**Author details**

#### Sean Stevens

**10.3. Tools for genome engineering**

346 Organ Donation and Transplantation - Current Status and Future Challenges

has been greatly facilitated [70].

**11. Conclusions and future prospects**

A number of novel enzymatic molecules have been created which help resolve the dilemma of targeted integration in porcine cells. Zinc Finger Nucleases (ZFN), Transcription Activator-Like Effector Nuclease (TALEN) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are all synthetic molecules based on genuine proteins which allow the precise targeting of genomic DNA based upon sequence [66–68]. In each case there are two functional components, a targeting module which recognizes a specific genomic sequence, and an enzymatic module which introduces a double-stranded DNA break at the target site. In the case of ZFN and TALEN, the targeting module is a complex array of protein sequences which have previously been shown to recognize specific DNA sequences and can be mixed in a modular way to bind to any desired sequence. Although both technologies have shown great success, the effort and cost required to identify a single functional molecule can be significant. In contrast, the relatively more recently recognized CRISPR, and related prokaryotic systems, is much more easily applied in mammalian cells. The DNA binding module in this case is RNA base-pairing to provide sequence specificity. The enzymatic module cleaves DNA, creating a double strand break similar to ZFN and TALEN. When heterologous DNA is present, the cellular repair machinery may use the synthetic DNA to repair the break, inserting the TG at the desired genomic site. It is important to note that all of these systems, ZFN, TALEN or CRISPR, are essentially the same in that they introduce double strand DNA breaks at a selected site in the genome and do not directly affect the rate of DNA insertion. Therefore, it is often necessary to include selection schemes for identification of the modified cells. The greater efficiency and ease of use of these systems, CRISPR in particular, has allowed targeted

insertion of DNA into genomes that were not previously able to be modified [69].

Due to the challenges of creating genomic modifications in porcine primary cells while maintaining their viability for SCNT, more efficient engineering methods are desirable. One approach to enhance efficiency is to target a specific region of DNA, called a landing pad, with multiple genes at once. By inserting a DNA vector bearing multiple therapeutic genes at once, a large amount of breeding and testing can be circumvented using a single event. This approach has the added advantage of avoiding inefficient crossbreeding necessary to bring loci from distinct chromosomes together in one lineage. When combined with the use of tools such as ZFN, TALEN and CRISPR more rapid progress in the genetic modification of animals

The increasing sophistication and accessibility of genome engineering toolsets and deeper understanding of immunological rejection mechanisms has allowed greater advancement in xenotransplantation than ever before. A key question is just how many genetic changes are required in order to make a pig organ suitable for transplantation? While the critical experimental data needed for such an assessment is still accumulating, it is clear that the number of alterations required for one organ may be different from another. For example, xeno-hearts with relatively minimal genetic modifications have demonstrated months to years survival in transplantation studies with non-human primates, whereas xeno-lungs with more extensive modifications have Address all correspondence to<EMAIL_ADDRESS>
Synthetic Genomics, Inc., La Jolla, CA, USA


*<IP_ADDRESS>. Area under the blood concentration-time curve (AUC)*

to be maintained within therapeutic range in order to achieve the optimal benefit of drug

The introduction of CsA in the early 1980s was immediately associated with an enhanced one year renal allograft survival; however, the argument for the therapeutic monitoring to optimize

Over the past two decades, there have been changes to recommended CsA dosing, changes in concomitant medications, and one major change to the oral drug formulation. Lately, there has also been the introduction of generic formulations of CsA [75]. In 1988, in a prospective study showed that although C0 (trough concentration) levels of CsA correlated poorly with dose, Cmax was significantly correlated with dose, Area Under the Curve (AUC) and elimi‐ nation half-life (t1/2). Those who suffered acute rejection had a significantly lower Cmax by 15– 31% [76]. The problem with this method for adjusting the dosage of CsA is that it relies on only one aspect of CsA pharmacokinetics, the predose or trough concentration. With the original formulation of CsA, Sandimmune®, this was the best practice, but during the conversion of patients from that formulation to the improved formulation, Neoral® the 2 h post-dose concentration has been advocated as a single concentration monitoring alternative to C0 [77]. The microemulsion formulation of CsA, Neoral®, makes CsA pharmacokinetics more predictable and reduces the effects of bile and food on absorption [78]. Nevertheless the predose concentration is still widely used in clinical practice. Currently, most transplant centers measure a single steady-state CsA concentration as either a C0 predose trough or 2 hours postdose, while some conduct multiple measurements to determine CsA AUC estimates [79]. The target predose concentrations varied not only with transplanted organ and time after transplant but also with the analytical method used. The therapeutic range of CsA used by clinicians varies greatly according to the type of assay used to measure CsA and whether blood

Thus, it has reported by high pressure liquid chromatography, monoclonal fluorescence polarization immunoassay (monoclonal TDx assay, Abbott Diagnostics®), or monoclonal radioimmunoassay (various manufacturers), the level of therapeutic concentrations in blood are 10-400 ng/mL. By high pressure liquid chromatography, monoclonal fluorescence polari‐ zation immunoassay (monoclonal TDx assay, Abbott Diagnostics®), or monoclonal radioim‐ munoassay (various manufacturers), the level of therapeutic concentrations in serum are 50-150 ng/mL. By polyclonal fluorescence polarization immunoassay (monoclonal TDx assay, Abbott Diagnostics®), or polyclonal radioimmunoassay (various manufacturers) the level of

efficacy and safety, has been discussed in the last 25 years and it is still debated [74].

therapy, rendering TDM necessary for these drugs.

324 Current Issues and Future Direction in Kidney Transplantation

**4.1. Cyclosporine (CsA)**

*4.1.1. Therapeutic monitoring*

*<IP_ADDRESS>. Trough concentration (C0) monitoring*

**4. Monitoring of individual immunosuppressive agents**

or serum concentrations are determined by the clinical laboratory.

The first steps towards the development of a more precise monitoring strategy for CsA resulted from the landmark studies by Lindholm and Kahan and Kahan et al., which identified a link between the pharmacokinetics of CsA and clinical outcomes in the individual transplant recipient [80,81]. The area under the concentration-time curve for CsA over a 12-hour drug administration interval (AUC0-12h) was a more precise predictor of graft loss and incidence of acute rejection than other parameters, including the C0. Since then, subsequent studies on the pharmacokinetics of CsA in renal transplant patients have identified that intrapatient varia‐ bility in AUC values over time was directly correlated with the risk of chronic rejection [77,82].

Proper calculation of AUC requires administration of a dose, followed by blood collection according to an intensive sampling strategy. Concentration values obtained are used to calculate AUC, usually by the trapezoidal method [78]. Some advantages of AUC monitoring are that it is the most precise indicator of drug exposure, can characterize abnormal absorption patterns, appears to be a predictor of clinical outcomes, generates a concentration-time profile, allows calculation of oral pharmacokinetic parameters, and reduces the problems associated with laboratory errors and single concentrations [74,83,84].

Despite its appealing potential advantages, the major disadvantage of AUC monitoring is its inherent need for multiple blood samples. The increased number of samples required, makes AUC monitoring impractical for routine clinical use, more expensive in the short term because of increased sample collection, analysis and interpretation of results, and inconvenient for patients, especially those in an outpatient setting [77,85]. AUC has been advocated as a better parameter to monitor than trough concentrations, because trough concentrations give no indication of exposure to CsA. For example, 2 patients could have the same trough concen‐ tration, but one could have a much lower AUC and, therefore, exposure to CsA. Unfortunately, AUC monitoring is not clinically feasible because of the added time, expense and inconven‐ ience required to collect a sufficient number of samples to properly calculate AUC. Although the full AUC for CsA has been demonstrated as being a sensitive monitoring tool, there may be an alternative approach to the determination of the degree and variability of CsA exposure in the individual patient [77,83].


About the Guest Editor

**Pierre Pichat**, as "Directeur de Recherche de 1ère classe" (first-class) with the CNRS (National Center for Scientific Research, France), has been active in heterogeneous photocatalysis for many years. He has founded a laboratory dealing with both basic investigations on this field and applications regarding self-cleaning materials and purification of air or water. He has published numerous research papers and several reviews of the domain. He has edited a book and special issues. He is a frequent invited lecturer at Conferences. He is a member of the International Scientific Committees of most of the International Conferences on photocatalysis. Over the years, he has served on CNRS-related Committees on diverse aspects of chemistry; he has been the coordinator or advisor of European Community projects on photocatalysis; he has evaluated projects on environmental chemistry for various countries. He has received an International Appreciation Award acknowledging his pioneering contributions to heterogeneous photocatalysis.


**3. Direct determination**

The direct determination of metals in oils can be carried out by sample solubilization in an organic solvent, an emulsification procedure in aqueous solutions in the presence of emulsifiers such as Triton X-100 or a solid sampling strategy.

#### **3.1 Dilution with organic solvent**

The procedure of the dilution with organic solvents is an easy way to sample pretreatment before detection, but has some requirements: special devices for sample introduction e. g. for FAAS (Bettinelli et al., 1995), the addition of oxygen as an auxiliary gas in ICP-OES or ICP-MS (Costa et al., 2001). The volatile organic solvents have been directly introduced into ICPs for many years, but this can cause plasma instability, less sensitivity, less precision and high cost. Al, Cr, Cd, Cu, Fe, Mn, Ni and Pb contents of olive oil were investigated using diethyl ether, methyl isobutyl ketone (MIBK), xylene, heptane, 1,4-dioxane as solvent and N,N-hexamethylenedithiocarbamic acid, hexamethyleneammonium (HMDC-HMA) salt as a modifier by ETAAS (Karadjova et al., 1998). A transverse heated filter atomizer (THFA) was employed for the direct determination of Cd and Pb in olive oil after sample dilution with nheptane (Canario & Katskov, 2005). Moreover, Martin-Polvillo et al. (1994) and List et al. (1971) determined trace elements in edible oils based on the direct aspiration of the samples, diluted in MIBK. In another research, the mixture of 2%lecithin-cyclohexane was used to introduce the oil samples to a polarized Zeeman GFAAS (Chen et al., 1999). Van Dalen was

Metal Determinations in Olive Oil 97

pretreatment procedures in common like: wet digestion, dry ashing, extraction and dilution with organic solvent in order to eliminate hard organic matrix. In the processing large numbers of samples, flow injection analysis (FIA) systems can be preferred for sample pretreatment. The FIA system for oil analysis is frequently based on the on-line preparation of oil-in-water emulsions by using ultrasonic bath with serious drawbacks in efficient preparation of stable emulsions. By this way, more concentrated emulsions (high oil concentration) can be introduced into the plasma and thereby the LODs were improved. A limited number of researches related to metal determination in oils by FIA systems have been presented. Jimenez et al. succeeded multi-element determination in virgin olive oil by flow injection ICP-MS using with HNO3 and Triton X-100 as emulsifying agents (Jimenez et al., 2003). A magnetic-stirring micro-chamber has been developed for on-line emulsification and has been successfully employed by Anthemidis et al. to detect Ag, Al, B, Ba, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, In, Mg, Mn, Ni, Pb, Tl and Zn in olive oil using flow injection ICP-OES (Anthemidis et al., 2005). The low concentration of analyte in the sample analysed and difficulty of obtaining stable emulsions with rich oil content were reported as the main problems. On-line emulsion preparation procedure was suggested as simpler, more effective, less time consuming, less labor intensive, less matrix interferences and less contamination risk over the other direct sample introducing procedures. The direct determination of Cu and Fe in edible oils based on the flow injection standard addition method by FAAS was performed

As mentioned above, various pretreatment procedure and detection techniques have been employed for the total determination of metals in olive oil. The researchers have dealt with metallic contents of olive oils during last few decades.  On the other hand, optimization of the particle size effect, slurry concentration and homogeneity are necessary in order to obtain good precision and recoveries with slurry techniques. In spite of optimization, complete destruction of the sample matrix in plasma and then liberation of analyte from the sample matrix are not always succeeded, causes unsatisfactory results. An alternative technique for introduction of oil sample directly into ICP is the on-line emulsification (Anthemidis et al., 2005). Direct introduction of oil samples in the form of emulsion into ICP facilitates the spray chamber and plasma torch owing to no need of extra oxygen or sophisticated desolvation device. In such a case, the use of stable emulsions with proper surfactant concentration is very important (Anthemidis et al., 2005).

Emulsification as sample preparation has been performed for the determination of trace metals in vegetable oils by ICP-OES (De Souza et al., 2005; Murillo et al., 1999), ICP-MS (Castillo et al., 1999; Jimenez et al., 2003), FAAS (List et al., 1971) and GF-AAS (Lendinez et al., 2001). Additionally, the use of microemulsion as sample preparation for vegetable oil analysis by High-Resolution Continuum Source FAAS (HR-CS FAAS) has been described by Nunes et al. (2011). The determination of Zn, Cd and Pb in vegetable oils by electrothermal vaporization in combination with ICP-MS (ETV-ICP-MS) was described in literature (Huang et al., 2001).

#### **3.3 Direct solid sampling**

Direct introduction of oil samples into the graphite furnace by solid sampling strategy is rarely used, providing an alternative methodology. Due to technical improvements in spectrophotometer and software capabilities of modern instrumentation, this method has not been entirely accepted (Sardans et al., 2010). Direct solid sampling has some advantages such as no sample dilution, satisfactory LOD levels, calibration probability with aqueous analytical solutions, simple analysis and no sample digestion or extraction. Other advantages of this method are reduced time and cost, required little amount of sample and the achievement of high sensitivity. Additionally, it reduces the risk of contamination due to the nonexistence of sample preparation and use of chemical reagents. Some disputes against the method are the difficulty of introducing small sample masses, faulty measurement of the results due to the heterogeneity of some natural samples and the limiting linear working range of AAS (Sardans et al., 2010). Despite these restrictions, direct solid sampling is a reasonable alternative for the determination of the total content of metals in oils, since it needs almost no sample preparation. A method for the direct determination of Ni and Cu in vegetable oils by GFAAS using the solid sampling strategy has been reported without sample dilution by Matos Reyes et al. (2006).

#### **3.4 Flow injection**

Various detection techniques like ETAAS, FAAS, ICP-OES, ICP-MS, voltammetry have been utilized for metal determination in oils. However, all of them have the need for sample

also used lecithin and the organopalladium modifier solutions for the injection of the edible

Taking into account parameters such as economy, safety, environment, time, and low risk of contamination, emulsification appears beneficial over microwave assisted acid digestion. On the other hand, optimization of the particle size effect, slurry concentration and homogeneity are necessary in order to obtain good precision and recoveries with slurry techniques. In spite of optimization, complete destruction of the sample matrix in plasma and then liberation of analyte from the sample matrix are not always succeeded, causes unsatisfactory results. An alternative technique for introduction of oil sample directly into ICP is the on-line emulsification (Anthemidis et al., 2005). Direct introduction of oil samples in the form of emulsion into ICP facilitates the spray chamber and plasma torch owing to no need of extra oxygen or sophisticated desolvation device. In such a case, the use of stable emulsions with proper surfactant concentration is very important (Anthemidis et al., 2005). Emulsification as sample preparation has been performed for the determination of trace metals in vegetable oils by ICP-OES (De Souza et al., 2005; Murillo et al., 1999), ICP-MS (Castillo et al., 1999; Jimenez et al., 2003), FAAS (List et al., 1971) and GF-AAS (Lendinez et al., 2001). Additionally, the use of microemulsion as sample preparation for vegetable oil analysis by High-Resolution Continuum Source FAAS (HR-CS FAAS) has been described by Nunes et al. (2011). The determination of Zn, Cd and Pb in vegetable oils by electrothermal vaporization in combination with ICP-MS (ETV-ICP-MS) was described in literature (Huang et al., 2001).

Direct introduction of oil samples into the graphite furnace by solid sampling strategy is rarely used, providing an alternative methodology. Due to technical improvements in spectrophotometer and software capabilities of modern instrumentation, this method has not been entirely accepted (Sardans et al., 2010). Direct solid sampling has some advantages such as no sample dilution, satisfactory LOD levels, calibration probability with aqueous analytical solutions, simple analysis and no sample digestion or extraction. Other advantages of this method are reduced time and cost, required little amount of sample and the achievement of high sensitivity. Additionally, it reduces the risk of contamination due to the nonexistence of sample preparation and use of chemical reagents. Some disputes against the method are the difficulty of introducing small sample masses, faulty measurement of the results due to the heterogeneity of some natural samples and the limiting linear working range of AAS (Sardans et al., 2010). Despite these restrictions, direct solid sampling is a reasonable alternative for the determination of the total content of metals in oils, since it needs almost no sample preparation. A method for the direct determination of Ni and Cu in vegetable oils by GFAAS using the solid sampling strategy has been

Various detection techniques like ETAAS, FAAS, ICP-OES, ICP-MS, voltammetry have been utilized for metal determination in oils. However, all of them have the need for sample

reported without sample dilution by Matos Reyes et al. (2006).

oils (Van Dalen, 1996).

**3.3 Direct solid sampling**

**3.4 Flow injection**

**3.2 Emulsification**

pretreatment procedures in common like: wet digestion, dry ashing, extraction and dilution with organic solvent in order to eliminate hard organic matrix. In the processing large numbers of samples, flow injection analysis (FIA) systems can be preferred for sample pretreatment. The FIA system for oil analysis is frequently based on the on-line preparation of oil-in-water emulsions by using ultrasonic bath with serious drawbacks in efficient preparation of stable emulsions. By this way, more concentrated emulsions (high oil concentration) can be introduced into the plasma and thereby the LODs were improved. A limited number of researches related to metal determination in oils by FIA systems have been presented. Jimenez et al. succeeded multi-element determination in virgin olive oil by flow injection ICP-MS using with HNO3 and Triton X-100 as emulsifying agents (Jimenez et al., 2003). A magnetic-stirring micro-chamber has been developed for on-line emulsification and has been successfully employed by Anthemidis et al. to detect Ag, Al, B, Ba, Bi, Ca, Cd, Co, Cr, Cu, Fe, Ga, In, Mg, Mn, Ni, Pb, Tl and Zn in olive oil using flow injection ICP-OES (Anthemidis et al., 2005). The low concentration of analyte in the sample analysed and difficulty of obtaining stable emulsions with rich oil content were reported as the main problems. On-line emulsion preparation procedure was suggested as simpler, more effective, less time consuming, less labor intensive, less matrix interferences and less contamination risk over the other direct sample introducing procedures. The direct determination of Cu and Fe in edible oils based on the flow injection standard addition method by FAAS was performed without sample dilution in a previous study (Carbonell et al., 1991 ).

As mentioned above, various pretreatment procedure and detection techniques have been employed for the total determination of metals in olive oil. The researchers have dealt with metallic contents of olive oils during last few decades. As can be seen in Table 6, the concentration range of total amount is given for many metals.

Metal Determinations in Olive Oil 99

Fractionation was defined as "the process of classification of analyte or a group of analytes from a certain sample according to physical (e.g., size, solubility) or chemical (e.g., bonding, reactivity) properties", and speciation of an element was also defined as "distribution of an element amongst defined chemical species in a system" by Templeton et al. (2000). The physicochemical form of an element, i.e. the actual species found in exposure medium and in the different body fractions, is frequently determinant in the evaluation of its bioavailability and toxicity (Flaten, 2002). An element can be found in various species: anionic or cationic inorganic forms, inorganic compounds, complex compounds with protein, peptide etc. Some organometallic compounds are much more toxic than the ions of the corresponding inorganic compounds. Hg, Pb and Sn obey this rule, for example, methyl-Hg and inorganic Hg are both toxic, but methyl-Hg show more toxicity than other (Templeton et al., 2000). In contrast to this, in the case of As and Se, most organo-arsenicals are less toxic than inorganic As species, organic forms of Se are ordinarily less toxic than

The determination of the total amount of an element in samples cannot give adequate information for understanding its bioavailability or toxicity, that's why the fractionation and speciation of metals in oils are increasingly gaining importance. The fractionation and speciation analysis are more informative than total element determinations for all type of

In general, many works dealing with the total amount of elements in oil samples are reported, but fractionation and/or speciation analysis in vegetable oils are less common in literature. To the best of our knowledge, magnesium fractionation analysis in olive and olive oil was cited firstly in 2004. The improvement of an analytical scheme for fractionation of magnesium in olive products and also the determination of Mg amounts absorbed in stomach and intestine was achieved by Bağdat Yaşar & Güçer (2004). It was reported that 3.37-8.47% of Mg was absorbed in the stomach (ionic and polar groups) and the remaining percentage of Mg was absorbed in the intestine (molecular and complexed structures) in olive oil. As can be seen, the Mg fraction in olive oil is almost absorbable in the intestine. This study can be accepted as a preliminary step for fractionation studies and the fractionation and/or speciation approach for other elements will be described in

Various researchers deal with determination of metals in oils at trace, ultra-trace levels using spectrometric and electrometric techniques. Mentioned detection techniques may be combined with some chromatographic systems. Oils have been analyzed for different metals using atomic absorption spectrometer (FAAS and GFAAS), inductively coupled plasma optical emission spectrometer (ICP-OES), inductively coupled plasma mass spectrometer (ICP-MS). ICP techniques have become more popular since the early 1990s. Although the use of AAS (flame, graphite furnace, hydride generation and cold vapour) has declined during the same period, it is still the most widely used technique (Rose et al.,

**4. Speciation and fractionation**

Se(IV) (Kot & Namiesnik, 2000).

samples.

the future.

2001).

**5. Detection techniques**

Table 6. The metal levels for olive oils.


Improved Postharvest Techniques for Fruit Coatings

*Chalermchai Wongs-Aree, Hanh Thi Nguyen and Sompoch Noichinda*

## **Abstract**

Fruits, particularly tropical fruits, have a high moisture content, distinct morphological characteristics, and physiological changes, all of which contribute to their high rate of perishability. Nonetheless, their organoleptic and nutritional qualities make them one of the most important horticultural products. Fruit coating, which imitates natural packaging, is a postharvest solution that is practical and cost-effective for a variety of applications, including on-shelf display, transportation, and storage in support of the supply chain of fruits and vegetables. Gas and moisture permeability, microbiological resistance, and esthetic enhancement are the coating functions. Using modified materials and procedures, edible coatings for fresh and freshly cut fruits are currently being developed. Edible coatings infused with essential oils or volatiles may help to prevent disease resistance while also providing consumers with a fragrant preference. When considering how to advance fruit coating technology when agricultural wastes are the primary source of new coating materials, composite coatings, nanoparticles, encapsulation, and multiple-layer coatings all hold a great deal of promise. Future research may center on the optimal material for particular fruits during the logistics phase.

**Keywords:** edible coating, modified materials, modified techniques, quality maintenance, hypoxia, tropical fruits

### **1. Introduction**

Fruits, especially tropical fruits, are highly perishable and quickly lose their quality after being picked. Fruit postharvest loss in tropical areas could be up to 50%. The high level of physiological changes that occur in fruit is mostly attributable to the high level of moisture content [1]. The amount of water that evaporates from fruit is a significant factor in determining both the quality of the fruit and the economic quantity losses [2]. As a result, packaging that includes coating has the potential to successfully limit the loss of water from the fruit that has been stored. On the other hand, standard packaging might not be appropriate for certain fruits or environments. For instance, the sharp spines of a durian can easily puncture the plastic film, or the headspace in the package can accumulate condensed water and cause the fruit to rot. Edible coatings are a simple kind of fruit packaging that is applied to individual fruits in order to facilitate easier postharvest handling [3]. However, for fruit coating to be successful, it is necessary to consider not just different coating processes but also the characteristics of the fruit itself.

#### **2. Background related to fruit-based coatings**

The extracellular barrier of cuticles protects the plant's surface and interior from environmental stresses. The cuticle, composed of polysaccharides and a lipid matrix termed "cutin" [4], can dramatically transform the epidermis's outer cell wall. Many factors are essential determinants of shelf life and storage capacity, including the cuticle's moderating effect on water transpiration, fruit dehydration, and vulnerability to rots, pests, and diseases. Fruit cuticles have been proven to be highly responsive to environmental factors, and their quality changes even after harvest. When a fruit matures, the cuticle grows thicker [5]. Cutin polymers can be formed from long-chain fatty acids (C20-C34) but are typically made up of esterified and oxygenated C16 and C18 fatty acids [6]. It may also contain trace amounts of glycerol, phenylpropanoids, primary and secondary alkanes, alcohols, aldehydes, ketones, etc. [7]. Fatty acids are essential for the biosynthesis of cutin and wax, which occurs primarily in chloroplasts [8]. The components of wax extracted from the fruit surface of guavas comprise fatty acids and primary alcohols as the major components, followed by sterols, *n*-alkanes, and aldehydes. Interestingly, the wax also contains triterpenoids, a natural pesticide against insects and pathogenic fungi [9]. Thus, understanding the components and function of the cuticle should be useful in improving the coating materials used in fresh fruit markets.

Fresh fruits are perishable due to their quick deterioration due to dehydration, softening, discoloration, microbiological decay, disorder, and loss of nutrients [1]. These properties result from the metabolic processes occurring within the fruit, which are accelerated mainly by an improper relative humidity and gas composition in the storage atmosphere. High water loss and rapid metabolic changes associated with ripening and senescence are the primary causes of fresh fruit deterioration after harvest [10–13]. Artificial coatings mimic the protective properties of the fruit's natural cuticle and can postpone ripening and senescence by acting as a barrier to water and gases. Currently, edible coatings that are safe for food or handling provide a sustainable and effective solution for preserving the high quality of fruit throughout the postharvest value chain [3]. It is generally accepted that edible coatings as a type of MAP (modified atmosphere packaging) add value to fruits by shielding them from contamination, improving their esthetics, and preventing the loss of flavorful volatiles during preparation and storage (**Figure 1**). For the success of a fruit coating business, a thorough understanding of fruit characteristics, nature, respiration, and transpiration behavior is essential.

#### **2.1 Fruit types**

Three distinct fruit categories can be identified based on their floral structure: simple fruit, aggregation fruit, and multiple fruit. Some fruits, like the papaya, the rose apple, and the chili, have an internal cavity despite having a variety of morphologies. In addition, some fruits produce an inner supplementary tissue called aril (mangosteen, durian). Fruits that are either an aggregate (sugar apple, strawberry) or multiple fruit (pineapple, jackfruit) are made up of many fruitlets that have fused

**Figure 1.** *Fruit coating on mature green "Nam Dok Mai" mango during air drying.*

together. Variations in fruitlet maturation might cause uncertain ripening of the entire fruit [14]. Fruit coating could improve the ripening quality of the uncertain ripening by maintaining moisture and gas diffusion in the whole fruit.

### **2.2 Fruit structure/fruit parts**

Dermal tissues (peel), cortical tissues (pulp), vascular bundles (veins), seeds, and intercellular spaces make up the anatomy of most fruits. The peel consists primarily of parenchyma, of which some are transformed into guard cells (**Figure 2A**) and the lenticular opening channel (**Figure 2B**). Fruit stomata and lenticels are responsible for gas and water vapor exchanges, which could be an issue for fruit coating. In rambutan (*Nephilium lappaceum* L.), the epidermal tissues, spinterns, include up to 100–200 apertures/mm2 of stomata [10] that are constantly open [15]. Thus, stomata on spinterns that connect up to 20 groups of vascular bundles in the mesocarp are primarily responsible for moisture loss from rambutan fruit (**Figure 2A**). Replacement of water lost by spinterns with water from the skin [16]. The aril is still edible, despite water loss from the pericarp, producing skin withering, spintern drying, and pericarp browning (**Figure 3**).

Vascular bundles (phloem and xylem tissues) are gathered at the fruit's stylar end (peduncle/pedicel) that connects the fruit to the mother plant. In tangerine fruit, the vascular bundles start from the stylar end through the albedo mesocarp flavedo under the peel (**Figure 4**). The maturing fruit's peel is typically covered with the cuticle. Consequently, the cut peduncle is where the fruit quickly loses its moisture to the air after being harvested. Fruit edible flesh could be derived from the ovary wall, some accessory organs (receptacle and petal), or the unique tissue of "aril" covering the seed, which comprises a majority of parenchymal cells carrying high water content, stored chemicals, and biological metabolisms. Furthermore, in some fruits, there is a fruit cavity (papaya, rose apple) or seed cavity (mango, apple) that is a gas container in the fruit. The fruit's intercellular spaces and cavity (**Figure 2B**) play crucial roles in the fruit's ability to transpire and respire. The gaseous atmosphere in the intercellular

#### **Figure 2.**

*Anatomical structure of cross-sectioned rambutan fruit showing spintern stomata and vascular bundle networks (A) and cross-sectioned papaya fruit showing lenticel and pericarp tissues (B).*

#### **Figure 3.**

*Fruit pericarps and arils of ordinary fruit (left) and dehydrated fruit (right).*

spaces and cavity can be modified depending on the respiration rate and metabolism of the cells. The portions of intercellular spaces in several tropical fruits are shown in **Table 1**. Moreover, in fruit cavities, such as papaya, the different varieties exhibit varying volumes of cavities [17]. Intercellular spaces are important for cellular modification under hypoxic conditions [18]. Thus, the higher portion of intercellular air spaces is more tolerant to hypoxic conditions caused by fruit coatings. For example, apples and rose apples are tolerant to hypoxia from fruit coatings, in contrast to tangerines and guavas, which contain tiny intercellular spaces. Excessive CO2 from respiration could accumulate in the room to reduce cellular toxicity, or O2 demand can be taken from the portions to delay hypoxic cellular conditions. Coating material types and concentrations must be carefully considered. An off-flavor may result if a wax coating effectively prevents water loss from fruits with few intercellular spaces, such as tangerines. Thus, the modulation of the proper coating formula for each fruit depends on the fruit characteristics of each cultivar.

*Improved Postharvest Techniques for Fruit Coatings DOI: http://dx.doi.org/10.5772/intechopen.110099*

#### **Figure 4.**

*Fruit structure of tangerine fruit when cross-sectioned (left) and longitudinally sectioned (right).*

#### **Table 1.**

*Percentages of intercellular spaces in some tropical fruits (own data).*

#### **2.3 Maturity, respiration, and ethylene production characters**

Fruits are classified as climacteric or non-climacteric based on their respiratory and ethylene production patterns during maturation. The success of fruit coatings depends on the fruit's maturation, transpiration, and respiration rates. After fruit setting and during fruit growth due to cell division, the respiration rate is at its highest; it then gradually decreases to its lowest point during the early stage of fruit maturation. During ripening, respiration and ethylene production rates in climacteric fruits rise sharply, peak, and drop off, while both rates are not apparent in non-climacteric fruits. Storage temperatures play important roles in the respiration (**Table 2**) and ethylene production rates of fruits (**Table 3**) [19]. When fruit is coated with exogenous waxes, natural gases, and humidity, exchanges between the fruit and its respective microclimates are disrupted. The respiration, ethylene production, and gas permeability of the coated fruit will alter the gas concentrations within the cellular fruit, decreasing O2 and increasing CO2 and C2H4. By measuring the concentration of gases in the fruit's intercellular spaces and/or cavity, the fruit gets into an equilibrium of internal gases (**Figure 5**). Ethylene and CO2 accumulation at different levels in "Solo" papaya during on-tree ripening [20] are shown in **Table 4**.

Many tropical fruits, such as mangosteen, longan, longkong, rambutan, durian, and salah, contain two parts: the pericarp (peel) and aril (flesh), which individually develop during fruit maturation. The aril of some tropical fruits is derived either from the funiculus (durian and lychee) or integument (mangosteen and rambutan) of the seed [14]. During maturation and ripening, the pericarp and the aril mature

#### **Table 2.**

*Respiration rates of some tropical fruits at 13°C and 25°C (adopted from Kosiyachinda and Tansiriyakul [19]).*

#### **Table 3.**

*Ethylene production rates of some tropical fruits at 13°C and 25°C (adopted from Kosiyachinda and Tansiriyakul [19]).*

independently at different levels. Fruits like the mango, whose flesh develops from the ovary wall, soften, change color, and release their natural aromas as they ripen regularly and consistently. However, the pericarp (dusk) of the durian fruit releases most of the climacteric ethylene during whole fruit ripening, which in turn causes the aril to ripen. Double climacteric blooming occurs in several cultivars (fruit ripening and dehiscence). The respiration and ethylene production rates of durian pulp during ripening are much lower than those from the pericarp [21]. The endogenous ethylene produced in the husk is required for whole fruit and pulp ripening [22]. Thus, a coating of fresh-cut durian must undergo proper ripening before husk removal because unripe pulp often fails to ripen regularly [23]. Interestingly, stages of maturity are crucial for fruit coating. In "Nam Dok Mai" mango, for example, the mature green fruit was induced to get anaerobic conditions and produce off flavor at a high concentration of composite coating, but the treated fruit was typical when the ripe fruit was coated at the same concentration [24].

### **3. Basic materials for fruit coatings**

After harvesting, coating fruits with shellac or fat-related substances was a frequent procedure in the past. Later, fat-based plant extracts from bananas, pineapple leaves,

*Improved Postharvest Techniques for Fruit Coatings DOI: http://dx.doi.org/10.5772/intechopen.110099*

#### **Figure 5.**

*Dynamic and metabolism changes in coated fruits and the surroundings.*

#### **Table 4.**

*Internal CO2 and C2H4 concentrations in the fruit cavity of "solo" papaya during fruit ripening (adopted from Akamine and Goo [20]).*

and carnauba trees were utilized. In contrast, coating materials often consist of a single complex molecule that is effective at preventing water loss but has poor gas exchange. Therefore, it is vital to understand the composition before searching for an acceptable covering. There are three major chemical properties of edible coatings.

#### **3.1 Polysaccharides**

The polysaccharide-based coating comes from modified or extracted polysaccharides from natural products, typically including many starches from plants (corn, wheat, rice, cassava, and potato), glucomannan, galactomannan, inulin, plant cellulose and pectin, plant gum, alginate from brown seaweeds, pullulan from the fungus *Aureobasidium pullulans*, and chitosan from shrimp. The edible film made of polysaccharides is transparent, strong mechanically, and impervious to lipids. From those

materials, chitosan, a plant disease elicitor, has been studied for coating many fruits. With stored longans (*Dimocarpus longan* Lour. cv. "Daw"), chitosan coating at 1.0% and 1.5% revealed a delay in pericarp browning and an effective retardant of disease growth with less than 4% disease incidence at 4°C [25]. Gac fruit (*Momordica cochinchinensis* Spreng) at the yellow stage coated with 0.5%, and 1.0% chitosan delayed fungal infection and enhanced fruit appearance [26]. Chitosan coatings have been successfully used to maintain the quality of many tropical fruits such as pineapple [27], banana [28], and papaya [29].

The polysaccharide-based coating is useful for coating freshly cut fruits because it can prevent fat remnants from leaking out. However, the constraint of the polysaccharide coatings is a lack of control over water loss and O2 and CO2 exchanges [30]. Moreover, if the logistics of handling fresh produce involve a high-temperature fluctuation, or if the fresh produce is immediately removed from the cold storage and placed at room temperature, the coating may be in a reversible phase, resulting in the peeling of the coating from condensed water droplets on the surface. Researchers have attempted to create an edible polysaccharide coating or film using antioxidants from plants. Typically, this is done to enhance the physical and chemical properties of the film and coating for safe consumption.

#### **3.2 Proteins/oligo peptides**

Proteins from plants, animals, and microorganisms are biopolymers that can be utilized to make films with tunable physical and functional properties when mixed with plasticizers or other components. The proteins such as gelatin, casein, collagen, whey proteins, egg white, etc. have been studied. Covalent unions (side chain cross-linking) and electrostatic or ionic interactions between protein chains contribute to coating formation [31]. Protein films' mechanical and hydrophobic barrier properties, and hence their suitability for food packaging applications, are strongly influenced by the final chain contacts and bonds. The major bonding processes are controlled by production conditions such as pH, salt addition, heating, enzyme action, drying, and reactions to food-grade chemicals [32]. Protein films have been enhanced with antimicrobials and antioxidants, among other active compounds. Some protein extracts have been studied for fruit coatings. Park et al. [33] used a corn-zein coating to delay fruit ripening in tomato fruit, while Avena-Bustillos et al. [34] used a casein (milk) protein coating to reduce weight loss in zucchini. Furthermore, zein and gelatin coatings could delay ripening in mangoes stored at 32°C [35].

Although the protein-based coating belongs to the hydrophilic group, the covalent cross-links, and electrostatic networks boost the coating's structural stability, limiting the coating's reversible phase when droplets condense on the surface during logistics.

#### **3.3 Lipids**

Fatty acid derivatives, as a significant component of the natural cuticle, play an important role in preventing water loss and gas exchanges in fruits. The properties of flexibility, hydrophobicity, and cohesion that edible films require are provided by lipids [36]. The quality of fruits can be maintained by edible coatings made of lipids, which are effective barriers against moisture, O2, and CO2, but not C2H4. Edible coatings such as carnauba wax, shellac, bee wax, and some plant oils based on lipids can cover fruits and vegetables.  Shellac and carnauba have long been applied to postharvest fruits. Ten percent shellac coating prevented fresh weight loss and disease infection, and reduced the respiration and ethylene production rates of gac fruit at 25°C storage, while 15% shellac coating led to a high accumulation of acetaldehyde since day nine [37]. Shellac and carnauba emulsions were coated on "Nova" mandarins (*Citrus reticulata*) at 20°C storage. The carnauba waxes resulted in minor weight loss compared to the uncoated control and shellac coating, but shellac-coated fruit showed the highest fruit shine. The highest levels of CO2 and the lowest level of O2 were found in shellac-coated fruit, resulting in the highest ethanol content in the juice due to induced anaerobic respiration [38].

Many lipid-based coatings can provide the best water transpiration prevention due mainly to their strong hydrophobic properties, but for fresh produce, the switch from aerobic to anaerobic respiration caused by too low O2 and/or high CO2 in the coated fruit must be considered. Although long fatty acid derivatives are the most abundant in natural cuticles, attachment to sterols, terpenoids, polysaccharides, and phenolics may modify the complex structure, increasing gas exchange permeability.


**Author details**

Fernanda-Isadora Corona-Meraz1,2, Jesus-Aureliano Robles-De Anda1,3, Perla-Monserrat Madrigal-Ruiz1 , Gustavo-Ignacio Díaz-Rubio3 , Jorge Castro-Albarrán1,4 and Rosa-Elena Navarro-Hernández1 \*

1 UDG-CA-701, Research Group on Immunometabolism and Complex Diseases, Department of Molecular Biology and Genomics, Health Sciences School, University of Guadalajara, Guadalajara, Jalisco, Mexico

2 Department of Biomedical Sciences, Health Sciences Division, CUTonalá, University of Guadalajara, Tonalá, Jalisco, Mexico

3 Doctoral Program in Molecular Sciences in Medicine, Department of Molecular Biology and Genomics, Health Sciences School, University of Guadalajara, Guadalajara, Jalisco, Mexico

4 Department of Health Sciences and Human Ecology, Regional Development Division, CUCSur, University of Guadalajara, Autlán de Navarro, Jalisco, Mexico

\*Address all correspondence to<EMAIL_ADDRESS>
© 2020 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**27**

*Adipose Tissue in Health and Disease*

**References**

*DOI: http://dx.doi.org/10.5772/intechopen.90559*

[1] Ross M, Wojciech P. Connective tissue. In: Histology: A Text and Atlas with Correlated Cell and Molecular Biology. Philadelphia: Wolters Kluwer/Lippincott Williams & Wilkins Health; 2015. p. 992

of adipose tissue. Cell Metabolism.

in health and disease. Trends in Pharmacological Sciences.

[12] Stenkula KG, Erlanson-Albertsson C. Adipose cell size: Importance in health and disease. American Journal of Physiology.

[11] Fasshauer M, Bluher M. Adipokines

Regulatory, Integrative and Comparative Physiology. 2018;**315**(2):R284-r295

[13] Goossens GH.

Obesity Facts. 2017;**10**(3):207-215

[15] Chang E, Varghese M,

Reports. 2018;**18**(9):69

2010;**72**(2):176-188

Springer; 2007. pp. 3-19

2012;**21**(11):827-830

Experimental Dermatology.

[19] Driskell RR et al. Defining dermal adipose tissue. Experimental Dermatology. 2014;**23**(9):629-631

[17] Totowa AO. In: Mazzone T, Fantuzzi G, editors. Nutrition and Health: Adipose Tissue and Adipokines in Health and Disease. Chicago, Il, USA:

[18] Schmidt B, Horsley V. Unravelling hair follicle-adipocyte communication.

2018;**25**(1):89

[14] Fitzgerald SJ et al. A new approach to study the sex differences in adipose tissue. Journal of Biomedical Science.

Singer K. Gender and sex differences in adipose tissue. Current Diabetes

[16] Blouin K et al. Effects of androgens on adipocyte differentiation and adipose tissue explant metabolism in men and women. Clinical Endocrinology.

2018;**27**(1):68-83

2015;**36**(7):461-470

[2] Haylett WL, Ferris WF. Adipocyteprogenitor cell communication that influences adipogenesis. Cellular and Molecular Life Sciences. 2019. Available from: https://doi.org/10.1007/

[3] Russo L, Lumeng CN. Properties and functions of adipose tissue macrophages in obesity. Immunology.

[4] Martinez-Santibanez G, Lumeng CN. Macrophages and the regulation of adipose tissue remodeling. Annual Review of Nutrition. 2014;**34**:57-76

[5] Khan T et al. Metabolic dysregulation and adipose tissue fibrosis: Role of collagen VI. Molecular and Cellular Biology. 2009;**29**(6):1575-1591

[6] Pasarica M et al. Adipose tissue collagen VI in obesity. The Journal of Clinical Endocrinology and Metabolism.

[7] Orr JS, Kennedy AJ, Hasty AH. Isolation of adipose tissue immune cells. Journal of Visualized Experiments.

[8] Rodriguez A et al. Revisiting the adipocyte: A model for integration of cytokine signaling in the regulation of energy metabolism. American Journal of Physiology. Endocrinology and Metabolism. 2015;**309**(8):E691-E714

[9] Ross M, Wojciech P. Adipose tissue. In: Histology: A Text and Atlas with Correlated Cell and Molecular Biology. Philadelphia: Wolters Kluwer/Lippincott Williams & Wilkins Health; 2015. p. 992

[10] Zwick RK et al. Anatomical, physiological, and functional diversity

2009;**94**(12):5155-5162

2013;**75**:e50707

s00018-019-03256-5

2018;**155**(4):407-417

*Adipose Tissue in Health and Disease DOI: http://dx.doi.org/10.5772/intechopen.90559*


**2. Secondary metabolites**

Secondary metabolites can be found in abundance in plants. They are the focus of several research since they have a wide range of biological actions. Scientific reports have demonstrated the medicinal value of various parts of plant species (*C. odorata*, *C. oblongifolius*, *T. cordifolia*, *M. malabathricum*, and *D. bulbifera*) against a wide range of human ailments. Only the cytotoxicity of various secondary metabolites from different plant species will be discussed in this study.