Asparagine-Glucose Amadori Compounds: Formation, Characterization, and Analysis in Dry Jujube Fruit.

Amadori rearrangement products of asparagine with glucose (Asn-Glc-ARP) were first prepared through Maillard model reactions and identified via liquid chromatography-mass spectroscopy. With the study on the effect of the reaction temperature, pH values, and reaction time, the ideal reaction condition for accumulation of Asn-Glc-ARP was determined at 100 °C for 40 min under pH 7. Asparagine (Asn) was prone to degrade from Asn-Glc-ARP in alkaline pH values within a lower temperature range, while in an acidic environment with high temperatures, deamidation of Asn-Glc-ARP to Asp-Glc-ARP (Amadori rearrangement products of aspartic acid with glucose) was displayed as the dominant pathway. The deamidation reaction on the side chain of the amide group took place at Asn-Glc-ARP and transferred it into the hydroxyl group, forming Asp-Glc-ARP at the end. Considering that lyophilization as pretreatment led to limited water activity, a single aspartic acid was not deamidated from Asn directly nor did it degrade from Asp-Glc-ARP even at 120 °C. The degradation of Asn-Glc-ARP through tandem mass spectrometry (MS/MS) analysis showed the obvious fragment ion at m/z 211, indicating that the stable oxonium ion formed during fragmentation. The structure of Asn-Glc-ARP was proposed as 1-deoxy-1-l-asparagino-d-fructose after separation and purification. Also, the content of Asn-Glc-ARP within dry jujube fruit (HeTianYuZao) was quantitated as high as 8.1 ± 0.5 mg/g.

Study the interaction of amino acids, sugars, thermal treatment and cooking technique on the formation of acrylamide in potato models.

This study unveiled the effect of the suspected precursors of acrylamide (asparagine, glutamine) combined/separated with different formulations of glucose, fructose, and sucrose. To better understand the interaction between acrylamide precursors, cooking technique (deep vs air frying), and temperature (170 °C vs 190 °C), seven potato models from starch, sugars, amino acids, water and hydrocolloids (alginate and agar) were formulated. In line with previous findings, the present results showed that asparagine, glucose and fructose played an important role in acrylamide formation in these synthetic potato models. Furthermore, glutamine and sodium alginate might have an inhibitory effect on acrylamide formation. A significant impact of frying technique was also revealed. On the other hand, GC-FID analysis detected acrylamide in only these three models, (glucose-fructose, sucrose and asparagine-glucose/fructose/sucrose models > LOD 333.33 µg.kg-1).

Efficient control of acrylamide in French fries by an extraordinarily active and thermo-stable l-asparaginase: A lab-scale study.

As a potential carcinogen, acrylamide (AA) widely exists in starch-rich foods during frying, triggering international health alerts. l-Asparaginase (l-ASNase, EC 3.5.1.1) could efficiently inhibit the AA by hydrolyzing its precursor l-Asparagine. Here, a novel recombinant l-ASNase from Palaeococcus ferrophilus was identified for the first time. The purified enzyme exhibited its highest activity at pH 8.5 and 95 °C and retained more than 70% relative activity after incubation at 80 °C for 2 h. Compared to untreated French fries, the AA content in the enzyme-treated (10 U/mL, 85 °C, 15 min) French fries was significantly reduced by 79%. Notably, the l-ASNase could remain over 98% of initial activity after three months of storage at 4 °C, suggesting good storage stability. These results demonstrated that P. ferrophilusl-ASNase could be a great candidate in controlling AA in the food industry, especially at high blanching temperature.

A New Strategy to Increase Production of Genoprotective Bioactive Molecules from Cotyledon-Derived Silybum marianum L. Callus.

There is a need to enhance the production of bioactive secondary metabolites and to establish new production systems, e.g., for liver-protective compounds of Silybum marianum seeds. Quantifying and identifying the produced phytochemicals, and examining their protective effects against genotoxic agents, is of great interest. This study established a protocol for the qualitative and quantitative production of hepatoprotective compounds in cotyledon-derived Silybum marianum callus through optimized supplementation of the MS medium with the growth regulators 2,4-D, benzylaminopurine, myoinositol, and asparagine. High-performance liquid chromatography (HPLC) coupled with electrospray ionisation mass spectrometry (ESI-MS) allowed for identification and quantification of the produced compounds. None of the growth medium combinations supported a detectable production of silymarin. Instead, the generated calli accumulated phenolic acids, in particular chlorogenic acid and dicaffeoylquinic acid, as revealed by HPLC and mass spectrometric analysis. 4-Nitro-o-phenylenediamine (NPD) was employed in the AMES-test with Salmonella typhimurium strain TA98 because it is a potent mutagen for this strain. Results revealed that callus extract had a high anti-genotoxic activity with respect to standard silymarin but more evident with respect seed extract. The callus produced chlorogenic acid and dicaffeoylquinic acid, which revealed higher bioactivity than silymarin. Both compounds were not formed or could not be detected in the seeds of Silybum marianum Egyptian ecotype.

Acrylamide determination during an industrial roasting process of coffee and the influence of asparagine and low molecular weight sugars.

The formation and partial degradation of acrylamide (AA), asparagine and low molecular weight sugars were evaluated during an industrial coffee roasting process, in which the temperature increased from 90° to about 215 °C. Arabica and Robusta varieties were roasted individually. AA content reached the maximum value at 10 min, corresponding to a temperature of 175-177 °C (1045 ±â€¯28 and 795 ±â€¯25 µg kg-1 for Arabica and Robusta, respectively). Successively, AA content decreased very quickly and at 14 min (203-205 °C) its concentration was lower than the benchmark level of 400 µg kg-1 for roast coffee set by the EU Commission Regulation (2017/2158). In the final product, AA content was close to 300 µg kg-1. Asparagine quickly decreased; contrary, the concentration of fructose and glucose increased reaching their maximum value at 12 min. Then, a quick degradation occurred; their increase could be mainly due to the hydrolysis of sucrose, which decreased in the same period.

Fungal L-asparaginase: Strategies for production and food applications.

L-asparaginase (L-asparagine amidohydrolase EC 3.5.1.1) is of great importance in pharmaceutical and food applications. This review aims to describe the production and use of fungal L-asparaginase focusing on its potential as an effective reducer of acrylamide in different food applications. Fungal asparaginases have been used as food additives and have gained importance due to some technical advantages, for example, fungi can grow using low-cost culture mediums, and the enzyme is extracellular, which facilitates purification steps. Research aimed at the discovery of new L-asparaginases, mainly those produced by fungi, have great potential to obtain cheaper enzymes with desirable properties for application in food aiming at the reduction of acrylamide.

Effect of buckwheat extracts on acrylamide formation and the quality of bread.

BACKGROUND: The presence of acrylamide in food has attracted wide attention and has raised concerns due to its potential toxic and carcinogenic effects. The phenolic compounds in buckwheat display strong antioxidant activity, which may affect the acrylamide levels. The aims of this study were to evaluate the effect of buckwheat extracts on acrylamide formation and the quality of the bread, and to investigate possible inhibitory mechanisms. RESULTS: The extracts from Tartary buckwheat seeds, Tartary buckwheat sprouts, common buckwheat seeds, and common buckwheat sprouts reduced acrylamide level in bread by 23.5, 27.3, 17.0, and 16.7%, respectively. In addition, all four buckwheat extracts significantly (P < 0.05) reduced acrylamide levels in the asparagine / glucose system. There were significant positive correlations between total phenolic compound content, the antioxidant activity of the extracts, and the reduction in the acrylamide level. Evaluation of the organoleptic and textural properties indicated that the addition of the extracts did not significantly affect the crust color, aroma, taste, crumb appearance, and hardness of the bread. CONCLUSION: This study showed that proper use of buckwheat extracts can reduce acrylamide levels in bread without having a significant impact on their properties. The study also revealed that a possible acrylamide formation inhibitory mechanism involved the Maillard reaction through the asparagine / glucose pathway. The study also provided useful information for the further application of buckwheat in improving food safety. © 2019 Society of Chemical Industry.

Reduction of acrylamide formation in fried potato chips by Aureobasidum pullulans L1 strain.

Acrylamide is a potential carcinogenic molecule formed during food heat processing at high temperature (Maillard reaction). In the present study, the ability of the yeast Aureobasidium pullulans to deplete the acrylamide precursor free asparagine in fresh potatoes was investigated. A. pullulans applied before final frying changes the free amino acid composition of potatoes, decreasing the content of free asparagine by 16% and reducing acrylamide by 83% in fried potatoes. Potato browning was also reduced by yeast treatment without negative drawbacks on chip taste. This yeast, commonly used in fruit postharvest disease control, can therefore also be applied in potato and bakery industries to reduce food acrylamide content.

Epicatechin Adducting with 5-Hydroxymethylfurfural as an Inhibitory Mechanism against Acrylamide Formation in Maillard Reactions.

This study aimed to investigate the inhibitory mechanism of epicatechin (EC) on the formation of acrylamide in Maillard reactions. The glucose + asparagine model is a typical chemical system used to investigate acrylamide formation. 5-Hydroxymethylfurfural (HMF) is an important carbonyl intermediate in Maillard reactions and can also react with asparagine to form acrylamide. Time courses showed that EC inhibited more HMF than acrylamide in the glucose + asparagine model heated at 180 °C. The reduction of EC on acrylamide formation in the HMF + asparagine model was about 70%, while that in the glucose + asparagine model was about 50%. Moreover, HMF decreased significantly faster when it was heated in the presence of EC. Liquid chromatography-mass spectrometry analysis revealed the formation of adducts between EC and HMF, and the dimeric adducts were verified in fried potato chips. These results suggested that the condensation of EC and HMF was one of the key steps leading to the inhibition of acrylamide. UV-visible spectra analysis showed that some polymerization products had absorption in the visible region and contributed to the development of browning, which was underestimated in the past.

Mitigation effects of proanthocyanidins with different structures on acrylamide formation in chemical and fried potato crisp models.

Proanthocyanidins with different structures were prepared from three condensed tannin-rich plant materials and then characterized using HPLC-ESI-MS coupled with thiolysis and MALDI-TOF MS. Their effects on acrylamide formation in asparagine-glucose models and fried potato crisps were determined. Results showed that all proanthocyanidins significantly and dose dependently mitigated acrylamide formation at concentrations of 50, 100 and 200 µg/mL in chemical model system. Using the food model, acrylamide formation were inhibited when potato crisps were soaked in 0.01-1 mg/mL proanthocyanidin solutions for 15 min at room temperature before frying. Proanthocyanidins reduced up to 44.2% of the acrylamide level as concentration was increased to 0.1 mg/mL, whereas higher concentration resulted in a decrease of inhibition. B-type proanthocyanidins had stronger inhibitory activity than A-type. Unit composition and DP had nearly no effect on their inhibitory activity based on same mass. This study showed that proanthocyanidins with various structures were promising additives to mitigate harmful acrylamide.

Effect of amino acids and frequency of reuse frying oils at different temperature on acrylamide formation in palm olein and soy bean oils via modeling system.

This work investigated the underlying formation of acrylamide from amino acids in frying oils during high temperatures and at different times via modeling systems. Eighteen amino acids were used in order to determine which one was more effective on acrylamide production. Significantly the highest amount of acrylamide was produced from asparagine (5987.5µg/kg) and the lowest from phenylalanine (9.25µg/kg). A constant amount of asparagine and glutamine in palm olein and soy bean oils was heated up in modelling system at different temperatures (160, 180 and 200°C) and times (1.5, 3, 4.5, 6, 7.5min). The highest amount of acrylamide was found at 200°C for 7.5min (9317 and 8511µg/kg) and lowest at 160°C for 1.5min (156 and 254µg/kg) in both frying oils and both amino acids. Direct correlations have been found between time (R2=0.884), temperature (R2=0.951) and amount of acrylamide formation, both at p<0.05.

Mechanistic Insight from Calorimetric Measurements of the Assembly of the Binuclear Metal Active Site of Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes.

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a binuclear metallohydrolase with a high affinity for metal ions at its α site but a lower affinity at its ß site in the absence of a substrate. Isothermal titration calorimetry (ITC) has been used to quantify the Co(II) and Mn(II) binding affinities and thermodynamics of the two sites in wild-type GpdQ and two mutants, both in the absence and in the presence of phosphate. Metal ions bind to the six-coordinate α site in an entropically driven process with loss of a proton, while binding at the ß site is not detected by ITC. Phosphate enhances the metal affinity of the α site by increasing the binding entropy and the metal affinity of the ß site by enthalpic (Co) or entropic (Mn) contributions, but no additional loss of protons. Mutations of first- and second-coordination sphere residues at the ß site increase the metal affinity of both sites by enhancing the binding enthalpy. In particular, loss of the hydrogen bond from second-sphere Ser127 to the metal-coordinating Asn80 has a significant effect on the metal binding thermodynamics that result in a resting binuclear active site with high catalytic activity. While structural and spectroscopic data with excess metal ions have indicated a bridging hydroxide in the binuclear GpdQ site, analysis of ITC data here reveals the loss of a single proton in the assembly of this site, indicating that the metal-bound hydroxide nucleophile is formed in the resting inactive mononuclear form, which becomes catalytically competent upon binding the second metal ion.

Unravelling effects of flavanols and their derivatives on acrylamide formation via support vector machine modelling.

This study investigated the effect of flavanols and their derivatives on acrylamide formation under low-moisture conditions via prediction using the support vector regression (SVR) approach. Acrylamide was generated in a potato-based equimolar asparagine-reducing sugar model system through oven heating. Both positive and negative effects were observed when the flavonoid treatment ranged 1-10,000µmol/L. Flavanols and derivatives (100µmol/L) suppress the acrylamide formation within a range of 59.9-78.2%, while their maximal promotion effects ranged from 2.15-fold to 2.84-fold for the control at a concentration of 10,000µmol/L. The correlations between inhibition rates and changes in Trolox-equivalent antioxidant capacity (ΔTEAC) (RTEAC-DPPH=0.878, RTEAC-ABTS=0.882, RTEAC-FRAP=0.871) were better than promotion rates (RTEAC-DPPH=0.815, RTEAC-ABTS=0.749, RTEAC-FRAP=0.841). Using ΔTEAC as variables, an optimized SVR model could robustly serve as a new predictive tool for estimating the effect (R: 0.783-0.880), the fitting performance of which was slightly better than that of multiple linear regression model (R: 0.754-0.880).

Reduction in Dietary Acrylamide Exposure-Impact of Potatoes with Low Acrylamide Potential.

Acrylamide forms primarily from a reaction between reducing sugars (e.g., glucose and fructose) and an amino acid (asparagine, Asn) formed naturally in foods, including potatoes. This reaction occurs when carbohydrate-rich foods are heated at temperatures above 120 °C. Multiple potato varieties were transformed with potato genomic DNA that results in down-regulation of the expression of the asparagine synthetase-1 gene (Asn1), significantly reducing synthesis of free Asn, and consequently lowering the potential to form acrylamide during cooking. These potatoes with low acrylamide potential (LAP) were tested in agronomic trials, and processed into French fries and potato chips. Decreased levels of acrylamide were measured in these cooked food products when derived from LAP potatoes compared with those derived from conventional potatoes. These reductions can be directly attributed to reduction in Asn levels in the LAP potatoes. The corresponding average reduction in exposure to acrylamide from French fry and potato chip consumption is estimated to be 65%, which would amount to approximately a 25% reduction in overall dietary exposure. Considering that children consume nearly three times more acrylamide than adults on a per kg body weight basis, they would experience the most impact from the reduced acrylamide associated with LAP potatoes. The potential public health impacts, in context of dietary acrylamide exposure reduction, are discussed in this study.

In Silico Investigation of Traditional Chinese Medicine for Potential Lead Compounds as SPG7 Inhibitors against Coronary Artery Disease.

Coronary artery disease (CAD) is the most common cause of heart attack and the leading cause of mortality in the world. It is associated with mitochondrial dysfunction and increased level of reactive oxygen species production. According to the Ottawa Heart Genomics Study genome-wide association study, a recent research identified that Q688 spastic paraplegia 7 (SPG7) variant is associated with CAD as it bypasses the regulation of tyrosine phosphorylation of AFG3L2 and enhances the processing and maturation of SPG7 protein. This study aims to identify potential compounds isolated from Traditional Chinese Medicines (TCMs) as potential lead compounds for paraplegin (SPG7) inhibitors. For the crystallographic structure of paraplegin, the disordered disposition of key amino acids in the binding site was predicted using the PONDR-Fit protocol before virtual screening. The TCM compounds saussureamine C and 3-(2-carboxyphenyl)-4(3H)-quinazolinone, have potential binding affinities with stable H-bonds and hydrophobic contacts with key residues of paraplegin. A molecular dynamics simulation was performed to validate the stability of the interactions between each candidate and paraplegin under dynamic conditions. Hence, we propose these compounds as potential candidates as lead drug from the compounds isolated from TCM for further study in drug development process with paraplegin protein for coronary artery disease.

The kinetics of the inhibition of acrylamide by glycine in potato model systems.

BACKGROUND: Acrylamide (AA) is a potential carcinogen which widely exists in heat-processed foods. The addition of glycine (Gly) has been shown to reduce the formation of AA. The objective of this work was to investigate the kinetics of the inhibition of AA by Gly in both asparagine (Asn)/glucose (Glc) and Asn/Glc/Gly potato model systems during heating at 160 °C, 180 °C, and 200 °C. RESULTS: The simplified two consecutive first-order kinetic model fitted well to the changes of AA in both systems. No significant difference in rate constant (kF) and apparent activation energy (EaF) was observed for AA formation between the two systems (P > 0.05). Whereas EaE and only kE at 200 °C for AA elimination in the Asn/Glc/Gly system was significantly higher than Asn/Glc system (P < 0.05). The elimination reaction between Gly and AA was confirmed by the identification of their major reaction product 2-((3-amino-3-oxopropyl)amino)acetic acid in the Asn/Glc/(15) N-Gly system. CONCLUSION: The reduction of AA by Gly is predominantly attributed to the elimination reaction between Gly and AA.

Investigation of the reactions of acrylamide during in vitro multistep enzymatic digestion of thermally processed foods.

This study investigated the fate of acrylamide in thermally processed foods after ingestion. An in vitro multistep enzymatic digestion system simulating gastric, duodenal and colon phases was used to understand the fate of acrylamide in bakery and fried potato products. Acrylamide levels gradually decreased through gastric, duodenal and colon phases during in vitro digestion of biscuits. At the end of digestion, acrylamide reduction was between 49.2% and 73.4% in biscuits. Binary model systems composed of acrylamide and amino acids were used to understand the mechanism of acrylamide reduction. High-resolution mass spectrometry analyses confirmed Michael addition of amino acids to acrylamide during digestion. In contrast to bakery products, acrylamide levels increased significantly during gastric digestion of fried potatoes. The Schiff base formed between reducing sugars and asparagine disappeared rapidly, whereas the acrylamide level increased during the gastric phase. This suggests that intermediates like the Schiff base that accumulate in potatoes during frying are potential precursors of acrylamide under gastric conditions.

In silico investigation of potential pyruvate kinase M2 regulators from traditional Chinese medicine against cancers.

A recent research in cancer research demonstrates that tumor-specific pyruvate kinase M2 (PKM2) plays an important role in chromosome segregation and mitosis progression of tumor cells. To improve the drug development of TCM compounds, we aim to identify potent TCM compounds as lead compounds of PKM2 regulators. PONDR-Fit protocol was utilized to predict the disordered disposition in the binding domain of PKM2 protein before virtual screening as the disordered structure in the protein may cause the side effect and downregulation of the possibility of ligand to bind with target protein. MD simulation was performed to validate the stability of interactions between PKM2 proteins and each ligand after virtual screening. The top TCM compounds, saussureamine C and precatorine, extracted from Lycium chinense Mill. and Abrus precatorius L., respectively, have higher binding affinities with target protein in docking simulation than control. They have stable H-bonds with residues A:Lys311 and some other residues in both chains of PKM2 protein. Hence, we propose the TCM compounds, saussureamine C and precatorine, as potential candidates as lead compounds for further study in drug development process with the PKM2 protein against cancer.

In silico investigation of potential TRAF6 inhibitor from traditional Chinese medicine against cancers.

It has been indicated that tumor necrosis factor receptor-associated factor-6 (TRAF6) will upregulate the expression of hypoxia-inducible factor-1α (HIF-1α) and promote tumor angiogenesis. TRAF6 proteins can be treated as drug target proteins for a differentiation therapy against cancers. As structural disordered disposition in the protein may induce the side-effect and reduce the occupancy for ligand to bind with target protein, PONDR-Fit protocol was performed to predict the disordered disposition in TRAF6 protein before virtual screening. TCM compounds from the TCM Database@Taiwan were employed for virtual screening to identify potent compounds as lead compounds of TRAF6 inhibitor. After virtual screening, the MD simulation was performed to validate the stability of interactions between TRAF6 proteins and each ligand. The top TCM compounds, tryptophan, diiodotyrosine, and saussureamine C, extracted from Saussurea lappa Clarke, Bos taurus domesticus Gmelin, and Lycium chinense Mill., have higher binding affinities with target protein in docking simulation. However, the docking pose of TRAF6 protein with tryptophan is not stable under dynamic condition. For the other two TCM candidates, diiodotyrosine and saussureamine C maintain the similar docking poses under dynamic conditions. Hence, we propose the TCM compounds, diiodotyrosine and saussureamine C, as potential candidates as lead compounds for further study in drug development process with the TRAF6 protein against cancer.

The inhibition of folylpolyglutamate synthetase (folC) in the prevention of drug resistance in Mycobacterium tuberculosis by traditional Chinese medicine.

Tuberculosis (TB) is an infectious disease caused by many strains of mycobacteria, but commonly Mycobacterium tuberculosis. As a possible method of reducing the drug resistance of M. tuberculosis, this research investigates the inhibition of Folylpolyglutamate synthetase, a protein transcript from the resistance association gene folC. After molecular docking to screen the traditional Chinese medicine (TCM) database, the candidate TCM compounds, with Folylpolyglutamate synthetase, were selected by molecular dynamics. The 10,000 ps simulation in association with RMSD analysis and total energy and structural variation defined the protein-ligand interaction. The selected TCM compounds Saussureamine C, methyl 3-O-feruloylquinate, and Labiatic acid have been found to inhibit the activity of bacteria and viruses and to regulate immunity. We also suggest the possible pathway in protein for each ligand. Compared with the control, similar interactions and structural variations indicate that these compounds might have an effect on Folylpolyglutamate synthetase. Finally, we suggest Saussureamine C is the best candidate compound as the complex has a high score, maintains its structural composition, and has a larger variation value than the control, thus inhibiting the drug resistance ability of Mycobacterium tuberculosis.