Autochthonous cultures to improve the quality of PGI Castellano cheese: Impact on proteolysis, microstructure and texture during ripening.

The aim of this research was to find out the effect of different combinations of starter and non-starter cultures on the proteolysis of Castellano cheese during ripening. Four cheese batches were prepared, each containing autochthonous lactobacilli and or Leuconostoc, and were compared with each other and with a control batch, that used only a commercial starter. To achieve this, nitrogen fractions (pH 4.4-soluble nitrogen and 12 % trichloroacetic acid soluble nitrogen, polypeptide nitrogen and casein nitrogen), levels of free amino acids and biogenic amines were assessed. Texture and microstructure of cheeses were also evaluated. Significant differences in nitrogen fractions were observed between batches at different stages of ripening. The free amino acid content increased throughout the cheese ripening process, with a more significant increase occurring after the first 30 days. Cheeses containing non-starter lactic acid bacteria exhibited the highest values at the end of the ripening period. Among the main amino acids, GABA was particularly abundant, especially in three of the cheese batches at the end of ripening. The autochthonous lactic acid bacteria were previously selected as non-producers of biogenic amines and this resulted in the absence of these compounds in the cheeses. Analysis of the microstructure of the cheese reflected the impact of proteolysis. Additionally, the texture profile analysis demonstrated that the cheese's hardness intensified as the ripening period progressed. The inclusion of autochthonous non-starter lactic acid bacteria in Castellano cheese production accelerated the proteolysis process, increasing significantly the free amino acids levels and improving the sensory quality of the cheeses.

Microbiological safety of dry-cured fish from the raw material to the end of processing.

The commercialization of processed fish products is rising in restaurants and small to medium enterprises. However, there is a lack of data related to the microbiological safety of such products. In this study total aerobic colony count and Enterobacteriaceae, as proxy of process hygiene criteria, and detection of Listeria monocytogenes and concentration of histamine, as food safety criteria, were investigated in Salmo salar (salmon), Xiphias gladius (swordfish) and Thunnus albacares (yellowfin tuna), before, during, and at the end of a dry-curing process, performed in a dedicated cabinet, at controlled temperature, relative humidity and ventilation, up to 240 h. The microbiological parameters were investigated in the tested fish products by culture methods and shotgun metagenomic, while the presence of histamine, and other biogenic amines, was quantified by High Performance Liquid Chromatography. In the raw material, and up to the end of the dry curing process, the concentration of Enterobacteriaceae was always lower than 10 CFU/g, while total aerobic colony counts ranged between 3.9 and 5.4 Log CFU/g in salmon; 5.5 and 5.9 Log CFU/g in swordfish; 4.4 and 4.8 Log CFU/g in tuna. The pH values were significantly different between fish species, in the raw materials and during processing except for T4, occurring 70 h after the start of the process for salmon and after 114 h for swordfish and tuna. Water activity was different at specific sampling points and at the end of processing. Overall, 79 % of the sequences identified in the tested fish samples were assigned to y bacteria. The most abundant phyla were Pseudomonadota, Bacillota and Mycoplasmatota. The microbial populations identified by shotgun metagenomic in the tested fish species clustered well separated one from the other. Moreover, the microbial richness was significantly higher in salmon and tuna in comparison to swordfish. Listeria monocytogenes was not detected in the raw material by using the reference cultural method and very few reads (relative abundance <0.007) were detected in swordfish and tuna by shotgun metagenomic. Histamine producing bacteria, belonging to the genera Vibrio, Morganella, Photobacterium and Klebsiella, were identified primarily in swordfish. However, histamine and other biogenic amines were not detected in any sample. To the best of our knowledge this is the first paper reporting time point determinations of microbiological quality and safety parameters in salmon, swordfish and tuna, before, during and at the end of a dry-curing process. The data collected in this paper can help to predict the risk profile of ready to eat dry-cured fish products during storage before consumption.

Microbial contribution to 14 biogenic amines accumulation in refrigerated raw and deep-fried hairtails (Trichiurus lepturus).

Biogenic amines (BAs) produced by microbial decarboxylation of amino acids are crucial toxic nitrogenous compounds in fish. An optimized ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method with simple pretreatment was established to detect 14 BAs in both raw (control check, CK) and deep-fried (DF) hairtails. This method exhibited a good linear relationship with average recoveries of 73.3-120.0 % and relative standard deviations of 2.5-10.0 %, respectively. The total BAs in CK and DF hairtails decreased sharply to 338.2 and 25.3 mg/kg on the 9th day, respectively. Four BAs, including cadaverine (CAD), histamine (HIS), tyramine (TYR), and putrescine (PUT) accounted for 92.5-99.9 % of total BAs were selected as the dominant BAs. Bacterial analysis showed that the abundance of DF was relatively low. Further correlation analysis proved that Vibrio had a significant (p < 0.05) positive correlation with total BAs and could be the main BA-producing bacterium in DF hairtail. This work provides new evidence of the accumulation of BAs in refrigerated hairtail.

The inhibitory action of lactocin 63 on deterioration of seabass (Lateolabrax japonicus) during chilled storage.

BACKGROUND: The bacteriocins, particularly derived from lactic acid bacteria, currently exhibit potential as a promising food preservative owing to their low toxicity and potent antimicrobial activity. This study aimed to evaluate the efficacy of lactocin 63, produced by Lactobacillus coryniformis, in inhibiting the deterioration of Lateolabrax japonicas during chilled storage, while also investigating its underlying inhibitory mechanism. The measurement of total viable count, biogenic amines, and volatile organic compounds were conducted, along with high-throughput sequencing and sensory evaluation. RESULTS: The findings demonstrated that treatment with lactocin 63 resulted in a notable retardation of bacterial growth in L. japonicas fish fillet during refrigerated storage compared with the water-treated and nisin-treated groups. Moreover, lactocin 63 effectively maintained the microbial flora balance in the fish fillet and inhibited the proliferation and metabolic activity of specific spoilage microorganisms, particularly Shewanella, Pseudomonas, and Acinetobacter. Furthermore, the production of unacceptable volatile organic compounds (e.g. 1-octen-3-ol, hexanal, nonanal), as well as the biogenic amines derived from the bacterial metabolism, could be hindered, thus preventing the degradation in the quality of fish fillets and sustaining relatively high sensory quality. CONCLUSION: The results of this study provide valuable theoretical support for the development and application of lactocin 63, or other bacteriocins derived from lactic acid bacteria, as potential bio-preservatives in aquatic food. © 2024 Society of Chemical Industry.

In-situ substitution and community dynamics modeling for enhanced safety in Chinese rice wine brewing.

This research paper focuses on the application of the "Design-Build-Test-Learn" framework to design and evaluate a synthetic microbial community aimed at studying the impact of Lactic Acid Bacteria (LAB) interactions and fitness on the formation of biogenic amines (BAs) in Chinese rice wine (CRW). The study reveals a close correlation between the assembly model of LAB and the accumulation of BAs in CRW, and multiple interactions were observed between amine-producing and non-amine-producing LAB, including commensalism, amensalism, and competition. The commensalism among amine-producing LAB was found to promote BAs accumulation through metabolic cross-feeding of amino acids. Moreover, the higher-order interaction community was designed to regulate the BAs formation effectively. For instance, the interference of Lactiplantibacillus plantarum (ACBC271) resulted in the elimination of amine-producing LAB viability, resulting in a 22% decrease (not exceeding 43.54 mg/L) in the total amount of BAs. Simulation of community dynamics models further suggests that LAB with quantitative social interactions can effectively control LAB accumulation in CRW by forecasting fluctuation in BAs generation through fitness competition and metabolic interference. Overall, this study provides valuable insights into the complex interaction networks within microbial communities in traditional fermentation ecosystems. It also proposes a novel approach for quality control of nitrogen food safety factors in fermented foods.

Screening and evaluating microorganisms with broad-spectrum biogenic amine-degrading ability from naturally fermented dry sausage collected from Northeast China.

This study aimed to screen autochthonous strains with broad-spectrum biogenic amine (BA) degradation ability from traditional dry sausages and to evaluate their BA-degrading ability in dry sausages. A total of 120 strains were isolated from dry sausages collected from various regions in Northeast China, and 35 of 120 isolates were identified as non-BA producing strains by the in vitro agar method. The random amplified polymorphic DNA polymerase chain reaction technique genotyped these 35 isolates into 18 biotypes. Moreover, high performance liquid chromatography (HPLC) quantification showed that six strains (Latilactobacillus sakei MDJ6; Lactiplantibacillus plantarum SH7; Weissella hellenica DQ9; Staphylococcus saprophyticus JX18 and SYS8; and Macrococcus caseolyticus SYS11) of the 18 biotypes exhibited broad-spectrum BA-degrading ability, all of which had various levels of amine oxidase activity with monoamine oxidase and diamine oxidase activities ranged of 6.60-619.04 and 26.32-352.81 U/mg protein, respectively. These six strains were subsequently inoculated into dry sausages and the results showed that they exhibited varying degrees of BA-degrading ability, of which strain Lat. sakei MDJ6 allowed to have less BA production on dry sausage with a final concentration of 61.33 mg/kg.

Phenylethylamine sensing at the electrified liquid-liquid interface. Can electrochemistry be used to follow the UHT milk spoilage process?

This study involved an investigation into the electrochemical characteristic of a few biogenic amines (BAs) occurring at the polarized interface between two immiscible electrolyte solutions (ITIES) with ion transfer voltammetry (ITV). The main focus of this research was the comprehensive electroanalytical and physicochemical analysis of phenylethylamine (PEA), allowing the determined of the formal Galvani potential of the ion transfer reaction (ΔorgaqΦ'), diffusion coefficients (D), formal free Gibbs energy of the ion transfer reaction (ΔG'aq→org) and water-1,2-dichloroethane partition coefficient (logPwater/DCEPEA). Furthermore, the collected data were employed to calculate analytical parameters, including voltametric detection sensitivity, limits of detection and the target analyte quantification. Moreover, the influence of the presence of 7 other BAs (histamine, spermine, spermidine, putrescine, cadaverine, tyramine and tryptamine) on the recorded signals originating from the PEA ion transfer was checked. The feasibility of the developed method was corroborated through experimentation with milk samples. Additionally, utilizing the devised methodology, an electrochemical assessment of the spoilage progression in milk samples was undertaken.

Development of a capillary electrophoresis method based on magnetic solid-phase extraction for simultaneous and sensitive detection of eight biogenic amines in foods.

BACKGROUND: Biogenic amines (BAs) in high concentrations are toxic and may cause a series of health symptoms. A sensitive measurement of BA levels is essential for human health. Capillary electrophoresis (CE) has emerged for the separation of eight BAs due to simple sample preparation and highly efficient separation. However, an important drawback for CE is low sensitivity. Magnetic solid-phase extraction (MSPE) has become a technique of interest owing to its brief operation and low solvent consumption. Hence, MSPE as a pretreatment has great potential to improve CE sensitivity for the analysis of BAs in complex food. RESULTS: Results showed that the Pt-Co-MWCNTs-COOH possessed strong magnetism, good reusability, and high adsorptive ability toward eight biogenic amines based on the hydrogen bonding between the -COOH of Pt-Co-MWCNTs-COOH and -NH2 groups of BAs. Using it as an adsorbent, a magnetic solid-phase extraction coupled with capillary electrophoresis (MSPE-CE) method was developed to effectively extract and sensitively analyze eight BAs. Under optimal conditions, the MSPE-CE method has wide linearities (10.0-1000.0 µg L-1 ) and low limits of detection (1.0-6.1 µg L-1 ). The accuracy of the developed method yielded recovery values from 82.07% to 102.58%. Meanwhile, the BAs contents in two samples were analyzed using the MSPE-CE method, with the results consistent with those detected by a high-performance liquid chromatography method. CONCLUSION: Given those advantages, the established MSPE-CE method promises the practical guidance of monitoring a variety of BAs and provides a foundation for the detection of other food hazards. © 2023 Society of Chemical Industry.

A three-in-one versatile sensor for concise detecting biogenic amines and beef freshness.

Biogenic amines (BAs), as important indicators for evaluating food spoilage caused by fermentation processes or microbial activities, present significant risks of food safety. Consequently, the development of a simple, sensitive, and selective detection method for amines is of great importance. In this study, we proposed a three-in-one sensor 3,6-bis(dimethylamino)-9-(ethylthio)xanthylium (PSE) for high sensitivity and selectivity detecting BAs with multimodal responses, including olfactory, colorimetric, and fluorescent signals, thus facilitating convenient real-time detection of BAs. Mechanism study indicated that the nucleophilic substitution of PSE with BAs induced such rapid multi-responses with a low detection limit (LOD = 0.03 µM). We further fabricated PSE loaded paper for portable detection of BAs vapors. And the accurate determination of BAs levels is achieved through analyzing the RGB color mode. Finally, we successfully applied these test strips for non-destructive assessing meat beef freshness with the assistance of a smartphone in on-site scenarios.

Variety and year: Two key factors on amino acids and biogenic amines content in grapes.

Amino acids have proved to play a key role in the development of volatile compounds present in wine with determining repercussions on the final wine bouquets. Biogenic amines originate from the chemical transformations of amino acids found in various foods, a phenomenon that has given rise to several health-related concerns among consumers. In the present research, the evaluation of two of the most influential factors: variety (genetic) and year (climatic conditions) on these compounds in grapes has been performed. Eight Vitis vinifera varieties have been collected during three years and the content of nineteen amino acids, two biogenic amines, and the ammonium ion has been quantified using the HPLC-PDA technique. The genetic factor has proved to be an influential variable (p-value < 0.05) with mean values of amino acids ranging from 896.89 to 1713.79 mg/L and of biogenic amines ranging from 10.61 to 22.28 mg/L. The climatic conditions have shown to be an influential factor as well (p-value < 0.05), being the low temperatures and rainfall and the high solar radiation favour the development of the amino acid and avoid biogenic amines accumulation in grapes.

Quality and Microbiome Analysis of Pickled Swimming Crabs (Portunus trituberculatus) during Storage at Two Alternative Temperatures.

The storage quality and microbiome analysis of pickled swimming crabs (Portunus trituberculatus) stored at 20 and 4 °C were investigated. It showed that samples stored at 4 °C had a longer shelf life, lower total viable count (TVC), pH, and total volatile base nitrogen (TVB-N) contents than those stored at 20 °C. The biogenic amine (BA) results demonstrated that tyramine (tyr), putrescine (put), and cadaverine (cad) were the dominant amines in all samples, and samples stored at 4 °C had lower BA contents. A microbiome analysis indicated that a salt-alcohol water mixture significantly inhibited the growth of Tenericutes. Firmicutes, Proteobacteria, Bacteroidetes, Acidobacteria, Actinobacteria, and Cyanobacteria were the dominant bacteria of stored pickled crabs, and storage at 4 °C significantly inhibited the growth of dominant bacteria, more than that of 20 °C. In conclusion, 4 °C storage guaranteed the quality of samples by inhibiting changes in biochemical properties and the growth of dominant bacteria, thereby prolonging its shelf life.

Functional Polymeric Membranes with Antioxidant Properties for the Colorimetric Detection of Amines.

Herein, the ability of highly porous colorimetric indicators to sense volatile and biogenic amine vapors in real time is presented. Curcumin-loaded polycaprolactone porous fiber mats are exposed to various concentrations of off-flavor compounds such as the volatile amine trimethylamine, and the biogenic amines cadaverine, putrescine, spermidine, and histamine, in order to investigate their colorimetric response. CIELAB color space analysis demonstrates that the porous fiber mats can detect the amine vapors, showing a distinct color change in the presence of down to 2.1 ppm of trimethylamine and ca. 11.0 ppm of biogenic amines, surpassing the limit of visual perception in just a few seconds. Moreover, the color changes are reversible either spontaneously, in the case of the volatile amines, or in an assisted way, through interactions with an acidic environment, in the case of the biogenic amines, enabling the use of the same indicator several times. Finally, yet importantly, the strong antioxidant activity of the curcumin-loaded fibers is successfully demonstrated through DPPH● and ABTS● radical scavenging assays. Through such a detailed study, we prove that the developed porous mats can be successfully established as a reusable smart system in applications where the rapid detection of alkaline vapors and/or the antioxidant activity are essential, such as food packaging, biomedicine, and environmental protection.

Insights into microbial communities and metabolic profiles in the traditional production of the two representative Hongqu rice wines fermented with Gutian Qu and Wuyi Qu based on single-molecule real-time sequencing.

Hongqu rice wine, a famous traditional fermented alcoholic beverage, is brewed with traditional Hongqu (mainly including Gutian Qu and Wuyi Qu). This study aimed to compare the microbial communities and metabolic profiles in the traditional brewing of Hongqu rice wines fermented with Gutian Qu and Wuyi Qu. Compared with Hongqu rice wine fermented with Wuyi Qu (WY), Hongqu rice wine fermented with Gutian Qu (GT) exhibited higher levels of biogenic amines. The composition of volatile flavor components of Hongqu rice wine brewed by different fermentation starters (Gutian Qu and Wuyi Qu) was obviously different. Among them, ethyl acetate, isobutanol, 3-methylbutan-1-ol, ethyl decanoate, ethyl palmitate, ethyl oleate, nonanoic acid, 4-ethylguaiacol, 5-pentyldihydro-2(3H)-furanone, ethyl acetate, n-decanoic acid etc. were identified as the characteristic aroma-active compounds between GT and WY. Microbiome analysis based on high-throughput sequencing of full-length 16S rDNA/ITS-5.8S rDNA amplicons revealed that Lactococcus, Leuconostoc, Pseudomonas, Serratia, Enterobacter, Weissella, Saccharomyces, Monascus and Candida were the predominant microbial genera during the traditional production of GT, while Lactococcus, Lactobacillus, Leuconostoc, Enterobacter, Kozakia, Weissella, Klebsiella, Cronobacter, Saccharomyces, Millerozyma, Monascus, Talaromyces and Meyerozyma were the predominant microbial genera in the traditional fermentation of WY. Correlation analysis revealed that Lactobacillus showed significant positive correlations with most of the characteristic volatile flavor components and biogenic amines. Furthermore, bioinformatical analysis based on PICRUSt revealed that microbial enzymes related to biogenic amines synthesis were more abundant in GT than those in WY, and the enzymes responsible for the degradation of biogenic amines were less abundant in GT than those in WY. Collectively, this study provides important scientific data for enhancing the flavor quality of Hongqu rice wine, and lays a solid foundation for the healthy and sustainable development of Hongqu rice wine industry.

Influence of lactic acid fermentation on the microbiological parameters, biogenic amines, and volatile compounds of bovine colostrum.

In this study we hypothesized that the relations between the bovine colostrum (BC) microbiota, biogenic amine (BA) as well as volatile compound (VC) profiles can lead to new deeper insights concerning the BC changes during the biological preservation. To implement such an aim, BC samples were collected from 5 farms located in Lithuania and fermented with Lactiplantibacillus plantarum and Lacticaseibacillus paracasei strains. Nonfermented and fermented BC were subjected to microbiological [lactic acid bacteria (LAB), Escherichia coli, and total bacteria (TBC), total Enterobacteriaceae (TEC) and total mold and yeast (M-Y) viable counts] and physicochemical (pH, color coordinates, BA content and VC profile) parameters evaluation, and the relationship between the tested parameters were also further analyzed. In comparison pH and dry matter (DM) of nonfermented samples, significant differences were not found, and pH of BC was, on average, 6.30, and DM, on average, 27.5%. The pH of fermented samples decreased, on average, until 4.40 in Lp. plantarum fermented group, and, on average, until 4.37 in Lc. paracasei fermented group. Comparing color characteristics among nonfermented BC groups, significant differences between lightness (L*) and yellowness (b*) were not detected, however, the origin (i.e., agricultural company), LAB strain used for fermentation and the interaction between these factors were statistically significant on BC redness (a*) coordinate. The microbial contamination among all the tested BC groups was similar. However, different LAB strains used for BC fermentation showed different effects toward the microbial contamination reduction, and specifically Lc. paracasei was more effective than Lp. plantarum strain. Predominant BA in BC were putrescine and cadaverine. The main VC in nonfermented and fermented BC were decane, 2-ethyl-1-hexanol, dodecane, 1,3-di-tert-butylbenzene, 3,6-dimethyldecane and tetradecane. Moreover, this study showed worrying trends with respect to the frozen colostrum storage, because most of the dominant VC in BC were contaminants from the packaging material. Additionally, significant correlations between separate VC and microbial contamination were obtained. Finally, these experimental results showed that the separate VC in BC can be an important marker for biological as well as chemical contamination of BC. Also, it should be pointed out that despite the fermentation with LAB is usually described as a safe and natural process with many advantages, control of BA in the end product is necessary.

Fluorescence turn-on mode of Eu3+ complex nanocomposite to detect histamine for seafood freshness.

Biogenic amines (BAs), which naturally occur as chemicals in seafood, are indicators of food freshness and quality. High concentrations of BAs can cause an undesirable inflammatory response. However, traditional detection methods cannot meet the needs of rapid analysis nowadays. It is essential to explore a simple and valid method to monitor the food quality. Herein, we design and prepare a nanoclay-based turn on fluorescent material with BAs response, which could be used for the real-time and visual detection of raw fish freshness. As the concentration of BAs increase, the sensor of the fluorescence signal is significantly enhanced. The sensor demonstrated wonderful response and sensitivity which showed a detection limit of 0.935 mg/L for typical BAs histamine within a linear range of 2-14 mg/L in an aqueous solution. More importantly, we developed a responsive BAs device by doping the sensor into polyvinyl alcohol (PVA), which is well applied as a rapid-responsive fluorescent marker for visual monitoring the freshness of raw fish.

Silver metallization-triggered liposome-embedded AIE fluorophore for dual-mode detection of biogenic amines to fight food freshness fraud.

To combat food freshness fraud, it is urgent to develop a method which could realize the detection of biogenic amines (BAs) present in food. In our study, we developed a colorimetric and ratiometric fluorescence dual-mode sensor which integrated with silver metallization-based response system of AIE liposome + OPD + RSM + Ag+ toward BAs in foods for fighting freshness fraud. With the hydrolysis from the alkaline of BAs to resorcinol monoacetate (RSM), the production resorcinol (RS) could metallize silver ion (Ag+) to silver atoms (Ag0) which could lead to a BAs concentration-dependent decrease of the oxidation product 2,3-diaminophenothiazine (DAP) of Ag+ to o-phenylenediamine (OPD). As a result, the dual-mode sensor has a low detection limit and wide linear range in the spiked detection of soy products, pork and milk samples for BAs. Thus, providing a reliable method for food safety and forestalling food freshness fraud.

Effect of alginate-gallic acid coating on freshness and flavor properties of Mackerel (Scomberomorus commerson) fillets under refrigerated storage.

This study investigates the effect of sodium alginate-gallic acid (ALG-GAL) coating on mackerel's flavor compounds and quality properties during cold storage at 4 °C for 12 days. To this end, freshness quality indicators, including biogenic amines (BAs), volatile organic compounds (VOCs), ATP-related compounds, K value, total viable counts (TVC), thiobarbituric acid (TBA), and sensory assessment, were measured. During storage, eight BAs, i.e., histamine (HIS), tyramine (TYR), putrescine (PUT), cadaverine (CAD), 2-phenylethylamine (2-PHE), agimation, spermine (SPM), and spermidine (SPD) were identified in control and treated samples. The biogenic amine index (BAI) for control samples was 56.25 at the time of sensory rejection (day 6). BAI for samples coated with ALG-GAL did not exceed 20 mg/100 g at the time of sensory rejection (day 12). The fillets treated with the ALG alone or incorporated with GAL possessed a different trend in the retardation of VOCs, including aldehydes, ketones, alcohols, and hydrocarbons. Seven key flavors VOCs, including 3-methylbutanal, phenylacetaldehyde, E-2-hexanal, 1-hexanol, 1-octen-3-ol, 2,3 pentanedione, and hydroxyl-2-butanone, were identified in control and coated samples. Samples coated with ALG and GAL were of significantly higher quality (p < 0.05) throughout storage, which could result in lower Inosine (HxR) concentrations and K values. The results of TVC showed that use ALG-GAL had lower bacterial counts compared to control (p < 0.05). The ALG-GAL-coated samples retarded the increase in the contents of TBA during storage. In addition, significant differences in sensory scores between ALG and ALG-GAL were observed (p < 0.05). In this study, aldehydes and hypoxanthine (Hx) were the main compounds in the formation of off-flavor. These results revealed that ALG coating combined with GAL improved the quality of refrigerated mackerel fillets by decreasing off-flavor compounds and TVC population.

Modulating Substrate Specificity of Rhizobium sp. Histamine Dehydrogenase through Protein Engineering for Food Quality Applications.

Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (Vmax) increased for variant Phe72Thr at the cost of an increased value for the Michaelis-Menten constant (Km) for histamine. The increased Km value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this enzyme class.

Cyanostilbene-based fluorescent paper array for monitoring fish and meat freshness via amino content detection.

The detection of biogenic amines released from degraded meats is an effective method for evaluating meat freshness. However, existing traditional methods like titration are deemed tedious, while the use of sophisticated analytical instruments is not amenable to field testing. Herein, a cyanostilbene-based fluorescent array was rapidly fabricated using macroarray synthesis on a cellulose paper surface to detect amines liberated from spoiled beef, fish, and chicken. The fluorescence changes of immobilized molecules from the interaction with gaseous amines were used to monitor changes in freshness levels. Thanks to the high-throughput nature of macroarray synthesis, a set of highly responsive molecules such as pyridinium and dicyanovinyl moieties were quickly revealed. Importantly, this method offers flexibility in sensing applications including (1) sensing by individual sensor molecules, where the fluorescence response correlated well with established titration methods, and (2) collective sensing whereby chemometric analysis was used to provide a cutoff of freshness with 73-100% accuracy depending on meat types. Overall, this study paves the way for a robust and cost-effective tool for monitoring meat freshness.

Structural basis of amine odorant perception by a mammal olfactory receptor.

Odorants are detected as smell in the nasal epithelium of mammals by two G-protein-coupled receptor families, the odorant receptors and the trace amine-associated receptors1,2 (TAARs). TAARs emerged following the divergence of jawed and jawless fish, and comprise a large monophyletic family of receptors that recognize volatile amine odorants to elicit both intraspecific and interspecific innate behaviours such as attraction and aversion3-5. Here we report cryo-electron microscopy structures of mouse TAAR9 (mTAAR9) and mTAAR9-Gs or mTAAR9-Golf trimers in complex with ß-phenylethylamine, N,N-dimethylcyclohexylamine or spermidine. The mTAAR9 structures contain a deep and tight ligand-binding pocket decorated with a conserved D3.32W6.48Y7.43 motif, which is essential for amine odorant recognition. In the mTAAR9 structure, a unique disulfide bond connecting the N terminus to ECL2 is required for agonist-induced receptor activation. We identify key structural motifs of TAAR family members for detecting monoamines and polyamines and the shared sequence of different TAAR members that are responsible for recognition of the same odour chemical. We elucidate the molecular basis of mTAAR9 coupling to Gs and Golf by structural characterization and mutational analysis. Collectively, our results provide a structural basis for odorant detection, receptor activation and Golf coupling of an amine olfactory receptor.