Enrichment of mayonnaise with a high fat fish oil-in-water emulsion stabilized with modified DATEM C14 enhances oxidative stability.

Enrichment of mayonnaise using delivery emulsions (DEs) containing 70% fish oil versus neat fish oil was investigated. DEs were produced with combined use of sodium caseinate, diacetyl tartaric acid esters of mono- and diglycerides (DATEM), and/or modified DATEMs with different length (C12 or C14) and covalently attached caffeic acid. Physical and oxidative stability of the mayonnaises were analyzed based on parameters including droplet size, viscosity, peroxide value, volatile compounds, and sensory properties. DEs addition to mayonnaise resulted in larger droplets and lower viscosity compared to neat fish oil. However, zeta potential was higher in mayonnaises with DEs containing DATEMs. Mayonnaise containing DATEM C14 had higher protein surface load leading to a thicker interfacial layer, lower formation of hexanal, (E)-2-hexenal, and (E)-2-heptenal as well as lower rancid odour intensity compared to mayonnaise containing DATEM and free caffeic acid, and thus benefitted from the location of the antioxidant at the interface.

Water-mediated catalyst-free synthesis of lysine-based ampholytic amphiphiles for multipurpose applications: Characterization and pH-responsive emulsifying properties.

A novel series of lysine-based ampholytic amphiphiles, with alkylsuccinic anhydrides of varying chain lengths as hydrophobic acylating agents, were synthesized in medium to high yield (50.23-90.15%) based on a facile, catalyst-free method in water medium; and structurally confirmed by mass spectrometry (MS), Fourier transform infra-red (FTIR) spectroscopy, and 1H/13C nuclear magnetic resonances (NMR) analysis. The resulting compounds were subjected to pH-dependent amphiphilic property, ferrous ion chelating, DPPH antioxidant capacity, and cytotoxicity analyses. Results showed that CMC values decrease, γ value increase, and emulsion stability increase with the increase of medium pH, suggesting that the surface activity of synthetic compounds at air/water and oil/water interface under neutral and alkaline conditions was remarkably higher than that under acidic condition. Lauryl O-acylated malic lysine (compound 4b) presented excellent foaming ability close to commercial detergent sodium dodecyl sulphate; dodecyl succinic lysine (compound 4a) afforded highly stable o/w nanoemulsion. Moreover, compound 4b displayed comparable ferrous ion chelating property to lysine and 2,2-diphenyl-1-picrylhydrazyl (DPPH) antioxidative capacity similar to a commercial food ingredient, diacetyl tartaric acid esters of mono- and di-glycerides (DATEM), indicating its multi-faceted functionalities. A cytotoxicity study of compounds 3b &4b showed that they were non-toxic. Thus, these novel ampholytic amphiphiles may find multi-purpose applications in food, detergent, pharmaceutical, and cosmetic industry.

Caffeoyl maleic fatty alcohol monoesters: Synthesis, characterization and antioxidant assessment.

HYPOTHESIS: Caffeoyl malate anhydride, as a good nucleophilic acceptor, can react with lipophilic fatty alcohols to yield interface-confined amphiphiles. The resulting novel molecules are hypothesized to deliver combined functionalities of parent natural building blocks, as emulsifier, stabilizer, ion chelator and free radical scavenger. EXPERIMENTS: Ring-opening reactions of caffeoyl malate anhydride with fatty alcohols of different chain lengths generated a new group of antioxidant amphiphiles. Structural verification was by MS (mass spectrometry), 1H/13C NMR (nuclear magnetic resonance) and FT-IR (Fourier transform infra-red) spectroscopy. Physicochemical characterization was done by use of DSC (differential scanning calorimetry), FT-IR, determinations of critical micelle concentrations (CMC) and calculations of HLB. Antioxidant activity was assessed by DPPH (2, 2-diphenyl-1-picrylhydrazyl) and hydroxyl radical scavenging activities. Dynamic light scattering (DLS) studies demonstrated surface-activity of G8-G18. Inhibition of iron- and thermally-accelerated lipid oxidation was monitored by thiobarbituric acid reactive substances (TBARS) assay. FINDINGS: Derivatization of caffeoyl malate anhydride with fatty alcohols maintained free radical scavenging activity, and improved hydroxyl radical scavenging activity of caffeic acid. Lipid oxidation at 22 °C was significantly inhibited (up to 3.5 times) in emulsions stabilized by G8-G18 with or without chitosan compared to emulsions stabilized by commercial emulsifiers and stabilizers. Thermal oxidation (at 80 °C) was 10 times less in emulsions facilitated by G8-G18 in combination with chitosan compared to emulsions stabilized by commercial emulsifiers and stabilizers. This study has developed a simple and straightforward approach for developing value-added compounds from underexplored fatty alcohols.

Mapping the location of DATEM in multi-phase systems: Synthesis and characterization of spin-label probe analogues.

Electron paramagnetic resonance (EPR) spectroscopy has emerged as a fast, reliable, non-invasive and sensitive method to determine the distribution, localization and reactivity of labelled ingredients in micro-heterogeneous systems. However, the commercially available probe molecules are very limited. In the present work, five new nitroxide [(4-hydroxy-2, 2, 6, 6-tetramethylpiperidin-1-oxyl (TEMPOL)] derivatives (1b-5b) of diacetyl tartaric acid esters of monoglycerides (DATEM) (1a-5a), a widely used food emulsifier, were synthesized under Steglich conditions and characterized by MS (mass-spectrometry), FT-IR (Fourier-transform infrared spectroscopy), EPR, NMR (nuclear magnetic resonance spectroscopy), fluorescence spectroscopy, and DSC (differential scanning calorimetry) analysis. Phase partitioning studies proved that the new spin labels are adequately capable of describing the localizations and partitioning of the corresponding DATEM in multi-phase systems. Findings disclosed in this work will provide new knowledge on ingredient reactivity and localization in multi-phase systems; which is vital to aid the design of more efficient delivery systems for bioactive compounds.

Aspartic-Acid-Based Ampholytic Amphiphiles: Synthesis, Characterization, and pH-Dependent Properties at Air/Water and Oil/Water Interfaces.

A facile and two-step strategy was employed to synthesize a series of novel aspartic-acid-based ampholytic amphiphiles from sustainable and commercially viable substances as starting materials. The molecular structures of the synthetic compounds were well-identified by mass spectrometry and 1H/13C nuclear magnetic resonance analysis, and the physicochemical, pH-dependent foaming, and emulsifying properties were evaluated by the use of multiple techniques, such as Fourier transform infrared spectroscopy, differential scanning calorimetry, Langmuir-Blodgett study, and fluorescence microscopy imaging. As a result of the co-existence of amino and carboxyl groups in the synthetic compounds, the compounds presented varying charges (cationic, ampholytic, and anionic) depending upon the pH of the medium compared to the dissociation constants (p Ka). Compounds with cationic (pH 1.0) and anionic (pH 9.0) forms had significantly higher γ0.1 and critical micelle concentration values than those with ampholytic forms (pH 7.0). sn-1-Lauroyl- sn-3-aspartic acid (compound 3) at neutral and alkaline conditions displayed comparable foaming properties, including foaming, calcium-tolerant, and temperature-resistant abilities, with commercial sulfonate sodium dodecyl sulfate (SDS), and thus might be a promising alternative to SDS, applied in personal care products and detergent formula. sn-1-Palmtoyl- sn-3-aspartic acid (compound 5a) with an ampholytic structure was proven as the most excellent stabilizer for the preparation of oil-in-water emulsions compared to palmityl aspartic acid (compound 5b), commercial food ingredient diacetyltartaric acid esters of mono- and diglycerides, and glyceride monopalmitate at aqueous phase pH 7.0. Thus, it has promising use as a pH-dependent emulsifying agent in various fields.

Modified phosphatidylcholine with different alkyl chain length and covalently attached caffeic acid affects the physical and oxidative stability of omega-3 delivery 70% oil-in-water emulsions.

This study investigated the effects of modified phosphatidylcholine (PC) with different alkyl chain lengths (PC_C14 and PC_C16) and covalently attached caffeic acid on the physical and oxidative stability of 70% fish oil-in-water emulsions. High fat emulsions were produced using different amounts of modified PCs in combination with sodium caseinate and soy-PC. Results showed that the physical stability of the emulsions was improved with increasing concentrations of modified PCs, due to their high surface activity. Emulsion stabilized with PC_C14 led to smaller droplets and higher viscosity, whereas PC_C16 had higher protein surface load, which may result in a thicker interfacial layer. Modified PCs enhanced the oxidative stability of the emulsions due to the attachment of caffeic acid to the glycerol backbone of PC, which brings the antioxidant in the vicinity of oil-water interface. PC_C16 led to less formation of primary and secondary oxidation products compared to PC_14 at their equivalent concentrations.

Grafting phenolics onto milk protein via conjugated polymerization for delivery of multiple functionalities: Synthesis and characterization.

A synthetic scenario for functionalization of ß-lactoglobulin (ßLg) with polymeric units containing caffeic acid (ßLg-polyCA) was developed; and all intermediates and final products were structurally confirmed using nuclear magnetic resonance spectroscopy, matrix assisted laser desorption ionization time-of-flight mass spectrometry, and physico-chemically characterized using differential scanning calorimetry and circular dichroism. The antioxidant properties and emulsion stability of ßLg, ßLg-CA conjugate and ßLg-polyCA based systems containing high percentage of fish oil (50%) were evaluated; and ßLg-polyCA presented the highest antioxidant and free radical-scavenging activity based on DPPH, ABTS and HS scavenging assays (92.4, 87.92 and 67.35% respectively). Thiobarbituric acid (TBARS) test demonstrated that compared to native ßLg, ßLg-polyCA afford up 4-5 fold of inhibition of oxidative rancidity and displayed drastic secondary structure changes. Compared to native ßLg based emulsions, ßLg-polyCA had larger oil droplet sizes, stronger negative zeta potentials (-69.9 mv), narrower size distributions (PDI: 0.22) and less creaming index.

Enzymatic alkylsuccinylation of tyrosol: Synthesis, characterization and property evaluation as a dual-functional antioxidant.

This work reports a novel approach to generate a new group of tyrosol-based amphiphilic lipid alkylsuccinylated tyrosol by lipase-catalyzed succinylation of tyrosol with alkylsuccinic anhydrides of varying alkyl chain lengths, in high yields (80-95%). The structures of the compounds were confirmed by MS, FTIR &1H NMR; and their properties were characterized by Temperature-Ramp FTIR, DSC & CMC measurements. The synthesized compounds integrate water-soluble phenylethanoid and hydrophobic alkyl into one molecule thus are endowed with dual functions: retaining the antioxidant property of tyrosol and entailing tyrosol with new surface-active property. The DPPH activity of tyrosol (13.77%) was significantly enhanced by 2-dodecen-1-ylsuccinylated tyrosol (16.01%). Compared to tyrosol-based emulsions (76.63%), the lipid oxidation is reduced to 21.57% and 42.32% in 2-octen-1-ylsuccinylated/2-dodecen-1-ylsuccinylated tyrosol emulsions, respectively. This work brings new members to the library of functional lipid excipients and open a novel and effective synthetic pathway for derivation of phenyl alcohols.

New phenophospholipids equipped with multi-functionalities: Regiospecific synthesis and characterization.

HYPOTHESIS: In multi-phase systems, many complex reactions take place at the interface where a molecule equipped with manifold functionalities is demanded. By taking advantage of the surface-active property of phosphatidylcholine (PC) scaffold and antioxidant properties of phenolic acids, new multifunctional molecules are generated, which are expected to confer physical and oxidative stability to sensitive bioactive ingredients in delivery systems. EXPERIMENTS: This work reports a successful synthesis of two new arrays of phenophospholipids sn-1-acyl(C12-C18)-sn-2-caffeoyl and sn-1-caffeoyl-sn-2-acyl phosphatidylcholines via mild scalable regiospecific pathways; as structurally verified by MS, 1H/13C NMR analyses, and characterized by critical micelle concentrations (CMC), FTIR, and DSC analysis. Synthesized phenophospholipids are subjected to stabilizing o/w emulsion, and antioxidation tests as demonstrated by TBARS (Thiobarbituric Acid Reactive Substances) and DPPH (2,2-diphenyl-1-picrylhydrazyl) assays. FINDINGS: This study has demonstrated that; (1) phenophospholipids with a broad spectrum of CMC are created, affording superior emulsion stability than soybean PC; (2) all phenophospholipids present improved oxidation inhibition and sn-2-caffeoyl phenophospholipids display superior performance to sn-1-caffeoyl phenophospholipids, soybean PC or admixture of caffeic acid and soybean PC; (3) incorporation of caffeoyl in PC scaffold does not sacrifice radical scavenging ability of caffeic acid, whilst the ion chelating capacity of sn-1-myristoyl(C14)-sn-2-caffeoyl PC enhance by 4.5 times compared to soy PC. Fluorescence Microscopy imaging verified the location of phenophospholipids in the interface as desired. Among synthetic phenophospholipids, sn-1-myristoyl(C14)-sn-2-caffeoyl PC commits the cut-off effect in most desired functionalities, which might be of great potential for multi-purpose applications.

Enhanced fish oil-in-water emulsions enabled by rapeseed lecithins obtained under different processing conditions.

It is hypothesized that rapeseed lecithins may have different emulsifying and antioxidant properties in delivering fish oil compared to soy lecithin based on previous studies. The results showed that in vitro antioxidant activities of rapeseed lecithins were stronger than those of soy lecithin. Emulsions stabilized by rapeseed based lecithins and DATEM were stable over 3 months at 4 °C, whereas the creaming of emulsions containing soy lecithin started immediately after its preparation. Zeta-potential of rapeseed lecithins was higher than soy lecithin and DATEM, which partially contributed to the emulsion stability. Although the particle sizes of emulsions prepared by rapeseed lecithins increased after 14 days storage, no creaming was observed. Lipid oxidation as indicated by TBARS values suggested that DATEM was the most unfavorable, followed by soy lecithin. It is concluded that rapeseed lecithins are better than soy lecithin and DATEM in terms of emulsion stability and antioxidant capability, respectively.

Combination of sodium caseinate and succinylated alginate improved stability of high fat fish oil-in-water emulsions.

Sodium caseinate (CAS) and commercial sodium alginate (CA), long chain modified alginate (LCMA) or short chain modified alginate (SCMA) were used in combination for emulsifying and stabilizing high fat (50-70%) fish oil-in-water emulsions. Physical (creaming, droplet size, viscosity and protein determination) and oxidative (primary and secondary oxidation products) stabilities of the emulsions were studied during 12 days of storage. Creaming stability was higher for emulsions produced with alginates and CAS compared to emulsions prepared with only CAS. Combined use of CAS + LCMA performed better in terms of physical stability compared to emulsions produced with only CAS. However, the oxidative stability of this emulsion was inferior probably due to the presence of an unsaturated carbon chain in LCMA structure. CAS + SCMA emulsions not only showed better physical stability such as smaller droplet size, lower creaming and higher viscosity, but also had an improved oxidative stability than emulsions produced with only CAS.

Effects of Modified DATEMs with Different Alkyl Chain Lengths on Improving Oxidative and Physical Stability of 70% Fish Oil-in-Water Emulsions.

The objective of this study was to produce oxidatively and physically stable 70% fish oil-in-water emulsions by combined use of sodium caseinate (CAS), commercial diacetyl tartaric acid esters of mono- and diglycerides (DATEM), and modified DATEM. First, the optimal formula was determined using DATEM and CAS. Subsequently, modified DATEMs (DATEM C12 and DATEM C14) were designed for investigating both the effects of different alkyl chain lengths and caffeic acid conjugation to the emulsifier on physical and oxidative stability of the emulsions. Emulsions produced with modified DATEMs showed better oxidative stability compared with emulsion using commercial DATEM plus an equivalent amount of free caffeic acid, confirming the advantage of having antioxidant covalently attached to the emulsifier. Results indicated that DATEM_C14 replaced more CAS compared with DATEM_C12 from the interface in 70% fish oil-in-water emulsion. Emulsions produced with DATEM_C14 had significantly decreased amounts of primary and secondary oxidation products compared with emulsions using DATEM_C12.

A new group of synthetic phenolic-containing amphiphilic molecules for multipurpose applications: Physico-chemical characterization and cell-toxicity study.

Nine synthetic amphiphilic phenolic lipids, varied in phenolic moiety (caffeoyl/dimethylcaffeoyl) and fatty acid chain lengths (8-18) were characterized by differential scanning calorimetry (DSC), temperature-ramp Fourier transform infra-red spectroscopy (FT-IR) and atomic force microscopy (AFM). FT-IR and DSC results revealed that the physical state and lateral packing of synthetic molecules were largely governed by fatty acyls. The critical micelle concentrations (CMC) of synthetic lipids was in the range of 0.1 mM to 2.5 mM, affording generation of stable oil-in-water emulsions; as evidenced by the creaming index (<5%) of emulsions stabilized by compounds C12‒C16, and C12a‒C16a after 7 days' storage. AFM analysis revealed that compound C14 formed stable double-layers films of 5.2 nm and 6.7 nm. Application studies showed that formulations stabilized by synthesized compounds containing 30% fish oil had superior physical and oxidative stability compared to formulations containing commercial emulsifiers or their mixtures with phenolic acids. Moreover, the synthetic compounds were non-toxic against in vitro transformed keratinocytes from histologically normal skin and Caco-2 cell lines. This study demonstrates the relevance of using a natural hydroxycarboxylic acid as a flexible linker between natural antioxidants, glycerol and fatty acids to generate multifunctional amphiphiles with potential applications in food, pharmaceutical and cosmetic industry.

ß-Glucosidase from Thermotoga naphthophila RKU-10 for exclusive synthesis of galactotrisaccharides: Kinetics and thermodynamics insight into reaction mechanism.

This work reports a novel thermophilic ß-glucosidase (TN0602) from Thermotoga naphthophila RKU-10, demonstrating exceptionally high catalytic selectivity (100%) for the exclusive synthesis of prebiotic galactotrisaccharides (GOS3) in a high volumetric production yield of 23.28gL-1h-1 (higher than the highest value ever reported) at pH 6.5 and 75°C, with milk processing waste lactose as both the galactosyl donor and acceptor. A comparative study with commercial ß-galactosidase from Aspergillus oryzae (AO) with respect to reaction kinetics, enzyme-substrate thermodynamic binding (substrate induced fluorescence quenching) and molecular docking simulation studies showed that ß-glucosidase TN0602 has a deep catalytic "pocket" with a narrow entrance that prevents simultaneous access of lactose and GOS3 to the catalytic site, explaining its distinct catalytic specificity and reaction kinetics. The findings revealed in this work offer an improved understanding of how enzyme protein structure determines catalytic specificity, which serves as new knowledge to engineer ß-glucosidase for the biosynthesis of designer GOS.

Adding functionality to milk-based protein: Preparation, and physico-chemical characterization of ß-lactoglobulin-phenolic conjugates.

Multi-functional phenolic emulsifiers were prepared by covalently coupling ß-Lactoglobulin (ßLg) to caffeic acid (CA) using crosslinker chemistry at different pH conditions (pH 2.5, 6.0, and 8.5). The resulting bioconjugates were characterized by MALDI-TOF MS, differential scanning calorimetry (DSC), fluorescence-quenching, infrared and circular dichroism spectroscopies. Furthermore, the emulsifying and antioxidant properties of ßLg-CA conjugates were evaluated and compared to native ß-Lactoglobulin and the non-covalent ß-lactoglobulin/caffeic complex (ßLg/CA). Results showed: 1) An optimal molar ratio (8:1) of caffeic acid to ßLg was obtained at pH 6; 2) DPPH activity of ßLg-CA increases as the number of CA units coupled increases; 3) ßLg-CA conjugates displayed comparable or superior water solubility than native ßLg and ßLg/CA. Moreover, DSC results showed that coupling of CA with ßLg significantly increased the thermal stability of ßLg. In summary, ßLg-CA conjugates can act as effective antioxidant emulsifiers and stabilizers and may find application in food and cosmetic industries.

A novel array of interface-confined molecules: Assembling natural segments for delivery of multi-functionalities.

HYPOTHESIS: Anionic surfactants can form stable monolayers around oil droplets via interactions with macromolecules thereby physically stabilizing high fish oil enriched emulsions (50-70% fish oil) while phenolic acids have antioxidant properties to prevent lipid oxidation. COSMO-RS (Conductor-like Screening Model for Real Solvents) is a powerful tool for the rational design of molecules with multi-functionalities. Therefore, it should be possible to assemble segments of natural molecules into a single multifunctional molecule using COSMO-RS to confer both physical and oxidative stability to fish oil enriched systems. EXPERIMENTS: COSMO-RS was used to predict the thermodynamic properties of series phenoleoyl malic acid esters of monoglycerides in comparison with commercial emulsifiers. A novel series of amphiphilic lipids, equipped with multi-functional groups from natural building blocks (fatty-acyl, glycerol, malic & phenolic acids), were then synthesized in a facile approach and characterized by various spectroscopy techniques. Oil-in-water emulsions stabilized by the amphiphilic lipids were formulated and characterized by dynamic light scattering measurements and fluorescence imaging. FINDINGS: An elaborate integration of multi-functions into a single molecule was achieved, displaying superior or comparable emulsion stability and antioxidant property, compared to a commercial emulsifier, phenolic acids and their combinations. This is the first report to holistically integrate the rational design, synthesis and functional characterization of natural-based multifunctional molecules for high capacity fish oil delivery systems.

Valorizing Dairy Waste: Thermophilic Biosynthesis of a Novel Ascorbic Acid Derivative.

l-Ascorbic acid (l-AA) is an essential nutrient that is extremely unstable and cannot be synthesized by the human body. Therefore, attempts have been performed to develop biologically active l-AA derivatives with improved stability. This work presents a facile, scalable, and efficient enzymatic transgalactosylation of lactose to l-AA using ß-glucosidase (TN0602) from Thermotoga naphthophila RKU-10. ß-Glucosidase TN0602 displays high transgalactosylation activity at pH 5.0, 75 °C, and l-AA/lactose ratio of 2:1 to form a novel l-AA derivative [2-O-ß-d-galactopyranosyl-l-ascorbic acid (l-AA-Gal)] with a maximal productivity of 138.88 mmol L-1 in 12 h, which is higher than most reports of enzymatic synthesis of l-AA-α-glucoside. Synthetic l-AA-Gal retains most l-AA antioxidant capability and presents dramatically higher stability than l-AA in an oxidative environment (Cu2+). In conclusion, this work reports a new way to valorize dairy waste lactose into a novel molecule l-AA-Gal, which could be a promising l-AA derivative to be used in a wide range of applications.