Active paper packaging material based on antimicrobial conjugated nano-polymer/amino acid as edible coating

2.2.1 Particle size distribution and zeta potential measurements

Tإhe particle size distribution and zeta potential of the prepared samples were measured, using Nicomp™ 380 ZLS size analyzer, USA. Leaser light scattering was used at 170° in case of particle size detection where zeta potential was measured at 18°.

3.2.1 Antimicrobial and MIC calculation

The antimicrobial activity of this prepared conjugate was tested against microbial strains with different concentrations. MIC was calculated with measuring the surrounded clear inhibition zone diameter which refers to conjugate activity against tested organism (s), as shows in (Table 2 and Fig. 1). The obtained data shows that novel conjugate has strongest inhibitory effect on broad spectrum bacteria (Gram positive and negative). Where the antimicrobial activity against gram positive with MIC 25 µg/ml for Bacillus subtilis (NCID-3610) and 12.5 µg/ml for Staphylococcus aureus (NCTC-7447). These values are highly prospective values according to (Basta et al, 2018). While, for the case of gram negative bacteria the MIC value for Escherichia coli (NCTC-10416) was 50 µg/ml, as well as for Pseudomonas aeruginosa (NCID-9016) was 12.5 µg/ml and this was in agreement with (El-Saied et al., 2003; Rowlinson et al., 2003). Moreover, this conjugate showed significant activity against unicellular fungi Candida albicans (NCCLS 11), where its MIC was 3.125 µg/ml. The antimicrobial activity of the investigated conjugate ascribed to the presence of bulkier PSS molecules, which interprets the metabolic pathway and makes cells lost their ability to progress over stopping the protein building up using the bulky group (PSS) to block the active side of amino acid binding site (Hyldgaard et al., 2014; Menninger, 1995). Thereby, resulting significant activity is appeared. This explanation is in agreement with that reported for the role of bulky group and the exact relationship between bulk structure of agent and its inhibition activity as well as the conjugate can be described as broad-spectrum antimicrobial agent.

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Fig. 1

MIC antimicrobial test for conjugate (well diffusion method).

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3.2.2 FTIR

The FT-IR spectra were illustrated in (Fig. 2), spectrum of PS-SO₃H powders were taken over the range of wavenumbers from 450 to 4000 cm⁻¹, but for the analysis of sulfonic groups, spectrum was shown over the range from 800 and 1400 cm⁻¹. Sulfonic group vibration bands are reported at approximately 1040 and 1180 cm⁻¹ (Martins et al, 2003). The characteristic infrared absorbance for PS-SO₃H have suggested the bonding of the sulfonic groups to the aromatic ring of PS (out of plane deformation bands assigned to substituted aromatic ring γ (Car-H) at wavenumbers approximately from 830 to 850 cm⁻¹). The absorption at 1040 cm⁻¹resulted from the symmetric stretching vibration of SO₃H groups and the absorption at 1127 cm⁻¹results from a sulfonate anion attached to a phenyl ring. Our results are in agreement with the literature ones (Pantelić et al., 2009). We verified that the ν_as(S—O) vibration at 1180 cm⁻¹ appears as a very broad band at approximately 1100 cm⁻¹–1350 cm⁻¹. AA shows the traditional amino acid IR spectra, however shows bands at 3316, 2944, 2104, 1576, and 1516 cm⁻¹ which refer to (O and N)—H, CH₂, C—H symmetrical deformation, and C⚌O respectively as well as fingerprint bands (Kitadai et al., 2009; Mostafa et al., 2017a,b). Consequence of after conjugation process, some function groups and new bands are appeared, as well as some are vanished. Conjugate spectra show bands positions as well as band sharpness were different than two started materials. Conjugate showed some changes in bands in the rang 4000–3000 cm⁻¹ which regarded to OH starching vibration, NH vibration in AA and C—H in PSS, this is in agreement with IR calculations (MHBS and Cr.I.) in (Table 3) which are increased in conjugate in result of OH starching vibration and NH vibrations, this is emphasized that the increased of MHBS value of conjugate which increased than AA and decreased than PSS. Cr.I. value of conjugate was duplicated, this results is in agreement with SEM image. Blue shift is appeared in conjugate band at 3457 cm⁻¹ which observed in PSS and AA at 3307 and 3358 cm⁻¹ respectively. This may lead to liberate the hydrogen bonds during synthesis process. The bands which are characterized to sulfonate group in PSS show some changes in follow bands; 1192, 1124, 1034, and 1009 which have become sharper and recorded insanity higher than PSS spectra, this may be related to formation of crystal as mentioned. The forgoing data illustrated that the reaction between PSS and AA was going out via —SO₃Na in PSS and —COOH group in AA and produces NaOH. Other evident is the pH of solution of conjugated material before washing was more than 8 whilst the pH of AA and PSS before reaction was less than 6.5 (Dhawan, 2002).

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Fig. 2

FTIR spectra for Amino acid, PSS, and conjugated compound.

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3.2.3 DSC

(Fig. 3) showed the three DSC curves for started materials and conjugate. PSS curve shows after adsorbed water region only one peak with onset 136.25 °C and offset 180.7 °C with as well as peak temperature (T_P) 158.5 °C. Similarly, in AA the DSC curve has one hump peak with T_P105.76 °C. Additionally, conjugate curves show completely difference from both started materials. Clear splitting of started materials main peak to two peaks was appearing in conjugate DSC curve this accentual the produce new conjugate (Hatakeyama and Hatakeyama, 1990). Moreover, no significant variation in onset and outset values as shown in (Table 4). However, heat capacity is recorded with high value 117.46 J/g in conjugate one, comparison with started materials PSS and AA are 14.99 and 4.909 J/g respectively. This emphasized that the reaction between PSS and AA give highly thermal stable conjugate with high activation energy value as stated before (Garg et al., 2005).

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Fig. 3

DSC curves for started materials and conjugate.

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3.2.4 DLS and zeta potential

DLS results were plotted in (Fig. 4), polydispersity index (PDI) and mean particle size were lasted in (Table 5). The PDI is a measure of dispersion homogeneity. PDI value is ranged from 0 to 1.0 where from 0 to 0.3 is refer to the highest homogeneity distribution state (Cheng et al., 2018). The narrow uniform Gaussian distribution can be pointed to low PDI especially for PSS, and AA. as shown in (Fig. 3). Additionally, conjugated coating still presents few particle size variations with little higher PDI in related to AA and PSS as lasted in (Table 5). The mean diameters were measured in aqueous solutions with relative concentration as 439, 340 and 527 nm for PSS, AA and conjugated coating particles, respectively. Additionally, zeta potential was calculated from the three main measured factors (cell current, phase shift and mobility) that depending on the charge over particles. Zeta measurements of PSS, AA and conjugated coating were tabulated in (Table 5) and all related factors. Sulfonated group play a vital role in the net charge of PSS to be −0.68 mV where AA presented average zeta potential −12.48 mV. Combined system was detected at −7.38 mV which pointed to the connection side from COO— that reduced the value from −12 to −7.38 mV. FTIR supported this assumption by little decrease in C⚌O bond in conjugated chart than AA peak.

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Fig. 4

DLS for Amino acid, PSS, and conjugated compound.

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3.2.5 Microstructure investigation

PSS polymer showed nano-polymer in scale around 25 nm however after conjugation interaction between protein and polymer give uniform aggregated particles with as shown in (Fig. 5). Conjugated coating paper uniform particle in micron rang size with core/shell form as shown in (Fig. 5-E and F). Core size was recorded 689 nm while shell diameter was 3675 nm which confirmed in result to drying sample and aggregates the particles as core/shell form and distribute in solutions as colloids. These results have nice agreements with particle size distribution over DLS for AA and conjugate. However the results of PSS which show in (Fig. 5-A and B) in opposed to the DLS data this may be takes place as result of highly surface area either aggregate phenomena of PSS in water (Sen et al., 2007) as well as water hydrophilic properties of PSS which performed to swelling and recorded particle size more than SEM topography.

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Fig. 5

SEM for PSS at low magnification (A), high magnification (B), amino acid at low magnification (C), high magnification (D), and Conjugate at low magnification (E), High magnification (F).

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3.3.1 Coating sheets properties

The mechanical properties, including tensile strength, Young’s modulus, roughness, and burst index were carried out for uncoated (blank) and coating papers sheets. The results were calculated and displayed in (Table 6). Tensile strength of the paper sheets coated with 1 layer 150 and 200 µm thickness was increased by about 2% and 17%, respectively in comparing to the blank paper sheets. Additionally, the Young’s modulus was also increased by about 19% and 22%, respectively. On the contrary were the both thickness (150 and 200 µm) of two and three layers sheets drawback are noticed in the tensile strength and Young‘s modulus. This is referring to the high swelling degree of paper fiber in two and three coating layers on papers sheets. This is due to water base coating conjugate which penetrates the fibers easily. Moreover, the hydrophilic coating gives the paper fiber highly microbial resistance. Where the active ingredient is adsorbed into fiber and distributed homogenously and kept out the fiber loaded with antimicrobial properties over all fibers areas. In one-layer paper sheets, paper fibers shape converted from flat form to round form, which help in compact the fibers together and became closed than in blank sheets. Antithesis in the two and three layers coated paper sheets, increasing in coating layers give significant draw back variations because paper fibers have over swell with coating matrix and become fragile.

On the other hand increasing the coating thicknesses gives significant variation in roughness values in comparison with reference sheets value (Abou-Zeid et al., 2018; Barhoum et al., 2014). One layer paper sheets roughness of 200 µm coating thickness increased with 13 times than 150 µm thickness. Additionally, double layers paper sheets with coating thickness 200 µm show increase in roughness value with 8 times than 150 µm thickness ones. Triple layers paper sheets showed less improvement with 4 times only. Burst index shows monotonous values, this may be takes place according to the swelling of fibers in all layers types lead to close all pours. However water absorption gives high values for coated paper sheets than blank one, the reason of this phenomena is similar to what happened in mechanical properties layers behaviors (Khwaldia Bet al., 2014). In addition, coating thickness and water absorption are directly proportion together. Air permeability in one layer paper sheets with coating thickness 200 µm was the lowest value, this is an advantage can synergized the antimicrobial properties of paper coated sheets (Basta et al., 2015; El-Gendy et al., 2017) as well as reinforce preservation characteristics behavior.

3.3.2 Coating sheets morphology

SEM image (Fig. 6) displays surfaces morphological structure of blank paper sheet (Fig. 6-A and B) and papers coated with one layer with 200 µm thickness (Fig. 6-C and D) and three-layer 150 µ thickness (Fig. 6-E and F) conjugate. Blank shows a typical morphology of the normal papers, which is a flat fiber network with free pores (Abou-Zeid et al., 2018). The fibers of coated papers were presented with thick diameter, that is can be attributed to highly soluble of conjugate which obtained from PSS (Essafi et al., 2009).

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Fig. 6

SEM for blank paper sheet at low magnification (A), high magnification (B), 1layer with 150 µ thickness at low magnification (C), high magnification (D), and 3 layers with 200 µ thickness. at low magnification (E), high magnification (F).

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Conjugate can be penetrating paper fibers and filling space of paper filament after drying. This is elucidating the roughness values as well as air permeability results. SEM images were emphasized the mechanical and physical measurements. In addition, this phenomenon enhanced the antimicrobial efficiency not only as a coating but also at active filler between cellulose fibers.