Harnessing the antimicrobial potential of natural starch and mint extract in PVA-based biodegradable films against staphylococcus aureus bacteria

Sustainable packaging solutions are of paramount importance in addressing the environmental challenges posed by conventional non-biodegradable materials. This study addresses this critical need by introducing a novel approach to crafting antimicrobial biodegradable polymer films. Leveraging the benefits of polyvinyl alcohol (PVA) as a base material, combined with corn-starch (CS) and mint extract (ME), these films offer a compelling synergy of eco-friendliness, antimicrobial efficacy, and mechanical strength. The antimicrobial property was imparted by adding mint extract, and boric acid (BA) was added as a cross-linker for better mechanical properties. All process was done by solution casting method followed by mechanical stirring. After 7 days, starch-PVA blend showed 50% weight loss; however, after adding mint extract, the action of microbes was reduced, and a 50% reduction in weight was observed after 12 days. The excellent mechanical properties were achieved by adding 10% aqueous solution of BA as a cross-linker. The confirmation of BA in the blend was done by the Fourier transform infrared spectroscopy (FTIR). Differential scanning calorimetry (DSC) was used to check the thermal properties of the films. Antimicrobial results showed that mint extract was resistant to staphylococcus aureus bacteria. These biodegradable films offer a multifaceted solution, aligning with sustainability objectives, showcasing antimicrobial potential, and demonstrating mechanical robustness. As such, they hold promise for a diverse array of applications, particularly in the realm of environmentally conscious food packaging. In the pursuit of greener alternatives, these films stand as a testament to innovative materials engineering that harmonizes functionality with ecological responsibility.


Introduction
Polymeric materials have a wide range of applications across various fields, ranging from synthetic replacements for damaged and missing body parts to disposable food packaging and utensils.The prevalence of these materials in our daily lives accentuates their significance.The widespread utilisation and manufacture of polymeric materials have raised significant environmental concerns due to their non-biodegradable nature under typical environmental conditions, thereby contributing significantly to pollution [1].Polyolefin such as polypropylene (PP) and polyethylene (PE) are the most commonly produced and widely used class of polymers for packaging applications.PE packing sheets are chemically stable and non-degradable under normal environmental conditions which produce waste filling landfills and oceans [2].Now, the world is facing the consequences of the excessive usage of single-use plastics (mostly in packaging applications) in the form of pollution and environmental impacts.Different techniques have been introduced to overcome plastic pollution and biodegradable plastics are one of them.Biodegradable plastics break down naturally by the action of bacteria into smaller compounds.PE, one of the most widely used packaging plastics can be designed to be biodegradable by preparing a blend of PE/PLA/PBAT, native starch, and calcium carbonate [3][4][5].
Beside olefins, some inherently biodegradable polymers are also utilised for packaging applications, such as polyvinyl Alcohol (PVA), starch, chitosan, polylactic acid, thermoplastic starch, and polybutylene adipate terephthalate (PBAT) [6][7][8].Biodegradable films are the need of the hour to lower the rate of plastic waste accumulation and its environmental impacts [9].These types of films have the characteristics of ordinary plastic as well as environment-friendly nature, they degrade easily under the action of different environmental conditions like sunlight, microbial action, splitting degradation, and environmental degradation.Rajeswari et al prepared a blend of cellulose acetate, alginate, and carrageenan to make environmentally friendly food packaging films [10].Extracts of mint and Artemisia are used in fish industry to increase the shelf life of the sardine fillets up to 7 days [11].Starch/PVA/CaCO 3 nanocomposites were prepared using solution casting method.The incorporation of CaCO 3 improves the thermal, fire retardant and tensile strength of the prepared films.The increase in CaCO 3 concentration reduced the oxygen permeability in the films which increased the fire retardant property along with tensile and thermal properties [12].
The development of biodegradable plastics sometimes involves trade-offs in terms of product quality (strength, flexibility etc).Thus, to improve the quality, different crosslinkers, plasticizers, and stabilizers are used [13].Borax and boric acid (BA) are most commonly used as crosslinking agents [14].There is a need to develop antimicrobial biodegradable packaging materials that can hinder the growth of microbes during their use to prevent microbial contaminations of food but readily degrade after their intended purpose.The aim of this research is to prepare biodegradable and antimicrobial films, based on PVA and corn-starch without compromising the quality.The mechanical properties were improved by adding BA as a cross-linker.Mint extract was used as an antimicrobial agent.The prepared films are recommended for food packaging applications.
2.2.Preparation of aqueous solution of PVA and gelatinization of starch 0.30 molar ratio of PVA was dissolved in 50 ml of deionized water at 95 °C and allowed stirring for one hour.0.30 molar ratio of corn-starch gelatinized solution was prepared at 90 °C then glycerol was added as a plasticizer with continuous stirring for 30 min.

Preparation of PVA-Starch-Mint Extract biodegradable film
Gelatinized starch (3 g) was then added to the PVA solution (3 g) and stirred for 30 min.Then mint extract (1 ml) was added to the solution dropwise.After 30 min aqueous solution of BA was added dropwise into the mixture.Transparent fluid was obtained in 15 min of stirring.Films were cast on a levelled glass plate (15 cm×15 cm) and dried for 24 h at 25 °C and then peeled off the dried films from the glass plate.For further drying, it was placed in the oven for 1 h at 60 °C.For comparative analysis, samples without mint extract and BA were also prepared.Table 1 shows the composition details of all prepared films and figure 1 shows the complete process of film fabrication and figure 2 shows the prepared films before degradation.

Characterization 2.4.1. Biodegradation test
A biodegradation test was performed under compost soil conditions at room temperature in soil that is microbially active (garden soil containing 22%-24% water content, 2% humus content, pH 6.5), stored in glass containers.The prepared films were exposed to soil and after every 3 days, test samples were extracted from containers followed by washing, cleaning with distilled water, drying, and weighing [15].

Fourier transform infrared spectroscopy (FTIR)
FTIR (PerkinElmer Spectrum Two) was used for the identification of functional groups present in the prepared sample in the range of 4000 cm −1 to 400 cm −1 .BA, cornstarch, and PVA functional groups were analyzed.

Tensile testing
Mechanical properties of films were measured using Universal Testing Machine (UTM) from Instron (3300) and according to ASTM D 882-01 [16].Tensile strength, % elongation, and modulus of the films were measured.

Antimicrobial test
The antimicrobial test was performed using Staphylococcus aureus and Escherichia coli bacteria.The disk diffusion susceptibility technique was used.The test was performed by applying approximately 1-2×10 5 CFU/ ml bacterial inoculum to the surface with a size 150 mm diameter Mueller-Hinton agar plate.

Differential scanning calorimetry (DSC
Differential Scanning Calorimetry (DSC) by Perkin Elmer was used for thermal analysis of the films from 30 °C to 280 °C at 10 °C per minute scan rate under N 2 atmosphere.

Scanning electron microscopy (SEM)
SEM was done to check the surface morphology of all the prepared films using (JEOL instrument JSM-6490A) instrument.At 3319 cm −1 the stretching vibration peak of -OH occurred, and the shape of the peak was similar to those of BA, mint extract, and PVA.This demonstrates that the hydrogen bonding in the prepared blends is similar to that in the individual component.The peak of -CH stretching in PVA is shifted to a higher wavenumber in the blend.The C=O group of mint extract and PVA is shifted to 1729 cm −1 in the blend.The absence of the B-O group of boric acid (BA) in the blend shows that the BA completely takes part in the cross-linking reaction.Second, the resemblance of the spectra of SPME-3, PVA, starch, and BA proved that the blending of all the components is achieved successfully.

Biodegradation test
The prepared films were degraded under the action of natural micro-organisms like fungi, bacteria, and algae.Degradation occurs in two steps, defragmentation caused by moisture, heat, and microbial enzymes, and in the second step of biodegradation, short carbon chains passed by microbes through their cell walls and are used as a food source.
The tendency of degradation of films under soil conditions was studied in this analysis.The humidity of the soil was maintained at approximately 20% to 40%.Film samples were cut into a rectangular strip (2.5 cm×3.0 cm).The biodegradation test was accomplished in customized soil pots.The films were buried 5 cm deep under the soil.After every three days' films were taken out from the soil, cleaned with deionized water,   Here W i and W f are the weights of the films before and after degradation.Physical conditions and changes were also observed as shown in figure 4. Figure 5 shows the degradation behaviour of different films.Sample SP degrade completely in 12 days and the sample SPME completely degrades in 18 days due to the incorporation of ME.The longer degradation time of SPME compared to SP caused by its antimicrobial activity in the prepared films which increases its lifetime.SPME-1 and SPME-2 show a little bit different behaviour and degrade in 21 and 23 days respectively due to cross-linker, which reacts with the PVA and starch to form a crosslinked structure as shown in figure 3.By increasing the amount of BA, increases in SPME-3 film, the degradation time increases to 27 days [11,12].

Tensile testing
The mechanical properties for instance, the tensile strength, modulus, and elongation at break of biodegradable films are compared in table 2. The sample SP shows a very low tensile strength of 1.53 MPa due to only starch and PVA in the blend.The sample SPME has improved mechanical properties due to the presence of OH groups (which increases resistance to degradation) as already discussed in FTIR.The blend SPME-1 shows an increase in tensile strength due to the addition of the crosslinker.The sample SPME-3 gives maximum tensile strength of 4.20 MPa which indicates that crosslinker improves strength but reduces the elongation at break as higher BA concentration restricts the motion under stress.The increase in the strength is due to the strong bonding between the PVA and BA, and the borate ions help to form a crosslinked structure by reacting with the alcoholic -OH group of PVA.BABA generates interfacial contacts with the polymer matrices, which leads to effective stress transfer, hence increasing the tensile strength.The flexibility of SPME-3 film decreases while that of SPME-1 film increases, as shown in figure 6.The slight increase of modulus of the films is due to the higher amount of BA.Glycerol performs the function of a plasticizer, creating only physical linkages with the biopolymer.The hydrogen bonding is generated between the hydroxyl groups of glycerol with the polymer  molecules at the carbonyl and hydroxyl sites.As a result, the plasticized biopolymer matrix transfers from brittle to leathery to rubber with increased extensibility and flexibility of the film [12].

Antimicrobial testing (Disc diffusion test)
A disc diffusion test was performed to test the antimicrobial activity of the packaging films containing mint extract.Two different bacteria Staphylococcus aureus and Escherichia coli were used.Figure 7(a) shows that SPME and SPME-2 samples have an inhibition zone of 6 mm against Staphylococcus aureus bacteria.While sample SP shows no zone of inhibition and bacteria grew all over the sample.In figure 7(b) for Escherichia coli, none of the samples show any zone of inhibition, and bacteria grows all over the samples and in a petri dish indicating mint extract is not effective against Escherichia coli.Starch has semi-crystalline microscopic granules that are joined together by a network of associated molecules that's why no sharp peaks of T g and T m were observed.PVA showed two small peaks of T g and T m at 130 °C and 194 °C respectively due to the crystalline microdomains of PVA.The blend of starch and PVA shows a sharp peak of T m at 140 °C.PVA crystallinity decreases with the addition of starch, because starch has amorphous structure, and when PVA is blended with the starch the crystalline structure of PVA and amorphous structure of starch mix together and it decreases the melting temperature of PVA.The expanded peak of the melting temperature shows that the PVA chains are restrained by the starch molecules which indicates the enhanced miscibility of these two components [14].
To explain the effect of BA on the thermal properties of blends, the peak of the SPME-1 blend shows T m at 118 °C which shows that with the addition of BA the chain motion of PVA and starch becomes slow.This is due to the inter and intramolecular chains of amorphous.
Regions linked with chemical reactions in starch, PVA, and BA in solution.Furthermore, BA restrained the growth of crystallite because crosslinking points are not included in crystalline domains [18].
3.6.Scanning electron microscopy (SEM) Figure 9 presents the SEM images of the prepared films before (a, c) and after (b, d) degradation.Before degradation, all formulations displayed almost smooth surfaces.There are no cracks or domains as visible in figures 9(a) and (c).The films show some irregularities due to the agglomerations of non-gelatinized starch, without compromising the properties of the material [19].The interactions between the polymers were due to the presence of hydroxyl groups of PVA and starch which leads to hydrogen bonding.
Reference [20] SEM results show some smoother patches of PVA-starch blends due to the plasticizer [21].Plasticizers reduce the intermolecular forces between the polymer chains and result in increased mobility of polymer chains, thus improving the flexibility of the film.PVA-starch blends have some irregular and rough surfaces when the PVA amount in the blends is equal to or more than that of starch.This behaviour is due to the formation of PVA-rich and starch-rich areas when the amount of PVA was equal to the starch content [22].Figure 9(b) shows the morphology of the SPME-1 blend after 5 days of degradation, minor cracks were produced on the surface of the films because of the less amount of cross-linking agent.Figure 9(d) shows the morphology of the SPME-3 blend in which cracks were starting to produce after 10 days, due to the high amount of crosslinking agent.It is seen that the biodegradation rate decreases with the addition of crosslinking agent.An optimum sample with the best mechanical and degradation properties, in this case is SPME-2.From SEM images, it is evident that the crosslinking agent significantly affects the degradation rate of the prepared films [23].

Conclusion
In conclusion, this study successfully engineered biodegradable polymer films based on PVA and corn-starch, enriched with the antimicrobial potency of mint extract.The incorporation of mint extract, along with the introduction of boric acid (BA) as a cross-linking agent, led to the production of films with desirable mechanical properties and antimicrobial functionality.Notably, the utilization of a 10% aqueous BA solution yielded remarkable mechanical strength, exemplified by the SPME-3 blend achieving a peak value of 4.20 MPa.The degradation behaviour of the films varied, with SP degrading within 12 days, SPME-1 and SPME-2 within 21 and 23 days respectively, and SPME-3 degrading in 27 days.The gradual degradation trend in SPME-3 can be attributed to the interaction between BA, PVA, and starch, resulting in a more robust matrix.Furthermore, antimicrobial assessments highlighted the resistance of mint extract to Staphylococcus aureus bacteria, while demonstrating susceptibility to Escherichia coli.The scanning electron microscopy (SEM) results indicated a correlation between crosslinking content and film degradation time, suggesting a potential avenue for tailoring the biodegradation rate.This research underscores the potential of these novel biodegradable films, blending eco-friendliness, antimicrobial efficacy, and mechanical integrity, thereby holding significant promise for sustainable food packaging applications and beyond.

Figure 1 .
Figure 1.Systematic Process for the fabrication of biodegradable packaging films.

3 .
Results and discussion 3.1.Fourier transform infrared spectroscopy (FTIR) The IR spectra of BA, starch, PVA, mint extract, and blend films are shown in figure 3 in the range of 3700-800 cm −1 .The peaks of BA are the sharp and wide vibration peak of the -OH group occurred at 3198 cm −1 .The peak at 1403 cm −1 is due to the asymmetric B-O stretching and at 1150 cm −1 the in-plane B-O-H bending can be seen.The peaks of starch are the flexible -OH vibration peak occurred at 3290 cm −1 .The peaks occurring from 1154 cm −1 to 1080 cm −1 were the asymmetric stretching vibrational peaks of C-O-C.The C-O stretching peaks occurred at 1017 cm −1 .The peaks of PVA are at 3328 cm −1 and 2917 cm −1 representing -OH and -CH 2 flexible groups respectively.A vibrational peak at 1729 cm −1 is due to the C-O bond in associating hydroxyl groups.The deforming vibration peaks of -CH 2 in -CH 2 OH appear from 1245 cm −1 to 1432 cm −1 , and the borate ions help to form a crosslinked structure by reacting with the alcoholic -OH group of PVA.Asymmetrical bending vibration peaks of C-O-C occurs at 1021 cm −1 .Infrared spectra of mint extract show OH group of alcohols and phenolic compounds at 3289 cm −1 and the peak of C=O at 1650 cm −1 .

Figure 2 .
Figure 2. Prepared biodegradable films (image captured before the start of biodegradation).

Figure 5 .
Figure 5.Comparison of % weight loss of different polymer films based on data obtained from soil burial test.(Higher concentrations of boric acid resulted in a reduction in biodegradability).

3. 5 .
Differential scanning calorimetry (DSC) Figure 8 represents the DSC curves of starch, PVA, SP, and SP-BA films.No obvious change is exhibited in the pure starch curve.A smaller endothermic peak is observed at 121 °C due to the agglomerate structure of starch.

Figure 6 .
Figure 6.Stress-strain curves of the prepared films.Samples tested at strain rate of 5 mm/min.(Increased boric acid concentration increased the tensile strength).

Figure 7 .
Figure 7. Disc diffusion method for the antimicrobial test of the prepared blends (a) Staphylococcus aureus & (b) Escherichia coli bacteria (inhibition zone of 6 mm against Staphylococcus aureus bacteria).

Figure 8 .
Figure 8.Comparison of the DSC thermograms of pure starch, PVA, SP, and biodegradable film (SPME-1) .Test was conducted at a heating rate of 10 °C under Nitrogen atmosphere.

Table 1 .
Composition details of all prepared films.

Table 2 .
Mechanical properties of PVA-Starch biodegradable films.