Fruits and vegetables are living tissues and well protected by their epidermis with water, gas, oil and bacteria barrier until the time they are cooked, consumed or processed. Minimally processed procedure like fresh cut can introduce tissue browning, softening and microbial problems to wounded surface and finally lead to the rejection from the consumer. The intensity of wounded response is influenced by various factors such as O2/CO2, enzymes, water vapor transfer, microbes, and the presence of inhibitors (Fig. 1). The concept of using edible coatings is to prevent fruit from exposing to a host of conditions that can cause changes in food quality and safety. To be specific, edible coatings can provide gas, water and bacterial barrier and result reducing browning rate, texture breakdown and bacterial problems[2-5].
Fig. 1Functional properties of an edible coating on fresh fruits and vegetables.Despite basic functionality of reducing physiological response, edible coatings can be designed to carry bioactive and functional ingredients such as antioxidants, nutrients and flavors to further enhance food quality and functionality. However, current edible coatings have limited uses due to their physical characteristics. For example, polysaccharide-based coatings is good gas barrier in high moisture environment but dry air can cause less effective protection or even peeling off from the surface of fruits. There is therefore a need to design novel multifunction edible coatings that can overcome shortcomings of conventional edible coatings.The layer-by-layer edible coatings are multifunction coatings that are fabricated by controlling of electronic deposition between different layers so it is possible to incorporate different active agents and nutriecuticals[6, 7] to protect minimally processed fruit and vegetables from environmental threats (water, O2 and bacteria).
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The overall objectiveof this project is to develop and characterize layer-by-layer edible coatings that could be used to protect minimally processed fruit. In particular, by controlling the chemical composition, thickness, number, orders of layers, it is possible to promote the antibrowning, antimicrobial and texture-maintaining capability of fruit and vegetables and offer extra healthy benefits by bioactive compounds. The proposed research is an important part of our long-term goal topromote the quality and safety of minimally processed fresh fruit and provide extra nutritional benefits. Our central hypothesisis that during storage,Layer-by-Layerbased edible coatings can protect the minimally processed fresh fruit from microbial threats and physiological response and add nutritional value on fruits and vegetables.
A.2. Specific Aims
Specific Aim 1:Characterization of properties of surface of minimally processed fresh fruit: The fabricated conditions of layer-by-layer edible coatings highly relies on properties of surface of minimally processed fruittherefore theircharacteristics will be studied (e.g., pH, charge, hydrophobicity and morphology), moreover, physical response of fresh-cut surface will also be investigated (e.g., browning rate, microbial contamination and texture change) to design Layer-by-Layer edible coatings.
Specific Aim 2:Development of methodology and characterization of Layer-by-Layer edible coatings:The fabrication procedures of layer-by-layer edible coatings are developed based on the principle of electronic deposition. The characteristics of multifunction edible coatings(e.g., thickness, hydrophobicity, antimicrobial activity, roughness, surface chemical structure)areexaminedby using modified silicon wafer as the replacement of fresh-cut fruit surface.
Specific Aim 3:Demonstration of protective capabilityof Layer-by-Layeredible coatings on minimally processed fruit:The potential for utilization of multilayers edible coating will be demonstrated by applying them to the actual fruit slice (e.g., apple slice). The functionality of layer-by-layer edible coatingswill be studied and the sensory test will also be performed by panelist to evaluate the acceptance of coated fruit.
A.3. Rational and Significance
According to the market report form USDA, the sales of production of fresh fruit and vegetables (e.g., fruit cups and salad mix) reached $9.4 billion in the U.S. market in 2011. Although, many conventional edible coatings are available for fruits and vegetables, the development of multifunction coatings has not been prevalent.Therefore, the Rationalfor this research is that various biopolymers and active agents (e.g., proteins, emulsion droplets and polysaccharides) can fabricate multiple protective layers onminimally processed fruit'ssurface by electrostatic deposition principle. Despite basic protective functionality (anti-browning,microbial barrier and texture enhancer), Layer-by-Layercan also provide extra nutritional benefits due to the incorporation of bioactive compounds.The significanceof the proposed research is thatit would provide fundamental knowledge that is currently lacking about the modulation of minimally processed fruit with multifunction edible coatings. This knowledge is critical for the development of food-based strategies to improve the quality and safety ofminimally processed fruit.
B. Background & Context
B.1. Physiological Properties of Minimally Processed Fresh Fruit and Vegetables
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Fruits play important role in human dietary by providing vitamins, minerals, and other nutraceuticals. Peels of fruits are protective layers that can prevent internal dedicated tissue from the influence of external factors such as bacteria, oxygen and water. But sometimes, this natural protection cause inconvenience for consumption therefore comparable fresh fruit product with minimally processed procedures like fresh-cut become welcomed in the grocery market. The quality and safety of these ready to eat products are commonly attributed to their marketability of fresh, appearance, color, texture, flavor, nutritional value and microbial safety that can generally be concluded as three issues: appearance, texture and safety.
Appearance.The change of appearance is a general but most direct index for consumers to evaluate the quality and safety of minimally processed fruit. The cut surface can introduce unstable changes in colorand appearance during storage and marketing. This phenomenon is caused by the reaction between the oxygen and the enzyme polyphenol oxidase (PPO). The presence of oxygen can convert the phenolic compounds into dark colored pigment that is recognized as "browning". Some studies have been done to reduce the browning rate as the inhibition of PPO activity or rendering a medium inadequate for the development of the browning reaction (modifying the storage atmosphere and removing phenolic compounds) or by adding another preferred substrate avoiding the formation of dark pigments.
Texture. It is a very important indicator for consumer to evaluate the quality and safety of a minimally processed fruit product that can be explained in terms as firmness, crispness, crunchiness and juiciness. The change of texture is a developing process that is caused by dehydration, respiration and microbial contamination. Many studied focused on the modification of storage conditions such as modified atmosphere package (MAP) that can reduce the respiration rate, or using texture enhancer like CaCl2which can interact with pectic polymers to form a cross-linked network that increases mechanical strength[12, 13]. However, edible coatings like alginate and chitosan with good gas and water barrier properties can provide effective protection to the minimally processed fruits, further, the texture enhancer can also incorporate with active agents of edible coatings such as chitosan to control physiological disorders in fresh-cut fruit.
Safety.It largely relies on the proper sanitation and handling procedures. Minimally processed fruits are more perishable than their whole uncut commodities due to wounded surface during processing. The exposure of cut surface can easily be contaminated by pathogens and spoilage bacteria therefore the application of antimicrobial coatings directly on the cut surface can provide most effective inhibition of microbial growth than other mechanical protections such as MAP. Currently, several kinds of antimicrobials can be potentially incorporated into edible coatings including polysaccharides (cellulous, alginate, chitosan, carrageenan), proteins(WPI, lysozyme, nisin), lipid-based agents (essential oil, bee wax) and others (antioxidants like Vc and VE). In addition, other factors like flavor, aroma and nutritional value also associate with the quality and safety of minimally processed fruit.
B.2. Principle of Layer-by-Layer
L2 (+)Layer-by-Layer is fabricated by depositing alternating layers of oppositely charged materials. In the Layer-by-Layer electrostatic deposition, a polyelectrolyte layer is formed on the two oppositely charged surface due to the strong oppositely charged droplets in solution, moreover, this outer layer is still charged because the total number of charges on the adsorbed surface is greater than required number of neutralization leading to reversal charge. This overconsumption is important because it can provide electronic repulsion force to stabilize the formation of monolayer structure and also ready for further absorption of oppositely charged polyelectrolytes on the top of the first layer, e.g., S-L1-L2, where S is the substrate, and L1 and L2 are two oppositely charged polyelectrolytes. Repetition ofbothadsorptionsteps leads to the formationofmultilayer structures: S-[L1-L2]n-L1-L2 or S-[L1-L2]n-L1. The polyelectrolyte that forms the outer layer usually determinesthe net charge on the overall interface(Fig. 2).
Fig. 2Schematic structure of Layer-by-Layer based on electronic deposition
Fig. 3Formation of multilayer emulsions based on the
Proteins (e.g., WPI, beta-lactoglobulin and lactoferrin) and some of hydrophilic polysaccharides (e.g., chitosan and alginate) are amphiphilic molecules with charges therefore they have good surface activity and can against environmental stress such as temperature, ionic strength and pH. For example, charged protein can either dissolve or anchor on the surface of substrate such as lipid droplets and form the original layer, then oppositely charged hydrophilicpolysaccharides can absorbon protein surface and the large steric force of polysaccharides can help to stabilize the structure. .
B.3. Definition and Composition of Edible Coatings
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Edible coating is a thin layer of multiple films containing edible materials that can cover the surface of food and can be eaten as part of the whole product. Therefore, the composition of edible coatings must be confirmed to the regulations of GSFA that apply to the food product concerned (http://www.codexalimentarius.net/gsfaonline/additives/index.html).According to their composition, edible coatings can be classified as hydrocolloids, lipids and composites. Hydrocolloids include protein and polysaccharides; lipids contain wax, fatty acid ester, glyceride and essential oil and composites contain both of hydrocolloid components and lipids such as protein-coated-lipid droplets (emulsions), so the combination can result enhancing advantages and lessening disadvantages of each.
Proteins can be used in the formation of edible coatings including animal resource (e.g., casein, whey protein) and plan resource (e.g., zein, soy protein). They exhibit good inhibition of oxygen, dioxide and lipids especially at low relative humility (RH), but not in high RH environment due to their hydrophilic nature. Plasticizers (e.g., glycerol, sorbitol) are commonly added into protein based edible coatings to form a flexible and extensible coating, because they can preventcoatings from brittle and crackingwith reduced cohesive energy densityof proteins. Heat denaturation can also produce insoluble protein coatings as a consequence of formation of intermolecular disulfide bonds [19, 20]. Proteins such as whey protein concentrated have also been evidenced as a inhibitor of enzymatic browning. Proteins with thoil-containing compounds can competitively combine with oxidative species such as quenions formed during the initial phase of enzymatic browning reactions and finally result reduced formation of colored compounds.
Polysaccharides are most widely used compounds for edible coatings on minimally processed fruits as they are inexpensive, biodegradable and easy to use. The main polysaccharides apply in edible coating including cellulose, chitosan, alginate, pectin and other modified starches. The formation ofpolysaccharides flexible film on the fruit surfacecan the internal atmosphere and reduce the respiration of plant tissue resulting extended shelf-life. So they all show better gas barrier properties rather than a water loss barrier due to their highly hydrophilic nature. However, there are some certain kinds of polysaccharides, in form of high-moisture gelatinous coatings, can offer effective moisture protection as a sacrificing agents.Some of polysaccharides can also be utilized to against pathogens or spoilage bacteria. Chitosan, extracted from shells of shrimp, is good gas barrier and also has a broad antimicrobial agent. It can produce a plant-defense enzyme, chitinase, in plant tissues which degrades microbial cell walls[23-25]. Researches show that chitosan coating can significantly decrease the spore germination, germ tube elongation, and growth of pathogens;further, its antimicrobial capability can be boosted by the incorporating with essential oils and wax[26, 27].
Emulsionis a kind of composites coating. With relatively uniformed particle size, emulsion coated edible coating can evenly distribution on the surface. Although they act less effective as a water vapor barrier, emulsion based edible coating gain increasing attention form the food industry due to one step procedure and extended characteristics. Emulsionsis formed by mixing of two immiscible phase (e.g., aqueous and lipid) with the function of emulsifer. Lipids can provide good moisture barrier with their hydrophobicity but thisnonpolymeric characteristic also limits the formation of cohesive coating on the surface of minimally processed fruit[28, 29]. Emulsifiers in the aqueous phase can decrease the Gibbs Energy of lipid droplets and form a hydrophilic out layer that allow emulsions to absorb on the hydrophilic fresh cut surface. In this way, emulsion based edible coating have combined advantages of lipid (e.g., low water vapor permeability) and emulsifier such as protein and polysaccharides (e.g., gas, microbial barrier). Recently studies also show that the water permeability of emulsion coatings is decreased with increased lipid concentration and the moisture barrier can also be improved with long hydrocarbon chain length of fatty acid alcohols and monoglycerides. In addition, the lipid polarity can also influence on the water vapor barrier of emulsion-based coatings.
Nutraceticalsincludes essential oil, vitamins and other nutritional ingredients commonly incorporate with main edible coating agents such as lipid, proteins and polysaccharides. Recent researches evidenced that essential oil can penetrate the cell membranes and enter mitochondira of bacteria leading to the disordering of physiological response and damaging the internal structure. In addition, proteins as the main ingredients of edible coatings can also have additional benefits. Lactoferrin from milk proteins is an antioxidant with antimicrobial capability by the function of ferric in the tertiary structure. Despite antimicrobial function, polysaccharides are effective gas barrier as well as a good resource of dietary fiber in our daily life; Vitamins such as Vc and VE are important antioxidants can help to against browning by the competitive reaction with the oxidative species from initial browning phase;the enhanced flavor from essential oil can also increase the consumer acceptance and enhance the healthy benefit for minimally processed fruit.
B.4. Characterization of Edible Coatings
The functionality of edible coatings are determined and influenced by their physical properties such as thickness, appearance, wettability and bioactivity. So currently, several techniques are applied to characterize the edible coatings.
Thickness.The thickness of edible coating highly associates with its gas permeability. The direct method is to dry and caste coatings on a glass plate, then peel them form plate surface and record the thickness with a handhold micrometer. Although this direct method is easy to handle and widely used, it is difficult to get accurate number, especially when coatings are applied to fruit surface. Ellipisometry is a sensitive measurement technique and provides unequalled capabilities for thin film metrology, so recently, ellipisometry become a promising technique in characterization of the thickness of edible coatings. The analysis is based on the change of polarization on reflection or transmission that is determined by the film's properties (thickness, complex refractive index or dielectric function tensor). The minimum scale of ellipisometry can be as low as a nanometer therefore currently it is widely used in semiconductor and material science[35, 36]. On the other hand, different kinds of microscopy techniques can also be used such as confocal Raman microspectrometry (CRM), surface enhanced Raman scattering (SERS) and fourier transform(FT)-Raman spectrometer.
Appearance.The appearance of minimally processed fruit directly influence the behavior of consumer by their visual evaluation, therefore the change of appearance of fruit should be minimized. As layers of coatings, color and gloss are key parameters to evaluate the change of appearance of minimally processed fruit. The change of color can be detected by using colorimeter which calculate chromatic parameters (L*,a*,b*) to reflect general appearance of fruit. The gloss of coated fruits can be measured using a flat surface glossmeter. The result are reported in gloss units (from 0 to 100) relative to a highly polished planar surface of black glass, which serves as a standard and is usually arbitrarily assigned with a gloss value of 100.The change of gloss is easily observed in emulsion based edible coatings therefore it is necessary to form transparent emulsions by using refractive index matching or fabricating nanoemulsions (d<wavelength).In addition, the information ofmorphology and arrange of edible coatings' surface on micron scale is obtained by applyingscanning electron microscopy (SEM), confocal and profilometry. The roughness of coatings can be scanned by atom force microscopy (AFM)and the scanning image can demonstrate the inter- and intra- interaction between layers.
Wettability. The wettability is evaluated by contact angle which is determined water and oil barrier and cohesive properties of edible coatings.By spreading coatings ingredients on glass plate, the wettabilityis analyzed. The equilibrium advancing and receding angle of a surface can present the hydrophobicity of material that the large contact angle indicates hydrophilic property. Therefore the balance between hydrophobicity and hydrophily of coatings need to be reached to gain good water resistance and well cohesive property. This method is suitable for all surfaces either whole fruit or well-tailored fresh-cut fruit. The final aim of decreasing wettability is to reduce the weight loss of fruit. The direct measurement is to scale the weight loss under controlled temperature and relative humidity conditions before and after storage and then the difference is used to evaluate the function of edible coatings. But this method is more suitable for tailored and easily determined fresh fruit-cut rather than irregular-shaped whole fruit.
Texture & Sensory.The instrumental measurements of minimally processed fruit texture are used to reflect comprehensively protective effect of edible coatings. The texture test includes puncture, texture profile analysis (TPA), and compression test. The puncture test is the most widely used method to characterize the change of firmness;in TPA test, the fruit sample is compressed twice imitating the action of the jaw and the parameters obtained from this test correlate well with sensory ratings and the compression test is also an indicator of hardness change of fruit. For sensory analysis,sensory panelists evaluate samples by visual inspection of appearance, odor, taste and over quality and the result are analyzed by statistic method (ANVON).
Microbiological analysis.The most common way to determine the antimicrobial capability of formulated edible coatings is to measure the inhibited zone of coatings . Typical food pathogens (e.g., L.plantarum, S.enteritidis, E.coli O157:H7, L. monocytogenes and S.aureus) are used as model organism to demonstrate antimicrobial activity of coatings, because of their importance in food safety. The agents of edible coatings are placed on the bacterial lawns for 24h incubation at 37â„ƒ. Then the "zone of inhibition" of the substrate is examined by measuring with a caliper.The other method is to use stomacher bag to determine the number of colonies on the coating surface. Sample pieces are washed with buffer and diluted. The suspension are pour-plated with tryptic soy agar (TSA) in triplicate at 37â„ƒfor 48hours then enumerated as recording as CFU/g.
C. Preliminary Studies
C.1. Characterization of Biopolymers and Emulsions
Over the past decade, we have learned the knowledge to fabricate oil-in-water emulsions by using high-energy method. From previous studies, we have examined characteristics of biopolymers (various proteins and polysaccharides) such as pI, charge, surface activity, thermal and other physicochemical properties and applied them in fabrication of emulsions and multilayer emulsions. We have optimized conditions of formulation of emulsions and characterized their physicochemical properties such as charge, particle size, surface interfacial thickness and permeability[43-47]. In addition, antioxidant and antibacterial capability of emulsions and multilayer emulsions have also been investigated to further application. So far, we have also successfully encapsulated various kinds of lipophilic components within oil-in-water emulsions, such as antimicrobials, ï¢-carotene and lycopene[48, 49]. All these works can provide guidelines and basic elements to built layer-by-layer edible coatings.
C.2. Characterization of Layer-by-Layer Model
We previous learned physicochemical characteristics of ingredients such as proteins and polysaccharides that can impact the formulation of layer-by-layer coatings.Generally, Layer-by-Layer model is based on the electrostatic interactions between different layers but these interactions can be influenced and determined by environmental stresses such as pH and salt concentration and physicochemical characteristics of ingredients like pI and structure. According to the principle, oppositely charged agents are selected under certain pH, for example, at pH 4, in the absence of salt, chitosan is positive charge and can absorb on the surface of negatively charged pectin, then negative alginate can form second layer on chitosan layer and positively charged bLg emulsion droplets followed to form third layer and continuous layers can keep forming by this order, finally, theirmorphology, microstructure, charge and thickness are examined. We have applied various agents intoLayer-by-Layer model and evidenced that this structure can enhance their antimicrobial and antioxidant capability.Our lab previously found that bLg-lactoferrin-alginate coatings have increased antioxidant capability and retard the lipid oxidation.
D. Experimental Approach
D.1. Specific Aim 1: Characterization of properties of surface of minimally processed fresh fruit
The objective of this part of the project is to understand characteristics of surface of apple slice and its physiological responses during storage. The result of this work can be used as guidelines inmodification of silicon wafer as replacement substrate in formulation and characterization of Layer-by-Layer edible coating. Further, according to the result of physical response in this study, suitable active agents will be chosen to form multifunction edible coatings.Various techniques will be applied in this study.
D.1.1. Characterization of surface of fresh-cut apple slice
Processing of apple slice:The fruits will be sanitized in NaClO and rinsed with water and dried before cutting.The sample slice of apple (Fuji) will be cut as shown as in Fig. 4. Apple wedges prepared from equator parts of the apples, then the wedges will be further cut into rectangular prisms with (2cmÃ-1.5cmÃ-0.5cm) and washed with double distilled water.
pH:Flat surface stick pH meter will be applied to measure the surface of fresh cut apple slice.
Hydrophobicity: The surface hydrophobicity of fresh-cut apple slice calculated from contact anglemeasurement using a goniometer (Kruss,Hamburg, Germany). Volume of2.5Î¼L hexane will be applied at a rate of 15 Î¼Lmin-1 with automatic dispenser on the surface of fresh-cut apple surface with dimensions of 2.0Ã-1.5 cm2 at 25â„ƒ. Hysteresis was obtained from the difference between advancing and receding. A total of three replicates will be performed on one slice and three measurements are done in very sample (9 testsper replicate).
Charge: Apple slice will be tailored to fit the dip cell and glued to the sample holder (NanoZS, Malvern Instruments, Malvern, Worcs, England). After alignment, the dip cell will insert into cuvette with standard solution (NanoZS, Malvern Instruments, Malvern, Worcs, England) and the test is triplicated. Macintosh HD:private:var:folders:9g:xftfh0h12ml3rn62jg5kymtm0000gn:T:com.tencent.qq:QQ20120730-1.png
Morphology: Profilometryis a stereomicroscope that it can provide the 3D image of apple slice's porous surface.The surface and internal characteristics of apple slice will be scanned by confocal laser microscope according to the method report from Fan et.al. After submerging into water-soluble fluoreseindiacetate, apple tissue will be dyed and show fluorescein image(Fig. 5).
D.1.2. Physiological properties of apple slice
Fig 4. The preparation of apple slice (Fuji)Texture: Firmness of fresh-cut apple slice is evaluated with a Texture Analyzer (Texture Technologies Corp. Scarsadale, N.Y. U.S.A). The prepared apple slice will be placed in the load cell and probe of 6-mm diameter probe is used to penetrate the center of slice to 10mm at 10mm/s with triplication. Maximum force is record and the result is analysis by software.Macintosh HD:private:var:folders:9g:xftfh0h12ml3rn62jg5kymtm0000gn:T:com.tencent.qq:QQ20120805-1.png
Browning rate:The browning rate is related to the change of appearance. The change of color of apple slice will be analyzed with certain frequency (0, 1, 3, 5, 7, 14 days). The tristimuluscolor coordinates (L*, a*, b*) of the sample will be measured using ColorFlex EZ (ColorFlex EZ, Hunter Lab, Virginia, US). Samples will be placed into a transparent flat-faced plate and covered by white cape, and then the color is recorded. The result can be reported as browning index (BI):
Fig 5.Confocal image of apple surficial and internal tissue
Respiration rate:For minimally processed products, the major obstacle is the activated respiration of live tissue and synthesis of ethylene which increment will lead to the growth of aerobic microbe and accelerated softening so the result can be used to predict the shelf life of minimally processed fruit. Samples will be placed in a sealed container with controlled temperature and relative humility (RH). During storage, we will determine concentrations of CO2and ethylene to monitor respiration rate with aShimadzu GC-2014 gas chromatograph equipped with a Shimadzu AOC-5000 headspace auto-injector.
Weight loss: It is an importantindicator ofoverall quality change of fresh-cut apple slice, so samples will be weighted after storage and the weight difference is reported.
All measurements are repeated three times and results are analyzed by statistic methods.
D.2. Specific Aim 2: Development of Methodology and Characterization of Layer-by-Layer Edible Coatings
The objective of this part of work is to develop multifunction coatings based on Layer-by-Layer principles and characterize properties of layer-by-layer edible coatings. Modified silicon wafer is used as replacement of actual fruit slice, becauseit is relatively easy to formulate Layer-by-Layer coatings and characterize their properties. The result of this work can offer the information about the formation and characterization of Layer-by-Layer edible coatings. In addition, the result can also provide theoretical supportto practical application.
D.2.1. Modification and Characterization of the Modified Silicon Wafer
The easy characterized silicon wafer surface is applied to form and analyze the edible coatings instead of original tissue surface of fruit. Therefore, we will modify the surface of silicon wafer to mimic features of fruit slice such as highly hydrophilic and negative charge surface as shown in Section D.1.
Modification of Silicon Wafer: The modification of silicon wafer is based on the reaction betweenorganofuncionalsilane and silicon wafer (Fig. 6). The apple slice is a highly hydrophilic. According to this feature,suitable silanewill be chosen to formself-assembledmonolayer (SAM) on the silicon wafer. After sonication and washing steps with ethanol and acetone, UV/Ozone will be applied to generate hydroxyl group on silicon surface and 3-(trihydroxysilyl)-1-propanesulfonic acid (3TPS),which is a hydrophilic silane with low pKa (â‰ˆ1), will be used to form SAM on the surface of silicon wafer (Fig. 5). The hydrophobicityof modified silicon wafer will bedetermined by equilibrium contact angle including advancing and recedingbya goniometer (Kruss, Hamburg, Germany).
Fig 5. The schematic reaction processof organofunctionalsilaneCharacterization of Modified Silicon Wafer:
Chemical Structure: The surface chemical structure change of the silicon wafer before and after the modification will be analyzed using Fourier Transform Infrared (FTIR) spectroscopy(Specac, Orpington Kent, UK) with a Monolayer Grazing Angle Accessory (Shimadzu, Tokyo, Japan). The areas of representative peaks will be analyzed with the IRsolution software (Shimadzu, Tokyo, Japan).
Surface Charge: Accroding to the result of surface pH of apple slice from Section D.1, the surface zeta-potential of modified silicon wafer will be measured (NanoZS, Malvern Instruments, Malvern, Worcs, England). Silicon wafer will be cut into required size and glued to the sample holder then the sample will be merged into a cuvette with stander solution (NanoZS, Malvern Instruments, Malvern, Worcs, England). The result will be analyzed and reported by NanoZS software (Malvern Instruments, Malvern, Worcs, England).
Surface Hydrophobicity: The modified silicon wafer is the substitution of fruit slice in this study, so the hydrophobicity of modified surface will be investigated with a goniometer (Kruss, Hamburg, Germany) to verify the modification.This measurement is also important because it can help to understand the cohesive and spreadable properties of coatings for followed coating steps.
D.2.2. Fabrication of layer-by-layer coatings
According to the result of primary studies in Section C.1,C.2and the result from Section D.1, suitable agents (e.g., Chitosan, Alginate and lactoferrin) are chosen to form ediblecoatings due to opposite charge. Moreover, the order of compositions is studied forpractical application since different functions aim to different orders in the formulation of Layer-by-Layer edible coatings.
Formation of Emulsions:Protein coated emulsion droplets will be formedbyMicrofluidizer (Model 110L Microfludics, Newton, MA), according to the method reported by Mao and McClements. The Dietary Reference Intakes (DRIs) of Vitamin E is 15mg/day. Usually, Vitamin E acetate is used in our nutrition supplementsbecause the Vitamin E acetate can be digested into Vitamin E in human body. So Vitamin E acetate will be added into the corn oil to form 10% oil-in-water emulsions which is reported to have significant barrier water barrier properties. The reflective index matching is applied by adding glycerol in protein emulsifier aqueous phase to obtain clear emulsions.
Charge& Particle Size Analysis:Charges of particles are determined based Dynamic Light Scattering principle (Mie theory). At pH from the from the result of Section D.1, zeta-potentials of coating agents will be measured (NanoZS, Malvern Instruments, Malvern, Worcs, England). The particle diameter of lactoferrin emulsions will be analyzed by Static Light Scattering (Master sizer 2000, Malvern Instruments, Malvern, Worcs, England).
Figure. 6 Example of a possible laminated coating assembled over a surface of modified silicon waferFormation of Layer-by-Layer coatings:Under the pH value from the result of Section D.1, the order of composition of layer-by-layer coatings is shown in Fig 6. First, the modified silicon wafer will be submerged into positively charged polysaccharides (e.g., chitosan) solution to form the first layer, due to their good inhibition of gas and bacteria;second, the wafer will be dipped into negative charge polysaccharides (e.g., alginate) to enhance the gas and bacteria barrier properties; then the wafer will be placed into positive charged protein-coated-Vitamin E emulsions (e.g., Lactoferrin emulsions) to form third layer. This emulsion layer can prevent water transfer with the lipid phase; the thiol-groups in specific protein canreduce the browning rate and active peptide (e.g., lactoferricin) in selected protein can also against bacteria. Besides, the incorporation of protein and Vitamin E can offer healthy benefit as antioxidant capability to human body. Another negative chargepolysaccharide layer will be used to form outer layer to enhance the water barrier properties and the negative charged shell can prevent the bacteria absorb on the surface. The washing is performed between each layer formation step (Fig 7).Macintosh HD:private:var:folders:9g:xftfh0h12ml3rn62jg5kymtm0000gn:T:com.tencent.qq:QQ20120810-2.pngMacintosh HD:private:var:folders:9g:xftfh0h12ml3rn62jg5kymtm0000gn:T:com.tencent.qq:QQ20120816-3.png
Fig 7. The preparation of Layer-by-Layer edible coating on modified silicon waferD.2.3. Characterization of Layer-by-Layer edible coatings
It is important to determine concentrations of active agents absorbed on the surface to understand possible function of Layer-by-Layer edible coatings. Their characteristics such as thickness, hydrophobicity and morphology will highly influence and determine the quality and safety of fruit products therefore, following experiments will analyze and characterizefunctionality of edible coatings.
Surface Chemical Structure Analysis: The surface chemical structure change is measured after each coating step and representative peaks of selected active agents (e.g., Chitosan, Alginate and Lactoferrin) are measured and analyzed by Fourier Transform Infrared (FTIR) spectroscopy (Shimadzu, Tokyo, Japan) with a Monolayer Grazing Angle Accessory (Specac, Orpington Kent, UK). The area of representative peak indicatesfinal concentration of active agents on each layer. The result will be calculated with the IRsolution software (Shimadzu, Tokyo, Japan) and compared with the control. In addition, in case representative peaks of different agents could be overlapped to provide limited information, the dye assay will be used as the backup strategies for confirmation of the formation of coatings. For example, AO7 will be applied to examine amine group from Chitosan layer; Toluidine blue O (TBO) dye will be used to determine the carboxyl acid group of alginate layer and the protein layer can be evidenced by protein assay like BCA assay.
Thickness Analysis: The thickness of coatings can affect functions of edible coatings such as water vapor resistance, gas barrier and appearance. In this study, before the determination of thickness, we will record the refractive index of modified silicon wafer with aby a digital refractometer (Sper Scientific modle 300034, sper scientific, Scottsdale, AZ).The Rudolph research model auto SL-II automatic ellipisometry (Rudolph Research Analytical, Hackettstown, NJ) will be used to measure the change of sample's polarization upon reflection and transmission. This change will be analyzed and reported as the thickness of coatings.
Hydrophobicity Analysis: The hydrophobicity highly associates with the water vapor resistance and adhesion of coatings. Socontact angle measurementwill show the change of Hysteresis (H) (advancing and receding) of each layer by using a goniometer (Kruss, Hamburg, Germany). According to the result, the capability of water barrier and coating spreadability of each layer can be determined.
Morphology & Roughness Analysis:The change of surface morphology with each coating step will be scannedby using scanning electron microscope (SEM) which can also provide the information of coatings' thickness. Since the incorporation of lipid/emulsions will change the roughness of edible coatings, the atomic force microscope (AFM) (Digital Instruments, Santa Barbara, CA) will be applied to analyze microstructure of each layer. AFM imageswill also provide relevant and specific information about thearrangement of layers and their interaction with light that can determine the optical properties.
Optical properties: The appearance of a food product is the one of most important quality attributes since it is the first sensory impression perceived by consumers, so the key factors of optical properties of edible coating: transparency and gloss are paramount to particle application. After the formation of coatings, the transparency of edible coatings is determined with a ColorFlex EZ (ColorFlex EZ, Hunter Lab, Virginia, US). According to the ASTM standard D-523 method,the gloss will be determined by usinga flat surface glossmeter (Multi, Gloss 268, Minolta, Germany).
Anti-microbial Test:Antimicrobial test of edible coatings will be conducted by zone inhibition method respectively with model bacteria(e.g., E.coli, S.aureus, yeast and mold). The coated silicon wafer will be placed on nutrient agar in petri dishes with the coated side facing the agar, which will be pre-seeded with bacterial cell suspensions. Then zone of inhibition of coatings will be examined after 24hour incubation at 37 oC. The area of whole zone will be calculated and reported as the capability of antibacterial.
All measurements are repeated three times and results are analyzed by statistic methods.
D.3. Specific Aim 3: Determination Protective Capability and Application of Layer-by-Layer Coatings.
The primary purpose of these experiments is to develop multifunction of Layer-by-Layer coatingson fruit slice. Initially, we will study the formation methodand examine characteristics of Layer-by-Layer coatings, then determine the functionality of coatings. In addition, Vitamin E acetate will be added into the lipid phase of protein-coated lipid droplet so this fortification can improve the antioxidant capability and health benefits of edible coatings.
D.3.1. Fabrication of layer-by-layer edible coatings on apple slice
Processing of apple slice:The fruits will be sanitized in NaClO and rinsed with water and dried before cutting. The sample slice of apple (Fuji) will be cut as shown in Section D1.1.Apple wedges prepared from equator parts of the apples, then the wedges will be further cut into rectangular prisms with (2cmÃ-1.5cmÃ-0.5cm) and followed by washing step.
Dipping Method: Dipping treatments is most important step in frabricating coatings. The apple silice will be dppied in polysacchrides, protein, and emulsion solutions by followingsame processdures desrcribed in Section D2.2 to frabricate multifunctional edible coatings on cut surface. The dipping time is around 3 minat room temperature; the washing stepwillbe followed by each dipping step (Fig. 8) and coated slice will be gently dried in an open air. Then, treated apple slices will be stored in sealed polyethylene bagsunder 4â„ƒup to random withdrawal for analysis.
Chemical Structure Analysis: The chemical structure of ingredients will be exmained by GA-FTIR (Specac, Orpington Kent, UK;Shimadzu, Tokyo, Japan). The effective concentration of active agent will be determined by the area of representative peak for each layer. The result can predict the function of edible coatings. According to the result from D2.3,dye assay (discribed in D2.3)will be kept as backup straitageto confirm the formationcoating by active agents.
Figure. 8 Schematic representation of coatings a minimall processed fruit with a Layer-by-Layer edible coatings with dipping treatment
D.3.2. Protective effect of layer-by-layer edible coatings on apple slice
Respiration Rate& Ethylene Synthesis Rate: During the storage, the gas sample will be taken form the sealed sample with certain frequency (1, 7, 14, 21days) and injected into a headspace auto-injector (Shimadzu AOC-5000). Then the concentration of CO2and ethylene will be determined with a gas chromatograph (Shimadzu GC-2014, Tokyo, Japan) to monitor respiration rateof wounded apple at different time point.
Texture: The change of the firmness largely determine the quality of fruit product so firmness of apple slices with and without coatings will be analyzed by TPA test method as described in Section D1.2. During the storage, the firmness will be measured at 1, 7, 14, 21days and the change of firmness will be used as a key indicator to evaluation the water barrier properties of coatings.
Optical Properties: The appearance is the first impression to accept a product for consumer so the color and gloss change of coated apple slice will be determinedwith a colorimeter (ColorFlex EZ, Hunter Lab, Virginia, US) at certain time frame (1, 7, 14, 21 days) and the result will be reported as browning rate. Since the browning rate is related to the activity of PPO, the browning rate can also be applied in the evaluation of antioxidant capability of edible coatings. The refractive index matching can change emulsions into transparent solution, therefore, the change of gloss would be minimized but the gloss will be still measured by ATSM method describedSection D2.3. The result of the sample will be compared with the control.
Antimicrobial: Total microbial count contains microbes, yeast and molds population that are most common contamination and spoilage bacteria in comparable package of apple slice. The storage condition will be controlled to mimic typical consumerhabit such as at ambient temperature with open air.After storage, Stomacher Lab Blender 400 (Seward medical, London, England) will be used to form homogenized suspension for thesample and control by agitation. Then Serial dilutions of suspension will be poured respectively in Tryticase Soy Agar (TSA) at 37Â±1â„ƒfor 2 days for bacteria counts. The colonies of sample and control apple slice will be reported as the antimicrobial activity of Layer-by-Layer edible coating.
Morphology& Roughness:The determination of morphology and roughness will be demonstrated by using SEM and AFM as mentioned in Section2.3. After dipping, SEM images will be taken to compare the morphology change between the coated sample and control. AFM images will be taken at each coating steps both for sample and control to analyze the inter- and intra- structure and interaction between fruit surface and different layers. In addition, the relationship between active agents such as oil and VE and morphology and roughness will be also investigated.
Weight loss: It is an important indicator of overall quality change of fresh-cut apple slice, so samples and controls will be compared during storage (1, 7, 14, 21 days) and the weight difference is reported.
Sensory Analysis: The sample and control will be tested at day1 and day 7 of storage to determine consumer acceptability of fresh-cut apple slice. Thirty untrained consumers, age between 20-65 years old, will attend the sensory test. The panelists will evaluate the acceptability of odor, color, taste, firmness and overall preference of samples and control and score from "0" indicated extreme dislike to "10" indicated extreme like.
All measurements are repeated three times and results are analyzed by statistic methods.
D.5. Tentative Schedule
Specific Aim 1:Characterization of properties of surface of minimally processed fruit
Specific Aim 2:Development of Methodology and Characterization of Layer-by-Layer Edible Coatings
Specific Aim 3: Determination of protective capability of layer-by-layer coatings on minimally processed fresh fruit