Characterization of enzymatically-synthesized erythorbyl laurate as a multi-functional (interfacial, antioxidant, and antibacterial properties) food additive = 효소적으로 합성된 다기능성(계면활성, 항산화성 및 항균성) 식품첨가물, 에리소르빌 라우레이트의 특성 규명

다국어 초록 (Multilingual Abstract)

An emulsion is a heterogeneous dispersion of two immiscible liquids (e.g. water and lipid) wherein droplets of one phase (dispersed or internal phase) are encapsulated within another phase (continuous or external phase) in the presence of surface-active agents (i.e. emulsifiers). For the emulsion-based products extensively used in food, cosmetic, and pharmaceutical industries, lipid oxidation and microbial contamination have been considered as the major hazards in terms of safety for human consumption. Under the strategy for simultaneously controlling the aforementioned hazards, I attempted to conducted lipase-catalyzed esterification between lauric acid and erythorbic acid to produce a novel multifunctional emulsifier with antibacterial and antioxidant activities.

Erythorbic acid, a stereoisomer of L-ascorbic acid and a food additive, has antioxidant activity and should be available at relatively low cost because calcium 2-ketogluconate is readily converted to erythorbic acid during the fermentation of glucose. On the other hand, lauric acid is a medium-chain fatty acid with a strong antimicrobial activities against a wide spectrum of food-borne pathogens. Therefore, erythorbyl laurate, which results from enzymatic esterification between hydrophilic erythorbic acid and lipophilic lauric acid, was anticipated to be an amphiphilic material with multi-functionalities. The lipase-catalyzed esterification between a hydroxyl group of erythorbic acid and the carboxyl group of lauric acid has been performed in an acetonitrile medium. A steady state (i.e. reaction equilibrium) was achieved after 8 h, from the initiation of the esterification, and the degree of esterification (molar conversion yield) reached 78.5%.

After the purification procedure based on solvent extraction, the resulting erythorbyl laurate was identified by mass spectrometry and structural analysis. The molecular mass of erythorbyl laurate was determined by LC-ESI-MS in full mode and the spectra gave a molecular ion at m/z = 359.2 [M1 + H]+ and 358.2 [M1], corresponding exactly to the molecular mass of erythorbyl laurate. This molecular mass was agreed with the major molecular ion at m/z 357.0 [M1–H]- from the MALDITOF MS in the negative ion mode. FT-IR spectra of erythorbyl laurate revealed a relatively strong peak at 1,739.4 cm-1 against erythorbic or lauric acids, indicating the newly introduced CO-O ester bond in erythorbyl laurate. The esterified position between a hydroxyl group of erythorbic acid and the carboxyl group of lauric acid was confirmed based on 1H- and 13C-NMR, which revealed the presence of an ester bond on C-6´ of the erythorbyl moiety (i.e. 6-O-lauroyl-erythorbic acid).

To optimize the conditions for the lipase-catalyzed synthesis of erythorbyl laurate, the response surface methodology (RSM) was adopted to both batch and continuous processes. A central composite experimental design with three independent variables and five levels was performed and then the statistical significance of the quadratic polynomial equation fitted from the experiments was determined by the analysis of variance (ANOVA). From the batch-type reaction, the optimum conditions were found as follows: enzyme content (2,994 PLU), molar ratio of lauric acid to erythorbic acid (24.23), and reaction emperature (53.03°C). Under these conditions, the molar conversion yield was 77.8%, which was not significantly different (p-value > 0.05) from the predicted value (76.9%) obtained from the quadratic model. In the continuous production employing a packed-bed enzyme reactor (PBER), temperature (X1), substrate molar ratio ([lauric acid]/[erythorbic acid], X2), and residence time (controlled by flow rate, X3) were selected as the independent variables. The three variables had significant effect on the molar conversion (%) and the observed and predicted values of molar conversion were fitted into the following quadratic polynomial equation:

Y = 2.51489 + 1.02661X1 + 3.97998X2 + 1.66831X3 -0.0104160X12-0.0763756X22-0.0218346X32+0.00914005X1X2+0.000715675X1X2-0.0460557X2X3 (p< 0.01)

The R2 value, the coefficient of multiple determination of the polynomial model, was 0.9768, indicating that the fitted model could explain 97.68% of the variability in the response. In addition, a relatively low coefficient of variation (CV = 1.96%) indicated good precision and reliability. The maximum molar conversion was 80.8% under the optimum conditions (temperature of 56.6°C, substrate molar ratio of 15, and a reaction residence time of 23.3 min), which verified the validity of the model designed in this RSM. In terms of catalytic performance of the enzymatic synthesis, the esterification activity of lipase in an organic medium is strongly influenced by the water content absorbed on lipase. If a large amount of water is bound to lipase or accumulates around lipase, the equilibrium of the reaction could shift from an esterification reaction toward a hydrolysis reaction. For this reason, an ion-exchange resin in potassium form (Dowex HCR-W2) was employed to improve the catalytic performance of the enzymatic esterification. The continuous water removal by adsorption on an ionexchange resin in a PBER improved operational stability, resulting in prolongation of half-life (2.02 times longer compared to the control without water-removal system). Furthermore, by the incorporation of an ion-exchange resin in the batch-type reactor, it could be found out the molar conversion yield at 240 min and the initial velocity of molar conversion also increased from 68.39 to 73.64% and from 1.58 to 2.04% per min, respectively. The effect of storage temperature on the thermal stability of erythorbyl laurate was examined by measuring its residual content during storage at various temperatures, which indicated the erythorbyl laurate stored below 50°C was unlikely to be degraded over a 30-day storage period.

Characterization of erythorbyl laurate was conducted to verify the multi functionalities, assessing the interfacial, antioxidant, and antibacterial properties. As a major concern for interfacial characteristics, the surface activity of erythorbyl laurate was evaluated by determining the critical micelle concentration (CMC), the concentration at which surface tension is independent of sample concentration. The CMC of erythorbyl laurate was 0.101 mM and the surface tension decreased to 35.3 mN/m at the CMC, compared with surface tension of 1% (v/v) DMSO solution as a control (73.2 mN/m). Furthermore, the foam stability of erythorbyl laurate (half-life 24.33±0.94 h) was markedly superior to that of Tween 20 (8.00±1.63 h) even though the foaming capacities of Tween 20 and erythorbyl laurate were not significantly different (18.67±0.94 and 18.00±0.82 mL, respectively, p-value > 0.05). The antioxidant activity of erythorbyl laurate was evaluated in the emulsion systems accelerated by thermal- or photo-oxidation. In the thermally induced oxidation at 60°C, the concentrations of lipid peroxides in Tween 20- and erythorbyl laurate-stabilized emulsions were 27.44 and 0.11 meq/kg oil after 8 days of incubation, respectively, which indicated that erythorbyl laurate effectively inhibited production of lipid peroxides. Similar to the result of thermal oxidation, the rate of photooxidation in the emulsion stabilized with Tween 20 was faster than that in the erythorbyl laurate emulsion (23.83 and 1.75 meq/kg oil for Tween 20 and erythorbyl
laureate during 12 h, respectively). Interestingly, the level of lipid peroxides in erythorbyl laurate-stabilized emulsion was relatively lower than that of Tween 20- stabilized emulsion containing the same moles of erythorbic acid. This phenomenon could be attributed to the fact that the localization of erythorbyl laurate as an emulsifier allowed the antioxidant molecules to be concentrated at the oil-water interface where oxidation is prevalent, which led to more effective retardation of lipid oxidation.

From the antibacterial susceptibility screening of erythorbyl laurate, it was confirmed that erythorbyl laurate had both bacteriostatic and bactericidal effects on Gram-positive pathogens such as Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus. Focused on Gram-positive bacterial strains, minimum inhibitory concentration (MIC) assessment employing the broth microdilution method was conducted, which revealed the MICs of erythorbyl laurate were from 0.48 to 0.88 mM and the MBCs were greater than those values of MICs (approx. 1.2-1.5 fold increase). In all cases of Gram-positive strains, the growth curves showed an increase in the lag phase (λ) accompanied by a decline in the specific growth rate (μmax) as the concentration of erythorbyl laurate increased. The corresponding values of increased λ and decreased μmax were concentration-dependent and significantly proportional to the increase in erythorbyl laurate concentration. Based on the results from crystal
violet uptake assay and LIVE/DEAD BacLight kit, it was plausible that the antibacterial mechanism of erythorbyl laurate was mainly attributed to the alteration in the permeability and the integrity of cell membrane. Moreover, it was evident that erythorbyl laurate had synergistic effects in combination with various antibacterial agents, which might be originated from the antibacterial mechanism of erythorbyl laurate (i.e. increase in the membrane permeability).

In conclusion, the results suggest that the erythorbyl laurate developed in this study could be a potential additive with multi-functionalities for the emulsion-based products in the food and cosmetic industries.

목차 (Table of Contents)

• Chapter I. General introduction 1
• I-1. Lipid oxidation and antioxidants 2
• I-2. Antibacterial activity of free fatty acids 5
• I-3. Emulsion and multi-functional emulsifier 7
• I-4. References 9
• Chapter II. Lipase-catalyzed synthesis and identification of erythorbyl laurate 16
• II-1. Introduction 17
• II-2. Materials and Methods 19
• II-2-1. Materials 19
• II-2-2. Lipase-catalyzed esterification 19
• II-2-3. Quantitative analysis by HPLC 20
• II-2-4. Purification procedure for the erythorbyl laurate 21
• II-2-5. Identification of the erythorbyl laurate 21
• II.3. Results and Discussion 23
• II-3-1. Progression of enzymatic synthesis of erythorbyl laurate in acetonitrile 23
• II-3-2. Identification and structural analysis of erythorbyl laurate 28
• II-4. Conclusions 38
• II-5. References 39
• Chapter III. Optimization of conditions for the lipase-catalyzed esterification in a batch synthesis 44
• III-1. Introduction 45
• III-2. Materials and Methods 48
• III-2-1. Materials 48
• III-2-2. Lipase-catalyzed esterification in batch synthesis 48
• III-2-3. Quantitative analysis of the esterification product 51
• III-2-4. Experimental design and statistical analysis 52
• III-3. Results and Discussion 53
• III-3-1. Analysis of central composite experiment design for the batch synthesis of erythorbyl laurate 53
• III-3-2. Effects of independent variables on the batch synthesis of erythorbyl laurate 58
• III-3-3. Optimization of the batch synthesis of erythorbyl laurate and verification of model 66
• III-4. Conclusions 67
• III-5. References 68
• Chapter IV. Continuous production of erythorbyl laurate through the lipase-catalyzed esterification in packed-bed enzyme reactor 71
• IV-1. Introduction 72
• IV-2. Materials and Methods 75
• IV-2-1. Materials 75
• IV-2-2. Lipase-catalyzed esterification on a packed-bed enzyme reactor 75
• IV-2-3. Determination of the bed void fraction and residence time 78
• IV-2-4. Experimental design and statistical analysis 78
• IV-2-5. Improving the operational stability and exhibition of enzyme activity using ion-exchange resin 80
• IV-2-6. Storage stability of erythorbyl laurate 81
• IV-3. Results and Discussion 82
• IV-3-1. Analysis of the central composite experimental design for the continuous production of erythorbyl laurate 82
• IV-3-2. Effects of independent variables on the continuous production of erythorbyl laurate 86
• IV-3-3. Optimization of the continuous production of erythorbyl laurate and verification of the model 95
• IV-3-4. Improvement of operational stability and exhibition of enzyme activity using an ion-exchange resin 95
• IV-3-5. Storage stability of erythorbyl laurate 100
• IV-4. Conclusions 102
• IV-5. References 103
• Chapter V. Interfacial characteristics and antioxidant activity of erythorbyl laurate 108
• V-1. Introduction 109
• V-2. Materials and Methods 111
• V-2-1. Materials 111
• V-2-2. Interfacial characteristics of erythorbyl laurate 111
• V-2-2-1. Surface tension and critical micelle concentration 111
• V-2.2.2. Foaming ability and stability 112
• V-2-3. Antioxidant properties of erythorbyl laurate 112
• V-2-3-1. Free radical scavenging activity 112
• V-2-3-2. Antioxidant activity of erythorbyl laurate in an emulsion system 113
• V-2-4. Statistical analysis 115
• V-3. Results and discussion 116
• V-3-1. Interfacial characteristics of erythorbyl laurate 116
• V-3-2 Foaming ability and stability 120
• V-3-3. Free radical scavenging activity 124
• V-3-4. Antioxidant activity of erythorbyl laurate in emulsion systems 128
• V-4. Conclusions 144
• V-5. References 145
• Chapter VI. Antibacterial activity of erythorbyl laurate and its mode of action 151
• VI-1. Introduction 152
• VI-2. Materials and Methods 155
• VI-2-1. Bacterial strains and culture conditions 155
• VI-2-2. Susceptibility screening (spot-on-lawn assay) 155
• VI-2-3. Bactericidal and bacteriostatic activities 156
• VI-2-4. Preparation of erythorbyl laurate stabilized emulsion 157
• VI-2-5. Crystal violet assay 158
• VI-2-6. Live/Dead BacLight bacterial viability assay 159
• VI-2-7. Transmission electron microscopy 160
• VI-2-8. Fluorescent microscopy 161
• VI-2-9. Synergistic effect with antibiotics and food preservatives 162
• VI-2-10, Statistical analysis 163
• VI-3. Results and discussion 164
• VI-3-1. Susceptibility screening and effective concentrations (MIC&MBC) 164
• VI-3-2. Effect of erythorbyl laurate on the permeability of cell membrane 172
• VI-3-3. Erythorbyl laurate-induced alteration of cell membrane integrity 176
• VI-3-4. Synergistic effects of EL in combination with antibacterial agents 180
• VI-4. Conclusions 183
• VI-5. References 184
• 국문초록188