Exploring the characteristics, digestion behaviors, and nutraceutical potential of the underutilized Chimonanthus praecox (L.) link kernel oil: A combined in vitro and in vivo study.

Chimonanthus praecox (L.) Link kernel oil (LMO) has the potential to expand the variety of nutraceutical plant oils available and provide support for the application of functional food. This study aimed to assess the edible potential of LMO by examining its physicochemical characteristics, digestion behaviors, and nutraceutical properties. The results revealed that LMO has a high oil content of 40.84% and is particularly rich in linoleic acid (53.37-56.30%), oleic acid (22.04-25.08%) and triacylglycerol (TAG) of linoleic acid -palmitoleic acid- oleic acid (10.57-12.70%). The quality characteristics and phytochemical composition of LMO were found to be influenced by variety and extraction methods used. In simulated in vitro digestion tests, LMO showed a better lipid release rate and degree. Animal studies further demonstrated that LMO led to better TAG and cholesterol excretion compared to soybean oil and camellia oleifera oil. Overall, this study highlights the potential of LMO as a high-quality edible oil.

Adenylation domains of nonribosomal peptide synthetase: A potential biocatalyst for synthesis of dipeptides and their derivatives.

Dipeptides and their derivatives are important functional compounds that can be applied to fields such as medicine and food. As biological macromolecules, the adenylation domains of nonribosomal peptide synthetase (NRPS) can recognize and activate various building blocks, such as amino acids, for the biosynthesis of nonribosomal peptides. In this way, the amide bond formation can be achieved through a nucleophilic reaction where the adenylation domain serves as a biocatalyst and is further used to conduct dipeptide synthesis. In this study, the adenylation domains (BAA2, BBA2, and BCA4) of bacitracin synthetase were predicted and expressed. The substrate evaluation results showed that adenylation domains displayed broad substrate selectivity for amino acids in vitro. Furthermore, the use of dipeptide synthesis in adenylation domains suggested that the polarity of amino acids could have an influence on nucleophilic reactions. Finally, L-alanyl-L-glutamine and aspartame were successfully synthesized through catalysis by the adenylation domains BAA2 and BCA4, respectively. This study expands on approaches to the synthesis of functional dipeptides and their derivatives based on the chemoenzymatic process.

Comprehensive lipidomic analysis of milk polar lipids using ultraperformance supercritical fluid chromatography-mass spectrometry.

Polar lipids in milk are receiving increasing interest due to their bioactivities. However, milk polar lipids present a wide range of physical-chemical properties at different concentrations, making their analysis challenging. In this study, we presented a comprehensive lipidomic method using ultraperformance supercritical fluid chromatography (UPSFC)-quadrupole-time of flight-mass spectrometry (Q-TOF-MS), which enabled the separation of 18 lipid classes (including nonpolar lipids, cholesterol, ceramide, glycerophospholipids, sphingomyelin, and gangliosides) within 10 min. The method was used to analyze the polar lipids in seven samples, including human milk, other mammalian milk and milk fat globule membrane ingredients, identifying 14 lipid classes containing 219 lipid molecular species. A mass spectrometry data processing strategy applicable for high-throughput studies was also developed and validated.

Triacylglycerol regioisomers containing palmitic acid analyzed by ultra-performance supercritical fluid chromatography and quadrupole time-of-flight mass spectrometry: Comparison of standard curve calibration and calculation equation.

Triacylglycerol (TAG) regioisomers containing palmitic acid (16:0) was identified using ultra-performance supercritical fluid chromatography and quadrupole time-of-flight mass spectrometry (UPSFC-Q-TOF-MS) and quantified using calibration curve method and calculation equation method. There were negative linear correlation between [RA-A]+/[RA-A]++[RA-B]+ and content of sn-A-B-A (%) for AAB/ABA type TAGs, [Rsn-1 FA-sn-3 FA]+/[RB-C]++[RA-C]++[RA-B]+ and content of fatty acid (FA) at sn-2 position (%) for BAC/ABC/ACB type TAGs. The difference between calculation equation and standard curve method was acceptable. The TAG regioisomers in human milk, mammalian milk, lard and fish oil were identified and quantified using the developed methods. This study provided a reliable and facile method for analysis of the TAG regioisomers, which was capable of the selection of oil materials for infant formula production.

Transcriptome Analysis of Bacillus licheniformis for Improving Bacitracin Production.

This study aims to find the targets that may influence the production of bacitracin based on RNA sequencing in Bacillus licheniformis. Transcriptional profiling revealed that (i) the expression of the bacT gene, encoding a type II thioesterase (TEIIbac), was positively correlated with bacitracin production and (ii) the oxygen uptake exhibited significant influence on precursor synthesis. The verified experiments showed that the overexpression of TEIIbac with an endogenous promoter increased the bacitracin A titer by 37.50%. Furthermore, the increase of oxygen availability through Vitreoscilla hemoglobin (VHb) expression increased the bacitracin A titer by 126.67% under oxygen-restricted conditions. From the transcriptome perspective, the results of this paper demonstrate that TEIIbac and oxygen supply are crucial to bacitracin production. This study also provides insights into the construction of chassis cells for the industrial production of secondary metabolites with a preference for aerobic conditions.

Combinatorial pathway engineering of Bacillus subtilis for production of structurally defined and homogeneous chitooligosaccharides.

Chitooligosaccharides (COSs) have a widespread range of biological functions and an incredible potential for various pharmaceutical and agricultural applications. Although several physical, chemical, and biological techniques have been reported for COSs production, it is still a challenge to obtain structurally defined COSs with defined polymerization (DP) and acetylation patterns, which hampers the specific characterization and application of COSs. Herein, we achieved the de novo production of structurally defined COSs using combinatorial pathway engineering in Bacillus subtilis. Specifically, the COSs synthase NodC from Azorhizobium caulinodans was overexpressed in B. subtilis, leading to 30 ± 0.86 mg/L of chitin oligosaccharides (CTOSs), the homo-oligomers of N-acetylglucosamine (GlcNAc) with a well-defined DP lower than 6. Then introduction of a GlcNAc synthesis module to promote the supply of the sugar acceptor GlcNAc, reduced CTOSs production, which suggested that the activity of COSs synthase NodC and the supply of sugar donor UDP-GlcNAc may be the limiting steps for CTOSs synthesis. Therefore, 6 exogenous COSs synthase candidates were examined, and the nodCM from Mesorhizobium loti yielded the highest CTOSs titer of 560 ± 16 mg/L. Finally, both the de novo pathway and the salvage pathway of UDP-GlcNAc were engineered to further promote the biosynthesis of CTOSs. The titer of CTOSs in 3-L fed-batch bioreactor reached 4.82 ± 0.11 g/L (85.6% CTOS5, 7.5% CTOS4, 5.3% CTOS3 and 1.6% CTOS2), which was the highest ever reported. This is the first report proving the feasibility of the de novo production of structurally defined CTOSs by synthetic biology, and provides a good starting point for further engineering to achieve the commercial production.

Combining Precursor-Directed Engineering with Modular Designing: An Effective Strategy for De Novo Biosynthesis of l-DOPA in Bacillus licheniformis.

3-Hydroxy-l-tyrosine (l-DOPA) is a promising drug for treating Parkinson's disease. Tyrosine hydroxylase catalyzes the microbial synthesis of l-DOPA, which is hindered by the efficiency of catalysis, the supply of cofactor tetrahydrobiopterin, and the regulation of the pathway. In this study, the modular engineering strategy in Bacillus licheniformis was identified to effectively enhance l-DOPA production. First, the catalytic efficiency of biocatalyst tyrosine hydroxylase from Streptosporangium roseum DSM 43021 (SrTH) was improved by 20.3% by strengthening its affinity toward tetrahydrobiopterin. Second, the tetrahydrobiopterin supply pool was increased by bottleneck gene expression, oxygen transport facilitation, budC (encoding meso-2,3-butanediol dehydrogenase) deletion, and tetrahydrobiopterin regeneration using a native YfkO nitroreductase. The strain 45ABvC successfully produced tetrahydrobiopterin, which was detected as pterin (112.48 mg/L), the oxidation product of tetrahydrobiopterin. Finally, the yield of precursor l-tyrosine reached 148 mg/gDCW, with an increase of 71%, with the deletion of a novel spliced transcript 41sRNA associated with the regulation of the shikimate pathway. The engineered strain 45ABvCS::PD produced 167.14 mg/L (2.41 times of wild-type strain) and 1290 mg/L l-DOPA in a shake flask and a 15 L bioreactor, respectively, using a fermentation strategy on a mixture of carbon sources. This study holds great potential for constructing a microbial source of l-DOPA and its high-value downstream pharmaceuticals.

Identification and Quantification of Triacylglycerols Using Ultraperformance Supercritical Fluid Chromatography and Quadrupole Time-of-Flight Mass Spectrometry: Comparison of Human Milk, Infant Formula, Other Mammalian Milk, and Plant Oil.

Triacylglycerol (TAG) components in human milk during different lactation periods, infant formulas with different fat sources, other mammalian milk (cow, goat, donkey, and yak milk), and plant oil (sunflower, rapeseed, corn, soybean, palm, palm kernel, and coconut oil) were analyzed and compared using ultraperformance supercritical fluid chromatography and quadrupole time-of-flight mass spectrometry (UPSFC-Q-TOF-MS). We identified 191 TAGs (86, 102, 101, and 54 TAGs in human milk, infant formula, mammalian milk, and plant oil, respectively). TAGs esterified with palmitic acid (16:0) were major TAG structures in human milk (59.08% of total TAGs) and contained 30 TAG types. The sn-O/P/O regioisomer constituted more than 80% of the O/P/O content of human milk, whereas the sn-O/O/P levels were higher in other samples. The carbon number (CN) 52 content was higher than the CN 54 content in human milk, with the opposite observed in infant formula. TAGs with CN < 40 content were abundant in cow, goat, and yak milk; donkey milk was rich in CN 52 content. TAGs composed of medium-chain fatty acids (MCFAs) and long-chain fatty acids (LCFAs) were rich in human milk, while TAGs with three MCFAs were rich in infant formula. The TAG characteristics of infant formula were directly related to its fat resource. TAGs with fewer double bonds were abundant in the plant oil formula; however, highly unsaturated TAGs were prominent in the cow and goat milk formulas, similar to plant oil and mammalian milk. Significant differences in the TAG distribution were observed among the different species.

Exploring the relationship between potato components and Maillard reaction derivative harmful products using multivariate statistical analysis.

The correlation between potato components and Maillard reaction derivative harmful products (MRDHPs) formation during heat-processing was assessed in nine commercial potato varieties in China. Principal component analysis (PCA) combined with canonical correlation analysis (CCA) approach was performed to explore their relationships. The variables contributing most to the PCA results were extracted for CCA, and the results indicated that several amino acids, including lysine, tryptophan, alanine, phenylalanine, aspartate, and glutamate, have significant impacts on acrylamide and ß-carboline heterocyclic amine formation. Moreover, 5-O-caffeoylquinic acid, 3-O-caffeoylquinic acid, α-solanine, and α-chaconine were also important factors associated with acrylamide and ß-carboline heterocyclic amine formation. Optimally using raw potato varieties based on the impacts of these factors can help control MRDHP formation during thermal processing. For the first time, such approach was applied, which may be a useful tool for discovering the correlation of food components and MRDHPs.

Characterization and determination of free phytosterols and phytosterol conjugates: The potential phytochemicals to classify different rice bran oil and rice bran.

Phytosterols are important beneficial compounds found in rice bran (RB) and rice bran oil (RBO). Although relationships have been confirmed between the forms of phytosterols and their bioactivities, the analysis of different forms of phytosterols in RB and RBO has been lacking. In this study, high temperature gas chromatography-mass spectrometry (HTGC-MS) was combined with the single standard to determine multi-components (SSDMC) method to determine free sterols (FSs) and steryl glycosides (SGs) in RB and RBO. High-performance liquid chromatography (HPLC) was used to determine steryl ferulates (SFs). There was clear variation in the composition of FS, SF and SG, indicating that different forms of phytosterols can discriminate between different RB and RBO. The developed method may be also useful for the detection of other compounds of interest in oils, oil seeds or cereals.

LC-MS/MS for simultaneous detection and quantification of Amadori compounds in tomato products and dry foods and factors affecting the formation and antioxidant activities.

In order to establish an efficient detection method to evaluate the formation of Amadori compounds (ACs) in food products and study the potential health effects, an ultra-high performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method using caffeine as internal standard was developed to determine eight ACs. The detection limits ranged from 0.0179 to 0.0887 mg/L for the ACs. The accuracy of the method was tested through measuring recovery of the spiked samples that varied from 81.90 ± 2.98% to 108.74 ± 2.34%. This method was further applied to detect ACs in 10 food products. Results showed that dry fruits and vegetables were rich in ACs, the total content of ACs varied from 1.36 ± 0.26 to 3415.91 ± 147.96 mg/100 g. The total amount of ACs in tomato juice heated under vacuum condition showed significant increment (P < 0.05) in 25 min at 80 °C comparing with that under atmospheric pressure due to the rapid loss of water. Besides, the amino acid content shows positive correlation with the corresponding AC formation in Maillard reaction during food drying. After heated at fixed water activity (Aw) for 4 hr by sous-vide process, the ACs content in tomato powder increased significantly and the antioxidant activity improved as well. PRACTICAL APPLICATION: Results of this study provided a valuable tool to evaluate the formation of ACs in complex dry food products, facilitated the quality control of food products. The knowledge obtained will offer useful information to food processors. The synthesized ACs would facilitate further study into the antioxidant activities and potential health effects of specified AC.

Caffeic acid assists microwave heating to inhibit the formation of mutagenic and carcinogenic PhIP.

The inhibitory effect of caffeic acid on the formation of 2-amino-1-methyl-6-phenylimidazo[4,5-b] pyridine (PhIP) was investigated in chemical model systems under microwave heating (MW). A mechanistic study was subsequently carried out to identify the inhibitory mechanism. The results showed that both for conductive heating (CV) and MW, the inhibition of PhIP increased with the concentration of caffeic acid but decreased with the prolongation of heating time. The inhibition on PhIP under MW was always higher than under CV, which were dominated by the difference in dielectric loss (ε″). UPLC-MS analysis showed that caffeic acid releases a CO2 molecule to produce 4-vinylcatechol which can form adducts with phenylacetaldehyde, thus reducing its availability for PhIP formation. The structure of adduct was characterized as 3-(3,4-dihydroxyphenyl)-2-phenylbutanal with a molecular weight of 256. The findings indicate that trapping of phenylacetaldehyde by 4-vinylcatechol is a key mechanism of caffeic acid in inhibiting PhIP formation.

Identification of α-Tocopherol and Its Oxidation Products by Ultra-Performance Liquid Chromatography Coupled with Quadrupole Time-of-Flight Mass Spectrometry.

This study aimed to determine α-tocopherol (α-T) and its thermal oxidation products simultaneously. A novel method based on an ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) was developed. This approach was achieved by means of a BEH C18 analytical column under gradient elution conditions with eluents of acetonitrile/isopropanol (1:9, v/v) and acetonitrile/water (4:6, v/v). Compounds were elucidated through exact molecular mass and fragmentation ions obtained from the Q-TOF-MS detector. Two oxidation products, α-tocopheryl quinone and 5-formyl-γ-tocopherol, were identified, and one new compound was determined. This approach offered a simple, precise, and reliable method to determine oxidation products of α-T, which may give a way to understand the mechanism of the thermal oxidative process of α-T.

Identification and quantification of triacylglycerols in human milk fat using ultra-performance convergence chromatography and quadrupole time-of-flight mass spectrometery with supercritical carbon dioxide as a mobile phase.

An ultra-performance convergence chromatography (UPC2) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF-MS), was developed for the determination of complicated triacylglycerols (TAGs) in human milk fat. The use of supercritical carbon dioxide as the mobile phase improved the chromatographic separation of the TAGs significantly. By optimizing the scan modes of Q-TOF-MS and selecting parent ions for MS/MS ionization, the fragment ions of each TAG including TAG isomers with overlapping retention time were adequately resolved for identification and quantification. A total of 95 different TAGs were identified in the human milk samples from Chinese mother volunteers, with O-P-L representing the main TAG, followed by O-P-O and O-L-L. In addition, the compositions and contents of TAGs in different fats and oils were successfully measured. The developed method can serve as an advanced and reliable analytical tool for the determination of complicated TAGs in various biological samples.

Acetonitrile extraction coupled with UHPLC-MS/MS for the accurate quantification of 17 heterocyclic aromatic amines in meat products.

This study proposed a simple and accurate acetonitrile extraction pretreatment method coupled with ultrahigh-performance liquid chromatography with tandem mass spectrometry for the simultaneous determination of 17 heterocyclic aromatic amines (HAAs) in meat products. With this new method, all 17 HAAs, including 11 polar and 6 nonpolarHAAs, were simultaneously extracted by acetonitrile and purified by one-step Oasis MCX cartridge purification. Compared with two different improved reference methods, the acetonitrile method could obtain higher recoveries (in the range of 42.5% to 99.0%) and better repeatability (lower than 12.2%). The limits of quantification were calculated between 0.028ngg-1and0.648ngg-1 with high correlation coefficients (r>0.9976) in wide linear ranges. The proposed acetonitrile method was successfully applied to the analysis of the HAAs levels in 10 commercial meat products with satisfactory recoveries.

Nε-(carboxymethyl)lysine and Nε-(carboxyethyl)lysine in tea and the factors affecting their formation.

The levels of Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL) in 99 tea samples from 14 geographic regions, including 44 green, 7 oolong, 41 black, and 7 dark teas were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The CML and CEL contents varied from 11.0 to 1701µg/g tea and 4.6 to 133µg/g tea, respectively. Dark tea presented the highest levels of CML and CEL, whereas green and oolong teas presented the lowest levels. Five kinds of catechins in the tea were also analyzed, and spearman's correlation coefficients showed that all the catechins negatively correlated with CML and CEL. The results suggested that withering, fermentation and pile fermentation may facilitate the formation of CML and CEL. Catechins might inhibit the formation of CML and CEL, but their inhibitory effects may be affected by tea processing. The results of this study are useful for the production of healthier tea.

Investigating the inhibitory activity and mechanism differences between norartocarpetin and luteolin for tyrosinase: A combinatory kinetic study and computational simulation analysis.

Flavonoids are an important type of natural tyrosinase inhibitor, but their inhibitory activity and mechanism against tyrosinase are very different because of their different structures. In this study, the inhibitory activity and mechanism differences between norartocarpetin and luteolin for tyrosinase were investigated by a combination of kinetic studies and computational simulations. The kinetic analysis showed that norartocarpetin reversibly inhibited tyrosinase in a competitive manner, whereas luteolin caused reversible noncompetitive inhibition. Both norartocarpetin and luteolin showed a single type of quenching and a static-type quenching mechanism. A computational simulation indicated that the hydroxyl groups of the B ring of norartocarpetin interacted with tyrosinase residues Asn81 and His85 in the active pocket, while the hydroxyl groups of the B ring of luteolin bound residues Asn81 and Cys83. HPLC and UPLC-MS/MS further confirmed that luteolin acted as a substrate or a suicide inhibitor, yet norartocarpetin acted as an inhibitor.

Inhibitory profiles of chilli pepper and capsaicin on heterocyclic amine formation in roast beef patties.

The inhibitory profiles of chilli pepper and capsaicin, as well as their relationship to the formation of heterocyclic amines (HAs) in roast beef patties were investigated using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) combined with principal component analysis (PCA). HAs including 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), 2-amino-1,6-dimethylimidazo[4,5-b]pyridine (DMIP), 2-amino-1,5,6-trimethylimidazo[4,5-b]pyridine (1,5,6-TMIP), 2-amino-3-methyl-3H-imidazo[4,5-f]quinoxaline (IQx), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), 2-amino-3,4,8-trimethyl-3H-imidazo[4,5-f]quinoxaline (4,8-DiMeIQx), 1-methyl-9H-pyrido[3,4-b]indole (harman) and 9H-pyrido[3,4-b]indole (norharman) were detected and quantified in beef patties. Different levels of chilli pepper and capsaicin had different inhibitory profiles on HA formation, but had no significant (P>0.05) effect on the texture of the patties. Furthermore, all levels of chilli pepper and capsaicin reduced total HA and PhIP concentrations dose-dependently, with the highest inhibitions of 80% and 98% at 2mg of capsaicin. Moreover, capsaicin inhibited all HAs more than chilli pepper, implying that ingredients other than capsaicin in chilli pepper may promote the formation of HAs. These results could be useful for the reduction of HA, during food processing procedures, by spices.

Identification of phospholipids classes and molecular species in different types of egg yolk by using UPLC-Q-TOF-MS.

Egg phospholipids (PLs) are currently the products of greatest commercial interest with major area of importance in various fields. Therefore, in this study, duck, hen and quail egg yolk PLs were isolated by solvent extraction with chilled acetone precipitation, and subsequently separated and identified by using ultra-performance liquid chromatography with quadrupole-time-of-flight mass spectrometry (UPLC-Q-TOF-MS). Egg PLs were separated on hydrophilic interaction liquid chromatography (HILIC) with ethylene bridged hybrid (BEH) column by gradient elution using acetonitrile/ammonium formate as a mobile phase, and detected by mass spectrometry (MS) under electrospray ionization in positive and negative ion mode. Structural characterizations of 57 molecular species of egg yolk PLs were identified based on MS/MS fragment ion information and elemental composition in MassLynx 4.1 software. The obtained results showed that phosphatidylcholine (16:0-18:1), phosphatidylethanolamine (18:0-20:4), phosphatidylinositol (18:0-18:2), phosphatidylserine (18:0-18:2), sphingomyelin (d18:1/16:0) and lysophosphatidylcholine (16:0) were the predominant species among the different classes of egg yolk phospholipids.

A novel isoflavone profiling method based on UPLC-PDA-ESI-MS.

A novel non-targeted isoflavone profiling method was developed using the diagnostic fragment-ion-based extension strategy, based on ultra-high performance liquid chromatography coupled with photo-diode array detector and electrospray ionization-mass spectrometry (UPLC-PDA-ESI-MS). 16 types of isoflavones were obtained in positive mode, but only 12 were obtained in negative mode due to the absence of precursor ions. Malonyldaidzin and malonylgenistin glycosylated at the 4'-O position or malonylated at the 4″-O position of glucose were indicated by their retention behavior and fragmentation pattern. Three possible quantification methods in one run based on UPLC-PDA and UPLC-ESI-MS were validated and compared, suggesting that methods based on UPLC-ESI-MS possess remarkable selectivity and sensitivity. Impermissible quantitative deviations induced by the linearity calibration with 400-fold dynamic range was observed for the first time and was recalibrated with a 20-fold dynamic range. These results suggest that isoflavones and their stereoisomers can be simultaneously determined by positive-ion UPLC-ESI-MS in soymilk.