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Lysine (Lys) glycated by xylose (Xyl) at α-NH 2 [ N α (1-deoxy-D-xylulos-1-yl)lysine ( N α -Xyl-Lys ARP)] or ε-NH 2 [ N ε -Xyl-Lys ARP. Furans and pyrazines formation during the degradation of the diglycated lysine [ N ε -Xyl-Lys ARP)] significantly impacted the thermal degradation pathways of Amadori rearrangement products (ARPs). N α -Xyl-Lys ARP was found to undergo retro-aldolization on the sugar fragment more readily to form glyoxal/methylglyoxal than N ε -Xyl-Lys ARP. Furans and pyrazines formation during the degradation of the diglycated lysine [ N -di-Xyl-Lys ARP and enabled glyoxal to actively participate in Strecker degradation. Thus, the production of furans and pyrazines was substantially increased, and the variety of pyrazines was expanded from three types to eight types. An appropriate increase to pH 7.5 effectively avoided the overprotonation of hydroxyl and amino groups (pH 5.5), simultaneously enhancing furans and pyrazines yield while minimizing the formation of pyridines under alkaline conditions.α , N ε -di(1-deoxy-d-xylulos-1-yl)lysine ( N α ,N ε -di-Xyl-Lys ARP)] was delayed at 120 °C relative to N ε -Xyl-Lys ARP. This was attributed to the complex degradation of N α ,N ε -di-Xyl-Lys ARP, which slowed the substantial formation of deoxypentosones and the effective release of Lys. At 140 °C, the dual glycated N α ,N ε -di-Xyl-Lys ARP was more conducive to promoting the redistribution of electrons and facilitating molecular rearrangement. This accelerated the efficient decomposition of dual glycated groups in N α ,N ε -di-Xyl-Lys ARP and enabled glyoxal to actively participate in Strecker degradation. Thus, the production of furans and pyrazines was substantially increased, and the variety of pyrazines was expanded from three types to eight types. An appropriate increase to pH 7.5 effectively avoided the overprotonation of hydroxyl and amino groups (pH 5.5), simultaneously enhancing furans and pyrazines yield while minimizing the formation of pyridines under alkaline conditions.

The formation pathway and mechanism of various pyrazines were investigated during the thermal treatment of the alanine-xylose Amadori compound (Ala-ARP) and exogenous alanine (Ala). 15 N -labeled Ala was used to coheated with Ala-ARP to clarify the nitrogen sources and the respective contributions of exogenous Ala and the regenerated Ala released from Ala-ARP to different pyrazine formation. It was found that exogenous Ala exhibited a priority in capturing glyoxal (GO) to form pyrazine during the thermal degradation of ARP. Compared to the Ala-methylglyoxal (MGO) model, a lower activation energy was required for the Ala-GO reaction, where the reaction dynamics of Ala-GO followed a zero-order model. In addition to forming pyrazine, the interaction between existing exogenous Ala and GO would accelerate the thermal degradation of Ala-ARP and retro-aldolization reaction of deoxyxylosones (DXs) to α-dicarbonyls. During this process, the release of regenerated Ala and MGO was promoted. Accordingly, as GO was expended by exogenous Ala during the initial stage of ARP-Ala degradation, the condensation between regenerated Ala and MGO became intensified, leading to the generation of methylpyrazine and 2,5-dimethylpyrazine. As a result, in the thermally treated mixture of Ala-ARP and exogenous Ala, 55% of the formed pyrazine originated from exogenous Ala, while 63% of the formed methylpyrazine and 57% of the formed 2,5-dimethylpyrazine were derived from regenerated Ala (120 °C, 30 min).

The involvement of exogenous alanine was observed to inhibit the generation of 2-furfural during the thermal degradation of the Amadori rearrangement product (ARP). To clarify the reason for the reduced yield of 2-furfural triggered by exogenous alanine, the evolution of the precursors of 2-furfural formed in the ARP model and ARP-alanine model was investigated, and a model including ARP and 15 N -labeled alanine was used to differentiate the role of endogenous and exogenous alanine in the degradation of ARP. It was found that the condensation between ARP and 3-deoxyxylosone could occur during thermal treatment. Nevertheless, the interaction of ARP with 3-deoxyxylosone exhibited an accelerated pace in the presence of exogenous alanine. In this way, exogenous alanine blocked the recovery of endogenous alanine while simultaneously enhancing the consumption of ARP and 3-deoxyxylosone during the Maillard reaction. Hence, the yield of 2-furfural was diminished with the interference of exogenous alanine. Furthermore, the promotion of the reaction between ARP and deoxyxylosone induced by exogenous alanine blocked their retro-aldolization reaction to short-chain α-dicarbonyls (α-DCs) and consequently resulted in a lack of pyrazine formation during the ARP degradation. The present study provided a feasible method for the controlled formation of 2-furfural during the thermal treatment of ARP derived from alanine.

2,5-Dimethylpyrazine (2,5-DMP) is a high-value-added alkylpyrazine compound with important applications in both the food and pharmaceutical fields. In response to the increasing consumer preference for natural products over chemically synthesized ones, efforts have been made to develop efficient microbial cell factories for the production of 2,5-DMP. However, the previously reported recombinant strains have exhibited low yields and relied on expensive antibiotics and inducers. In this study, we employed metabolic engineering strategies to develop an Escherichia coli strain capable of producing 2,5-DMP at high levels without the need for inducers or antibiotics. Initially, the biosynthesis pathway of 2,5-DMP was constructed that realized 2,5-DMP production from glucose. Subsequently, efforts focused on enhancing 2,5-DMP production by improving the availability of the cofactor NAD + and precursor l-threonine. Additionally, the supply and conversion of l-threonine were balanced by optimizing the copy number of the key gene tdh on the chromosome and by modifying the l-threonine transport system. The final engineering strain D19 produced 3.1 g/L of 2,5-DMP, which is the highest titer for fermentative production of 2,5-DMP using glucose as the carbon source up to date. The strategies used in this study lay a good foundation for the production of 2,5-DMP on a large scale.

Long-term storage Baijiu has an appealing sensory property, yet the chemical makeup is rarely reported. This study investigated a 30-year-old Jiangxiangxing (JXX) Baijiu and recognized and measured 69 aroma compounds. 3-Methyl-2,4-nonanedione (2.76 μg/L), 2,5-dimethyl-4-hydroxy-3(2 H )-furanone (HDMF, 46.2 μg/L), 2-ethyl-4-hydroxy-5-methyl-3(2 H )-furanone (HEMF, 61.7 μg/L), and piperitone (3.66 μg/L) were detected for the first time in Baijiu. Compared with 3-year-old JXX Baijiu, 24 compounds were significantly higher in the 30-year-old, mainly including furans, pyrazines, and aromatics. Notably, 4,5-dimethyl-3-hydroxy-2(5 H )-furanone (sotolon), HDMF, HEMF, vanillin, acetovanillone, and alkyl pyrazines in 30-year-old JXX Baijiu were 2-7 times higher than those of a 3-year-old, and they increased steadily during aging for 3, 15, and 30 years, assumed to be associated with the aging aroma. Following 24 months of storing JXX Baijiu under different conditions, the pottery significantly promoted the synthesis of sotolon, HDMF, HEMF, and alkyl pyrazines. These findings suggest that pottery is a potential catalyst for enhancing aged Baijiu.

Jinhua dry-cured ham (JDH) is a traditional fermented Chinese meat product. We studied the dynamic sensory and emotional profiles of JDHs obtained by five preparation methods and the corresponding release of sodium ions (Na + ), potassium ions (K + ), and volatile organic compounds (VOCs) during oral processing. The VOCs with salty taste enhancement abilities were screened based on the correlations of VOCs with salty flavor and concentration of Na and K ions with salty flavor. A trained sensory panel evaluated the saltiness enhancements of selected VOCs by using static and dynamic sensory methods. The results revealed that Na + , K + , and selected VOCs were mainly released during 0-10 s of the chewing process. The release of Na + and K + in JDH residue samples exhibited consistently decreasing trends, while in saliva, their concentrations increased. The VOCs showing a high correlation with Na + and K + and salty flavor have saltiness enhancement abilities in both NaCl solutions and NaCl + MSG mixtures. Odor-induced saltiness was pronounced at low salt concentrations (0.2% NaCl). The investigation demonstrated 16 VOCs exhibiting saltiness enhancement abilities, including 4 pyrazines, 5 acids, 4 sulfur-containing compounds, and 3 other compounds. The sensory evaluation suggested pyrazines and sulfur-containing compounds as good saltiness enhancers. 2-Furfuryl mercaptan significantly enhanced the salty sensation in the NaCl + MSG solutions when compared with MSG alone ( p < 0.05). This research provides evidence that certain odorants identified in JDHs exhibit salty-enhancing properties, indicating their potential for salt reduction at the industrial level.

The contribution of glutathione (GSH) and free amino acids degraded from GSH to the generation of pyrazines and 2,3-butanedione was illustrated during their interaction in the thermal treatment of the Amadori compound of alanine and xylose (ARP). GSH-degraded amino acids, glutamic acid (Glu), cysteine (Cys), and glycine (Gly), but not pyroglutamic acid (pGlu), could effectively capture α-dicarbonyls to facilitate the formation of pyrazines when ARP was heated with GSH. Deoxypentosones, the precursors of 2,3-butanedione, were largely consumed in the ARP-GSH model by the interaction with GSH and its degradative Cys compared with the ARP model. The addition of GSH and deoxypentosones inhibited the further degradation of deoxypentosones, resulting in less formation of 2,3-butanedione and other α-dicarbonyl compounds. Meanwhile, the reaction between GSH-degraded Cys and deoxypentosones to form sulfur-containing compounds such as thiols accelerated the consumption of deoxypentosones; thereby, the formation of 2,3-butanedione was severely interfered. However, this inhibition was compensated for by the GSH-degraded Gly through the addition between Gly and MGO and the subsequent deamination. The involvement of exogenous GSH could simultaneously boost the yields of 2,3-butanedione and pyrazines compared with those of ARP heated alone. As the degree of GSH degradation strengthened in the ARP-thermal-degraded GSH models, the yields of both pyrazines and 2,3-butanedione steadily increased.

The involvement of exogenous threonine during the degradation of l-threonine-d-xylose Amadori rearrangement product (Thr-ARP) was found to promote the formation of pyrazines. A model including Thr-ARP and 15 N-labeled l-threonine was applied to reveal the role of free threonine in Thr-ARP conversion to pyrazines. Quantitative analyses of pyrazines in the model of Thr-ARP/ 15 N-labeled threonine showed a precedence of the endogenous threonine (formed by the degradation of Thr-ARP) over the exogenous threonine in pyrazines formation, and the ratio of 15 N to 14 N content in pyrazines increased significantly over time. According to the observed occurrence of the Heyns rearrangement products (HRP) derived from 15 N-threonine, as well as the sharp decrease of 15 N-threonine content and a rapid increase of 14 N endogenous threonine at the initial stage of heat treatment, it was proposed that aldimine condensation between exogenous threonine and Thr-ARP followed by the hydrolysis led to the endogenous threonine and the generation of HRP. Then, the HRP underwent dehydration followed by hydrolysis to form exogenous threonine and deoxyxyosones, and the dehydration and hydrolysis of deoxyxyosones to form organic acids was inhibited, but the retro-aldolization of deoxyxyosones was promoted, facilitating the generation of reactive α-dicarbonyl compounds. In this way, exogenous threonine accelerated the release of endogenous threonine and α-dicarbonyl compounds and the pH decline was slowed down, which was favorable for the formation of pyrazines.

Maillard reaction products obtained from the model system of binary amino acids (asparagine and proline) with glucose were first studied. GC-MS results showed that proline-specific aromatic compounds, 2,3-dihydro-1 H -pyrrolizines and cyclopent[ b ]azepin-8(1 H )-ones, were dominant among overall products, followed by pyrazines at different temperatures. Aspartic acid was first applied to model reactions as the precise control of asparagine deamidation, and lysine was further introduced into model systems for improving pyrazine formation. Quantitative results of model reaction products demonstrated that pyrazines were not significantly increased in deamidated states (Asn-Asp-Pro and Asp-Pro) while proline-specific compounds had a rapid enhancement at the same time. With excellent ability to form pyrazines, lysine did help to increase the formation of pyrazines, but still far fewer than pyrrolizines and azepines. It was assumed that proline would preferentially react with α-dicarbonyl compounds in Maillard reaction cascades with lower activation energies.

Maillard flavor compounds, such as 2,3,5-trimethylpyrazine, have been frequently identified in thermally processed food products, such as popcorn and peanuts. However, the origin of the carbon atoms in 2,3,5-trimethylpyrazine has not been clearly elucidated. Herein, a model reaction showed that precursor methylglyoxal and intermediates glyoxal and formaldehyde contributed to the formation of 2,3,5-trimethylpyrazine via a conversion reaction between methylglyoxal and glyoxal. In addition, carbon module labeling technology and model response validation experiments indicated that this transformation reaction between methylglyoxal and glyoxal brought formaldehyde into the methyl group carbon atoms of the 2,3,5-trimethylpyrazine ring. The proposed novel route provides a new perspective for approaches to control the formation of flavor compounds, such as 2,3,5-trimethylpyrazine.

The Amadori compound of glucose and glycyl-l-glutamine (Gly-Gln-ARP) was prepared and characterized by UPLC-MS/MS and NMR. Gly-Gln-ARP could be thermally degraded into Gly-Gln and other secondary reaction products like glycyl-l-glutamic acid and its ARP via deamidation. The thermal processing temperature exerted a tremendous influence on the flavor formation of ARP. Furans were mainly formed at 100 °C, while an elevated temperature of 120 °C facilitated the massive accumulation of α-dicarbonyl compounds through the retro-aldolization of deoxyglucosone, and then increased the formation of pyrazines. The extra-added amino acids further promoted the formation of pyrazines at 120 °C, especially Glu, Lys, and His, further increasing the total concentration of pyrazines to 457 ± 6.26, 563 ± 65.5, and 411 ± 59.2 μg/L, respectively, exceeding the pure heated control at 140 °C (296 ± 6.67 μg/L). The total concentration of furans was enhanced to (20.7 × 10 3 ) ± 8.17 μg/L by extra-added Gln. Different increasing effects were observed on the type and flavor intensity of formed pyrazines and furans from different extra-added amino acids.

The influence of pH was studied on volatile flavor formation during thermal treatment of an Amadori rearrangement product (ARP) with or without the addition of cysteine (Cys). The formation of thiols and sulfides or 2-acetylthiazole and pyrazines induced by Cys during thermal degradation of ARP was pH-dependent. At low pH levels, the hydrolysis of Cys to hydrogen sulfide (H 2 S) was promoted, giving rise to the increase of thiols and sulfides with an obvious meaty aroma. However, alkaline conditions were beneficial for enhancing the cyclization or transformation of imine to the enol structure, which strengthened the formation of 2-acetylthiazole and pyrazines with a roasted and nutty aroma. The imine was derived from the nucleophilic addition of Cys and methylglyoxal (MGO) and subsequent decarboxylation. At pH 8, Cys-induced variation of the flavor profile was weakened during thermal degradation of ARP. Accordingly, the combinational effect of pH and added Cys could be beneficial for achieving the desirable flavors during thermal processing of ARP.

The intervention of cysteine (Cys) on the formation of 2,3-butanedione and pyrazines was evaluated during the thermal processing of the alanine-xylose Amadori compound (AX-ARP). With the involvement of Cys, the competitive formation of 2,3-butanedione and pyrazines was induced. The formation of 2,3-butanedione in the AX-ARP/Cys model was suppressed due to the inhibitory effect of the precursors of 2,3-butanedione like deoxypentosones, while the added Cys in the AX-ARP/Cys model competed with the recovered alanine (Ala) to capture glyoxal and methylglyoxal to make up for the absence of pyrazines in the AX-ARP model at an initial pH value of 7. The content of pyrazines increased from 0 up to 16.48 μg/L (120 °C, 120 min). Exogenous Cys itself showed lower reactivity with 2,3-butanedione through the Strecker degradation reaction; while the pH was increased to 8, the degradative products of Cys were facilitated to consume the residual 2,3-butanedione giving rise to the formation of 2,4,5-trimethylthiazole at 120 °C. It was the degradative products of Cys that accelerated the reaction for consumption of 2,3-butanedione rather than Cys itself. Additionally, the inhibitory effect of Cys on 2,3-butanedione formation was weakened under a basic condition, while the promotional effect on the formation of pyrazines was further boosted. With more Cys participating in the process of AX-ARP thermal degradation, the formation of 2,3-butanedione was further inhibited, while the yields of pyrazines were increased.

In this study, it was found that extra-added cysteine (Cys) became involved in volatile compound formation during the Maillard reaction of the glycine-ribose Amadori rearrangement product (GR-ARP). The priority of the Cys reaction with different α-dicarbonyls and its dependence on the Cys dosage were investigated. At the same concentrations of methylglyoxal (MGO) and glyoxal (GO), it was found that 2-acetylthiazole was the dominant product when the molar ratio of Cys to MGO was 1:1, while formation of pyrazines was improved when the Cys percentage increased. Cys preferentially reacted with MGO first rather than GO to exclusively generate 2-acetylthiazole at a high yield. The concentration of 2-acetylthiazole quickly increased up to a plateau and remained stable during further heat treatment. When MGO was totally consumed, remaining Cys began to react with GO through the predominant pathway where the keto form of carbonylcysteimine derived from Cys and GO was hydrolyzed to recover GO with cysteamine formation, whereas the hydrolysis reactivity of enolized carbonylcysteimine as the Strecker pathway for generation of pyrazines was relatively low. During the heat treatment of GR-ARP, the constantly lower ratios of α-dicarbonyls to Cys led to inhibited formation of 2-aminopropanal, which accounted for the decreased methylpyrazine yields.

Aroma extract dilution analysis was performed on volatile fractions extracted from a freshly prepared Dahongpao (DHP) tea infusion using solvent-assisted flavor evaporation, yielding 65 odor-active domains with flavor dilution factors ranging between 32 and 32,768. In addition, six aromatic substances were captured by headspace analysis. Quantitation of 54 compounds by an internal standard method and stable isotope dilution assays revealed that the concentrations of 32 odorants exceeded their respective orthonasal odor threshold values in tea infusion. The results of odor activity values (OAVs) suggested that 2-metylbutanal (malty) and γ-hexalactone (coconut-like) had the highest OAVs (248 and 154). Eight odorants including γ -hexalactone (OAV 154), methyl 2-methylbutanoate (59), phenylacetic acid (7.2), acetylpyrazine (5.7), 2-methoxyphenol (3.4), p -cresol (2.7), 2,6-diethylpyrazine (2.7), and vanillin (1.8) were newly identified as key odorants in DHP tea infusion. An aroma recombination model in a non-volatile matrix extracted from tea infusion satisfactorily mimicked the overall aroma of DHP tea infusion, thereby confirming the identification and quantitative experiments. Omission experiments verified the obvious significance of 6-methyl-5-hepten-2-one (OAV 91), 2-ethyl-3,5-dimethylpyrazine (19), 4-hydroxy-2,5-dimethylfuran-3(2 H )-one (13), and acetylpyrazine (5.7) as key odorants for the special roasty and caramel-like aroma of DHP tea.

An Amadori rearrangement product (ARP) derived from ribose (Rib) and glutathione (GSH) was prepared and identified as N -(1-deoxy-d-ribulos-1-yl)-glutathione by ultraperformance liquid chromatography-tandem mass spectrometry and NMR. Thermal treatment of the ARP aqueous solution was conducted, and a relatively high temperature was found to accelerate the degradation of the ARP. The concentration of furans formed at 120 °C was more than 6.39 times that at 100 °C, and especially, the high temperature favored the formation of furfural and 4-hydroxy-5-methyl-3(2 H )-furanone through deoxyosone dehydration. The promoting role of extra-added GSH or its constituent amino acids was investigated in the volatile formation during thermal processing of the ARP. Both, the added GSH and its constituent amino acids, could timely capture glyoxal (GO) and methylglyoxal (MGO) to facilitate Strecker degradation, which improved pyrazine formation. Compared with glycine and glutamic acid, cysteine was the most effective extra-added amino acid to react with GO and MGO to produce pyrazine and methylpyrazine. More importantly, the cysteine degraded from extra-added GSH effectively accelerated the generation of sulfur-containing volatile compounds through the reaction of cysteine degradation products with furans and shorter-chain α-dicarbonyl compounds.

The biosynthesis of 3-isobutyl-2-methoxypyrazine (IBMP) in bell pepper fruits ( Capsicum annuum L.) was investigated by in vivo feeding experiments with stable isotope-labeled precursors. Volatiles were extracted using headspace solid-phase microextraction (HS-SPME) and analyzed by comprehensive two-dimensional gas chromatography (GC×GC) coupled to a time-of-flight mass spectrometer (ToF-MS). Feeding experiments revealed incorporation of l-leucine and α-ketoisocaproic acid (α-KIC) as well as glycine and glyoxylic acid into IBMP. Furthermore, it has been shown that de novo biosynthesis of IBMP occurs in pericarp tissues of unripe bell pepper fruits, whereas pericarp tissues of ripe bell pepper fruits showed no capability of IBMP biosynthesis.

Theanine is a non-proteinogenic amino acid found in the tea plant Camellia sinensis . At an elevated temperature (>90 °C), it released two major volatile compounds 1-ethyl-1,5-dihydro-2 H -pyrrol-2-one and N -ethylsuccinimide. Other products were identified, including 10 pyrroles and 12 amides/imides. The formation of the two major compounds was proposed to be initiated by the deamination of theanine and through the intermediate α-keto acid. In the presence of glucose, the two major products and many other volatiles from theanine thermal degradation were accelerated and further Maillard reactions occurred. A total of 56 compounds were identified in the model system of theanine and glucose, including 12 amides/imides, 16 pyrazines, 16 pyrroles and other N -heterocycles, and 12 furans and other O -heterocycles. Although most of the reaction products were detected in tea leaves and in their aqueous extract with or without the addition of theanine under the same experiment conditions, imides and amides were considerably suppressed, left only minute amounts, or were even no longer detectable. Pyrazines and pyrroles were also shown at reduced concentrations as a result of the interaction with tea components but to a lesser extent. A total of 16 and 12 pyrazines were identified in the theanine/glucose reaction system and tea leaves/aqueous extract after roasting, respectively. The results indicated that pyrazines and other main volatiles in roasted tea leaves were formed from the Maillard reactions of the aqueous fraction of tea leaves. Theanine participated in the formation of pyrazines in tea leaves under roasting conditions.

The effect of ellagic acid on the formation of pyrazine, methylpyrazine, 2,3-methylpyrazine, 2,6-methylpyrazine, 2,5-methylpyrazine, and trimethylpyrazine in the xylose-glycine Maillard reaction model was researched. Ellagic acid could either inhibit or promote pyrazine formation, depending on its addition time point and the pH of the system. The addition of ellagic acid during the accumulation period of an Amadori compound inhibited pyrazine formation by capturing the Amadori compound in the xylose-glycine Maillard system and decreasing the pyrazine precursors. The inhibitory effect of ellagic acid on pyrazine formation got more obvious with an increase in the pH of the system. However, when ellagic acid was added at the beginning of the xylose and glycine Maillard system and when the oxidizing substances such as glyoxal and methylglyoxal were significantly formed in the Maillard system, its oxidation could promote the formation of pyrazines.

Alkylpyrazines are important heterocyclic compounds used as flavorants in food and beverage industries. In this study, a regioselective semibiocatalytic process was developed to synthesize 2-ethyl-3,5-dimethylpyrazine (235-EDMP) over its 3-ethyl-2,5-dimethyl pyrazine (325-EDMP) isomer and vice versa. We initially explored how sterics could direct the coupling orientations between diamines and diketones to access 235- or 325-EDMP selectively. Also, the physical parameters of the reaction conditions were changed, such as reduced temperature, the order-of-addition of the reactants, and supplementation with chiral zeolites to template the orientation of the coupling partners to direct the reaction regiochemistry. Each reaction trial resulted in 50:50 mixtures of the EDMP isomers. An alternative approach was explored to control the regioselectivity of the reactions; α-hydroxy ketones replaced the diketones as the electrophilic coupling reactant used in previous trial experiments. The hydroxy ketone reactants were made biocatalytically with pyruvate decarboxylase. The coupling reaction between 2-hydroxypentan-3-one and propane-1,2-diamine resulted in the desired 235-EDMP at >70% (∼77 mg) relative to 325-EDMP in the mixture. The 3-hydroxypentan-2-one congener was biocatalyzed and reacted with propane-1,2-diamine as a proof of principle to synthesize 325-EDMP (∼60% relative abundance, ∼73 mg) over 235-EDMP. These results suggested a mechanism that was directed by the hydroxy ketone electrophilicity and the sterics at the diamine nucleophilic centers.

The major non-volatile reaction products formed from free amino acids during the early stage of coffee roasting were investigated using biomimetic in-bean experiments with labeled and unlabeled free amino acids. Comprehensive untargeted screening by ultra-high performance liquid chromatography-electrospray-ionization-quadrupole time-of-flight-tandem mass spectrometry (UHPLC-ESI-QToF-MS) in data-independent acquisition (DIA) mode was carried out and modeling by orthogonal partial least-squares discriminant analysis (OPLS-DA) helped in revealing 11 pyrazine structures identified in coffee for the first time. 2-(2',3',4'-Trihydroxybutyl)-(5/6)-methyl-pyrazine ( 1 ) and 2,(5/6)-bis(2',3',4'-trihydroxybutyl)-pyrazine ( 2 ) were the most prominent compounds, while 2-(3',4'-dihydroxybutyl)-(5/6)-methyl-pyrazine ( 5 ) and 2-(2',3',4'-trihydroxybutyl)-(5/6)-(2'-hydroxyethyl)-pyrazine ( 10 ) were not even previously identified in other food matrices. The structures could be verified by means of additional biomimetic in-bean experiments with labeled sucrose leveraging the carbon module labeling (CAMOLA) approach. Based on these results, plausible formation pathways could be formulated fitting into the known Maillard reaction mechanisms. Sucrose was highlighted as the predominant precursor of the carbon backbone of all identified pyrazines butonly 33-55% of the nitrogen atoms originated from free amino acids.

2,5-Dimethylpyrazine (2,5-DMP) is an important pharmaceutical intermediate and an important essence. Conventional chemical synthesis methods are often accompanied by toxic substances as by-products, and the biosynthesis efficiency of 2,5-DMP is insufficient for industrial applications. In this study, the tdh and soaao genes were overexpressed to enhance enzymatic and nonenzymatic reactions in metabolic pathways, and kbl was knocked out to block competitive branching carbon flow metabolic pathways. Finally, a genetically engineered Escherichia coli strain with the highest carbon recovery rate (30.18%) and the highest yield reported to date was successfully constructed, and 9.21 g·L -1 threonine was able to produce 1682 mg·L -1 2,5-DMP after 24 h. At the same time, an expression regulation strategy and whole-cell biocatalysis helped to eliminate the damage to cells caused by 2,5-DMP, aminoacetone, and reactive oxygen species generated by aminoacetone oxidase from S. oligofermentans , and the negative effect of 2-amino-3-ketobutyrate CoA ligase on the yield of 2,5-DMP in E. coli was also demonstrated.

Using environment-friendly catalysts to convert biomass into compounds with high values is one of the central topics of green chemistry. In this work, [Ch][Pro] (cholinium as the cation and l-proline as the anion) ionic liquid was synthesized and applied as a model catalyst in the production of deoxyfructosazine (DOF) and fructosazine (FZ) from d-glucosamine (GlcNH 2 ). The 13 C NMR chemical shift titration experiments and the diffusion-ordered NMR spectroscopy (DOSY) measurements showed that, when the [Ch][Pro] interacted with GlcNH 2 , the l-proline anion ([Pro] - ) played a major catalytic role instead of cholinium cation ([Ch] + ). The effects of the reaction temperature and the amount of [Ch][Pro] on the product yields were surveyed. The experimental results showed that the highest DOF yield (33.78%) was obtained after 30 min at 100 °C when the molar ratio of [Ch][Pro]/GlcNH 2 was 1. Moreover, in situ 1 H NMR and in situ 13 C NMR experiments were applied to monitor the reaction process with [Ch][Pro] as the catalyst. The reactive intermediate, dihydrofructosazine, was clearly detected by these in situ techniques. Accordingly, a possible reaction pathway was proposed. By applying other amino acids as the anions, we also prepared five other [Ch][AA] ionic liquids, and they showed different catalytic activities and selectivity in the GlcNH 2 self-condensation reaction.