Conversion of hydroxycinnamic acids by Furfurilactobacillus milii in sorghum fermentations: Impact on profile of phenolic compounds in sorghum and on ecological fitness of Ff. milii.

The conversion of phenolic compounds by lactobacilli in food fermentations contributes to food quality. The metabolism of phenolics by lactobacilli has been elucidated in the past years but information on the contribution of specific enzymes in food fermentations remains scarce. This study aimed to address this gap by disruption of genes coding for the hydroxycimmanic acid reductase Par1, the hydroxycinnamic acid decarboxylase Pad, the hydrocinnamic esterase EstR, and strains with disruption of all three genes in Furfurilactobacillus milii FUA3583. The conversion of phenolics by Ff. milii and its isogenic mutants in sorghum fermentations was studied by LC-UV and LC-UV-MS/MS analyses. Ff. milii FUA3583 converted hydroxycinnamic acids predominantly with Par1. Vinylphenols were detected only in mutants lacking par1. A phenotype for the estR defective mutant was not identified. The formation of pyrano-3-deoxyanthocyanidins was observed only after fermentation with strains expressing Pad. Specifically, formation of these compounds was low with Ff. milii FUA3583, substantially increased in the Par1 mutant and abolished in all mutants with disrupted pad. Competition experiments with Ff. milii FUA3583 and its isogenic mutants demonstrated that expression of one of the two metabolic pathways for hydroxycinnamic acids increases the ecological fitness of the strain. Disruption of EstR in a Δpar1Δpar2Δpad background improved ecological fitness, indirectly demonstrating a phenotype of the esterase in Ff. milii. The documentation of the functionality of genes coding for conversion of hydroxycinnamic acids may support the selection of starter cultures for improved quality of fermented cereal products.

Profiling of phenolic compounds in desiccation-tolerant and non-desiccation-tolerant Linderniaceae.

INTRODUCTION: Craterostigma plantagineum and Lindernia brevidens are resurrection plants, so these plants can tolerate desiccation of their vegetative tissues. Different components and mechanisms contribute to desiccation tolerance and secondary plant metabolites, like phenolic compounds, may play a role during these processes. OBJECTIVES: Secondary plant metabolites of the two resurrection plants, C. plantagineum and L. brevidens as well as the closely related desiccation sensitive species, L. subracemosa, were investigated regarding the polyphenol profile. MATERIAL AND METHODS: Secondary plant compounds were extracted with acidified methanol and analysed with ultra-high-performance liquid chromatography electrospray ionisation mass spectrometry (UHPLC-ESI-MS). Phenolic compounds were identified by comparing of ultraviolet (UV) and MSn -spectra with published data. All compounds were quantified as verbascoside equivalents by external calibration at the compound specific wavelength. RESULTS: In total, eight compounds that belong to the subclass of phenylethanoid glycosides and one flavone, luteolin hexoside pentoside, were identified. Two of these compounds exhibited a fragmentation pattern, which is closely related to phenylethanoid glycosides. The predominantly synthesised phenylethanoid in all of the three plant species and in every stage of hydration was verbascoside. The total content of phenolic compounds during the three stages of hydration, untreated, desiccated, and rehydrated revealed differences especially between C. plantagineum and L. brevidens as the latter one lost almost all phenolic compounds during rehydration. CONCLUSION: The amount of verbascoside correlates with the degree of desiccation tolerance and verbascoside might play a role in the protective system in acting as an antioxidant.

Methylation of Cyanidin-3-O-Glucoside with Dimethyl Carbonate.

The approach presented in this study is the first for the hemisynthesis of methylated anthocyanins. It was possible to obtain cyanidin-3-O-glucoside derivatives with different degrees of methylation. Cautious identification of 4'-, 5-, and 7-OH monomethylated derivatives was also accomplished. The methylation agent used was the "green chemical" dimethyl carbonate (DMC), which is characterized by low human and ecological toxicity. The influence of the temperature, reaction time, and amount of the required diazabicyclo[5.4.0]undec-7-en (DBU) catalyst on the formation of the products was examined. Compared to conventional synthesis methods for methylated flavonoids using DMC and DBU, the conditions identified in this study result in a reduction of reaction time, and an important side reaction, so-called carboxymethylation, was minimized by using higher amounts of catalyst.

Chemical Hemisynthesis of Sulfated Cyanidin-3-O-Glucoside and Cyanidin Metabolites.

The metabolism of anthocyanins in humans is still not fully understood, which is partly due to the lack of reference compounds. It is known that sulfation is one way of the complex phase II biotransformation mechanism. Therefore, cyanidin-3-O-glucoside and the cyanidin aglycone were chemically converted to their sulfates by reaction with sulfur trioxide-N-triethylamine complex in dimethylformamide. The reaction products were characterized by UHPLC coupled to linear ion trap and IMS-QTOF mass spectrometry. Based on MS data, retention times, and UV-Vis spectra, the compounds could tentatively be assigned to A-, C-, or B-ring sulfates. Analysis of urine samples from two volunteers after ingestion of commercial blackberry nectar demonstrated the presence of two sulfated derivatives of the cyanidin aglycone and one sulfated derivative of the cyanidin-3-O-glucoside. It was found that both the A ring and the B ring are sulfated by human enzymes. This study marks an important step toward a better understanding of anthocyanin metabolism.

Development and Validation of Methods for the Determination of Anthocyanins in Physiological Fluids via UHPLC-MSn.

Recent in vitro and in vivo studies on anthocyanins confirmed numerous health-promoting effects in humans. Daily anthocyanin intake can be estimated via food databases, but the amount absorbed by the organism still remains uncertain because anthocyanin bioavailability is yet to be elucidated in its entirety. For this purpose, suitable and validated methods of sample preparation and analysis are required. Therefore, a sample preparation method for anthocyanin metabolite analysis in plasma was successfully established and validated. The validation yielded acceptable results for the anthocyanins in terms of recovery (54-108%) and precision (coefficient of variation (CV) < 15%). The UHPLC-MS method used in the consecutive reaction monitoring (CRM) mode was sufficiently sensitive, resulting in limits of detection <2.3 ng/mL and limits of quantification < 8.1 ng/mL with associated repeatability of the MS system with CVs of <5.1%. In addition, a method for the sum parameter determination of anthocyanidins in urine comprising solely the evaporation of acidified samples was developed, validated, and successfully applied to real samples. The results showed that this method is applicable for the methylated anthocyanidins, but not for the hydroxylated anthocyanidins, due to the chosen CRM modes required for optimum selectivity.

Hemisynthesis of Anthocyanin Phase II Metabolites by Porcine Liver Enzymes.

The aim of this work was to obtain phase II metabolites of cyanidin-3- O-glucoside and its aglycone using porcine liver enzymes. For this purpose, anthocyanins extracted from blackberry concentrate and containing mostly cyanidin-3- O-glucoside were incubated with the S9, microsomal, and cytosolic fractions of porcine liver. The reactions were targeted to the direction of the respective phase II transformation by the addition of activated cofactors. LC-MS n and LC-IMS-QTOF-MS analyses showed that one methylated, three glucuronidated and three sulfated metabolites of cyanidin-3- O-glucoside were generated. The aglycone, cyanidin, was sulfated and glucuronidated by the liver enzymes. In addition, both were glucuronidated and methylated simultaneously. The detected compounds and the generated data like exact masses, mass spectra, and CCS values may serve as a basis in the search for metabolites formed in vivo. As their effects are largely unexplored, the described synthesis may contribute to a better understanding of the metabolism of anthocyanins.

An Innovative Approach to the Preparation of Plasma Samples for UHPLC-MS Analysis.

A new sample processing method for analyzing flavonol metabolites in plasma using enzymatic proteolysis was developed and validated. Four endopeptidases were examined regarding their influence on the analyte recovery of quercetin-3- O-glucuronide (Q3GlcA). Methanol was added to inactivate and precipitate the enzymes, and samples were concentrated via evaporation prior to UHPLC-MS analysis. Quercetin-3- O-rutinoside (Q3Rut) was used as an internal standard. The selectivity and accuracy of the established UHPLC-ESI-MS n method showed a coefficient of variation (CV) of the repeatability of the measuring instrument of 1.7% for Q3GlcA. The average recovery of Q3GlcA was approximately 67% with an interday method precision of 24% and r = 46.9 as its repeatability. Therefore, enzymatic proteolysis has proven to be a suitable alternative to the methods previously described in the literature, such as solid-phase extraction (SPE). Still, the method has only been validated for Q3GlcA, but its applicability to other substance classes seems possible.

Polyphenols and Metabolites Enhance Survival in Rodents and Nematodes-Impact of Mitochondria.

(1) Background: Polyphenols (PP) play an important role in the prevention of non-communicable diseases and may contribute to healthy aging. To investigate the molecular and cellular aspects of PP metabolites on longevity with a focus on mitochondrial function, we applied a pre-fermented mixture of polyphenols (Rechtsregulat®, RR) to rodents and nematodes. (2) Methods: The lifespans of Navar Medical Research Institute (NMRI) mice and C. elegans were recorded. The heat-stress resistance (37 °C) of C. elegans N2 was measured using nucleic staining. Respiration and membrane potential (ΔΨm) were measured in isolated mitochondria. The energetic metabolites adenosine triphosphate (ATP), lactate, and pyruvate were determined in lysates. Expression levels of longevity related genes were determined using quantitative real time polymerase chain reaction (qRT-PCR). Phenolic compounds were identified using ultra high performance liquid chromatography-diode array detection-Iontrap-multiple stage mass spectrometry (UHPLC-DAD-Iontrap-MSn). (3) Results: Several phenolic metabolites including protocatechuic acid (PCA) were identified in RR. Feeding of mice with RR resulted in a significantly increased lifespan. Heat-stress resistance (RR *** p = 0.0006; PCA **** p < 0.0001), median lifespan (NMRI: RR ** p = 0.0035; C. elegans RR * p = 0.0279; PCA **** p < 0.0001), and activity of mitochondrial respiratory chain complexes (RR *-** p = 0.0237 - 0.0052; PCA * p = 0.019 - 0.0208) of C. elegans were significantly increased after incubation with RR (10%) or PCA (780 µM). PCA significantly improved nematodes ΔΨm (* p = 0.02058) and ATP levels (* p = 0.029). RR significantly up-regulated lactate levels, indicating enhanced glycolysis. The expression levels of longevity related genes daf-16, sir-2.1, and skn-1 were significantly upregulated after PCA, and partially after RR administration. (4) Conclusion: Phenolic metabolites such as PCA have the potential to enhance health and lifespan and mitochondrial function, and thus may contribute to healthy aging.

Polyphenol Phase-II Metabolites are Detectable in Human Plasma after Ingestion of 13 C Labeled Spinach-a Pilot Intervention Trial in Young Healthy Adults.

SCOPE: After intrinsic labeling of spinach (Spinacia oleracea L., Chenopodiaceae) with 13 CO2 , we investigated if labeled polyphenol metabolites were detectable in human plasma. METHODS AND RESULT: In a pilot intervention trial, five healthy men consumed 5 g freeze-dried 13 C labeled spinach, including a total amount of 160 µmol methoxyflavonols, including 70 µmol 5,3',4'-trihydroxy-3-methoxy-6,7-methylendioxyflavone-4'-glucuronide. Plasma samples of all subjects were analyzed with regard to their 13 C/12 C ratio. Additionally, 13 C labeled metabolites of patuletin, spinacetin, and 5,3',4'-trihydroxy-3-methoxy-6,7-methylendioxyflavone (TMM) were analyzed in plasma samples in a subgroup of three subjects. TMM-glucuronide, TMM-sulfate, and spinacetin-glucuronide-sulfate, the latter as 12 C113 C16 and 13 C17 isotopologs, were tentatively identified. Plasma concentration of TMM-glucuronide and TMM-sulfate reached cmax from 19.1-54.3  and 22.5-125.5 nmol L-1 , respectively, 7-9 h post-ingestion. CONCLUSION: It seems likely that 13 C labeled TMM-glucuronide and TMM-sulfate are phase-II metabolites which were converted after colonic transformation. Variations in plasma kinetics were observed for these two metabolites and may be attributed to the individual composition of the microbiota. We conclude that 13 C labeled polyphenol metabolites are detectable and quantifiable in human plasma.