Effect of the C-Ring Structure of Flavonoids on the Yield of Adducts Formed by the Linkage of the Active Site at the A-Ring and Amadori Rearrangement Products during the Maillard Intermediate Preparation.

Flavonoids (dihydromyricetin, dihydroquercetin, epicatechin, and epigallocatechin) were applied to indicate the critical formation condition of the Amadori rearrangement product (ARP) in Maillard reaction performed under a two-step temperature rising process in the threonine-xylose model system. Threonine-ARP (Thr-ARP) was mixed with dihydromyricetin (DM), dihydroquercetin (DQ), epicatechin (EC), and epigallocatechin (EGC) before the heat treatment; then, the mixture was tested by liquid chromatography-mass spectrometry (LC-MS). The results showed that these flavonoids trapped the ARP and generated adducts. The A-ring of flavonoids (the meta-polyhydroxylated benzene ring) was the functional group to capture the Thr-ARP. The relative contents of the adducts of DM-Thr-ARP, DQ-Thr-ARP, EC-Thr-ARP, and EGC-Thr-ARP were compared with each other, and it was found that the structure of the C-ring of the flavonoids (the carbonyl group on C-4) significantly impeded the formation of adducts with Thr-ARP, while the number of hydroxyl groups on the B-ring had little influence. The formation of adducts delayed the degradation of Thr-ARP, decreased the production of α-dicarbonyl compounds, and suppressed Maillard browning. In this way, the flavonoids might trace the critical formation conditions of ARP during the two-step temperature rising process.

The preparation of phytosteryl succinyl sucrose esters and improvement of their water solubility and emulsifying properties.

Phytosterols have gained much attention due to their outstanding cholesterol-reducing effect, while the insolubility in water limits their application. The aim of this study was to synthesize a novel hydrophilic phytosteryl derivatives-phytosteryl succinyl sucrose esters (PSSEs) and investigated their water solubility and emulsifying properties. PSSEs were synthesized by esterifying phytosterol hemisuccinates with sucrose through a mild chemical reaction. PSSEs were characterized by fourier transform infrared spectroscopy, mass spectroscopy, and nuclear magnetic resonance spectroscopy. The yield of PSSEs exceeded 84% in N,N-dimethylformamide for 36 h of reaction under the selected conditions: 100 mmol/L phytosteryl hemisuccinates, 150 mmol/L sucrose, 110 mmol/L 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochlide, 10 mmol/L 4-dimethylaminopyridine and 10 mmol/L p-toluenesulfonic acid. The water insolubility of phytosterols was overcome and the water solubility of PSSEs achieved 2.13 mg/mL. The emulsifying activity of PSSEs was 2.5 times that of phytosterols, reaching 0.95 mg/mL. PSSEs with better water solubility and emulsification properties could facilitate the widespread use of phytosterols in foods.

Dependence and Conversion Mechanism for Selective Preparation of a Xylose-Diglycine Amadori Compound and a Cross-linking Product in an Aqueous Maillard Reaction.

The structure of the prepared Amadori rearrangement product of xylose-glycylglycine (XGG-ARP) and a cross-linking product (XGG-CP) was first characterized by liquid chromatography-mass spectrometry (LC-MS)/MS and NMR analysis, respectively. The dependences were then studied for the formation of XGG-ARP and XGG-CP in an aqueous Maillard reaction of a xylose-glycylglycine model system. The influence factors were the reaction temperature, pH, molar ratio of reactants, and the reaction time. It was found that XGG-ARP would acquire the highest yield of 73.8% when the thermal reaction was carried out at 70 °C and pH 8.0 for 10 min. A higher temperature and a lower pH might enhance the yield of the formation of XGG-CP. Combining the low-temperature dehydration reaction with the high-temperature aqueous-phase Maillard reaction increased the XGG-CP yield to 43.54%. The efficient and selective preparation of XGG-ARP and XGG-CP was controllable through the adjustment of reaction conditions. Moreover, the pathway of XGG-CP formation from XGG-ARP and Gly-Gly by the aldimine condensation reaction was also proposed. The high stability of an XGG-CP molecule enhanced by its p-π-p conjugated structure inhibited the occurrence of the Amadori rearrangement and led to a non-keto structure, blocking the further Maillard reaction of XGG-CP.

Adducts Derived from (-)-Epigallocatechin Gallate-Amadori Rearrangement Products in Aqueous Reaction Systems: Characterization, Formation, and Thermolysis.

The interaction mechanism of (-)-epigallocatechin gallate (EGCG) with Amadori compound (Amadori rearrangement product, ARP) in xylose-alanine model reaction systems was investigated. The adducts between ARP and EGCG were identified as two ARP-EGCG isomers, two ARP-EGCG-H2O isomers, and multiple ARP-deoxypentosone (DP)-EGCG isomers. The structure of an isolated and purified ARP-EGCG adduct was analyzed by means of Fourier transform infrared spectroscopy, ultraviolet-visible spectroscopy, liquid chromatography-time-of-flight (TOF)-mass spectrometry (LC-TOF-MS), and nuclear magnetic resonance (NMR). Using the two-dimensional NMR analyses, the structure of ARP-EGCG adducts was clarified to consist of a covalent linkage between the C12 position of the ARP and the C8 position of the A-ring of EGCG, presumably generated by the nucleophilic nature of the EGCG or aromatic substitution reactions. The results showed that slightly alkaline pH and higher temperature could facilitate this reaction. Additionally, the thermal stability of ARP-EGCG and its degradation products revealed that the decomposition pathways of this adduct altered the classic decomposition pathway of ARP, resulting in a lower browning rate and blocking the subsequent Maillard reaction.

Preparation of phytosteryl ornithine ester hydrochloride and improvement of its bioaccessibility and cholesterol-reducing activity in vitro.

In this work, synthesis of phytosteryl ornithine ester hydrochloride was studied for the first time using an intermediate phytosteryl N,N'-bis[tert-butoloxycarbonyl(BOC)]-ornithine ester. This method also involved esterification of phytosterols with N,N'-bis(BOC)-ornithine and deprotection. The maximum yield was 90% and deprotection of BOC group was more than 99% using the HCl/ethyl acetate method. As a result, thermal stability and water solubility as well as emulsifying activity and stability of phytosterols were improved through coupling with ornithine, which is favorable for their application in water-based food systems. We also observed increased bioaccessibility of phytosteryl ornithine hydrochloride (4.5%) and 65% of phytosteryl ornithine hydrochloride was hydrolyzed in vitro. These results indicated that ornithine phytosteryl ester hydrochloride can reduce dissolution capacity of cholesterol in vitro, representing improved cholesterol-reducing activity, which will further expand the applications of phytosteryl ornithine ester hydrochloride.

Direct and selective enzymatic synthesis of trehalose unsaturated fatty acid diesters and evaluation of foaming and emulsifying properties.

Trehalose diesters are Gemini-type surfactants that might have better surface activity than conventional surfactants. A one-step method for the preparation of trehalose unsaturated fatty acid diesters has been successfully developed. The yield of trehalose diester of different unsaturated fatty acids was between 78 % and 88 % under optimal conditions: 25 mmol/L trehalose, 100 mmol/L unsaturated fatty acid, 60 g/L 3 Šmolecular sieves and 20 g/L lipase at 150 rpm and 50 °C for 42 h in 15 mL of acetone. Additionally, trehalose diester was the sole product obtained with Novozym 435 in acetone. The chemical structures of 6,6'-di-O-oleoyltrehalose, 6,6'-di-O-linoleoyltrehalose, 6,6'-di-O-eicosenoyltrehalose and 6,6'-di-O-erucoyltrehalose were confirmed by FTIR, MS and NMR. Moreover, the hydrophile-lipophile balance (HLB) values, foaming properties and emulsifying properties of trehalose diesters were assessed, showing the potentials of these diesters as naturally derived surfactants for the food industry.

Preparation of N-(1-Deoxy-Α-D-Xylulos-1-Yl)-Glutamic Acid via Aqueous Maillard Reaction Coupled with Vacuum Dehydration and Its Flavor Formation Through Thermal Treatment of Baking Process.

Amadori rearrangement product (ARP) derived from glutamic acid (Glu) and xylose (Xyl) was prepared by aqueous Maillard reaction. Subsequently, ion exchange chromatography, MS, and NMR were used for purification and identification, confirming that the molecular formula of ARP was C10 H17 NO8 , namely N-(1-deoxy-α-D-xylulos-1-yl)-glutamic acid, with a molecular mass of 279 Da. To improve the aqueous yield of ARP, a thermal reaction coupled with vacuum dehydration was used and the yield of ARP was increased from 2.07% to 75.11%. Furthermore, flavor formation capacity of ARP by a thermal treatment simulated to a baking process was compared with Maillard reaction products, Maillard-dehydration reaction products, and Glu-Xyl mixture. The results indicated that a larger amount of volatile flavor compounds and a biscuit-like, burnt aroma was generated rapidly from the mixture of ARP and unreacted Glu-Xyl, which could be a potential flavor enhancer for baked foods. PRACTICAL APPLICATION: Maillard reaction performed in aqueous medium through thermal reaction combined with vacuum dehydration is a novel and practical technology that could be widely used to produce Maillard reaction intermediates (MRIs), such as Amadori or Heyns rearrangement products, which are regarded as significant nonvolatile aroma precursors and have stable physical and chemical properties compared with Maillard reaction products (MRPs). MRI derived from glutamic acid and xylose is a potential substitute of MRPs for flavorings preparation and shows a great capacity to generate fresh flavors in a short time at high temperature, which meets the requirements of baking foods. Therefore, the new developed method could be a promising tool for MRI preparation and application in food and flavoring industries.

Interaction of Added l-Cysteine with 2-Threityl-thiazolidine-4-carboxylic Acid Derived from the Xylose-Cysteine System Affecting Its Maillard Browning.

The Maillard reaction under a stepwise increase of temperature using l-cysteine as the indicator was performed to determine the formation conditions for the preparation of 2-threityl-thiazolidine-4-carboxylic acid (TTCA) which was the main Maillard reaction intermediate (MRI) derived from the xylose (Xyl)-cysteine (Cys) model system in aqueous medium. To clarify the indicating mechanism of Cys for the TTCA formation, the thermal model systems of TTCA-Cys and TTCA solutions were investigated. The browning of the final Maillard reaction products (MRPs) and concentration of downstream degradation products of MRIs indicated that the added Cys could react with TTCA to inhibit the formation of visible color via preventing the generation of dicarbonyl compounds derived from MRIs. Through HPLC analysis, it was demonstrated that added Cys affected the normal reaction pathway from TTCA to ARP and other downstream products by restoring TTCA to sugar and amino acid under heat treatment.

Timely Addition of Glutathione for Its Interaction with Deoxypentosone To Inhibit the Aqueous Maillard Reaction and Browning of Glycylglycine-Arabinose System.

The inhibitory effects of glutathione (GSH) and oxiglutathione (GSSG) on Maillard browning were compared, and it was clarified that free sulfhydryl was the key substance for the inhibition. The Amadori rearrangement product (ARP) derived from glycylglycine (Gly-Gly) and arabinose (Ara) was prepared by aqueous Maillard reaction, and LC-MS/MS was used to investigate the reaction products of GSH and purified ARP. Reaction between GSH and deoxypentosone (DP) was found to alter the pathway of aqueous Maillard reaction, which reduced the production of glyoxal, methylglyoxal, and furfural and thereby inhibited the formation of melanoidins. To determine the optimal conditions for browning inhibition, a stepwise increase of temperature was used to prepare Maillard reaction products (MRPs). The results showed that the optimum browning inhibitory effect was obtained by adding GSH after Gly-Gly and Ara heating at 80 °C for 60 min.

Effective Mechanism of (-)-Epigallocatechin Gallate Indicating the Critical Formation Conditions of Amadori Compound during an Aqueous Maillard Reaction.

The formation conditions of the Amadori compound (ARP) N-(1-deoxy-d-xylulos-1-yl)-alanine were determined in an aqueous Maillard reaction between l-alanine and d-xylose under a two-step temperature rising process with (-)-epigallocatechin gallate (EGCG) as an indicator followed by browning intensity detection of the final Maillard reaction products (MRPs). To clarify the mechanism of EGCG indication on the ARP formation, the change in the concentration of some key products generated during the Maillard reaction with EGCG addition was investigated. Results showed an inhibition effect of EGCG on the browning precursor formation through the generation of ARP-EGCG adducts and deoxyosone-EGCG adducts, which was proposed as an important pathway to inhibit browning during the Maillard reaction and to indicate ARP formation.

Mild and Efficient Preparation of Phytosteryl Amino Acid Ester Hydrochlorides and Their Emulsifying Properties.

The aim of this work was to produce a series of phytosteryl amino acid ester hydrochlorides by a two-step method, which involved esterification of phytosterols with N- tert-butoxycarbonyl (BOC) amino acid and deprotection of the BOC group. The highest yield of over 95.0% was obtained when the catalysts were the mixtures of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochlide, 4-dimethylaminopyridine, and triethylamine. It was found that batch charging of the reactants and catalysts was conducive to improving the yield. In addition, over 99.0% of the BOC group deprotection degree was achieved using the HCl/ethyl acetate deprotection method. All of the compounds were characterized by fourier transform infrared spectroscopy, mass spectroscopy, and nuclear magnetic resonance spectroscopy. The emulsifying properties of phytosterols and phytosteryl amino acid ester hydrochlorides were also investigated. The results showed higher emulsifying properties of phytosteryl amino acid ester hydrochlorides, which could favor its wide application in food systems.

Synergistic Effect of a Thermal Reaction and Vacuum Dehydration on Improving Xylose-Phenylalanine Conversion to N-(1-Deoxy-d-xylulos-1-yl)-phenylalanine during an Aqueous Maillard Reaction.

The synergistic effect of a thermal reaction and vacuum dehydration on the conversion of xylose (Xyl) and phenylalanine (Phe) to a Maillard-reaction intermediate (MRI) was researched. The yield of N-(1-deoxy-α-d-xylulos-1-yl)-phenylalanine was successfully improved and increased from 13.62 to 47.23% through the method combining a thermal reaction and vacuum dehydration. A dynamic process was involved in the transformation of Xyl and Phe (Xyl-Phe) to N-substituted d-xylosamine and in the transformation of N-substituted d-xylosamine to N-(1-deoxy-α-d-xylulos-1-yl)-phenylalanine during the initial stage of dehydration; then, only the transformation of N-substituted d-xylosamine to N-(1-deoxy-α-d-xylulos-1-yl)-phenylalanine occurred during the final stage. Furthermore, the MRI was prepared under optimized conditions (90 °C and pH 7.4), and the obtained MRI was characterized and confirmed by ESI mass spectrometry and NMR.

A new approach for facile synthesis of phytosteryl phenolates.

In this study, a one-step method for producing phytosteryl phenolates, namely phytosteryl cinnamate and ferulate, has been successfully developed and their chemical structures were confirmed by FT-IR, MS and NMR. The highest yield of phytosteryl ferulate (85.7%) was obtained at 100 °C for 2 h after optimization. A Lewis acid catalyst scandium triflate was selected as the catalyst, and it turned out that it could be reused for at least five times without significant loss of activity. Meanwhile, it has been demonstrated that the solubility of phytosteryl phenolates in soybean oil was much higher than in both phenolic acid and phytosterol, which was conducive to expand their applications in oil-based food. The research finding helps realize convenient, green and efficient synthesis of phytosteryl phenolates.

Sodium sulfite pH-buffering effect for improved xylose-phenylalanine conversion to N-(1-deoxy-d-xylulos-1-yl)-phenylalanine during an aqueous Maillard reaction.

The yield of the Maillard reaction intermediate (MRI), prepared in aqueous medium, is usually unsatisfactory. However, the addition of sodium sulfite could improve the conversion of xylose-phenylalanine (Xyl-Phe) to the MRI (N-(1-deoxy-d-xylulos-1-yl)-phenylalanine) in aqueous medium. Sodium sulfite (Na2SO3) showed a significant pH-buffering effect during the Maillard reaction, which accounted for its facilitation of the N-(1-deoxy-d-xylulos-1-yl)-phenylalanine yield. The results revealed that the pH could be maintained at a relatively high level (above 7.0) for an optimized pH-buffering effect when Na2SO3 (4.0%) was added before the reaction of Xyl-Phe. Thus, the conversion of Xyl-Phe to N-(1-deoxy-d-xylulos-1-yl)-phenylalanine increased from 47.23% to 74.86%. Furthermore, the addition moment of Na2SO3 and corresponding solution pH were crucial factors in regulating the pH-buffering effect of Na2SO3 on N-(1-deoxy-d-xylulos-1-yl)-phenylalanine yield. Based on the pH-buffering effect of Na2SO3 and maintaining the optimal pH 7.4 relatively stable, the conversion of Xyl-Phe to N-(1-deoxy-d-xylulos-1-yl)-phenylalanine was successfully improved.

Enzymatic synthesis of phytosteryl lipoate and its antioxidant properties.

In this work, an enzymatic route for synthesizing phytosteryl lipoate was successfully set up for the first time. The structure of final product phytosteryl lipoate was determined by Fourier Transform Infrared (FTIR), Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR). The maximum conversion of 71.2% was achieved when the following conditions were employed: 150mmol/L phytosterol, 1: 2.5M ratio of phytosterol to lipoic acid, 10g/L of 4Å molecular sieves and 60g/L Candida rugosa in 2-methyl-2-butanol/n-hexane (1:1, v/v) at 55°C for 96h. The physicochemical properties including solubility and antioxidant ability of phytosteryl lipoate in oil were assessed. The results revealed that phytosteryl lipoate possessed over twice as much oil solubility as free phytosterol and also showed better antioxidant ability. Investigation on its biological functions will be the main object in the future work.

Coupled effects of salt and pressure on catalytic ability of Rhizopus chinensis lipase.

BACKGROUND: Both high pressure and environmental factors could influence the catalytic abilities of enzymes. This work investigated coupled effects of pressure and salts on Rhizopus chinensis lipase (RCL) to provide significant information for its further applications. RESULTS: The maximum activity of RCL was observed under 200 MPa at 40 °C. The highest activity was achieved at concentrations of 0.06-0.1 mol L-1 for tested salts. The effect of monovalent cations on RCL activity followed the Hofmeister series (K+ > Na+ > Li+ ) at 0.1 MPa but the order of Na+ and K+ was changed under 200 MPa. Meanwhile, the effects of anions did not follow the Hofmeister series. KCl slightly improved the thermostability of RCL at moderate concentration. At 60 °C, LiCl only stabilised RCL at 0.1 mol L-1 . The pre-transition unfolding point was shifted from 4.5 to 3.5 mol L-1 with pressure increasing from 0.1 to 600 MPa. In addition, KCl could not change the lipase's extrinsic fluorescence evolution versus pressure. CONCLUSION: Pressure and salts could improve catalytic ability and stability of RCL under appropriate conditions. The effect of high pressure on RCL was influenced by salts. Meanwhile salts cannot prevent high pressure-induced damage to RCL. © 2017 Society of Chemical Industry.

Mechanism of Formation and Stabilization of Nanoparticles Produced by Heating Electrostatic Complexes of WPI-Dextran Conjugate and Chondroitin Sulfate.

Protein conformational changes were demonstrated in biopolymer nanoparticles, and molecular forces were studied to elucidate the formation and stabilization mechanism of biopolymer nanoparticles. The biopolymer nanoparticles were prepared by heating electrostatic complexes of whey protein isolate (WPI)-dextran conjugate (WD) and chondroitin sulfate (ChS) above the denaturation temperature and near the isoelectric point of WPI. The internal characteristics of biopolymer nanoparticles were analyzed by several spectroscopic techniques. Results showed that grafted dextran significantly (p < 0.05) prevented the formation of large aggregates of WD dispersion during heat treatment. However, heat treatment slightly induced the hydrophobicity changes of the microenvironment around fluorophores of WD. ChS electrostatic interaction with WD changed the fluorescence intensity of WD regardless of heat treatment. Far-UV circular dichroism (CD) and attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopies confirmed that glycosylation and ionic polysaccharide did not significantly cause protein conformational changes in WD and ChS (WDC) during heat treatment. In addition, hydrophobic bonds were the major molecular force for the formation and stabilization of biopolymer nanoparticles. However, hydrogen bonds slightly influenced their formation and stabilization. Ionic bonds only promoted the formation of biopolymer nanoparticles, while disulfide bonds partly contributed to their stability. This work will be beneficial to understand protein conformational changes and molecular forces in biopolymer nanoparticles, and to prepare the stable biopolymer nanoparticles from heating electrostatic complexes of native or glycosylated protein and polysaccharide.

A novel chemo-enzymatic synthesis of hydrophilic phytosterol derivatives.

In this study, a novel method was developed for chemo-enzymatic synthesis of hydrophilic phytosterol derivatives, phytosteryl polyethylene glycol succinate (PPGS), through an intermediate phytosteryl hemisuccinate (PSHS), which was first chemically prepared and subsequently coupled with polyethylene glycol (PEG) through lipase-catalyzed esterification. The chemical structure of intermediate and goal product were finally confirmed to be PSHS and PPGS by FT-IR, MS and NMR, suggesting that hydrophilic phytosterol derivatives were successfully synthesized. The effects of various parameters on the conversion of PSHS to PPGS were investigated and the highest conversion (>78%) was obtained under the selected conditions: 75 mmol/L PSHS, 1:2M ratio of PSHS to PEG, 50 g/L Novozym 435, 120 g/L 3 Å molecular sieves in tert-butanol, 55 °C, 96 h and 200 rpm. The solubility of phytosterols in water was significantly improved by coupling with PEG, facilitating the incorporation into a variety of foods containing water.

Stable nanoparticles prepared by heating electrostatic complexes of whey protein isolate-dextran conjugate and chondroitin sulfate.

A simple and green method was developed for preparing the stable biopolymer nanoparticles with pH and salt resistance. The method involved the macromolecular crowding Maillard process and heat-induced gelation process. The conjugates of whey protein isolate (WPI) and dextran were produced by Maillard reaction. The nanoparticles were fabricated by heating electrostatic complexes of WPI-dextran conjugate and chondroitin sulfate (ChS) above the denaturation temperature and near the isoelectric point of WPI. Then, the nanoparticles were characterized by spectrophotometry, dynamic laser scattering, zeta potential, transmission electron microscopy, atomic force microscopy, and scanning electron microscopy. Results showed that the nanoparticles were stable in the pH range from 1.0 to 8.0 and in the presence of high salt concentration of 200 mM NaCl. WPI-dextran conjugate, WPI, and ChS were assembled into the nanoparticles with dextran conjugated to WPI/ChS shell and WPI/ChS core. The repulsive steric interactions, from both dextran covalently conjugated to WPI and ChS electrostatically interacted with WPI, were the major formation mechanism of the stable nanoparticles. As a nutrient model, lutein could be effectively encapsulated into the nanoparticles. Additionally, the nanoparticles exhibited a spherical shape and homogeneous size distribution regardless of lutein loading. The results suggested that the stable nanoparticles from proteins and strong polyelectrolyte polysaccharides would be used as a promising target delivery system for hydrophobic nutrients and drugs at physiological pH and salt conditions.

Efficient synthesis and characterization of ergosterol laurate in a solvent-free system.

Ergosterol and its derivatives have attracted much attention for a variety of health benefits, such as anti-inflammatory and antioxidant activities. However, ergosterol esters are advantageous because this compound has better solubility than the free ergosterol. In this work, ergosterol laurate was efficiently synthesized for the first time by direct esterification in a solvent-free system. The desired product was purified, characterized by Fourier transform infrared spectroscopy, mass spectrometry, and nuclear magnetic resonance, and finally confirmed to be ergosterol laurate. Meanwhile, the effect of various catalysts, catalyst dose, reaction temperature, substrate molar ratio, and reaction time were studied. Both the conversion of ergosterol and the selectivity of the desired product can reach above 89% under the selected conditions: sodium dodecyl sulfate + hydrochloric acid as the catalyst, 2:1 molar ratio of lauric acid/ergosterol, catalyst dose of 4% (w/w), 120 °C, and 2 h. The oil solubility of ergosterol and its laurate was also compared. The results showed that the solubility of ergosterol in oil was significantly improved by direct esterification with lauric acid, thus greatly facilitating the incorporation into a variety of oil-based systems.