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Twin Function of Zein-Zinc Coordination Complex: Wheat Nutrient Enrichment and Nano-Shield Against Pathogenic Infection Badal Kumar Biswal, mahmoud elsadany, Divya Kumari, Poonam Sagar, Nitin Kumar Singhal, Sandeep Sharma, Tsering Stobdan, and Vijayakumar Shanmugam ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b04174 • Publication Date (Web): 19 Mar 2018 Downloaded from http://pubs.acs.org on March 19, 2018

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ACS Sustainable Chemistry & Engineering

Twin Function of Zein-Zinc Coordination Complex: Wheat Nutrient Enrichment and Nano-Shield Against Pathogenic Infection Badal Kumar Biswal,,‡1Mahmoud El Sadany,‡,ʃ1Divya kumari,‡1Poonam Sagar,2 Nitin Kumar Singhal,2Sandeep Sharma,1Tsering Stobdan3 and Vijayakumar Shanmugam*1 1

Institute of Nano Science and Technology, Habitat Centre, Phase- 10, Sector- 64, Mohali, Punjab – 160062, India.

2

National Agri-Food Biotechnology Institute, C-127, Industrial Area, S.A.S. Nagar, Phase 8, SahibzadaAjit Singh Nagar, Punjab 160071, India. 3

Defence Institute of High Altitude Research, Leh, India.

*Corresponding authors E-mail: [email protected] ABSTRACT

Cereal grains undergo huge loss in storage, which is significantly due to microbial contamination, on the other hand nutrient deficiency also coexists. Grain moisture is the key for microbial contamination to occur, hence we envisage to coat the grain with edible hydrophobic moisture barrier “zein”. But the challenge in coating zein is to have control over

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the zein film thickness, until now there is no report of zein film thickness of less than 50 µm in nonplanar surface. However, coating thickness lesser than 50 µm, has appeared on planar surface. In the present work, zinc is coordinated with the zein (Zinc@Zein), which unwraps the hydrophobic zein domain and also reduces the viscosity of non-Newtonian zein fluid to form nano-layer coating. Zinc coordination furnishes anti-microbial property as well as nutrient supplementation. The nano-layer of Zinc@Zein on Triticum aestivum proves efficient protection from seed borne pathogen Pseudomonas syringae infection and increases available zinc by 4.5 times in simulated gastric digestion and Caco 2 cell model study. Approximately ~2-3 g of zein and 80 mg of zinc will be sufficient for coating 1 Kg of grain to enhance mineral availability and anti-pathogenic effect.

INTRODUCTION Grains are the major energy source for human and they also act as the seed for propagation. In cereal grain up to 60% is lost in storage,1 mostly due to microbial contamination, which reduce the nutrition value and cause quarantine issue.2–7 Fumigation is the widely practiced method to prevent microbial contamination in storage grains, but often results in residual effect. Hence, the solution of

eco-friendly plant smoldering has been developed

recently.8 Chitosan and other coatings were also employed for protecting seeds from microbial contamination.9,10 Here, the coating solution has to be prepared in water, hence being limited to the seed treatment just before sowing and not compatible for pre-storage processing. For the long term storage of wheat, yeast coating has been recommended, but this led to changes in the wheat chemical composition.11


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Microorganisms gain opportunity to infect stored grain through the following openings, 1. natural or manmade openings in the grain, 2. hilum, 3. micropyle and 4. grain accessory structure like hair.12–14 However, the bacterial diseases can be transmitted from the grain that do not have internal infection, which meant the presence of the bacteria on the surface is sufficient.14 The grain infections are directly proportional to the relative humidity (RH).15,16 Still major part of the world, store the grain in ventilated open go-down,1 hence often the grain moisture content fluctuates to maintain equilibrium with the relative humidity of the environment. Apart from stored pathogens, agricultural pathogen like Pseudomonas syringae have been found to contaminate grain, which have the ability to spread, even by air and exist in extreme cold conditions.17,18 Further, the wheat seeds infected by P. syringae were known to cause up to 50% infection in the plants germinating in field, irrespective of resistance and susceptible variety.19 Recently, toxins viz.,syringopeptin and syringomycin synthesized by P. syringae were reported to cause haemolysis.20,21 Hence, hydrophobic edible coating is the desirable solution for grains. The zein protein with 3.8: 1 ratio of hydrophobic to hydrophilic surface area that disperses well in 70-80 % ethanol and is compatible to form hybrid structure could be a perfect match.22–31 But the challenge in zein coating has been to get continuous coating, since plasticizers were found to be essential even for a continuous coating on planar surface. The plasticizer like oil may increase stickiness and eventually favors infection.32 Whereas the non-sticky plasticizer like glycerol, compromises the water barrier property.33,34 Hence, hardly any study has taken forward the concept of zein coating to test food preservation. Further, the reported zein films were in micron scale measuring >120 µm,35 150-180 µm,36 79 µm,37 1 µm,38 which were not pursued on food.


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Since, tensile strength and thickness are indirectly proportional,39 it is important to control the thickness. On the other hand, zinc deficiency is an alarming problem and known to cause brain malfunction, immune system failure and physical growth impairment.40 Further, zinc also have anti-microbial property both in ionic as well as oxide forms, which can supplement zein in grain protection.41 Biomass has good metal ion adsorption properties, which has been recently adopted for water purification applications by T. Pradeep and coworkers in 2016.42 The zein film has been proved to support the activity of the loaded active ingredients like silver ions,43 lysozymes,36 and antioxidant.30,44 Such hybrid materials are known to give excellent size control, whereas the polymer without metal support led to poor size control.45 Hence, in this study zein biopolymer composite with the zinc ion have been formed and coated on Triticum aestivum to form nanofilm. Such nanofilm coatings on grain have never been tested for pathogen control. (Scheme 1). This coating has been found to resist the seed borne Pseudomonas syringae NCIM 5102 infection. The flour of the wheat coated with Zinc@zein has been found to increase available zinc in simulated gastric digestion.


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Scheme 1.Schematic diagram showing anti-pathogenic effect expressed by the zinc-zein coating on wheat as compared to pathogen infection in the uncoated wheat seed. EXPERIMENTAL SECTION Materials. Zein (as per the manufacturer the purity is 99.54 % ), ZnSO4, pepsin, bile salt, pancreatin and ethanol were purchased from TCI chemicals. Peptone, malt extract, yeast extract, dextrose and beef extract was purchased from HIMEDIA. Sodium hydroxide, glycerol, tris base, SDS,

ammonium

persulphate

(APS),

TEMED,

acrylamide,

bis-acrylamide,

glycine,

bromophenol blue and coomassie brilliant blue were purchased from Sigma-Aldrich. At necessary places de-ionised water from the Milli-Q was used. Dulbecco's modified Eagle's medium, fetal bovine serum and HEPES from HiMedia and antibiotic penicillin-streptomycin solution from Gibco. Chelex-100 was purchased from Bio-Rad Laboratories, Hercules, CA and 12,000 kda molecular weight cut-off dialysis membrane from Sigma-Aldrich. Piperazine-N,N′bis-[2-ethanesulfonic acid] was purchased from SRL chemicals ltd., hydrocortisone and insulin from Sigma-Aldrich, selenium from Alfa Aesar, triiodothyronine from Sigma-Aldrich and epidermal growth factor from MP biomedicals. Coating of wheat grain. Zein coating on Triticum aestivum grain was performed by solvent evaporation process, by simply allowing the solvent to evaporate at constant magnetic stirring of 5 gram grain in X% zein solution (value of “X” is varied in descending order from 2 to 0.2 to 0.02% to identify the critical value to achieve uniform but thin coating) in 10 mL


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volume (9:1 ratio ethanol: water) (for brevity this will be represented as TaS@Z). Similarly, for the zinc coordinated zein composite (Zinc@Zein) coating, 1mg of ZnSO4 was mixed in 0.2% of zein in 10 mL of 9:1 ratio ethanol: water for 2 h. If any other ratio of zinc to zein had been used, the same is mentioned in the result. Following this, Zinc@Zein was coated on wheat grain by following the same solvent evaporation method as mentioned above for zein coating. This will be represented as TaS@Z-Z. Surface characterization. The scanning electron microscopy images of the coated and uncoated wheat grains were observed with JEOL JSM 1500 (USA). Three-D surface profile of the coated and uncoated wheat grains were measured with non-contact optical profilometer (Model: Bruker GT-KO). Surface charge of the wheat grain before and after coating was measured with Surface Zeta Potential Cell (ZEN1020). Fragmentation of protein. Fragmentation of protein was studied by the SDS-PAGE gel electrophoresis. One percent of zein solution was prepared with and without ZnSO4 in 80% aqueous ethanol and mixed with the sample buffer in the ratio of 80:20. Sample buffer was prepared by mixing 20% glycerol, 0.5 M Tris-HCl of pH 6.8 and 0.5% bromophenol blue. Stacking gel (4% polyacrylamide, 1.5 M Tris-HCl of pH 6.8, 10% SDS, 0.1% ammonium persulphate (APS), 0.01% TEMED) and separating gel (15% polyacrylamide, 1.5 M Tris-HCl of pH 8.8, 10% SDS, 0.1% APS, 0.01% TEMED) were prepared in a gel–casting plate and submersed in tank buffer (0.25 M Tris, 1.92 M glycine, 1% SDS). The gel was run at 80-120 mv and the fragmentation of protein was observed by Bio-rad geldoc. Circular dichroism. The secondary structure of the zein protein with and without ZnSO4 was analyzed by circular dichroism spectrometer (JASCO J-1500-150). Samples were prepared


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by dissolving zein and Zinc@Zein in 1mL of 80% aqueous ethanol with the same amount of zein in both the samples. Viscosity. Viscosity of zein solution was measured at 25 °C with the viscometer (Anton paar MCR 302) at spindle speed of 100 rpm. Viscosity were recorded at different time (1 min, 7 min, 15 min, 30 min, 45 min, 60 min) after spindle rotation had been initiated. Viscosity measurement of zein and Zein@Zinc samples were taken in 80% ethanol at the same concentrations of zein in both the samples. Zinc estimation. The concentration of zinc was measured from the digested sample obtained from the grain flour, by using the inductively coupled plasma mass spectrometry (ICPMS, Agilent 7700 series). Fourier Transform Infra-red Spectroscopy. Fourier transform infrared (FT-IR) spectrum analysis was carried out by using the Perkin Elmer, Spectrum 100 FT-IR spectrophotometer (Agilent technologies) in ATR mode. Moisture dynamics. To enumerate the ability of the different coating to act as the moisture barrier, 5 g of grains were placed at 75% relative humidity at which generally the grains are prone to microbial contamination. The grains were heated at 130 °C for 1 h and incubated in the BOD incubator for different time intervals up to a month with 75% relative humidity. The difference in the weight of the grain before and after incubation is calculated as percent moisture content with reference to the initial grain weight. Moisture (%) = (final weight – initial weight/ final weight) x 100


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Water resistance. To check the effect of hydrophobic zein layer in resisting water entry onto the grain, different treatment of grains were dipped in water and the water was decanted after a short period of 20 and 600 seconds incubation. Then, the moisture content in the seed was estimated as above after a brief uniform air drying for 10 minutes. Bioavailability by dialysate method. The bioavailability of zinc ions from the wheat grain, with and without coating was enumerated with the method described by Luten et al., 1996.46 Briefly, the powdered sample of the grain (1 g) was allowed for digestion with pepsin (300 mg/10 mL) which was adjusted to pH 2 with 6 M HCl. This mixture was incubated for shaking for 2 h at 37 °C. Following the digestion, 1 mL of the aliquot was taken to check the titratable acidity with 0.5 M NaOH required to bring the pH to 7.5. In a dialysis tube, 12.5 mL of NaHCO3 equivalent to the titrated mole of NaOH was packed and dialyzed against the remaining pepsin digests aliquot. Following this incubation for 30 minutes, pancreatin (0.5 g)–bile (0.3 g) mixture was added for further digestion for another 2 h. Subsequently, the dialysates present in the tube was mixed with 5% nitric acid and centrifuged at 10,000 rpm. Finally, the zinc ion concentration in the supernatant was analyzed with ICP-MS. Bioavailability in Cell culture. Caco-2 cells were obtained from NCCS Pune and passage number 39 was used in these experiments. Cells were seeded at a density of 50,000 cells/cm2 in 6-well plates and grown in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 25 mmol HEPES and 1% antibiotic penicillin-streptomycin solution. The cells were maintained in an incubator 37 °C with 5% CO2. The medium was changed every 2 days and the cells were used for the zinc uptake experiments after 20 days post seeding.


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In vitro digestion: For peptic digestions, 0.24 g pepsin was dissolved in 3 mL of 0.1M HCl and 3.0 g of Chelex-100 and shaken gently for 30 min at room temperature and centrifuged at 8000 RPM for 5 min. The supernatant was collected and additional 3.0 mL of 0.1M HCl was added to the solution. For the intestinal digestion, 0.04 g pancreatin and 0.24 g bile extract were dissolved in 20 mL of 0.1M NaHCO3. To this mixture, 10 g of Chelex-100 was added and mixed gently for 30 min and then centrifuged at 8000 RPM for 5 min. To this supernatant, additional 10 mL of 0.1 M NaHCO3 was added. Treatment of both solutions via above described method didn’t affect the activity of enzymes. Both peptic and intestinal digestions were taken in an incubator shaker and maintained at 37 °C with 5% CO2. To start the peptic digestion, 1g of the sample was transferred to a 50-mL centrifuge tube and 0.5 mL of the pepsin solution was added. The pH of each sample was adjusted to pH 2.0 with 6M HCl and the volume of each sample was made 8 mL by using pH 2.0 buffer (120 mmol/L NaCl and 5 mmol/L KCl). The tube was capped, placed horizontally and incubated for 60 min on incubator shaker at 37 °C. For the intestinal digestion step, the pH of the sample (the above peptic digest) was raised to pH 6.5 by dropwise addition of 1N NaHCO3. Then, 2.5 mL of pancreatin-bile extract mixture was added and pH was again adjusted to pH 6.5. The volume was finally made up to 15 mL by using pH 6.7 buffer (120 mmol/L NaCl and 5 mmol/L KCl). The intestinal digestion was carried out in the upper chamber of a two-chamber system in 6-well plates, with the cell monolayer attached to the bottom surface of the lower chamber. The upper chamber was formed by fitting the bottom of an appropriately sized Transwell insert ring


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(corning) with a 12,000 molecular weight cut-off dialysis membrane. The membranes were soaked in deionized water before use. The dialysis membrane was held in place with a silicone ring (Fitco Seals). After the dialysis membrane was fitted to the insert ring, the entire unit was sterilized in 0.1 N HCl at 4 °C overnight, afterwards washed in sterile water thrice and then kept in sterile water for 1 h before use. Preparation of the 6-well culture plates with cell monolayers Immediately before the intestinal digestion period, the growth medium was removed and replaced with 37 °C Minimum Essential Medium (pH 7). This MEM was chosen because it contained no added Zn. The MEM was supplemented with 10 mM PIPES (piperazine-N,N′-bis[2-ethanesulfonic acid], 1% antibiotic penicillin-streptomycin solution, hydrocortisone (4 mg/L), insulin (5 µg/ml), selenium (5 ng/ml), triiodothyronine (34 μg/L) and 20 ng/mL epidermal growth factor. A fresh 1.0-mL aliquot of MEM covered the cells during the experiment. A sterilized insert ring, fitted with a dialysis membrane, was then inserted into the well, forming two-chamber system. Then, a 1.5 mL aliquot of the intestinal digest was pipette into the upper chamber. The plate was covered and incubated at 37 °C with 5% CO2 atmosphere for 120 minutes. When the intestinal digestion was terminated, the insert ring and digest were removed. An additional 1 mL of MEM was added to each well after removing inserts. The cell culture plate was then returned to the incubator for an additional 22 h, after which the cells were harvested for analysis. Exactly 24 h after the start of the intestinal digestion period, the cell monolayers were harvested after washing with 2 mL of a chilled 50 mM PBS (pH 7.4) with cell scrapper in the presence of


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800 µl of 50 mM PBS. The collected cells were then sonicated (30 strokes for 2-3 times at intervals) at 4 °C. Experiments were replicated three times for each treatment. The treatments include wheat, wheat coated with zein and wheat coated with zein coordinated with zinc, along with blank composed of all components of in vitro digestion protocol but no added ZnSO4 or ascorbic acid. In addition, ZnSO4 was taken as negative control, while for positive control ZnSO4 along with 200 mM ascorbic acid was used. Zinc content of samples was analyzed using an inductively coupled plasma emission spectrometer (Agilent technologies 7700). Antimicrobial activity. The Seed borne pathogen of wheat, Pseudomonas syringae NCIM 5102 was obtained from NCIM culture collection from NCL, Pune and grown in Nutrient broth medium at 250 RPM at 28±2 °C. The infection of the wheat seed with the bacteria was done by following the standard protocol as mentioned earlier. Wheat grains (5 g) were incubated in 10 mL of bacterial culture at log phase (OD value approximately 0.6 at 600 nm), and infiltered with vacuum for 20 minutes and allowed for 24 h of incubation. Following this, the grains colony count was enumerated with the seed flour after serial dilution. RESULTS AND DISCUSSION Grain coating and characterization


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Figure 1. SEM image showing surface morphology of Triticum aestivum after different coating material treatment (a) Control without coating (b) 0.2% zein coated (c) 0.02% zein coated (d) 0.2% zein+1mg ZnSO4 coated. Scale of insets a and d are 50 µm. In this study, Triticum aestivum (wheat) grains were coated with the biopolymer viz., zein protein, with and without zinc, to reduce microbial contamination during storage and to improve nutrition value, through solvent evaporation technique. The coating was tested with 2% zein solution in 10 mLvolume (9:1 ratio ethanol: water)/ 5 g of grain. Here, the solvent viz., aqueous ethanol mixture used in the coating method is non-toxic and can simultaneously sterilize the grain. The coated samples were observed by the SEM to confirm the coating. The coating solution at 2% zein concentration, resulted in a thick layer ranging at ≥10 µm (Figure S1).


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Hence, zein concentration was reduced to 0.2% and 0.02%. The coating from these concentrations resulted in merged appearance of the transverse and longitudinal ridges (Figure 1b, 1c respectively),which is found clearly distinct in the control (Figure 1a and inset); thus confirming thinner coating compared to the grain coated with 2% zein solution. However, the coating with 0.2 and 0.02% zein solution resulted in incomplete surface coating that is marked with arrows, which causes the film thickness to increase beyond 2 µm. In comparison, the grain coating with 0.2% zein solution resulted in maximum coverage, hence 0.2% was fixed as the optimum coating precursor concentration for further experiments. This sample will be referred as TaS@Z (Zein coated Triticum aestivum)] for brevity. For the mineral (zinc) fortification in wheat grain, zinc sulphate (3 mg) was added to 0.2% zein solution, followed by 1 h incubation before coating. The 1HNMR spectra of the zein incubated with the zinc showed peak broadening and down shift (~0.03 ppm) of the amine peak in zein at 8.0 ppm. The thiol peak in zein at around 1 to 1.5 ppm, also shows broadening and up shift in the maxima (~0.02 ppm) with the addition of zinc. This could be due to the closer binding of the zinc with the electron donors like nitrogen and thiol groups in zein (Figure S2).47 Supporting this fact, earlier reports found that the metal ions bind to the protein through N, O and S groups.43,48 This down or up shift and broadening in the 1HNMR peak happens due to the co-ordination bond formation (this composite of zein and zinc will be referred as Zinc@Zein in the following text).49,50 The test for zinc leaching from Zinc@Zein complex at 20 mg of zein to 1 mg of ZnSO4 ratio showed negligible zinc loss, hence this ratio have been fixed for further studies except when noted. Similar to zein coating on wheat grain, the Zinc@Zein was also coated on wheat grain by solvent evaporation technique, for brevity this sample will be referred


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as TaS@Z-Z (Zinc@Zein coated Triticum aestivum) in the following text. In SEM, TaS@Z-Z

Figure 2. Surface profile of Triticum aestivum after different coating treatment (a) Control without coating (b) 0.2% zein coated (c) 0.2% zein+1mg ZnSO4 coated (d) Roughness plot of different coating treatments (Control: Without coating; TaS@Z(0.2%): 0.2% zein coated; TaS@Z(0.02%):0.02% zein coated; TaS@Z-Z(0.2%): 0.2% zein +1mg ZnSO4 coated; TaS@ZZ(0.02%): 0.02% zein +1mg ZnSO4 coated). coating (Figure 1d) have been found to be well assembled on the grain surface as compared to TaS@Z. Further, the thickness of the coating has been found to be around 700 to 900 nm in the


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cross-section image (Figure 1d inset). Although, this is not homogenous around the grain, especially in the grain furrows- the coating thickness increases marginally, still this is much thinner compared to the earlier zein coatings, which resulted in several microns,44 Practically, this control in the coating thickness gives maximum zinc ions exposure with minimal zinc content, which will improve protection from the pathogen (vide infra). The elemental EDAX mapping of zinc shows that TaS@Z-Z sample have increased number of zinc spots as compared to the control without coating (Figure S3a and b). In corroboration to the EDAX mapping, the spot EDAX also shows significant increase in the zinc content in TaS@Z-Z, as compared to the control (Figure S3a and b). The SEM images also confirmed that the coated grain have smooth surface as compared to the grain without coating. Hence to quantify this feature, the surface profile of the wheat grains was studied with the optical surface profiler. Since, the profilometer can give better resolution to these kinds of heterogeneous rough surfaces that vary in the range of nano to micron scale. The results from the profilometer study have been plotted in the Figure 2. In corroboration to SEM images, the profilometer shows marginal reduction in the roughness of the zein coated grain as compared to the control grain. However, with Zinc@Zein coating on the grain viz., TaS@Z-Z, the roughness increased marginally to 49.5.


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Figure 3. Characterization of Zein and Zinc@Zein coordination complex with (a) SDS-PAGE Gel documentation (b) UV-Circular dichroism spectra (c) Viscosity at different time intervals of evaporation (d) Contact angle. Effect of zinc on the properties of zein Viscosity is the major factor in deciding the coating film thickness;51-54 hence the viscosity of the zein and Zinc@Zein was measured for 1 h at different intervals (Figure 3a). This incubation time is the period during which most of the solvent evaporates in the coating experiment, leading to crowding of protein and increase in viscosity.55-57 After 15 minutes, there is a reduction in the viscosity of the zein without zinc, this may be attributed to the structure


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realignment due to the change in solvent ratio, possibly because of faster ethanol evaporation. However, this viscosity control by the structure realignment is short lived, since generally such change is dynamic and only the ionic strength is stable.58 Interestingly, Zinc@Zein shows significant control in viscosity in the course of solvent evaporation, although the initial Zinc@Zein precursor had higher viscosity than zein precursor. Such observations were also reported in other proteins,59,60 and is attributed to the weak or strong electrostatic strength causing attraction or repulsion respectively, when brought closer below the critical distance.61-65 Supporting this report, in the Zinc@Zein sample, the ionic strength was found greater than zein (Figure S4). This increase in the viscosity will proportionally cause shear thickening, hence eventually uneven and thicker coating is evidenced in the control zein coating. Whereas, the viscosity control in Zinc@Zein may have resulted in thinner and closer coating, which led to slight increase in roughness of TaS@Z-Z grain morphology as compared to TaS@Z grain. Since, in such non-Newtonian fluid like zein solution, shear thickening can be controlled by viscosity. Apart from the charge, the viscosity has also been controlled by the molecular structure and size, and hence the dichroism and molecular size were documented.66-73 Since there is a chance of non-enzymatic cleavage with the physical changes like high ionic concentration, the confirmation of the molecular weight is necessary.74-84 The CD spectra shows the typical negative maxima of α-helix structure at 207 and 222 nm (Figure 3b). This α-helix of zein protein diminished with the addition of the zinc ion. This pattern may be due to the partial denaturation of zein protein, due to the cleavage in disulphide and hydrogen bonds (amine and carboxyl group assisted) that have participated in the coordination bond formation with zinc ion. This reduction in the α-helix may also favor improvement in the tensile strength,85 which may complements the


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life of grain coating. The SDS-PAGE analysis, confirms that both zein and Zinc@Zein complex has the characteristic band at 19 and 22 kDa (Figure 3c). This confirms that the viscosity control has not been due to the undesired non-enzymatic cleavage. With a closer examination of the bands, a slight increase in the molecular weight of the Zinc@Zein at both 19 and 22 kDa region could be recognized, which may be attributed to the metal binding. The absence of band at lower molecular weight, confirms that the interaction of zinc on zein in Zinc@Zein didn’t fragment the protein. The higher molecular weight bands in the gel has been reported because of the native character of zein to form dimer.53

In spite of the above modifications, the hydrophobic property needs to be retained to avoid the increase in grain moisture content. Hence to quantify the hydrophobicity, the contact angle of the zein films with and without zinc was measured (Figure 3d). Interestingly, the contact angle of the Zinc@Zein has been found to increase more than zein alone, which is in contrast to earlier non-sticky plasticizer that reduces contact angle. This increase in the contact angle has been attributed to the unwrapping of the hydrophobic domain. Such exposure of hydrophobic domain with the decrease in α-helix was also shown earlier.86 Unlike the metal ions induced hydrophilic core and hydrophobic shell formation,48 the CD, viscosity and contact angle measurements confirms stretching of protein, which supports thinner coating.

Further, to confirm the zein coating on wheat, the FTIR spectroscopy of the grain surface with and without the coating were documented in ATR mode (control, TaS@Z and TaS@Z-Z) (Figure 4a). In wheat, the outer bran layer extends for few micron depth, which is constituted of the cellulose, hemicellulose and lignin.87 The characteristic multiple peaks of cellulose at 978,


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988, 1009, 1021, 1029 and 1036 cm-1 were observed in the control uncoated wheat grain. The wheat protein i.e., gluten is located deep inside the endodermis of the grain, hence protein signal at 1655 cm-1 from control wheat grain surface is not prominent. In case of TaS@Z and TaS@Z-Z grain samples, the cellulose peaks have slightly shifted to higher wavenumber,88 and amide 1/2 around 1655/1524 cm-1,which is characteristic to C=O and C-N-H vibrations are also present with slight broadening.88,89 Suspecting that the charge could be the possible force for the wheat grain coating, the surface charge dynamics of the wheat grain before and after coating has been studied. The whole wheat grain (not powdered but cut into half; the cut surface glued in the space specified on the platform of ZEN 1020) without coating, shows the surface charge to be of -46 mV. This native negative grain surface strength has been reduced to -15 mV with 0.2% zein coating, which may be due to the partial neutralization by the amine group (Figure 4b). Next, in the grains coated with 0.2% Zinc@Zein, the surface zeta potential switched to positive charge (+18 mV). This could be because of the binding of the zinc ion on the carboxylate group, that cause the neutralization of glutamate in zein.90 Zinc has been known to form transient coordination with the protein through thiol and glutamate.91 In support of this observation, the zeta potential of the zein dispersion in 9:1 ethanol: water with the pH 6.5 was found to be +5 mV (Figure S4). Similar weak positive charge on zein at pH ≤10.5 has been reported.90 This positive potential has been found to be further strengthen with the zinc ion binding (~+6 mV). This proves that the coating have been driven by electrostatic attraction in addition to the solvent evaporation assisted assembly. This degree of positive charge has been known to support the horizontal arrangement of the zein rather than the vertical arrangement, since such arrangements were identified to contribute to smoother coating here.53


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Figure 4. Characterization of Triticum aestivum grain with different coating treatments (a) FTIR (ATR mode) spectra of uncoated grain, zein coated grain (TaS@Z), zein powder, Zinc@Zein coated grain (TaS@Z-Z) and Zinc@Zein powder (b) Surface zeta potential of uncoated grain, TaS@Z and TaS@Z-Z (c) Total zinc content in 1 kg of grain.


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Nutrient Enrichment and bioavailability After the above confirmation of zein coating, we estimated the amount of zein and zinc required for coating 1 Kg of grain (Figure 4c). It is found that approximately ~2-3 g of zein will be sufficient for coating 1 Kg of grain, since in the optimized 0.2% zein coating procedure, almost half of the zein was found to stick to the walls of the flask. With reference to zinc fortification in 0.2% zein coating procedure, it is estimated that a maximum of ~70 mg of zinc could be fixed in 1 Kg of grain. This is ~5 times of the zinc present in the native grain; this zinc amount fortified is within the WHO limit.92 Finally, the availability of the zinc from the fortified grain (TaS@Z-Z) and the control grain was compared with the simulated gut condition (Figure 5a). Here, the treatment with TaS@Z 0.2%-Z shows a 4.5 times increase in the availability as compared to the control. The availability of zinc in TaS@Z grain has been ignored as there is no change in total zinc content with reference to control. Zein has pH responsive drug release behavior,93 hence the zein coating may have assisted in mineral release with the stimuli from the gastric enzyme. Following the dialysate test, the bioavailabilty of zinc has also been verified in the Caco 2 cell model in comparison with the uncoated wheat and zein coated wheat along with the ZnSO4 and ZnSO4+Ascorbic acid as the negative and positive control respectively (Figure 5b). Here, the fortified grain (TaS@Z-Z) showed ~0.2 µg increase in the zinc availability as compared to the uncoated wheat and zein coated wheat. The increase in the zinc uptake more than the positive control, could be due to the protein.94 Grain Moisture dynamics The grain moisture content is the detrimental factor for grain storage as microbial contamination is prone to happen at 70% relative humidity.95-97 Hence, the grain coatings were


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tested for moisture resistance, by incubating the grain samples with and without coating at 70% RH. Although, the results showed that the zein coating resisted moisture increase, it was not significant. Hence, the efficiency of grain coating to withstand flooding condition was tested, by drowning the grains briefly in water. After this quick incubation time, water was decanted quickly and the grain was spread on bloating paper followed by air drying for few minutes uniformly, before estimating the moisture content (Figure 5c). The coated samples TaS@Z and TaS@Z-Z found to show less moisture as compared to control, which may be due to the hydrophobic zein moisture barrier.98


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Figure 5.Effect of coating on the zinc availability, moisture resistance and anti-pathogenic property. (a) Available zinc content from 1 kg of grain estimated by dialysate method (b) Bioavailable zinc content estimated by Caco 2 cell model (c) Grain moisture content of the samples (uncoated, TaS@Z and TaS@Z-Z grain) after a brief soaking into water for 20 and 600 S and followed by even air drying (d) Anti-pathogenic effect of TaS@Z-Z in comparison to uncoated and TaS@Z by colony count method after exposing to Pseudomonas syringae culture at log phase for 20 min and incubation for 24 h. Antipathogenic effect: The anti-microbial effect of the grain coating was tested with the seed borne bacterial pathogen Pseudomonas syringae, which have been known to produce toxins (Figure 5d). The wheat with different coating treatment and control was allowed to incubate in the log phase P. syringae broth culture. After the incubation, the broth was decanted to enumerate the bacterial colony in the wheat grain. The TaS@Z-Z wheat grains were found to show ~80% reduction in the pathogen colony compared to the control and TaS@Z. Zinc ions released from the zinc oxide nanoparticles were found to cause bactericidal activity, but this release was too less at the most favorable dissolution condition.99-102 Compared to this low availability of zinc in oxide nanoparticles, in TaS@Z-Z, the zinc ions are readily available. Further, the thickness control of the grain coating enhances the zinc exposure, hence the anti-bacterial effect has been expressed very well at least concentration. The anti-pathogenic activity of the zinc has been attributed to the intracellular reactive oxygen species synthesis, DNA damage, cationic charge and also by the imbalance in zinc homeostasis.103-106 The anti-pathogenic effect could be rationalized with the matching surface area of 3 nm zinc oxide nanoparticle (1.5 m2/100 mg ZnO),107 and 1 Kg wheat (1 m2), which has been coated with ~80 mg of zinc.108 In zein coated wheat grain (TaS@Z) also,


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slight reduction in the colony as compared to the control without coating has been achieved. This could be because, the hydrophilic grain surface has been covered by the zein coating.105 But, considering the higher moisture absorbance rate in the zein coating, in the later stage (Figure 5a), the hydrophobic coating may not be the cause for the inhibition of infection, in our condition of infection with the culture broth (not understand). Hence, in the context of infection with bacterial pathogen culture, the enhancement in smoothness with the zein treatments may have contributed for reduction in bacterial infection. Since, the surface roughness aid in the microbial adhesion and growth.109,110 CONCLUSIONS Zinc with the edible polymer viz.,zein forms a complex formation, which shows increase in hydrophobicity and decrease in viscosity. These properties enable the complex to favor thin (nanoscale) uniform coating that protect from moisture flux and pathogen along with mineral enrichment. Since, the nanoscale film ensures maximum surface area with the zinc at limited zinc concentration, for efficient pathogen control. This technology could help to maintain phytosanitary quality in seed storage. The cost of grain preservation by this method may be nil, as there is a bonus in nutrition value. ASSOCIATED CONTENT Supporting Information. Electronic Supplementary Information (ESI) available: SEM image, NMR spectra, EDX mapping and Zeta potential is available free. AUTHOR INFORMATION Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. ʃ NAM fellow, INST, Mohali from Public Health, Molecular Carcinogenesis & Occupational Medicine, Department of Environmental Studies, Institute of Graduate Studies and Research, University of Alexandria. Funding Sources Nanomission, Department of Science and Technology, Government of India (SR/NM/NS1434/2014(G)) ACKNOWLEDGMENT P.S.V. is thankful to DST Nano mission for financial aid. M.E.S is thankful for the financial support NAM S&T centre, New Delhi, India. Authors wish to thank Prof. Santanu Kumar Pal, of IISER Mohali for readily granting access for Surface Zeta Potential Cell (ZEN1020) and Prof. Pijush Ghosh, of IIT, Chennai for readily granting access to contact angle measurement. Dr. Senthil Nathan, IIT Chennai, for the FTIR spectra in ATR mode. REFERENCES (1)

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Monoclonal

Antibody

Solutions:

Correlation

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Interaction

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Table of contents graphics

Anti-pathogenic effect expressed by the zinc-zein coating on wheat compared to pathogen infection in the uncoated wheat seed


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