Description:FIELD OF INVENTION:
The present invention relates to the utilization of lignin, gelatin, and tannic acid for the development of a composite biopolymer film for sustainable packaging and seed protection.

BACKGROUND OF INVENTION:
Accelerated lifestyles of urban consumers and their relatively high levels of health and hygiene awareness has become a determining factor for the rise in the demand of packaged food items over their unpackaged alternatives. Globalisation and urbanisation have led to the increase in brand-consciousness and choice of quick and healthy food items, among the old and young population alike. The constant demand for packaged products originates from adapting to newer food trends and imitating other cultures. Such changeover from traditional to modern cultural environment has been in vogue which continues to create vast demands for packaged food products.

Packaged foods offer the benefit of being convenient and timesaving and prove to be a boon for busy individuals and families. Packaging technologies and materials help extend the shelf life of food products, reducing food wastage and allowing consumers to stock up on non-perishable items. Proper packaging ensures food safety and hygiene, protecting products from contamination and maintaining their quality throughout the supply chain. Packaging designed for on-the-go consumption, such as single-serve snacks and beverages, caters to the modern mobile lifestyle. However, maintaining food safety while minimising the use of preservatives remains a challenge in food packaging.

Packaging is an essential part of food manufacturing. Food packaging is a crucial tool for protecting, preserving, and presenting food products for distribution, sale, and consumption. The primary purposes of food packaging are typically to preserve food passively, chemically, biologically, and physically from environmental impacts, mechanical damage during storage and transit, secondary contamination, and obtaining light, air, either moisture or UV radiation.

The food industry is adapting to evolving consumer demands for “healthy” and premium quality food by reducing the adverse effects of food packaging through innovative advancements in active and intelligent packaging technologies. These smart innovations offer diverse and creative ways to enhance food product quality and safety while extending shelf life. Sustainable food packaging aims at reducing the environmental footprint of packed food by using plants, biodegradable materials, and nanomaterials to help mitigate negative impact on the environment. Innovative sustainable packaging reduces food waste by improving food quality, as well as food safety issues such as food borne diseases and food chemical contamination. It also addresses the issue of plastic waste, as well as saving oil and food material resources. Emerging techniques are also improving the passive aspects of food packaging systems, such as thermal stability, barrier effectiveness, and mechanical strength. There is a need to develop multipurpose food packaging systems by integrating intelligent, environmentally friendly, and active packaging technologies.

Since the last few years, polymers have been the preferred choice of food packaging material due to its high tensile strength to weight ratio. However, majority of the polymers used are derived from non-renewable sources which have poor degradability and recyclability, thus causing long-term pollution and waste accumulation in landfills and oceans.

Consequently, in recent years, biodegradable food packaging materials have emerged as a sustainable alternative to address the problems associated with conventional packaging. Biopolymers offer outstanding advantages of biodegradability, biocompatibility, and minimal environmental footprint. Among these gelatin, lignin and tannic acid stand out for their exceptional properties and abundant availability. These environmentally friendly biopolymers can be either chemically synthesized from bio-derived monomers or directly extracted from biomass or industrial wastes. Thus, the development, characterization, and use of these bio-based and biodegradable polymers as the basis of design and manufacture of packaging material is the need of the hour.

Gelatin, a protein obtained from collagen, has excellent film-forming capabilities, edibility, and flexibility, making it quite effective for the production of biodegradable films. Gelatin films have excellent oxygen barrier properties and mechanical strength, which makes it a suitable candidate for various applications including food packaging. Lignin, a complex aromatic polymer, demonstrates high rigidity, mechanical strength, and barrier properties against gases and water vapor. The UV absorbing property of lignin is an outstanding characteristic that makes it suitable for fabricating biofilms with a wide range of applications where light-induced degradation is crucial. Tannic acid, a naturally occurring polyphenol is renowned for its antibacterial and antioxidant properties, which can enhance the shelf life and stability of biofilms. The mechanical strength of Tannic acid is attributable to its ability to create crosslinking with proteins and polysaccharides.

A lot of research has been done in recent years on the one-to-one advantage of gelatin, tannic acid, and lignin.

Patent application no. IN202341066770A relates to the development of a biodegradable packaging film comprising of a biopolymer selected from a combination of cellulose, polysaccharides or other carbohydrates combined with a biodegradable gauze coated with fatty acid, the packaging film displaying high mechanical strength, surface hydrophobicity, and barrier properties.

Patent application no. US8105667B2 discloses an environmentally friendly multi-layer flexible film having barrier properties, comprising an outer layer of a bio-based film of polylactide, an adhesive layer adjacent to the outer layer and a product side layer comprising a metalized polyolefin.

Another patent application no. IN202211056564A relates to an antimicrobial biocomposite packaging film developed from reinforced fibre derived from agriculture waste. The packaging film is made from starch biocomposite incorporated with antimicrobial agent with antimicrobial properties that extends the shelf life of packaged food and pharmaceutical products.

Su Jin Lee et.al., 2023, developed a multifunctional chitosan/tannic acid composite films with improved anti-UV, antioxidant, and antimicrobial properties for active food packaging.

However, none of the above studies are focussed on the development of biopolymer based biofilms utilizing the synergistic potential of gelatin, lignin and tannic acid as packaging and seed coating material.

OBJECT OF THE INVENTION:
In order to obviate the drawbacks of the existing state of the art, the present invention discloses a hydrogel-based biopolymer film having applications in food packaging and seed preservation.

The main object of the present invention is to provide a strong, resilient hydrogel-based composite biopolymer biofilm comprising of gelatin (GA), lignin (LG) and tannic acid (TA) having antimicrobial properties and UV-blocking potential ideal for food packaging and seed coating.

Yet another object of the invention is to provide a hydrogel-based composite biopolymer biofilm as a viable and superior alternative to conventional materials in various applications.

Yet another object of the invention is to provide a sustainable hydrogel-based composite biopolymer biofilm exhibiting synergistic interaction of gelatin, lignin and tannic acid.

Yet another object of the invention is to provide a method of fabrication of the composite hydrogel-based biopolymer biofilm, such that it can be used as a biodegradable and ecofriendly food packaging and seed coating material for long-term storage of food items without losing the nutritional properties.

SUMMARY OF THE INVENTION:
The demand for eco-friendly, biodegradable, and recyclable packaging materials has increased in recent times due to the growing concern among the consumers about the environmental impact of material used for packaging food material. This underscores the need for innovative advancements in active, intelligent and biocompatible packaging technologies which are environment friendly and at the same time, retain the nutritional quality of food. Despite advancements in biopolymer research, the currently available biocompatible polymers used as packaging films for food items, face challenges such as limited antimicrobial property, inadequate UV blocking capabilities and insufficient mechanical strength, which can lead to reduced shelf life and compromised quality food and seed products, making them less effective for packaging and preservation purposes.
The present invention addresses this potential gap by developing a novel biofilm combining the properties of biopolymers, gelatin, tannic acid and lignin, having applications in food packaging and seed preservation. The present invention is the first attempt of its kind, to portray the benefits of the three biopolymers in real-time packaging applications as a viable and superior alternative to conventional materials in various applications through synergistic interaction between gelatin, lignin, and tannic acid.

The synergistic effect of the integrated biopolymers results in a material with enhanced mechanical performance, intrinsic antibacterial properties, and superior barrier performance. The biopolymer exhibits remarkable antimicrobial properties, effectively inhibiting the growth of common food-borne pathogens like Staphylococcus aureus and Escherichia coli. Additionally, the biofilm exhibits exceptional UV-blocking ability safeguards against critical ultraviolet radiation that can deteriorate the nutritional value and quality of food products. These qualities make the composite polymer biofilm of the invention a superior alternative to conventional materials, ensuring extended viability and protection for perishable goods.

BRIEF DESCRIPTION OF DRAWINGS:

Fig 1(a): depicts the FT-IR spectra of LGT, GA, LG and TA (b): depicts the
FT-IR spectra of LG/GA/TA biofilms synthesized using different compositions

Fig. 2: depicts the transmittance spectrum of lignin-gelatin - tannic acid
biofilms

Fig. 3: depicts the UV blocking ability of different biofilms

Fig. 4: depicts the a) Stress-Strain curve of 2% lignin with varying tannic
acid composition, b) Stress-Strain curve of 4% lignin with varying tannic acid composition, c) Stress-Strain curve of 6% lignin with varying tannic acid composition.

Fig. 5: depicts the TGA curves of the fabricated biofilms.

Fig. 6: depicts the DTA curves of the fabricated biofilms

Fig. 7: depicts the self-standing nature of synthesized biofilms

Fig. 8: depicts the comparison of the swelling percentage of biofilms prepared with different lignin to tannic acid ratios

Fig. 9: depicts the antimicrobial activity of LGT00, LGT22 and LGT68.

Fig. 10: depicts the Antifungal analysis of LGT00, LGT22 and LGT68 in
Aspergillus niger and Candida albicans

Fig. 11: depicts the Comparison of seeds with and without coating. The
seeds on the left are coated with biofilm and the right side are uncoated.

Fig. 12a: depicts the Preservation of raw beef over time using biofilm, Beef
wrapped in the biofilm from day 1 to 6.

Fig. 12b: depicts the microscopic images of beef pieces after 6 days; A)
without biofilm packaging, B) with biofilm packing.

Fig. 13: depicts the preservation of mushrooms overtime using biofilm. Top
row: mushrooms wrapped in the biofilm on day1 and day3. Bottom row: Comparison of mushrooms wrapped with and without biofilm after three days

DETAILED DESCRIPTION OF THE INVENTION:
The present invention relates to a method and system for developing a hydrogel based composite biofilm comprising of lignin (LG), gelatin (GA), and tannic acid (TA), as a functional and eco-friendly alternative to conventional packaging materials. In the present context, the term ‘hydrogel-based’ refers to a biofilm that incorporates a significant amount of water, either in its structure or as a key component of its matrix.

The composite biofilm is designed and developed for food packaging applications and demonstrates barrier properties, mechanical strength, antimicrobial efficacy, and biodegradability. The biofilm is versatile and agriculturally sustainable thus being effective in seed protection applications. The invention is a step forward towards a more sustainable future for both packaging industries and agricultural practices.

The composite hydrogel based biofilm of the invention is fabricated from Gelatin, Lignin and Tannic acid. The film is prepared in vitro using commercially available raw material. For fabricating the biofilm in vitro, Alkali lignin (LG) was purchased from Sigma-Aldrich, Bangalore, and Gelatin (GA) and Tannic acid (TA) were purchased from Sigma Aldrich, USA. All chemical reagents were used without further purification.

Fabrication of hydrogel-based biofilm:
The composite hydrogel based biofilm was prepared by dissolving a a specified amount of alkali lignin, and tannic acid in deionized water. Gelatin was dispersed in alkali lignin through ultrasonication for 10 minutes, followed by the addition of tannic acid. The reaction mixture was stirred magnetically for 3 hours at 80°C. Thereafter, the film was cast in a petri dish with a diameter of 9 cm and dried at room temperature for 24 hours. The resulting biofilm, with a uniform thickness of approximately 100 µm, was peeled off to obtain the final product. The biofilm thus obtained, was single layered. Biofilms fabricated with varying composition of GA, LG and TA is depicted in Table 1.

Table 1: Biofilms with different compositions of TA, GA and TA
Sample name Gelatin (g) Lignin (%) Tannic acid (%)
LGT00 2 0 0
LGT22 2 2 2
LGT24 2 2 4
LGT28 2 2 8
LGT42 2 4 2
LGT44 2 4 4
LGT48 2 4 8
LGT62 2 6 2
LGT64 2 6 4
LGT68 2 6 8

Characterization of Biofilm:
The fabricated hydrogel-based composite biofilm was characterized for different parameters which define its mechanical strength and preservation potential such that it can be used as a potential food packaging and seed coating material for long-term storage of food items without depleting their nutritional properties.

Thickness: Thickness of the film was assessed using digital vernier calliper following a standardized procedure after calibration. The Biofilm samples were placed securely between the jaws of the calliper and measurements were taken at multiple points across the surface, ensuring representative data collection. The obtained thickness of the samples was recorded accurately, from which the average thickness was calculated and noted as the thickness of the biofilm.

UV Transmittance: The synthesized biofilm was characterized for UV transmittance, after drying the samples by Fourier Transform Infrared Spectroscopy (FTIR). FTIR spectrum was recorded on PerkinElmer spectrum 100 ATR-FTIR spectrophotometer in the range of 4000 cm-1 to 400 cm-1. Lambda-25 UV-Visible spectrophotometer (Perkin Elmer, USA) in transmittance mode was used to measure the transmittance of the composite lignin-gelatin-tannic acid biofilm within the wavelength range of 200-800 nm. It is used to detect the UV rays transmitted from the films for analyzing their UV-blocking ability of films which is essential in packaging applications.

Figure 1(a) depicts the FTIR spectra of the biofilm samples. Pure GA characteristic bands were observed such as C=O stretching at 1678 cm-1 of amide 1, N-H bending at 1522 cm-1 of amide 2 and C-N stretching at 1235 cm-1. These characteristic bands of GA were also observed in the GA composite biofilms with varying concentrations of LG and TA. The spectra of LG (fig. 1a) show characteristic bands for aromatic C=C stretching at 1593 cm-1, aromatic C-H stretching at 3078 cm-1, and methoxy groups at 2935 cm-1 respectively. The characteristic band of TA for O-H stretching is the broad peak around 3200-3600 cm-1, aromatic C=C stretching at 1539 cm-1, and C=O stretching at 1710 cm-1.

Comparison of the FTIR spectrum of the composite biofilm shows several key differences compared to GA, LG, and TA indicating significant interaction between the LG, TA, and GA in the fabricated biofilms. Notably, new peaks and shifts in the position indicate the possibility of interactions and linkages between TA, GA, and LG. The shift of the amide 1 peak from 1678 cm-1 to 1629 cm-1 suggests the possibility of the formation of hydrogen bonds. This also supports the amide linkages between the carboxylic group of tannic acid and the amine group of gelatin. The aromatic C=C stretch peaks around 1500-1600 cm-1 show changes in intensity and slight shifts, indicating the interaction between the aromatic rings of tannic acid and lignin. When TA, LG, and GA are combined, GA molecules interact with TA and LG through hydrogen bonding which changes the molecular environment around the amide linkages typically seen at 1679 cm-1.

It is clear from the spectra that the peak is shifted to a lower wavenumber of 1636 cm-1 indicating that the interactions between carbonyl groups in the amide group of GA with TA make them more stable and reduce their stretching frequency. This could probably be due to the formation of hydrogen bonding between carbonyl groups of gelatin and the hydroxyl groups present in the TA and LG, which confirms the effective crosslinking ability of TA. The broad O-H stretches at 3200-3600 cm-1 in the LGT show broadening and slight shifts towards higher frequency. This indicates increased hydrogen bonding between LG, TA, and GA. Shifts in the amide 1 region, the appearance of the new ester peak, and the broadening of the peaks corresponding to O-H and N-H vibrations collectively indicate the formation of new hydrogen bonds, amide linkages and ester linkages. These findings provide an insight into the possible structural and chemical interactions within the biofilm.

Figure 1(b) compares the FT-IR spectra of different biofilms synthesized using different compositions of lignin and tannic acid which is indicated in Table 1. The GA is primarily composed of collagen which contains amide linkages typically seen at 1679 cm-1, upon addition of tannic acid and lignin they can interact with gelatin molecules through hydrogen bonding, leading to changes in the molecular environment around the amide linkages as discussed above. The peaks are shifted to a lower wavenumber of 1636 cm-1 indicating that the interactions likely involve the carbonyl groups of the amide linkages, making them more stable and reducing their stretching frequency. Similar presence of hydrogen bonding, amide, and ester linkages can be observed in all the cases which confirms effective crosslinking interaction between TA, GA and LG in each case irrespective of the loading.

UV Blocking: The UV-Visible radiation from the sunlight is mainly divided into three categories based on the radiation wavelength: UVC, UVB and UVA corresponding to 220-280nm, 280-320nm and 320-400nm respectively. As per previous reports, blocking of UVC and UVB are the most important because of the fact thatthey can induce photo oxidation, photo carcinogenesis and photo ageing. Many of the polymers are transparent to these types of radiations and are therefore, not safe for applications concerning food articles like food packaging, and coatings. Both the petroleum derived films as well as the biofilms also lack the UV radiation blocking efficiency in most of the cases. For good UV blocking materials, the transmittance should be near to zero in the entire range of UV spectrum. From the existing literature, it is understood that the gelatin films have only very poor efficiency to block UV radiation. On the other hand, lignin has excellent UV blocking ability due to its unique chemical structure which exhibits a variety of aromatic rings and phenolic hydroxyl groups that can absorb and dissipate ultraviolet radiations.

The transmittance spectra of the gelatin films modified with lignin is displayed in Figure 2. Here, the transmittance spectrum is considered instead of the absorbance spectrum since the reflection of light from the sample has to be regulated while measuring the UV absorbance. The spectrum under consideration ranged from 200 nm to 800nm range which includes UV and visible region. Considering the visible range of electromagnetic spectrum, lignin-modified gelatin film exhibit a transmittance efficiency of up to 80 percent. This coincides with the transparency of visible light ranging from 65% - 80% considered effective, as per the state of the art.

Usually, it is quite difficult to achieve a completely transparent biofilm with good UV-blocking efficiency. Notably, pure gelatin films are almost transparent and exhibit 95%-100% percent transparency, therefore, they have no UV blocking ability. However, the transparency decreases with the inclusion of lignin and tannic acid in the gelatin matrix. The transparency level of higher-loaded biofilms reaches up to 75% at the end of the region and lower-loading biofilms reach up to 80% as shown in Figure 3. This level of transparency is considered good enough for packaging applications. The thickness of the biofilm is another determinant for transparency of the biofilm, such that a higher loading of lignin with different tannic acid content decreases the transparency.

Every sample represented in Figure 3 having different lignin content can block UVC and UVB. The biofilms LGT62, LGT64 and LGT68 show zero transmittance to UV radiations up to 400 nm range, which means they can block the entire range of UV radiations includes UVA, UVB and UVC. The LGT42, LGT44 and LGT48 biofilms have transmission up to 370 nm in UVA and up to 400 nm reaching 24% transmittance. The LGT22, LGT24 and LGT28 maintain a zero transmission up to 340 nm after which the transmission rapidly increases to 40% at 400 nm. The UV blocking efficiency of the fabricated biofilms of the present invention make them a good candidate for packaging food materials as they prevent radical oxidation caused by UV radiation which preserves the seeds from damage enabling generation of superior plants from them.

Mechanical Properties: The mechanical properties of the films were studied to understand their suitability for packaging applications. Tensile strength, elongation at break, and Young’s modulus were measured using a Universal Testing Machine, Tinius Olsen with slightly modified ASTM D882. The tension test was conducted at normal room temperature with a crosshead speed of 2mm/min. Every test was performed in triplicate to ensure reproducibility and the average considered.

The stress-strain curves of different biofilms prepared using LG, TA, and GA is depicted in Fig. 4. The curves in the graph portray the balanced strength and flexibility of the biofilm. The graph indicates an initial portion showing a linear relationship between stress and strain for all the biofilms. This represents the elastic deformation of the biofilms, where the biofilm returns to its original shape upon the removal of stress. After that the curves start to deviate, showing the plastic deformation region of the biofilms. Thus, it indicates that before breaking, all the gelatin films show linear and plastic deformation.

The stress-strain curve of 2% lignin with 2%, 4%, and 8% tannic acid content relative to gelatin is shown in Figure. 4(a). For LGT22 sample the stress increases steadily with strain and the sample shows a tensile strength of about 18.6MPa±0.03. The curve shows a typical elastic-plastic behavior where the biofilm behaves initially elastically and then plastically before breaking. In the case of LGT24, the stress increases more sharply when compared to LGT22 with a maximum of 22.4±0.05MPa. The behavior of this film is almost like LGT22 but has a higher peak stress, indicating an optimal combination for strength in this series. In LGT28, the stress is significantly lower, which means that the film is more flexible, peaking around 1.8±0.02MPa. The curve flattens out rapidly showing that the material can stretch more easily before breaking. The tensile strength increases first with the TA content up to 4wt% but reduces at 8%.

Figure 4b. shows the stress-strain curve of biofilms with 4% lignin and 2%,4% and 8% tannic acid with respect to gelatin. LGT42 sample shows a steady increase in stress peaking around 19.6±0.01MPa. This trend shows a more balanced strain before failure as compared to the 2% series. It is also clear that with increasing LG content, the tensile characteristics of the gelatin films increase irrespective of the loading of TA up to 4%. The dragged strain in the biofilm suggests that an increase in lignin content improves the ductility of the biofilm. The LGT44 biofilm peaks around 29.2±0.04MPa. Here the stress increases sharply, suggesting a more balanced strength and flexibility in this series. This characteristic flexibility and strength make it more suitable for robust applications. In the case of LGT48, the stress is significantly lower, peaking around 2.5±0.05MPa. This trend is in good agreement with the previous series, reduced strength with increased tannic acid content.

Figure 4c. shows the stress-strain curve of biofilms with 6% lignin content and 2%,4% and 8% tannic acid content. However, it is important to note that the tensile strength reduces with the incorporation of 6% of LG. Thus, tensile strength increases with the filler loading of LG up to 4% after which negatively affects the tensile properties. However, a moderate increase in stress around 20±0.01MPa can be seen in LGT62. This biofilm shows a decent strength and high flexibility. Meanwhile, in LGT64 the stress increases sharply peaking around 34±0.03 MPa. This composition maintains an improved strength compared to the LGT62 with good flexibility. The stress of the LGT68 sample is very low, around 9.02±0.04MPa.

The stress-strain data gives a significant idea about the mechanical properties of the biofilm which are influenced by the relative concentration of lignin and tannic acid i.e., by increasing the amount of tannic acid in the system the tensile strength gets diminished. These conclusions highlight the importance of tailoring the ratio of lignin and tannic acid in achieving a film with desired balance of strength and flexibility. The tensile strength of samples of different biofilms is denoted in Table 2.

Table 2. Tensile strength of different biofilms.
Composition Tensile Strength (MPa)
LGT22 18.6±0.03
LGT24 22.4±0.05
LGT28 1.8±0.02
LGT42 19.6±0.01
LGT44 29.2±0.04
LGT48 2.5±0.05
LGT62 20±0.01
LGT64 34±0.03
LGT68 9.02±0.04

Thermal analysis: The Differential thermal analysis (DTA) of the fabricated biofilms was performed to evaluate their thermal properties. The thermal stability and phase transitions were determined to assess the two steps of degradation, first one at initial time due to moisture and the second is due to the decomposition of the components in the biofilm. Thermal analysis of the biofilm was performed using PerkinElmer Pyris Diamond TG/DTA to study the thermal behavior and decomposition nature of the biopolymer films. Thermal stability and decomposition nature of the biofilms were investigated using Thermo Gravimetric Analysis (TGA) and Differential Thermal Analysis (DTA). TGA was conducted from room temperature to 700°C at a heating rate of 10°C /min under nitrogen atmosphere to prevent oxidation. The weight loss as a function of temperature was recorded and the data was evaluated to determine the decomposition temperatures and residual weights. DTA was performed simultaneously with TGA to identify the exothermic and endothermic transitions in the biopolymer films. The differential temperature between the sample and a reference material was recorded as a function of temperature.

Figure 5 depicts the TGA of the fabricated biofilms LGT00, LGT28, LGT48, and LGT68 showing different thermal degradation profiles. As shown in the figure, LGT00 sample shows a steady decrease in weight, similarly, LGT28, LGT48, and LGT68 also show a significant weight loss, but a different trajectory compared to LGT00. The LGT00 sample appears to lose weight slightly slowly in the initial stage, indicating that the thermal stability of LGT00 is slightly more than the others. The addition of LG in the GA films deteriorates the degradation temperature. The LGT68 film starts slightly degrading around 58°C and the second degradation starts at 380°C. A similar trend is also observed in other lignin compositions also which is due to the inherent characteristics of lignin. The inclusion of LG leads to less char yield than the LGT00 film. LGT00 achieves a char yield of 0.99% at 750°C meanwhile the LGT68 achieves 0.020% char yield at 698°C. These differences highlight the influence of lignin and tannic acid on the thermal behaviour of the biofilms and concludes that the thermal stability of the biofilm slightly decreases with an increase in LG and TA.

Figure 6 depicts the endothermic peaks of the biofilms at 159°C corresponding to the dehydration of gelatin films in the Differential Thermal Analysis (DTA). The second DTA curve at 339°C is due to the decomposition of the gelatin biofilm. The reason for this decrease is probably due to the disintegration of the intermolecular side chain and main chain of the gelatin. As the amount of lignin and gelatin increases, the second peak shifts to the lower temperature indicating the reduction in the thermal stability of the biofilm with the LG and TA content.

The fabricated biofilms of the invention were also analysed for their resilience and self-standing properties, which is indicative of their ability to maintain their structure without the need for additional support. Fig. 7 depicts the self-standing property of the fabricated biofilms. The self-standing ability of a film refers to its capacity to maintain structural integrity and shape without external support. It is a critical property for various applications, particularly in packaging, coatings, and biomedical fields, where the film must function independently under practical conditions. This characteristic property is particularly advantageous for applications requiring free-standing films.

Swelling studies: The swelling properties of biopolymer films are critical for seed preservation and food packaging applications. The swelling behaviour of fabricated biofilms was investigated with different weight ratios of lignin, gelatin and tannic acid.

The assessment of swelling properties of the biofilm revealed the crosslinking efficiency of the gelatin film with respect to LG and TA. The swelling percentage of different biofilms is correlated in Figure 8 which indicates that as the amount of tannic acid increased in the biofilm, the extent of crosslinking also increased. This observation is in line with the fact that tannic acid is known for its ability to crosslink, thereby modifying the physical properties of the biofilm network. This enhanced crosslinking results in a denser polymer network, which significantly influences the swelling properties of the biofilm. . In every set of studies, as the crosslinking agent increased, the swelling decreased. As can be seen in Figure 8, maximum swelling is observed for LGT22, and minimum swelling is observed for LGT68. This impact in the biofilm swelling resulted in a decrease in free volume or space between the network to absorb water. Hence better crosslinked samples show reduced swelling percentage.

Mechanical strength is believed to have a good agreement with the swelling behaviour of biofilms. The increased crosslink density enhances the mechanical strength and rigidity of the biofilm, making it more difficult for the network to expand when exposed to a solvent. Considering the thermodynamic side of the hydrophilicity and crosslinking, while tannic acid and lignin may contribute to the hydrophilicity of the films due to its hydroxyl groups, the predominant effect of elevated crosslinking overshadows this. The dense network restricts water uptake despite potential hydrophilic sites. This becomes more important since for food packaging and seed preservation coating applications, a lesser swelling biofilm with higher lignin and tannic acid content is suitable.

Water vapor permeability: The water vapor permeability of the biofilms was measured over a period of 7 hours wherein the water vapor permeability of the samples with different compositions of lignin and tannic acid in gelatin matrix were evaluated. Results of the assessment are summarized in table 3.

Table 3. Water vapor permeability of different biofilms with varying lignin and tannic acid concentrations
Samples WVP (g.m/Kpa.m2.h)
LGT22 0.0829
LGT24 0.0484
LGT28 0.04005
LGT42 0.06295
LGT44 0.04780
LGT48 0.03666
LGT62 0.07272
LGT64 0.03783
LGT68 0.02876

The water vapor permeability results of different biofilms indicate the clear trend associated with the composition of lignin and tannic acid. As the amount of tannic acid and lignin increases, the water vapor permeability decreased. This pattern is clear in samples LGT44, LGT48, LGT62, LGT64, and LGT68, which exhibited smaller vapor permeability values as compared to the other samples. The decrease in water vapor permeability is attributed to the enhanced network strength because of the increased crosslinking created by the higher amount of lignin and tannic acid. The high networking within the biofilm matrix reduces the free volume within the biofilm, resulting in the limiting of water vapor passage through the biofilm.

For fresh food packaging, the water vapor permeability should be around 2-5g.m/kPa.m2.day and for dried or moisture-sensitive foods, a low water vapor permeability of almost 0.01g.m/kPa.m2.day is crucial to prevent moisture. The results in Table 3 are in good agreement with the existing state of the art. This indicates towards the efficiency of the fabricated biofilm synthesized with higher lignin and tannic acid contents for seed preservation coating and food packaging. Maintaining low moisture levels prevents drying out and avoids excessive moisture, thus giving an extended shelf life for packaged food. In seed preservation coatings, lower moisture content can prevent premature germination of seed, protection against mold and fungi and long-term storage stability, thus, biofilm with low water vapor permeability can provide a good barrier against water vapor.

Antimicrobial activity: Antimicrobial activity was studied using the agar-disc diffusion method. The antimicrobials present in the samples were allowed to diffuse out into the medium and interact in a plate freshly seeded with the test organisms. The resulting zone of inhibition is visible as a uniformly circular zone within a confluent lawn of growth of the microorganism. The antimicrobial efficiency of the samples, LGT00, LGT22, LGT68 were analyzed with 3 replicas to ensure accuracy. Petri plates containing 20ml Muller Hinton Agar Medium were seeded with the bacterial culture of E. coli and Staphylococcus aureus (growth of culture adjusted according to McFarlands Standard, 0.5%). Plates were placed with sterile material having respective test samples. Plates were then incubated at 37°C for 24 hours and the antibacterial activity was assayed by measuring the diameter of the inhibition zone formed around the discs.

The development of antibacterial biofilms for food packaging and seed preservation application is very crucial in maintaining the shelf life and preventing contamination. Previous research has reported that pure gelatin film does not show any antimicrobial efficiency. However, it has been reported that tannic acid and lignin have very good levels of antimicrobial resistance to both gram-positive and gram-negative bacteria.

The activity of the fabricated biofilms towards gram-positive and gram-negative bacteria was studied especially with respect to Staphylococcus aureus and E. coli. Table 4 depicts the antimicrobial activity of the biofilms comprising of varying levels of gelatin, lignin and tannic acid. From the table, it is clear that as the composition of lignin and tannic acid increases, the antibacterial efficiency also increases. The biofilm having the highest composition of lignin and tannic acid has a zone of 23mm, at the same time the lowest composition of lignin and tannic acid has a zone of 11mm (figure 9.). This suggests the potential application of these films as sterile food packaging material and seed preservation coating.

Table 4. Antibacterial activity of LGT00, LGT22 and LGT68
Organism Sample code Zone of inhibition (mm)
E. coli Streptomycin(100µg) 30
LGT 00 Nil
LGT 22 11
LGT 68 23
Staphylococcus aureus Streptomycin(100µg) 28
LGT 00 Nil
LGT 22 11
LGT 68 23

The antifungal activity was determined by Agar well diffusion method. The antifungals present in the samples are allowed to diffuse out into the medium and interact in a plate freshly seeded with the test organisms. For testing the antifungal properties of the biofilm, Potato Dextrose agar plates (PDA) were prepared and overnight grown species of fungus, Aspergillus niger and Candida albicans were swabbed. Plates were placed with sterile film having respective test samples. The zone of inhibition was measured after overnight incubation at room temperature and compared with that of standard antimycotic as control.

The results of antifungal activity of fabricated biofilms are demonstrated in Figure 10, wherein, no fungal growth is visible on the surface of the LG/GA/TA biofilm samples, indicating their potential antifungal properties in preventing colonization on their surface. The absence of a zone of inhibition suggests that while the biofilms do not release antifungal agents into the surrounding medium, they possess surface properties that inhibit fungal growth. This property can be particularly beneficial in applications where direct contact inhibition is desired, such as in food packaging and seed preservation. Only one sample namely, LGT00 containing pure gelatin exhibited fungal growth on the surface, indicating lack of antifungal properties.

Water uptake: The water uptake capacity of the biofilms was analyzed for samples with dimensions 1cm x 1cm. The films were dried at 600C for 3 hrs. after which the dry weight was recorded, and the film pieces placed in 10 ml of distilled water. The weight change was measured every 10 minutes, by swiping the films using filter paper to remove the excess water on the surface and the weight measured till constant weight was obtained. The water vapor permeability of the biofilms was estimated gravimetrically using slight modifications in the standard method ASTM E96/E96M-12. The films were cut into circular pieces of radius 3.15 cm and placed safely on the top of the water vapor permeability measuring cells. The level of water was kept 1cm below the biofilm. The weight of all cells was measured before placing them in a desiccator having anhydrous silica crystals, with a relative humidity of 30% and an internal temperature of 25°C. The cell weight was measured every hour over a period of 7 hours. The water vapor permeability of the biofilms was calculated in gm./kPa.m2.h using the following equation:
WVP=(W.L)/(A.t.∆P) (1)
Where W is the weight of permeated water through the cell (g); ΔP is the water vapor pressure difference between the two sides of the film (KPa); A is the area of the biofilm(m2) and L is the thickness of the biofilm.

Efficiency in seed coating: The real-time application of biofilm as an effective packaging material was studied in packing red meat and common edible button mushrooms. Further, the efficiency of the material to act as a coating in seed preservation was evaluated using the seed of Momordica charantia (bitter gourd). The red meat was packed using biofilm and the packed as well as unpacked material was monitored for about 6 days with the help of optical microscopy. The seeds of Momordica charantia were dried properly and coated with the biofilm, after which they were left for drying at room temperature for 24 hrs. The coated as well as uncoated seeds were monitored for fungal growth for 2 weeks. Figure 11 depicts that the seeds coated with the biopolymer film remained fresh even after 6 days whereas the uncoated seeds turned dry and stale in 6 days. Moreover, the coated seeds show no visible signs of fungal contamination, in contrast the uncoated seeds exhibit significant fungal growth on their surface.

Real packaging of beef raw meat with gelatin-tannic acid-lignin films- Raw beef pieces were packaged with LGT68 films at normal room temperature for 6 days in an open atmosphere to evaluate its properties of UV protection, antimicrobial action, biodegradability, and moisture control on raw meat. Figure 12a depicts the effect of biofilm coating on the surface of beef pieces after six days of packaging. The surface of coated meat was analyzed using a microscope. The microscopic image of Figure 12b shows that there was significant microbial growth on the surface of the beef without packaging film which is evident by the appearance of numerous colonies of bacteria or fungi, indicating contamination. On the other hand, the beef with packaging film shows that the surface remained largely intact and free from extensive spoilage, suggesting that the biofilm packaging effectively inhibited microbial contamination and growth. This observation highlights the potential benefits of the LGT68 biofilm packaging in extending the shelf life of beef by inhibiting microbial growth and spoilage.

The packaging efficiency of the fabricated biofilms was also assessed in common edible button mushroom. LGT48 biofilm was used for wrapping mushrooms, and compared with the mushrooms without wrapping, after both wrapped and unwrapped mushrooms were kept for two days in an open space. The colour of the wrapped mushrooms remained intact whereas the colour of the unwrapped mushrooms changed, indicating that they started decaying after two days as shown in Figure 13.

The biofilm forms a protective barrier that inhibits fungal growth and UV damage, thereby enhancing the shelf life and safety of the seeds. The effectiveness of the biofilm coating can be attributed to the antifungal property of lignin and tannic acid, as well as the film forming capacity of gelatin, which together create an environment that is hostile to fungal proliferation.

The present invention is focussed on the synthesis of a novel biopolymer film which is strong, flexible, robust, having low water absorption and UV absorption properties and prevents the growth of bacteria and fungi. These properties of the biofilms are attributed to the synergistic effect of its components namely gelatin, lignin and tannic acid in different combinations, which makes is an ideal material for packaging food items and as coating material for seeds. The present invention focusses on the use of commercially available lignin, gelatin, and tannic acid for the fabrication of hydrogel based biofilms to maintain consistency in fabrication and ease of procurement of raw material. However, these materials can also be sustainably sourced from biowaste. Lignin can be extracted from lignocellulosic agricultural residues such as coconut shells, wood chips, or crop waste through methods like alkaline extraction or organosolv processing. Gelatin, a protein derived from collagen, can be extracted from animal waste such as bones, skins, and connective tissues using hydrolysis techniques. Similarly, tannic acid can be isolated from natural sources like tree barks, nut shells, and certain leaves through aqueous or alcohol-based extractions. By developing efficient extraction protocols, future work can focus on utilizing these biowaste sources to reduce dependency on commercially available materials and enhance the sustainability of the developed products

The fabricated biofilms contribute to the global effort in reducing plastic waste thus promoting sustainability. The synthesis of biofilms by leveraging the excellent properties of lignin, gelatin and tannic acid prioritizes the fulfilment of the demand of modern preservation and packaging technologies, thus aligning with the SDG goals SDG 2 (Zero Hunger), SDG 3 (Good Health and Well-being).

, Claims:WE CLAIM:
1. A hydrogel based bio based film for food packaging and seed coating, said biofilm comprising a composite polymer comprising Lignin, Gelatin and Tannic acid (TA),
characterized in that the synergistic interaction of Lignin, Gelatin and Tannic acid in pre-determined compositions provides strength, resilience, antimicrobial properties and UV-blocking potential ideal for food packaging and seed coating.
2. The biofilm, as claimed in claim 1 wherein, the composition of gelatin in the said biofilm ranges from 85%to 95% by weight.
3. The biofilm, as claimed in claim 1, wherein the composition of lignin in the said biofilm ranges between 1% to 10% (w/w) and the composition of tannic acid in the said biofilm ranges between 1 to 12 percent.
4. The biofilm, as claimed in claim 1, wherein the said biofilm comprises at least 2% (w/w) of tannic acid.
5. The biofilm, as claimed in claim 1, wherein the thickness of the biofilm ranges from 0.05mm to 3mm.
6. The biofilm, as claimed in claim 1, wherein gelatin, lignin and tannic acid are cross-linked by forming hydrogen bonds, amide and ester linkage.
7. A method of fabrication of the biofilm as claimed in claim 1, the method comprising the steps of:
- suspending gelatin in 1 to 10 percent by weight alkali lignin dispersed in deionized water using ultrasonication for 10 minutes,
- adding 1 to 12 percent by weight tannic acid dissolved in deionized water, to the gelatin-lignin suspension,
- magnetically stirring the reaction mixture for 3 hrs. at 800C,
- casting the reaction mixture in a dry petri dish for 24 hours,
- peeling-off the cast mixture to obtain the resulting composite biofilm.