Synthesis of new multifunctional linolenic acid vanillyl ester and investigation of antioxidant and antibacterial activities

Vanillyl alcohol (VA) possesses potent antioxidant activity, yet its applicability is hindered


Introduction
Antibacterial compounds play a crucial role in various industries including food, cosmetics, and medicine by inhibiting or eliminating pathogenic and spoilage-causing bacteria.Recently, there has been a growing interest in developing antibiotic substitutes derived from lipid substances, such as medium-chain saturated fatty acids and long-chain polyunsaturated fatty acids, which bacteria do not readily develop resistance to [1,2].For instance, certain plantderived oils rich in polyunsaturated fatty acids exhibit notable antibacterial activity, owing to their higher degree of solubility in water, surpassing that of saturated fatty acids [1].
Linolenic acid (C18:3), abundant in linseed oil (LO), has demonstrated bacteriostatic and bactericidal effects against both gram-positive and gram-negative bacteria [1,2], making it a versatile ingredient in industries ranging from paints and varnishes to biodiesel production [3].
Pathogens such as Pseudomonas fluorescens, is known for causing bacteremia in humans while also contaminating transfusion products and ready-to-eat foods.This pose significant health risks due to their wide temperature tolerance and resistance to temperature fluctuations [4,5].Similarly, Bacillus coagulans can induce allergic reactions when ingested via contaminated food sources [6], while the emergence of multidrug-resistant bacteria like Alcaligenes faecalis and Bacillus subtilis underscores the pressing need for novel antibacterial compounds [7,8].
In parallel, antioxidants serve a pivotal role in mitigating human aging and disease by counteracting free radicals and lipid peroxidation [9].Phenolic compounds derived from plants, such as vanillyl alcohol (VA), exhibit potent antioxidant properties [10].However, the hydrophilic nature of most natural antioxidants poses challenges in their solubility within emulsions or non-polar solvents, thus prompting research into phenolic lipid compounds that combine hydrophilic phenolic rings with lipophilic aliphatic chains [11].
Phenolic lipid compounds, found in plants, bacteria, and fungi, offer dual functionality with both antibacterial and antioxidant properties [12,13].Their amphipathic nature allows for incorporation into emulsions or liposomal membranes [11,12].While chemical synthesis of phenolic lipids poses environmental concerns due to high temperatures, energy consumption, and by-products, enzymatic synthesis offers a greener alternative [14].Lipases, widely utilized in biodiesel and ester compound synthesis, has an important role in producing phenolic lipids [15][16][17].
In this study, we present the synthesis of α-linolenic acid vanillyl ester (LAVE), a novel phenolic lipid compound, via transesterification between LO and VA using Candida antarctica B (CalB) lipase.Optimization of reaction parameters, including temperature, substrate concentration, and reaction time, was undertaken to achieve efficient LAVE synthesis.The antioxidant and antibacterial activities of LAVE were evaluated in emulsion and solvent systems, and its antibacterial mechanism was elucidated.

Optimization of enzyme reaction
The synthesis of vanillyl fatty acid ester was conducted using a lipase-catalyzed reaction (Scheme 1).The reaction mixture consisted of 30 mM VA, 30 mM LO, and 30 mg Candida antarctica B (CalB) lipase dissolved in 2 ml of acetone.The reaction was carried out at temperatures of 30°C, 40°C, and 50°C with agitation at 220 rpm for 20 h, after which the reaction products were analyzed.
To determine the optimal substrate concentration, various concentrations ranging from 30 to 600 mM of VA and LO, along with 30 mg of CalB lipase, were added to 2 ml of acetone.
The reaction was conducted at 40°C for 20 h, and the resulting products were subsequently analyzed.
In another set of experiments, a fixed concentration of 500 mM each of VA and LO, along with 30 mg of CalB lipase, was added to 2 ml of acetone, and the reaction was allowed to proceed for 24 h.Samples were collected at different time intervals to monitor the progress of the reaction, and the resulting products were analyzed accordingly.

HPLC analysis
The substrates and products present in the reaction mixture were analyzed with Agilent 1100 series HPLC system.A Cogent Bidentate C18 column (4.6 mm x 250 mm, 5 µm particle size; microSolv Technology Corp., USA) was employed for chromatographic separation.The column oven temperature was maintained at 40°C throughout the analysis.Elution was carried out using a solvent mixture comprising 5% H2O, 47.5% MeOH, and 47.5% EtOH at a flow rate of 1 ml/min.Detection of vanillyl ester compounds in the reaction mixture was achieved through monitoring absorbance at 280 nm.The quantification of ester compounds was determined based on the reduction in the amount of VA.

Purification of Product A
Product A was purified through preparative LC utilizing an XBridge C18 column (50 mm x 250 mm, 5 µm particle size; Waters Corp., USA).Elution was carried out with a solvent composed of 5% H2O, 47.5% MeOH, and 47.5% EtOH at a flow rate of 70 ml/min.Fractions eluting between 7 and 8 min were collected and subsequently concentrated using a centrifugal evaporator (EYELA N1000V, Japan) to obtain a viscous and colorless liquid.
The purity of Product A was assessed by HPLC using the aforementioned analysis conditions.NMR spectra were acquired on an Avance III 300 MHz instrument (Bruker BioSciences Corp., USA) using deuterated chloroform (CDCl3) as solvent.Additionally, LC-MS analysis was performed utilizing a LC-MS 8050 system (Shimadzu) in positive electron spray ionization (ESI) mode, with a mass scan range of m/z 100-1500.

Determination of DPPH radical scavenging activity
The DPPH radical scavenging activity test was conducted on VA, LO, and LAVE following a previously reported protocol [11,18].Solutions containing 0.5 mM of VA, LO, and LAVE in methanol, 2-propanol, 1-butanol, and toluene were mixed with DPPH radical solution (1 ml) in corresponding solvents (0.06 mM DPPH in methanol, 0.2 mM DPPH in 2-propanol, 0.19 mM DPPH in 1-butanol, and 0.2 mM DPPH in toluene).The reaction mixtures were incubated in the dark at room temperature for 30 min.Subsequently, the decrease in absorbance was measured at 517 nm using an SV1200 Vis spectrophotometer (Abbott, USA).
The residual DPPH radical percentage was calculated using the formula: where Asample represents the absorbance of sample at 517 nm, and Acontrol is the absorbance of the negative control at 517 nm.

Determination of ABTS radical scavenging activity
The ABTS radical scavenging activity test was conducted on VA, LO, and LAVE following a previously reported method [11,18].The ABTS radical cation (ABTS•+) was generated by incubating 7 mM ABTS stock solution in water with 2.5 mM potassium persulfate in the dark at 25°C for 16 h prior to use.The resulting ABTS•+ solution was then diluted in methanol, 2propanol, a mixture of DMSO/1-butanol (2/8, v/v), or a mixture of DMSO/dichloromethane (3/7, v/v) until the absorbance reached 0.7 at a wavelength of 734 nm.Subsequently, solutions containing 0.05 mM of VA, LO, and LAVE dissolved in methanol, and 0.5 mM of the test compounds dissolved in 2-propanol, DMSO/1-butanol, and DMSO/dichloromethane were prepared.These samples were then mixed with the ABTS•+ solution and reacted at room temperature for 30 min with total reaction mixture volume of 1 ml.The decrease in absorbance was measured at 734 nm using an SV1200 Vis spectrophotometer.The residual ABTS radical percentage was calculated using the following equation: where Asample represent the absorbance of sample at 734 nm, and Acontrol is the absorbance of the negative control at 734 nm.

Determination of conjugated diene and triene in menhaden oil-in-water emulsion
The capacity of VA, LO, and LAVE to inhibit the formation of conjugated diene (CD) and conjugated triene (CT) in menhaden oil (MO)-in-water emulsions was assessed in accordance with previously documented methods [2,17].
The MO-in-water emulsion, comprising 1% Tween 20 and 10% MO, was prepared by sonication in an ice bath for 10 min.Each test compound (1 mM) in acetone (2 ml) was added to an empty glass vial, followed by the removal of acetone using centrifugal evaporator.Subsequently, 2 ml of the MO-in-water emulsion was added to each vial, and the mixtures were sonicated for 2 min.
All emulsions were then incubated in the dark at 35°C with agitation at 160 rpm for 5 days.Absorbance measurements were conducted over time at 237 nm for CD and 270 nm for CT.The results were quantified as mmol of CD or CT per milliliter of emulsion, utilizing the Beer-Lambert Law: where c represents the concentration of CD or CT in emulsion, A is absorbance,  denotes the extinction coefficient of CD or CT in ethanol at RT (28,000/M.cmfor CD and 38,000/M.cmfor CT) and l is the path length (cm) of the light.

Colony forming unit (CFU) analysis
LAVE (or LO) was combined with TCN to achieve a concentration of 180 mM, while VA was dissolved in H2O to reach a concentration of 180 mM.This process was carried out in a shaking incubator for 1 h at 50°C under a nitrogen gas atmosphere.Afterwards, the mixtures were cooled to room temperature for 1 h, and any undissolved residues were removed by filtration through a syringe filter (Nylon, 0.45 μm).The concentration of each sample was determined through HPLC analysis.
Subsequently, TCN with samples (LAVE or LO) were mixed with H2O in a 1:1 (v/v) ratio, along with 8% Tween 40.These mixtures were sonicated at 4°C for 5 min followed by vortexing for 1 min.Meanwhile, H2O containing VA was mixed with TCN in a 1:1 (v/v) ratio using sonication, which then separated by centrifugation.After centrifugation, the TCN phase was collected and mixed with H2O in a 1:1 (v/v) ratio, along with 8% Tween 40.The resulting mixture was subjected to the same sonication and vortexing steps as before.
To evaluate the antibacterial activity of VA, LO, and LAVE, four model strains of spoilage bacteria (Bacillus coagulans, Bacillus subtilis, Pseudomonas fluorescens, and Alcaligenes faecalis) were utilized.These bacterial strains were cultured for 24 h at 30°C (B.subtilis and P. fluorescens) and 37°C (A.faecalis and B. coagulans) in 5 ml LB broth medium.Subsequently, the inoculum of each strain was adjusted to a concentration of 5 x 10 6 CFU/ml.Then, 10 μl of each inoculum was added to a mixture containing 50 μl of emulsion and 40 μl of LB broth.The cultures were then incubated for 24 h.The number of viable cells was determined by counting the CFU/ml value using LB agar.

Cell growth curve analysis
Growth curve analysis was conducted on four strains of spoilage bacteria to evaluate the inhibitory effects of LAVE on bacterial growth.Initially, all bacterial strains were cultured for 24 h in 5 ml LB broth medium.Subsequently, a bacterial culture (1%, v/v) was inoculated into 100 ml LB broth supplemented with LAVE (0.125 mM) dissolved in acetone (2.5%).
The cultures were then incubated at 120 rpm for 48 h.
For the negative control, a culture without LAVE (or acetone) was prepared under the same conditions.Growth curves were monitored over time by measuring absorbance at 600 nm.

Zeta-potential analysis
Four bacterial strains were cultured for 16 h and subsequently washed twice with phosphatebuffered saline (PBS).The washed cells suspended in PBS were adjusted to an optical density at 640 nm of 0.2 and then diluted 500-fold.LAVE (1 mM) was added to the bacterial cell suspensions, and the mixtures were incubated for 4 h at 30°C.Afterward, the bacterial cells were centrifuged at 10,000 g for 5 min at 4°C.The resulting cell pellets were resuspended in PBS.As controls, bacterial cells treated with acetone (2.5%) were also prepared.These suspensions were then loaded into a folded capillary zeta cell of a particle electrophoresis instrument (Zetasizer Nano ZA, Malvern Instruments Ltd, UK).

Propidium iodide (PI) staining assay
The propidium iodide (PI) uptake assay was conducted to assess the effect of LAVE treatment on P. fluorescens cells.Initially, P. fluorescens cells were cultured for 16 h and subsequently washed twice with PBS.The washed cells were then adjusted to an optical density at 640 nm of 0.2.Following this, the cells were treated with LAVE (0.125 mM) and incubated for 4 h at 30°C.Subsequently, the bacterial cells were centrifuged, and the resulting cell pellets were suspended in PBS.PI solution (3 μM) was added to the cell suspension, and the mixture was incubated in the dark for 20 min at room temperature.After incubation, the suspensions were centrifuged, and the cell pellet was resuspended in PBS.
The fluorescence of the suspensions was measured using a fluorescence spectrophotometer (Synergy MX, BioTek, USA), with excitation at 544 nm and emission at 620 nm.
Fluorescence microscopy analysis P. fluorescens cells were subjected to PI uptake assay with three different treatments: phosphate-buffered saline (PBS) as a control, acetone (2.5%), and LAVE at a concentration of 0.125 mM.Subsequently, 1 μM of PI was added to each bacterial suspension, followed by incubation for 10 min in the dark.The suspensions were then centrifuged, and the resulting cell pellet was resuspended in PBS.Observation of the cells was carried out using fluorescence microscopy (Nikon Eclipse Ti, Nikon Corporation, Japan), and images were digitally recorded using NIS-Elements AR software (V.4.0).

Effects of reaction temperature on transesterification
VA and LO were utilized in the synthesis of fatty acid vanillyl esters as illustrated in Scheme 1.When employing CalB lipase in the transesterification process, several peaks were discerned in the HPLC chromatogram (Fig. 1).The two prominent peaks at the forefront correspond to VA and acetone, eluting within the range of 2.3 to 3 min.Subsequently, three distinct peaks labeled as A, B, and C were observed.This outcome was anticipated since LO comprises three primary fatty acids: oleic acid (18.5-22.6%),linoleic acid (14.2-17%), and α-linolenic acid (51.9-55.2%).Among these, Product A, exhibiting the largest peak area, was presumed to be LAVE due to the abundance of α-linolenic acid within LO.Subsequent identification of Product A was accomplished using NMR and LC-MS methodologies.To scale up the synthesis of Product A, a reaction optimization process was undertaken.
The optimization of reaction temperature was conducted within the range of 30-50°C (Fig. 2A).Temperatures exceeding 50°C were not explored due to the boiling point (56°C) of acetone, the solvent utilized.The concentrations of Product A produced at 30°C, 40°C, and 50°C were determined to be 10.79 mM, 12.50 mM, and 10.97 mM, respectively.
The reaction temperature plays a critical role in influencing the stability and activity of the substrate, lipase, and solvent.Generally, elevated temperatures enhance the solubility of the substrate and augmented lipase activity.However, excessive temperatures may lead to diminished lipase stability and enzyme denaturation [2].As the highest production of Product A was achieved at 40°C, subsequent experiments were continued at this temperature.

Effects of substrate concentration on Product A production
Optimization of substrate concentration was conducted across a range of 30-600 mM (Fig. 2B).The findings revealed a positive correlation between higher concentrations of VA and LO with increased product concentrations.Notably, at a substrate concentration of 500 mM, a remarkable yield of 223.8 mM Product A was achieved, representing approximately 84% conversion of the total α-linolenic acid to Product A. However, no significant enhancement in Product A concentration was observed at 600 mM substrate concentration.This outcome could be attributed to potential limitations such as substrate solubility in the solvent or potential inhibition of enzyme activity by the high substrate concentration.
Consequently, to achieve substantial production of Product A, subsequent experiments were conducted with substrate concentrations set at 500 mM.

Time course of Product A production
The reaction spanned a 24-h period, during which sampling was conducted at various intervals (Fig. 2C).Initially, as the reaction proceeded, the product concentration exhibited a concurrent increase.The initial production rates of the products were determined as follows: 1.22 M/h/g CalB for product A, 0.28 M/h/g CalB for product B, and 0.69 M/h/g CalB for product C.However, beyond the 8-h mark of reaction time, no significant increase in Product A concentration was observed.These findings indicate that the optimal time for Product A production was determined to be 8 h.

Product A purification and identification
Product purification was carried out using a preparative LC system, followed by analysis utilizing an HPLC system (Fig. 3A).The purification process was undertaken with the assumption that Product A, was the target product derived from α-linolenic acid.
The structural elucidation of Product A was conducted via 300 MHz NMR analysis.By comparing the anticipated NMR results with the experimental findings (supplementary Fig. S1), it was conclusively verified that LAVE had been successfully purified.Subsequent analysis was performed using LC-MS, which revealed that the purified LAVE exhibited major peaks corresponding to [LAVE + Na]+ ions at an m/z of 437.2.This aligns with the relative molecular weight of 414.2 (Fig. 3B).

Antioxidant assay
The DPPH radical scavenging activity of LAVE, LO, and VA was evaluated across four organic solvents: methanol, 2-propanol, 1-butanol, and toluene (Fig. 4A-D).Notably, LAVE and VA exhibited significant antioxidant activity in all solvents, whereas LO did not demonstrate activity in any of the solvents.The antioxidant activity of LAVE is attributed to the presence of phenol rings in VA, facilitating the relocation and stabilization of unpaired electrons within their structure, thereby enabling electron and hydrogen atom transfer from the hydroxyl groups [19].
The ABTS assay results confirmed that LAVE and VA possessed high antioxidant activity, even in water-miscible organic solvents.
Additionally, lipid oxidation analysis revealed that LAVE significantly inhibited the formation of conjugated dienes (CD) and conjugated trienes (CT) compared to VA (Fig. 5A     and B).In the absence of added compounds, the rate of CD and CT formation in the emulsion was observed to be 2.96 mM CD/d/mM compound and 0.47 mM CT/d/mM compound, respectively.However, upon the addition of butylated hydroxytoluene (BHT), LAVE, VA, and LO, the rate of CD formation decreased to 0.3, 2.02, 2.4, and 2.73 mM CD/d/mM compound, respectively.Similarly, the rate of CT formation decreased to 0.08, 0.28, 0.33, and 0.41 mM CT/d/mM compound, respectively.While weaker than BHT, LAVE significantly attenuated the rate of CD and CT formation.
It is established that the oxidation of fatty acids in emulsions occurs at the interface between oil and water.Previous studies have suggested that the distribution of antioxidants at the interface significantly influences the level of lipid oxidation [21].The hydrophiliclipophilic balance (HLB) values of VA, LAVE, Tween 20, and menhaden oil (MO) are 18.0, 7.4, 16.7, and 12, respectively.Due to its high HLB value, VA appears to be unable to disperse or distribute on the emulsion surface effectively.Conversely, LAVE, with a lower HLB value, is primarily distributed in the emulsion core, with some molecules dispersed on the emulsion surface.Given that lipid oxidation primarily occurs at the emulsion surface, the presence of LAVE on the surface is instrumental in preventing lipid oxidation.

Antibacterial assay using emulsion system
The antibacterial activity was assessed using an emulsion system containing Tween 40 and tributyrin (TCN).LO and LAVE were effectively dissolved in TCN at a concentration of 180 mM.However, VA exhibited poor solubility in TCN but readily dissolved in water.
Consequently, VA was dispersed in the aqueous phase rather than within the emulsion.
The colony-forming units per milliliter (CFU/ml) of bacterial cells declined with the presence of LAVE and LO.Notably, LAVE demonstrated superior inhibition of bacterial cell growth against Gram-negative bacteria (Alcaligenes faecalis and Pseudomonas fluorescens) compared to Gram-positive bacteria (Bacillus subtilis and Bacillus coagulans) (Fig. 6).
Moreover, LO exhibited significant inhibition against A. faecalis.Long-chain unsaturated fatty acids are recognized for their antibacterial properties [22].While the precise mechanism remains unclear, it has been proposed that α-linolenic acid might exert antibacterial effects by inhibiting bacterial enoyl-acyl carrier protein reductase (Fab I), an essential enzyme involved in bacterial fatty acid synthesis [23].Additionally, antibacterial lipids are known to induce destabilization of cell membranes through membrane fission and partial solubilization [24].This phenomenon may contribute to LAVE's enhanced antibacterial activity against Gramnegative bacteria relative to Gram-positive bacteria.
In the case of LO, the emulsion displayed limited antibacterial activity against P. fluorescens, B. subtilis, and B. coagulans.This could be attributed to the hydrophobic nature of LO, which tends to partition into the emulsion's interior rather than onto its surface, thereby minimizing contact with the target bacterial cells.However, intriguingly, the LO emulsion exhibited strong inhibition of A. faecalis growth.Further investigations are warranted to elucidate the mechanism underlying the inhibition of A. faecalis growth by LO emulsions.

Antibacterial activity of LAVE for bacterial growth
The antibacterial efficacy of LAVE was demonstrated through bacterial growth curve analysis, as depicted in Fig. 7. LAVE, dissolved in 2.5% acetone at a concentration of 0.125 mM, exhibited notable inhibitory effects on bacterial growth.After 48 h of cultivation, LAVE inhibited bacterial growth by 72.4% and 59.8% in B. coagulans and B. subtilis suspensions, respectively.Similarly, LAVE inhibited 68.2% and 97.3% of bacterial growth in A. faecalis and P. fluorescens suspensions, respectively.Furthermore, we assessed and compared the growth rates during the logarithmic phase.
Previous studies have highlighted that antibacterial activities are influenced by the degree of unsaturation, with higher degrees correlating with increased fatty acid solubility and enhanced antibacterial efficacy [1].Specifically, α-linolenic acid has demonstrated bactericidal activity against Gram-negative bacteria, such as H. pylori [25], by inserting into bacterial cell membranes and inducing leakage of cytoplasmic contents.In contrast, Grampositive bacteria possess a thick peptidoglycan layer in their cell wall, which poses a barrier to antimicrobial agents.However, amphiphilic molecules like LAVE can potentially affect the outer membrane and cytoplasmic membrane of Gram-negative bacteria [26], as observed in our experiments, where LAVE exhibited more potent antimicrobial activity against Gramnegative bacteria, particularly P. fluorescens.

Notably, LAVE displayed robust inhibitory effects on A. faecalis growth in an emulsion
state, whereas it effectively inhibited the growth of P. fluorescens when dissolved in a solvent.Thus, the choice of using LAVE as an antibacterial agent should be tailored based on the specific target bacteria.

Zeta potential analysis
To investigate the potential binding of LAVE to cell membranes and its effects on membrane properties such as expansion, fluidity, and permeability, we conducted zeta potential measurements on four bacterial strains.The results revealed a notable increase in the surface charge towards more negative values upon the addition of LAVE for all bacterial strains (Fig. 8).This shift can be attributed to the negative charge carried by the hydroxyl group of the phenolic compound present in LAVE [27].Specifically, treatment with LAVE at a concentration of 1 mM led to negative charges of (-40.8 and -37) mV for B. coagulans and B. subtilis, respectively.In contrast, P. fluorescens and A. faecalis exhibited even more pronounced increases in negative charges, measuring (-41.4 and -45.4) mV, respectively.Notably, the surface charge shifts (ΔmV) induced by LAVE were (25.4 and 25.5) mV for B. coagulans and B. subtilis, while for P. fluorescens and A. faecalis, they were (33.7 and 38.5) mV, respectively (Fig. 8E).
These findings suggest a stronger interaction of LAVE with the cell membrane of Gramnegative bacteria, as evidenced by the more significant change in surface charge observed in these strains.

PI staining
Propidium iodide (PI) staining was conducted on P. fluorescens, which exhibited the most significant growth inhibition by LAVE, to assess whether LAVE could induce membrane damage.PI is commonly employed as a membrane-impermeable dye capable of binding nucleic acids in deceased cells [26].In this experiment, LAVE (0.125 mM) dissolved in acetone (2.5%) was utilized.
As depicted in Fig. 9, the addition of acetone resulted in fluorescence levels 16.2 times higher than the control (P.fluorescens in PBS).However, upon the addition of LAVE, the fluorescence intensified to 50.6 times higher than the control and 3.1 times higher than that observed in acetone-treated cells.These findings indicate that LAVE facilitated greater permeation of PI through the cell membrane, leading to increased intracellular accumulation of the dye.

Fluorescence microscopy
Additional experiments were conducted to visually assess PI staining on bacterial cells.
Optical and fluorescence microscopes were employed for this purpose.When cells were treated with PBS, no red fluorescent cells were observed (Fig. 10).This is attributable to the inability of PI dye to penetrate living cells.Upon addition of acetone (2.5%),only a few fluorescent cells were observed under the fluorescence microscope.However, following the addition of LAVE (0.125 mM), a significant number of fluorescent cells were visible under the fluorescence microscope.These results provide visual evidence that LAVE induces membrane disruption by enhancing membrane fluidity and permeability, ultimately leading to cell death.

Conclusions
LAVE was synthesized through a lipase-mediated transesterification reaction.It emerges as a novel multifunctional molecule with antioxidant and antibacterial properties, alongside amphiphilic characteristics.Consequently, LAVE holds promise as an antioxidant or antibacterial agent in the cosmetic and food industries, particularly in the form of emulsions.

Fig. 1
Fig. 1 HPLC analysis of reaction products.a Vanillyl alcohol and linseed oil were mixed

Fig. 2
Fig. 2 Optimization of LAVE production.a The effects of reaction temperature on the

Fig. 3
Fig. 3 Purification of product a and LC-MS analysis.a HPLC chromatogram following

Fig. 6
Fig.6 Antibacterial assay of compounds through emulsion system.Antibacterial activities

Fig. 8
Fig. 8 Zeta potential measurements.Zeta potential values of LAVE were measured after 4 h

Fig. 9
Fig. 9 PI uptake assay using Pseudomonas fluorescens.Levels of PI uptake were measured

Fig. 10 Scheme 1
Fig. 10 PI staining of Pseudomonas fluorescens analysed with light and fluorescent

Fig. 1 Fig. 2 Fig. 3 Fig.Fig. 5 Fig. 6 Fig. 7 Fig. 8 Fig. 9 Fig. 10
Fig. 1 HPLC analysis of reaction products.a Vanillyl alcohol and linseed oil, CalB lipase was added in acetone and 0 h-reaction mixture was analyzed by HPLC.b After 8 h-reaction, reaction mixture was analyzed.Product peaks were indicated as A, B, and C according to the elution order.