival [118].

In the last decade, different LAB species were proposed as biocontrol agents in minimally-processed fruits and vegetables (see Table 3). In 2004, Scolari and Vescovo [111] performed several challenge tests on salad leaves by simultaneously inoculating *Lactobacillus casei* and various pathogens (*S. aureus*, *Aeromonas hydrophila*, *E. coli* and *L. monocytogenes*). A significant inhibitory effect by the LAB towards all of the pathogenic strains was observed and confirmed by the same authors during a subsequent study about the influence of *Lb. plantarum* on the growth of *S. aureus* [119]. Trias *et al.* [103] found five strains of LAB (some *Leuconostoc* spp. strains) that were able to inhibit *L. monocytogenes* and *Salmonella* Typhimurium in cut iceberg lettuce leaf, but were not effective in reducing the amount of *E. coli*. More recently, Siroli *et al.* [108] showed the good performance of a nisin-producing strain of *Lc. lactis*, which was able to inhibit *L. monocytogenes*, *E. coli* and the total mesophilic species when added at a level of 7 log CFU/mL in the washing solution of minimally-processed lamb's lettuce and combined or not with thyme essential oil. In 2015, the same authors proposed two other LAB strains (*Lb. plantarum* V7B3 and *Lb. casei* V4B4) to be used as biocontrol agents alone or in combination with thyme essential oil (EO) in lamb's lettuce [99]. In this work, the use of the *Lb. plantarum* V7B3 strain (6 log CFU/mL) during the washing phase of fresh-cut lettuce increased product shelf-life and safety; *L. monocytogenes* and *E. coli* viabilities were significantly reduced over the nine days of refrigerated storage. Promising results were also obtained for biopreservation of minimally-processed golden delicious apples packaged in a modified atmosphere alone or in combination with natural antimicrobials (2-(E)-hexenal/hexanal and 2-(E)-hexenal/citral) [99]: a strain of *Lb. plantarum* (CIT3) was able to increase the safety of sliced apples, when inoculated at levels of 6–7 log CFU/g in the washing dipping solution, both alone or in combination with natural antimicrobials.

Other non-lactic acid bacteria and yeasts were also proposed as biocontrol agents (Table 3) For example, the growth of *L. monocytogenes* and *Salmonella* Enterica in fresh-cut apples has been prevented using fungal antagonists [106]. When inoculated at a low level, *L. monocytogenes* cell loads were greatly reduced (from 5.7 to 6.0 log units after seven days) by strains of *G. asaii*, *Candida* spp., *D. fagi* and *M. pulcherrima*. At high pathogen inoculum levels, only *G. asaii* and *Candida* spp. reduced the cell load of *L. monocytogenes* population to non-detectable levels. Abadias *et al.* [101] found that the application of the fungal postharvest antagonist *C. sake* CPA-1 reduced the growth of a mixture of *E. coli* strains in fresh-cut apples at 25 ◦C, whereas Alegre *et al.* [107] isolated a new strain of *Enterobacteriaceae* that reduced the growth of *Salmonella*, *L. monocytogenes* and *E. coli* O157:H7 on fresh-cut apples and peaches. *P. graminis* was found be able to reduce or slow down the development of foodborne pathogens on minimally-processed fresh-cut apples and peaches [105]; in this case, the inhibitory effect of the antagonists on the foodborne pathogens was not instantaneous and became apparent after 6 days at 5 ◦C. This strain was also proposed for fresh-cut melon, reducing *Salmonella* and *L. monocytogenes* growth during storage at 5, 10 and 20 ◦C [109]. The strain effectiveness depended on the pathogens' concentration and on storage temperature. At a low pathogen concentration and 20 ◦C, *L. monocytogenes* growth was reduced between 2.1 and 5.3 log CFU/g after two days of storage and *Salmonella* growth between 2.0 and 7.3 log CFU/g.

Although research on the use of biocontrol agents in minimally-processed fruits and vegetables has increased in recent decades, the standardization of a biopreservative approach is still difficult to realize. Independent of the species and strains proposed, in fact, the various studies available in the literature clearly highlight that the efficacy of biocontrol agents is affected by different factors, such as the inoculation level, the presence of other bacteria, the physico-chemical and compositional features of the products and the storage conditions. Further investigations are required, especially considering that process conditions have to be taken into account, during the scaling up at the industrial level.

An interesting modern challenge is to incorporate probiotic bacteria into coated processed fruits and vegetables in order to improve their shelf-life (biopreservation), while providing new non-dairy functional foods. As is well accepted, edible films/coatings may serve as carriers of food additives, such as anti-browning agents, antimicrobials, colorants, flavors, nutrients and spices [24]. Some authors [114,120] proved that the coatings were also good carriers for antioxidant agents, such as cysteine, glutathione and ascorbic and citric acids; thus, the addition of LAB to obtain functional edible films and coatings could have successful implications. Several studies were already conducted on different fruits, as is shown in Table 3. This new trend has arisen from more considerations. As just mentioned, the inclusion into coatings of cultures with inhibitory properties could improve the shelf-life and safety of minimally-processed products, while reducing the need to use increasing levels of chemical additives [118]. In addition, the steps of minimally-processing vegetables, such as peeling and cutting, promote the release of cellular content rich in minerals, sugars, vitamins and other nutrients, creating ideal conditions for microbial growth: this characteristic allows the use of fruit and vegetable food products as probiotic carriers. According to Soccol *et al.* [121], in fact, minimally-processed fruits and vegetables are very good matrices providing ideal substrates for probiotics, since they contain minerals, vitamins, antioxidants and fibers. Moreover, due to their cellulose content, fruits, such as apples and pears, may also exert a protective effect on the probiotic microorganisms during passage through the intestinal tract [122], allowing these microorganisms to reach the colon and benefit the host. It has been reported that the optimum probiotic growth temperature is between 35 and 40 ◦C, and the best pH is between 6.4 and 4.5, ceasing when a pH of 4.0–3.6 is reached [123]. This situation could be solved by using some supports, such as agar, polyacrylamide, calcium pectate gel, chemically-modified chitosan beads and alginates, to provide a physical barrier against unfavorable conditions [124–126].

One of the first reports about this issue is the study of Tapia *et al.* [114]; fresh-cut apple and papaya cylinders were coated with 2% (w/v) alginate or gellan film-forming solutions containing viable Bifidobacteria, namely *Bifidobacterium lactis* Bb-12. The bifidus-containing coatings were more permeable to water vapor than the corresponding films without probiotics, and gellan coatings were more resistant to water transfer than the alginate ones. The most important result was that the edible coatings were efficient in supporting *Bb. lactis* Bb-12 on fresh-cut apple and papaya. In 2010, Rößle *et al.* [112] applied a probiotic microorganism (*Lactobacillus rhamnosus* GG; LGG) to fresh-cut apple wedges (cultivar Braeburn). All samples were able to maintain a probiotic load of about *ca*. 10<sup>8</sup> CFU/g over 10 storage days, which is sufficient for a probiotic effect, and this is comparable to the counts of probiotic bacteria in commercially-available dairy products. It is also important to underline that the physico-chemical properties of the apple wedges containing LGG compared to the control (without probiotics) remained stable over the observation period. In a subsequent work [113], the effectiveness as a biocontrol agent of the same probiotic strain against *Salmonella* and *L. monocytogenes* on minimally-processed apples throughout storage, as well as its effect on apple quality and natural microflora was evaluated. The obtained results showed that *Salmonella* was not affected by co-inoculation with LGG, whereas the *L. monocytogenes* population was 1-log unit lower in the presence

of probiotic population maintained over recommended levels for probiotic action (106 CFU/g) along 14 days.

Although still unexplored, this new challenge appears to be highly advantageous, since minimally-processed fruits and vegetables are a food category rich in nutrients, intended for consumption by all individuals and widely accepted among consumers [127]. Thus, edible coatings, including probiotic bacteria, when applied to minimally-processed products, open new possibilities to improve their shelf-life and safety while providing innovative functional foods.
