Effects of pickle brine and glycine addition on biogenic amine production in pickle fermentation.

This study investigated the effects of different pickle brines and glycine additions on biogenic amine formation in pickle fermentation. The results showed that the brines with higher biogenic amine content led to the production of more biogenic amines in the simulated pickle fermentation system. This was related to the abundance of biogenic amine-producing microorganisms in the microbial communities of the brines. Metagenome analysis of the brines and metatranscriptome analysis of the fermentation systems showed that putrescine was primarily from Lactobacillus, Oenococcus, and Pichia, while histamine and tyramine were primarily from Lactobacillus and Tetragenococcus. Addition of glycine significantly reduced the accumulation of biogenic amines in the simulated pickle fermentation system by as much as 70 %. The addition of glycine had no inhibitory effect on the amine-producing microorganisms, but it down-regulated the transcription levels of the genes for enzymes related to putrescine synthesis in Pichia, Lactobacillus, and Oenococcus, as well as the histidine decarboxylase genes in Lactobacillus and Tetragenococcus. Catalytic reaction assay using crude solutions of amino acid decarboxylase extracted from Lactobacillus brevis showed that the addition of glycine inhibited 45 %-55 % of ornithine decarboxylase and tyrosine decarboxylase activities. This study may provide a reference for the study and control of the mechanism of biogenic amine formation in pickle fermentation.

Identification and validation of N-acetylputrescine in combination with non-canonical clinical features as a Parkinson's disease biomarker panel.

Parkinson's disease is a progressive neurodegenerative disorder in which loss of dopaminergic neurons in the substantia nigra results in a clinically heterogeneous group with variable motor and non-motor symptoms with a degree of misdiagnosis. Only 3-25% of sporadic Parkinson's patients present with genetic abnormalities that could represent a risk factor, thus environmental, metabolic, and other unknown causes contribute to the pathogenesis of Parkinson's disease, which highlights the critical need for biomarkers. In the present study, we prospectively collected and analyzed plasma samples from 194 Parkinson's disease patients and 197 age-matched non-diseased controls. N-acetyl putrescine (NAP) in combination with sense of smell (B-SIT), depression/anxiety (HADS), and acting out dreams (RBD1Q) clinical measurements demonstrated combined diagnostic utility. NAP was increased by 28% in Parkinsons disease patients and exhibited an AUC of 0.72 as well as an OR of 4.79. The clinical and NAP panel demonstrated an area under the curve, AUC = 0.9 and an OR of 20.4. The assessed diagnostic panel demonstrates combinatorial utility in diagnosing Parkinson's disease, allowing for an integrated interpretation of disease pathophysiology and highlighting the use of multi-tiered panels in neurological disease diagnosis.

Characterizing arginine, ornithine, and putrescine pathways in enteric pathobionts.

Arginine-ornithine metabolism plays a crucial role in bacterial homeostasis, as evidenced by numerous studies. However, the utilization of arginine and the downstream products of its metabolism remain undefined in various gut bacteria. To bridge this knowledge gap, we employed genomic screening to pinpoint relevant metabolic targets. We also devised a targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) metabolomics method to measure the levels of arginine, its upstream precursors, and downstream products in cell-free conditioned media from enteric pathobionts, including Escherichia coli, Klebsiella aerogenes, K. pneumoniae, Pseudomonas fluorescens, Acinetobacter baumannii, Streptococcus agalactiae, Staphylococcus epidermidis, S. aureus, and Enterococcus faecalis. Our findings revealed that all selected bacterial strains consumed glutamine, glutamate, and arginine, and produced citrulline, ornithine, and GABA in our chemically defined medium. Additionally, E. coli, K. pneumoniae, K. aerogenes, and P. fluorescens were found to convert arginine to agmatine and produce putrescine. Interestingly, arginine supplementation promoted biofilm formation in K. pneumoniae, while ornithine supplementation enhanced biofilm formation in S. epidermidis. These findings offer a comprehensive insight into arginine-ornithine metabolism in enteric pathobionts.

Discovery of a Novel Bioactive Compound in Orange Peel Polar Fraction on the Inhibition of Trimethylamine and Trimethylamine N-Oxide through Metabolomics Approaches and In Vitro and In Vivo Assays: Feruloylputrescine Inhibits Trimethylamine via Suppressing cntA/B Enzyme.

This study compares the inhibitory effects of orange peel polar fraction (OPP) and orange peel nonpolar fraction (OPNP) on trimethylamine (TMA) and trimethylamine N-oxide (TMAO) production in response to l-carnitine treatment in vivo and in vitro. Metabolomics is used to identify bioactive compounds. The research demonstrates that the OPP effectively regulates atherosclerosis-related markers, TMA and TMAO in plasma and urine, compared to the OPNP. Our investigation reveals that these inhibitory effects are independent of changes in gut microbiota composition. The effects are attributed to the modulation of cntA/B enzyme activity and FMO3 mRNA expression in vitro. Moreover, OPP exhibits stronger inhibitory effects on TMA production than OPNP, potentially due to its higher content of feruloylputrescine, which displays the highest inhibitory activity on the cntA/B enzyme and TMA production. These findings suggest that the OPP containing feruloylputrescine has the potential to alleviate cardiovascular diseases by modulating cntA/B and FMO3 enzymes without directly influencing gut microbiota composition.

A study on phytogenotoxicity induced by biogenic amines: cadaverine and putrescine.

Among the compounds present in necro-leachate, a liquid released during the process of decomposition of the human body, are the biogenic amines cadaverine and putrescine. Although some studies on necro-leachate have indicated a potential ecotoxicological and public health risk associated with it, the research on this type of contamination is still rather limited. This study presents information about the phytotoxic and cytogenotoxic potential of cadaverine and putrescine, evaluated separately and within a mixture. Phytotoxicity was evaluated through a germination test, the initial growth of seedlings with Lactuca sativa, and cytogenotoxicity through chromosomal aberration and micronucleus tests with Allium cepa. The L. sativa results showed a phytotoxic effect for the evaluated amines, by reducing root (> 90%) and hypocotyl (> 80%) elongation. The co-exposure of cadaverine and putrescine potentiated cytogenotoxic activity by aneugenic action in the meristematic cells of A. cepa. From this result, it is possible to infer the eco-toxicogenic potential of cadaverine and putrescine. This study not only highlights the importance of the phytotoxic and cytogenotoxic effects of these amines but also emphasizes the urgent need for further investigation into contamination originating from cemetery environments. By evaluating the risks associated with necro-leachate, this research is aimed at informing global efforts to protect ecological and public health.

Physiological, Metabolic, and Transcriptomic Analyses Reveal Mechanisms of Proliferation and Somatic Embryogenesis of Litchi (Litchi chinensis Sonn.) Embryogenic Callus Promoted by D-Arginine Treatment.

D-arginine (D-Arg) can promote embryogenic callus (EC) proliferation and increase the rate of somatic embryo induction of litchi (Litchi chinensis Sonn.), yet the mechanism underlying the processes is incompletely understood. To investigate the mechanism, physiological responses of polyamines (PAs) [putrescine (Put), spermidine (Spd), and spermine (Spm)] were investigated for D-Arg-treated litchi EC and enzyme activity related to polyamine metabolism, plant endogenous hormones, and polyamine- and embryogenic-related genes were explored. Results showed that the exogenous addition of D-Arg reduces the activity of diamine oxidase (DAO) and polyamine oxidase (PAO) in EC, reduces the production of H2O2, promotes EC proliferation, and increases the (Spd + Spm)/Put ratio to promote somatic embryo induction. Exogenous D-Arg application promoted somatic embryogenesis (SE) by increasing indole-3-acetyl glycine (IAA-Gly), kinetin-9-glucoside (K9G), and dihydrozeatin-7-glucoside (DHZ7G) levels and decreasing trans-zeatin riboside (tZR), N-[(-)-jasmonoyl]-(L)-valine (JA-Val), jasmonic acid (JA), and jasmonoyl-L-isoleucine (Ja-ILE) levels on 18 d, as well as promoting cell division and differentiation. The application of exogenous D-Arg regulated EC proliferation and somatic embryo induction by altering gene expression levels of the WRKY family, AP2/ERF family, C3H family, and C2H2 family. These results indicate that exogenous D-Arg could regulate the proliferation of EC and the SE induction of litchi by changing the biosynthesis of PAs through the alteration of gene expression pattern and endogenous hormone metabolism.

Caldomycin, a new guanidopolyamine produced by a novel agmatine homocoupling enzyme involved in homospermidine biosynthesis.

An extreme thermophilic bacterium, Thermus thermophilus produces more than 20 unusual polyamines, but their biosynthetic pathways, including homospermidine, are not yet fully understood. Two types of homospermidine synthases have been identified in plants and bacteria, which use spermidine and putrescine or two molecules of putrescine as substrates. However, homospermidine synthases with such substrate specificity have not been identified in T. thermophilus. Here we identified a novel agmatine homocoupling enzyme that is involved in homospermidine biosynthesis in T. thermophilus. The reaction mechanism is different from that of a previously described homospermidine synthase, and involves conjugation of two molecules of agmatine, which produces a diamidino derivative of homospermidine (caldomycin) as an immediate precursor of homospermidine. We conclude that there is a homospermidine biosynthetic pathway from agmatine via caldomycin synthase followed by ureohydrolase in T. thermophilus. Furthermore, it is shown that caldomycin is a novel compound existing in nature.

Cadaverine and putrescine exposure influence carbon and nitrogen cycling genes in water and sediment of the Yellow River.

The response patterns of microbial functional genes involved in biogeochemical cycles to cadaver decay is a central topic of recent environmental sciences. However, the response mechanisms and pathways of the functional genes associated with the carbon (C) and nitrogen (N) cycling to cadaveric substances such as cadaverine and putrescine remain unclear. This study explored the variation of functional genes associated with C fixation, C degradation and N cycling and their influencing factors under cadaverine, putrescine and mixed treatments. Our results showed only putrescine significantly increased the alpha diversity of C fixation genes, while reducing the alpha diversity of N cycling genes in sediment. For the C cycling, the mixed treatment significantly decreased the total abundance of reductive acetyl-CoA pathway genes (i.e., acsB and acsE) and lig gene linked to lignin degradation in water, while only significantly increasing the hydroxypropionate-hydroxybutylate cycle (i.e., accA) gene abundance in sediment. For the N cycling, mixed treatment significantly decreased the abundance of the nitrification (i.e., amoB), denitrification (i.e., nirS3) genes in water and the assimilation pathway gene (i.e., gdhA) in sediment. Environmental factors (i.e., total carbon and total nitrogen) were all negatively associated with the genes of C and N cycling. Therefore, cadaverine and putrescine exposure may inhibit the pathway in C fixation and N cycling, while promoting C degradation. These findings can offer some new insight for the management of amine pollution caused by animal cadavers.

Chemosensory detection of polyamine metabolites guides C. elegans to nutritive microbes.

Much is known about molecular mechanisms by which animals detect pathogenic microbes, but how animals sense beneficial microbes remains poorly understood. The roundworm Caenorhabditis elegans is a microbivore that must distinguish nutritive microbes from pathogens. We characterized a neural circuit used by C. elegans to rapidly discriminate between nutritive bacteria and pathogens. Distinct sensory neuron populations responded to chemical cues from nutritive Escherichia coli and pathogenic Enterococcus faecalis, and these neural signals are decoded by downstream AIB interneurons. The polyamine metabolites cadaverine, putrescine, and spermidine produced by E. coli activate this neural circuit and elicit positive chemotaxis. Our study shows how polyamine odorants can be sensed by animals as proxies for microbe identity and suggests that, hence, polyamines might have widespread roles brokering host-microbe interactions.

Electrostatic Repulsion Hydrophilic Interaction Liquid Chromatography (ERLIC) for the Quantitative Analysis of Polyamines.

Highly polar low molecular weight organic molecules are still very challenging to analyze by liquid chromatography. Yet, with the steadily increasing application of metabolomics and similar approaches in chemical analysis, separating polar compounds might be even more important. However, almost all established liquid chromatography techniques (i.e., normal and reversed phase, hydrophilic interaction liquid chromatography (HILIC), ion chromatography) struggle with either carry-over, low sensitivity, or a lack of retention. For improving these shortcomings, electrostatic repulsion hydrophilic interaction chromatography (ERLIC) might be an alternative. By combining a HILIC mobile phase, that is highly organic with a low water content, and an ion exchange column, a distinct layer system develops. When the analyte's charge is of the same direction as the stationary phase, retention and elution are determined by two antagonistic forces: electrostatic repulsion and hydrophilicity. One prominent group of challenging polar analytes are the polyamines cadaverine, putrescine, spermidine, and spermine. Carrying charges from +2 to +4 at physiological pH, these compounds are essential cell constituents and found in all living organisms. However, they are still notoriously challenging to analyze via the established liquid chromatography methods. In the present work, an ERLIC tandem mass spectrometry method has been exemplarily developed, optimized, and validated for the quantitative determination of cadaverine, putrescine, spermidine, and spermine. This method enables symmetrical peak shapes and good separation of analytes with different charges while simultaneously selectively detecting the co-eluting diamines by MS/MS. Furthermore, high linearity (R > 0.998) and sensitivity (LODs ≤ 2 ng/mL) have been proven. Thus, ERLIC may be interesting for both targeted and untargeted analysis approaches of highly charged low molecular weight organic molecules.

Functional divergence of two arginine decarboxylase genes in tropane alkaloid biosynthesis and root growth in Atropa belladonna.

Putrescine, produced via the arginine decarboxylase (ADC)/ornithine decarboxylase (ODC)-mediated pathway, is an initial precursor for polyamines metabolism and the root-specific biosynthesis of medicinal tropane alkaloids (TAs). These alkaloids are widely used as muscarinic acetylcholine antagonists in clinics. Although the functions of ODC in biosynthesis of polyamines and TAs have been well investigated, the role of ADC is still poorly understood. In this study, enzyme inhibitor treatment showed that ADC was involved in the biosynthesis of putrescine-derived metabolites and root growth in Atropa belladonna. Further analysis found that there were six ADC unigenes in the A. belladonna transcriptome, with two of them, AbADC1 and AbADC2, exhibiting high expression in the roots. To investigate their roles in TAs/polyamines metabolism and root growth, RNA interference (RNAi) was used to suppress either AbADC1 or AbADC2 expression in A. belladonna hairy roots. Suppression of the AbADC1 expression resulted in a significant reduction in the putrescine content and hairy root biomass. However, it had no noticeable effect on the levels of N-methylputrescine and the TAs hyoscyamine, anisodamine, and scopolamine. On the other hand, suppression of AbADC2 expression markedly reduced the levels of putrescine, N-methylputrescine, and TAs, but had no significant effect on hairy root biomass. According to ß-glucuronidase (GUS) staining assays, AbADC1 was mainly expressed in the root elongation and division region while AbADC2 was mainly expressed in the cylinder of the root maturation region. These differences in expression led to functional divergence, with AbADC1 primarily regulating root growth and AbADC2 contributing to TA biosynthesis.

Host-Guest Chemosensor Ensembles based on Water-Soluble Sulfonated Calix[n]arenes and a Pyranoflavylium Dye for the Optical Detection of Biogenic Amines.

Biogenic amines (BAs) are biologically active nitrogen-containing compounds formed during the food spoilage process and are often related as key markers of food quality, safety, and freshness. Because their presence in foods at high levels can cause significant health problems, researchers have been focused on developing novel strategies and methods for early detection and capture of these analytes. Herein, water-soluble sulfonated calix[n]arene macrocycles (SC4, SC6, and SC8) and a pH-sensitive dye (4'-hydroxy-10-methylpyranoflavylium) were investigated as host-guest systems for BA sensing. The hosts were able to bind the flavylium cation of the dye with association constants of 103 to 104 M-1. The dye complexation also allowed tuning its pKa from 6.72 (free) toward high values: 7.68 (SC4), 7.79 (SC6), and 8.45 (SC8). These data were crucial to optimize the host-guest complexes as optical sensing systems for putrescine/tyramine (pH 7.2-7.6), yielding a colorimetric redshift from yellow to red. The BA sensing was also demonstrated by fluorescence quenching for the calix[n]arene/dye complexes and fluorescence recovery after the addition of BAs. 1H NMR spectroscopy was used to demonstrate the interaction mode, confirming an encapsulation-driven mechanism. Overall, these host-guest systems demonstrated great potential for the detection of BAs, one of the main key markers of food spoilage.

Concentration-tuned diverse response to selective biogenic amines using a reusable fluorophore: monitoring protein-rich food spoilage.

Maintaining the freshness of food is essential for a healthy and quality life. Nevertheless, it remains a global challenge. Hence, an easy detection and monitoring protocol would be highly desirable. A cyanoacrylic acid (CAA)-based fluorophore is manifested as a reusable platform that responds diversely against different concentrations of selective aliphatic biogenic amines (BAs) in both solution and vapor phases. Slow spoilage of the protein-rich food is progressively monitored through emission shifts visible to the naked eye. This fluorophore provides easy and naked-eye detection of the BA vapor through a change in emission, i.e., red → orange → orange-yellow → cyan → green and quantum yield enhancement, which occur in stepwise increments of vapor concentrations. The probe design includes π-conjugated functionalized fluorescent molecules linked to multiple twisting sites, resulting in both solid and solution-state emission. The attached carboxylic acid responds quickly with selective BAs, mainly putrescine (PUT), cadaverine (CAD), and spermidine (SPM), where the concentration-based emission variation has appeared to be distinct and prominent against PUT [sensitivity (µM): 2 (solution); 3.3 (vapour)]. The selectivity towards diamine can be clarified by the formation of carboxylic acid salts and the consequent proton exchanges between free and protonated amines. In addition, -CN···H interaction is likely to develop within this ammonium carboxylate system, providing extra stability. Such ammonium carboxylate salt formation and gradual change in the molecular arrangement, resulting in symmetry development, are validated by FT-IR and wide-angle X-ray diffraction studies. Besides, this fact is supported by DFT studies that validate intramolecular H-atom exchange between free amine and ammonium salt units. A fluorophore-coated coverslip, filter paper, or silica gel-coated Al-plate is fruitfully utilized to detect the freshness of fish and chicken, which reveals the potential of this probe to prevent food waste and control food safety.

Pholiotic acid promotes apoptosis in human metastatic melanoma cells.

Mushrooms produce a great variety of secondary metabolites that can be successful in both prevention and treatment of various cancers. In particular, higher Basidiomycete mushrooms contain various types of biologically active low-molecular compounds in fruiting bodies with suggested anticarcinogenic effects. The polyamine analogue {(2R)-2-[(S)-3-hydroxy-3-methylglutaryloxy] putrescine dicinnamamide} indicated with the name pholiotic acid, isolated for the first time by us from the fruiting bodies of the Basidiomycete Pholiota spumosa (Fr.) Sing. (Strophariaceae), inhibited the viability of human prostate cancer cells, such as other polyamine synthetic analogues that have shown antitumor activity in several types of cancer, including melanoma. Melanoma is an aggressive skin cancer that can metastasize to other organs and presents a high resistance to conventional therapies. In light of these considerations, the present study was therefore designed to assess whether this putrescine derivative could inhibit the growth of human metastatic melanoma cell lines, M14 and A2058. The results obtained demonstrate that this natural compound, at 12.5-50 µM concentration, was able to reduce cell viability of both cancer cells inducing cell death by intrinsic apoptotic pathway that probably involves PTEN activity, inhibition of Hsp70 expression and reactive oxygen species production. On the other hand, the increased expression of enzymes involved in polyamine catabolism trigger apoptotic cell death leading to polyamine depletion and generation of reactive oxygen species as by-products. In conclusion, these findings, starting point for further investigation, implement available our data to support pholiotic acid as an attractive potential chemopreventive agent, and provide a basis for further research into the use of this polyamine derivative as potential anticancer agent for melanoma in combination with existing therapies to improve treatment efficacy and overcome the obstacle of drug resistance.

High-Resolution Comparative and Quantitative Proteomics of Biogenic-Amine-Producing Bacteria and Virulence Factors Present in Seafood.

The presence of biogenic amines (histamine, tyramine, putrescine, and cadaverine) in seafood is a significant concern for food safety. This review describes for the first time a shotgun quantitative proteomics strategy to evaluate and compare foodborne strains of bacteria that produce biogenic amines in seafoods. This approach recognized 35,621 peptide spectrum matches, belonging to 20,792 peptides, and 4621 proteins. It allowed the determination of functional pathways and the classification of the strains into hierarchical clusters. The study identified a protein-protein interaction network involving 1160 nodes/10,318 edges. Proteins were related to energy pathways, spermidine biosynthesis, and putrescine metabolism. Label-free quantitative proteomics allowed the identification of differentially regulated proteins in specific strains such as putrescine aminotransferase, arginine decarboxylase, and l-histidine-binding protein. Additionally, 123 peptides were characterized as virulence factors and 299 peptide biomarkers were selected to identify bacterial species in fish products. This study presents the most extensive proteomic repository and progress in the science of food biogenic bacteria and could be applied in the food industry for the detection of bacterial contamination that produces histamine and other biogenic amines during food processing/storage.

The Deficiency of Hypusinated eIF5A Decreases the Putrescine/Spermidine Ratio and Inhibits +1 Programmed Ribosomal Frameshifting during the Translation of Ty1 Retrotransposon in Saccharomyces cerevisiae.

Programmed ribosomal frameshifting (PRF) exists in all branches of life that regulate gene expression at the translational level. The eukaryotic translation initiation factor 5A (eIF5A) is a highly conserved protein essential in all eukaryotes. It is identified initially as an initiation factor and functions broadly in translation elongation and termination. The hypusination of eIF5A is specifically required for +1 PRF at the shifty site derived from the ornithine decarboxylase antizyme 1 (OAZ1) in Saccharomyces cerevisiae. However, whether the regulation of +1 PRF by yeast eIF5A is universal remains unknown. Here, we found that Sc-eIF5A depletion decreased the putrescine/spermidine ratio. The re-introduction of Sc-eIF5A in yeast eIF5A mutants recovered the putrescine/spermidine ratio. In addition, the Sc-eIF5A depletion decreases +1 PRF during the decoding of Ty1 retrotransposon mRNA, but has no effect on -1 PRF during the decoding of L-A virus mRNA. The re-introduction of Sc-eIF5A in yeast eIF5A mutants restored the +1 PRF rate of Ty1. The inhibition of the hypusine modification of yeast eIF5A by GC7 treatment or by mutating the hypusination site Lys to Arg caused decreases of +1 PRF rates in the Ty1 retrotransposon. Furthermore, mutational studies of the Ty1 frameshifting element support a model where the efficient removal of ribosomal subunits at the first Ty1 frame 0 stop codon is required for the frameshifting of trailing ribosomes. This dependency is likely due to the unique position of the frame 0 stop codon distance from the slippery sequence of Ty1. The results showed that eIF5A is a trans-regulator of +1 PRF for Ty1 retrotransposon and could function universally in yeast.

CsCuAO1 Associated with CsAMADH1 Confers Drought Tolerance by Modulating GABA Levels in Tea Plants.

Our previous study showed that COPPER-CONTAINING AMINE OXIDASE (CuAO) and AMINOALDEHYDE DEHYDROGENASE (AMADH) could regulate the accumulation of γ-aminobutyric acid (GABA) in tea through the polyamine degradation pathway. However, their biological function in drought tolerance has not been determined. In this study, Camellia sinensis (Cs) CsCuAO1 associated with CsAMADH1 conferred drought tolerance, which modulated GABA levels in tea plants. The results showed that exogenous GABA spraying effectively alleviated the drought-induced physical damage. Arabidopsis lines overexpressing CsCuAO1 and CsAMADH1 exhibited enhanced resistance to drought, which promoted the synthesis of GABA and putrescine by stimulating reactive oxygen species' scavenging capacity and stomatal movement. However, the suppression of CsCuAO1 or CsAMADH1 in tea plants resulted in increased sensitivity to drought treatment. Moreover, co-overexpressing plants increased GABA accumulation both in an Agrobacterium-mediated Nicotiana benthamiana transient assay and transgenic Arabidopsis plants. In addition, a GABA transporter gene, CsGAT1, was identified, whose expression was strongly correlated with GABA accumulation levels in different tissues under drought stress. Taken together, CsCuAO1 and CsAMADH1 were involved in the response to drought stress through a dynamic GABA-putrescine balance. Our data will contribute to the characterization of GABA's biological functions in response to environmental stresses in plants.

Decarboxylase activity of the non-starter lactic acid bacterium Loigolactobacillus rennini gives crack defects in Gouda cheese through the production of γ-aminobutyric acid.

Ten Gouda cheese wheels with an age of 31 weeks from six different batch productions were affected by a crack defect and displayed an unpleasant off-flavor. To unravel the causes of these defects, the concentrations of free amino acids, other organic acids, volatile organic compounds, and biogenic amines were quantified in zones around the cracks and in zones without cracks, and compared with those of similar Gouda cheeses without crack defect. The Gouda cheeses with cracks had a significantly different metabolome. The production of the non-proteinogenic amino acid γ-aminobutyric acid (GABA) could be unraveled as the key mechanism leading to crack formation, although the production of the biogenic amines cadaverine and putrescine contributed as well. High-throughput amplicon sequencing of the full-length 16S rRNA gene based on whole-community DNA revealed the presence of Loigolactobacillus rennini and Tetragenococcus halophilus as most abundant non-starter lactic acid bacteria in the zones with cracks. Shotgun metagenomic sequencing allowed to obtain a metagenome-assembled genome of both Loil. rennini and T. halophilus. However, only Loil. rennini contained genes necessary for the production of GABA, cadaverine, and putrescine. Metagenetics further revealed the brine and the rennet used during cheese manufacturing as the most plausible inoculation sources of both Loil. rennini and T. halophilus.IMPORTANCECrack defects in Gouda cheeses are still poorly understood, although they can lead to major economic losses in cheese companies. In this study, the bacterial cause of a crack defect in Gouda cheeses was identified, and the pathways involved in the crack formation were unraveled. Moreover, possible contamination sources were identified. The brine bath might be a major source of bacteria with the potential to deteriorate cheese quality, which suggests that cheese producers should regularly investigate the quality and microbial composition of their brines. This study illustrated how a multiphasic approach can understand and mitigate problems in a cheese company.

The highly selective rhodol-based putrescine probe and visual sensors for on-site detection of putrescine in food spoilage.

Putrescine (Butane-1,4-diamine) has been regarded as a vital marker of spoiling protein-rich foods, especially meat and seafood. The detection of putrescine in food is considered a convenient and powerful method for evaluating the degree of spoilage of protein-rich foods. Herein, a novel rhodol-based fluorescent probe RSMA (formyl-rhodol Schiff base with methoxyaniline) was developed to detect putrescine. RSMA exhibited excellent linearity (R2 = 0.9912) in the concentration range of 0-45 µM of putrescine with a detection limit as low as 0.45 µM. Although RSMA had moderate responses to some aliphatic diamines, the selectivity of RSMA for putrescine was one of the best reported in the literature so far. Moreover, RSMA was successfully fabricated to solid-state sensors for on-site detection of putrescine in shrimp, that demonstrated its application in monitoring food spoilage.

Molecular Insights into the Synergistic Effects of Putrescine and Ammonium on Dinoflagellates.

Ammonium and polyamines are essential nitrogen metabolites in all living organisms. Crosstalk between ammonium and polyamines through their metabolic pathways has been demonstrated in plants and animals, while no research has been directed to explore this relationship in algae or to investigate the underlying molecular mechanisms. Previous research demonstrated that high concentrations of ammonium and putrescine were among the active substances in bacteria-derived algicide targeting dinoflagellates, suggesting that the biochemical inter-connection and/or interaction of these nitrogen compounds play an essential role in controlling these ecologically important algal species. In this research, putrescine, ammonium, or a combination of putrescine and ammonium was added to cultures of three dinoflagellate species to explore their effects. The results demonstrated the dose-dependent and species-specific synergistic effects of putrescine and ammonium on these species. To further explore the molecular mechanisms behind the synergistic effects, transcriptome analysis was conducted on dinoflagellate Karlodinium veneficum treated with putrescine or ammonium vs. a combination of putrescine and ammonium. The results suggested that the synergistic effects of putrescine and ammonium disrupted polyamine homeostasis and reduced ammonium tolerance, which may have contributed to the cell death of K. veneficum. There was also transcriptomic evidence of damage to chloroplasts and impaired photosynthesis of K. veneficum. This research illustrates the molecular mechanisms underlying the synergistic effects of the major nitrogen metabolites, ammonium and putrescine, in dinoflagellates and provides direction for future studies on polyamine biology in algal species.