Synthesis and Characterisation of Novel Cross Linked Biopolyesters from Olive Oil as Eco-friendly Biodegradable Materials

Novel cross linked biopolyesters were synthesised from naturally available olive oil, oligomeric polytriglyceride fumarate (o–PTF) was prepared by glycerolysis of the olive oil followed by condensation with maleic anhydride. The (o–PTF) was characterised by spectral (FTIR, UV, NMR) and physicochemical properties (specific gravity, viscosity, saponification value, iodine value). The molecular weight of o-PTF was estimated using gel permeation chromatography (GPC). The cross linked biopolyesters were prepared by treating o-PTF of olive with N-vinyl-2-pyrrolidone and vinyl acetate. The cross linked biopolyesters were evaluated for spectral properties (FT IR), cross linked density, thermal properties, mechanical properties and biodegradation. The potential antifungal and anti bacterial activities of the newly prepared biopolyesters were evaluated. The environment degradation was assessed by soil burial test. The outcome of the studies has revealed that the newly prepared cross linked biopolyesters are potential biodegradable material for various consumer application like package materials and agricultural applications.


Introduction
The replacement of petroleum-based raw materials by renewable resources constitutes a major contemporary challenge in terms of both economical and environmental aspects 1 .
Natural vegetable oils are considered to be one of the most important classes of renewable sources because of the wide variety of possibilities for chemical transformations, universal availability and low price and they are preferred by the chemical industry as alternative [2][3][4] .The main components of the triglyceride vegetable oils are saturated and unsaturated fatty acids which in its pure form are also available as platform chemicals for polymer synthesis 4,5 .Plant oils are triglycerides (tri-esters of glycerol with long chain fatty acids), with varying composition of fatty acid depending on the plant, the crop, the season and growing conditions 6 There has been renewed interest recently in developing new polymers from soybean and other plant oils as offer a renewable feed stock against finite and unreliable petroleum sources.Larock et al 7 have developed a family of chemically modified plant oils which can be polymerised using free radical initiators, into rigid composites, rubbers and adhesives 7 .Sperling et al 8 have prepared soft rubbers from epoxidised oil cured with acid anhydrides like maleic anhydrides and used the interpenetrating networks to apply on roof tops 8 .Cross linked biodegradable polymers have been prepared from edible oils with unsaturated anhydrides or acids have useful applications as adhesives, fillers and elastomers 9 .These renewable resources have proved to be useful basis for the synthesis of a variety of monomers as well as linear and cross linked polymers of different types (e.g.Polyolefin, polyesters, polyurethane resins and others) 10 .The present study explore the potential utility of the edible oil olive oil for the preparation of the novel cross linked biopolyesters as eco-friendly biodegradable material of various consumers application like packaging material and agricultural application.

Synthesis of o-PTF resins
Glycerolysis of olive oil were carried out using 30% hydrogen peroxide and formic acid, in ice water bath.The reaction was vigorously stirred over night.The resulting product was poured into a separator funnel and extracted with ether and the ether extracted product was then reacted with maleic anhydride and the mixture was refluxed for 5 h at 70 0 C -80 0 C at 160 0 C for 30 minutes under vacuum condition using rota mantle to yield a yellow transparent liquid o-PTF resins.

Synthesis of novel cross linked biopolyesters
The four biopolysters were prepared by free radical polymerisation of o-PTF resin of olive oil with cross linking co-monomer N-vinyl-2-pyrrolidone and vinyl acetate at room temperature with benzoyl peroxide as free radical indicator and dimethyl aniline as accelerator.To prevent oxygen free radical reaction the resin was purged with nitrogen gas prior to curing and then casted on a clean silicone oil spreaded glass plate, cured for six hours at room temperature.All the cured material showed high toughness, elastomeric and good transparency.

Characterisation of o-PTF resins
The o-PTF resins were subjected to extensive analysis for the determination of specific gravity, iodine value and sap value as per the ISI standard 840 -1964.The molecular weight was determined by GPC using µ styragel column, 100 A 0 and 500 A 0 , UV detector and 280 nm filter.The o-PTF were analysed by UV, IR, NMR spectral analysis.

Characterisation of biopolyesters
The AT -IR spectral analysis of the four biopolyesters was done by perkin 597 infrared spectrophotometer.The Tensile strength of the biopolyesters were determined using Dumbbell shaped cut from the specimen using ISO 527 -2:1993 (E) specimen type SA die.The values represented are an average mean of about 6-10 samples.The thermal analysis of the biopolyesters were determined by simultaneous DTA -TGA and DSC using SDT -2960 TA instruments INC derived from ASTME 1131 -03.

Soil burial test
The replicate pieces of the sample (5x3 cm) were buried in the garden soil at the depth of 30 cm from the ground surface for 3 months, inoculated with the sewage sludge having ability to adhere and degrade the polymer film.The test specimen was periodically removed from the soil and the specimen was then gently washed to remove attached soil and dust after being dried in vacuum oven.The extent of degradation was examined by weight loss and surface observation.

Microbial studies
Bacterial adhesion and antimicrobial activity were evaluated by agar diffusion method the microbial strains used are Escherichia coli (gram positive bacteria) and Candida albicans (fungi).

Results and Discussion
Olive oil is a mixed glyceride of unsaturated components (linoleic acid, oleic acid) and saturated compound (palmitic and stearic acid), since the concentration of unsaturated compounds are higher the mixed unsaturated triglyceride molecule offers a number of reactive sites, C=C bonds, the carbon alpha to the ester group for functionalisation 11,12 .Under the present experimental conditions hydroxylation takes place at the double bonds of the triglyceride units.The reaction of hydroxylated olive oil with maleic anhydride catalysed by morpholine lead to the formation of oligomeric-poly triglyceride fumarate (o-PTF) resin, the analysis of these resins reveals the formation of o-PTF resins.The analytical data are given in Table 1, finally the cross linking of o-PTF resins involves the reaction of unsaturated sites in the polymer chain with a co-monomer which may be of vinyl type such as vinyl acetate and N-vinyl-2-pyrrolidone to form biopolyesters of olive oil.Iodine value represents the degree of unsatuaration (= bonds) in the triglyceride oil.The high iodine value of olive oil indicates the higher concentration of unsaturated compound in the parent oil.In the hydroxylated resin iodine value is decreased drastically revealing that the double bond is replaced by hydroxyl group.The hydroxyl value determined the number of hydroxyl groups.The iodine values is increased significantly in the o-PTF resins due to the unsaturated fumarated side chain in the o-PTF resins Saponification is applied to the hydrolysis of fatty acid ester under alkaline condition.Saponification number is used for the determination of the size, average molecular weight of the fatty acid and to estimate the non fatty impurity if present.Therefore the molecular weight of the compounds were determined from the relation molecular = weight = 168,000 / Sap value.The molecular weight determined by this method match the calculated value.The saponification value reveals that the molecular weight of o-PTF resin has increased compared to the oil

Spectral analysis of o-PTF resin
The UV spectrum of olive oil shows an absorption peak at about 230 nm (ester).The UV spectrum show a shift of peak from 230 nm for oil to 259 for hydoxylated resin due to the hyper chromic effect which is attributed to the substitution of the unsaturated group.In the o-PTF resins the peak is again shifted back to 235 nm due to hypo chromic effect which is attributed to the distortion of geometry by the introduction of fumarate group 13 .

IR spectral analysis
The IR spectra of olive oil, HTO resins, (o-PTF) resins are recorded between 500 cm -1 to 4500 cm -1 (Figure 1 4. The responses at 2925 cm -1 is due to the aliphatic C-H group in the chain, the peak at 1730 cm -1 (C=O stretch in esters) and peak at 1454 cm -1 is due to CH 2 linkages and the peak at 1372 cm -1 is due to CH 3 group and 3500 cm -1 is due to OH group.  5.The spectrum shows responses at 2924 cm -1 due to the aliphatic -CH groups in the chain, the peak at 1723 cm -1 C=O stretch in esters and peak at 1645 cm -1 is due to CH bending of trans CH=CH and the peak at 1021 is due to C=C stretching.
The possible peaks in 1 H NMR for quantitating unsaturated fatty acids are those of olefinic protons (5.3-5.4 ppm), protons attached to the bis-allyllic carbons (2.7-2.8 ppm), protons attached to alliylic carbon (2.0-2.1 ppm), methylene -CH 2 -(1.2-1.4 ppm) and the terminal methyl protons (0.8-0.9 ppm) shown in the (Figure 6).The corresponding olefinic protons peak in the glycerolysis product has almost disappeared (Figure 7), showing the double bonds in oil is replaced by the hydroxyl group.This peak is shifted to 3.1-3.2ppm in the o-PTF resins due to the deshielding effect of hydroxyl and carboxylate ester linkages (Figure 8).4).

Mechanical properties
The data of mechanical properties of all the cure samples are shown in the Table 3.The higher tensile strength and modulus are observed in vinyl acetate biopolyesters than N-vinyl-2pyrrolidone biopolyesters.The modification with MA increases the chemical functionality of the biopolyester resins results in a increasing cross linked density thus from these results the modulus and strength of triglyceride based biopolyesters can be increased by simply increasing the chemical functionalities of the triglycerides 16 .The high strength and high modulus reflect the strong and hard character of these poly esters (Table 5)

Soil burial test
The biopolyester films 6x3 cm were buried in soil for 6 months for isolation of degrading microbe.It was found that a remarkable degradation was indicated in the sample (Figure 11).The percentage of weight loss was low between 30 and 40 days After 40 days the degradation was rapid from this results triglycerides cross linked with hydrolysable bonds such as esters remain biodegradable 17 .

Aerobic test
The aerobic biodegradability of polymeric resins in a aqueous medium was evaluated by determining the oxygen requirement in a closed respirometry (Germany standard ISO 9408 .1999) the degradation rate = BOD / COD x 100 was calculated.From the results (Table 6) the degradation rate is faster after 45 days i.e. longer the sample is subjected to the aerobic test, the faster the degradation rates.When compared to soil burial test aerobic degradation is much faster.

Bacterial adhesion test
The biopolyesters were incubated on mineral salt agar medium inoculated with isolated bacterial and fungi strain.Degradation of several of these polymers proceeds through adsorption of the micro organism to the polymer surface followed by hydrolytic cleavage.S.aureus species degrades polyesters.

Antimicrobial activity
The newly prepared biopolyesters have been studied against bacterial and fungi strains (Figure 12).The N-vinyl-2-pyrrolidone biopolyesters showed potential anti bacterial and anticandidal activity against micro organisms (Table 8).

Conclusion
Natural oils are expected to be inexpensive renewable resources development of new polymeric materials from vegetables oil is highly desirable.The purpose of this work is to prepare high molecular weight polymers and it would be alternative petroleum based polymeric materials such as plastic and rubber materials.The present method of in situ glycerolysis of the oil followed by condensation with maleic anhydride and the Four cross linked biopolyesters were prepared by treating o-PTF of olive oil with N-vinyl-2-pyrrolidone and vinyl acetate.The outcome of those studies has revealed that the newly prepared cross linked biopolyesters are potential biodegradable material for various consumer application like package materials and agricultural applications.
-3).In hydroxylated triglyceride resins, the IR spectra showed a strong absorption band at 3465.84 cm -1 , due to the present of free -OH group in the molecule.The corresponding peak is completely reduced in the o-PTF resins indicates the -entire OH group get substituted.A strong absorbance band in 2925.81cm -1 is due to the presence of C=0 of the glycerol ester moiety.A strong band in 2854.45 cm -1 is due to the symmetrical −C−H bond in −CH 2 −group of the side chain.

Figure 1 .
Figure 1.IR spectrum of olive oil

Figure 2 .
Figure 2. IR spectrum of hydroxylated olive oil

Figure 3 .
Figure 3. IR spectrum of o-PTF resin olive oil The IR spectrum of o-PTF -olive oil -co-vinyl acetate polyesters are shown in the Figure4.The responses at 2925 cm -1 is due to the aliphatic C-H group in the chain, the peak at 1730 cm -1 (C=O stretch in esters) and peak at 1454 cm -1 is due to CH 2 linkages and the peak at 1372 cm -1 is due to CH 3 group and 3500 cm -1 is due to OH group.

Figure 11 .
Figure 11.Analysis of soil burial test

Table 1 .
Physical properties

Table 2 .
Chemical shift and assignment of the signal in the 1 H NMR spectrum of olive oil in pyridine-d 5 / chloroform -d solvent

Table 5 .
Mechanical properties

Table 8 .
Antimicrobial studies with zone of inhibition (mm) Figure 12.Antimicrobial studies of OLINVP with zone of inhibition