Search Results (67)

The relationship between protein structure and function is intrinsically interconnected, as the structure of a protein directly determines its functional properties. To investigate the effects of temperature and pressure on protein function, this study employed ethyl carbamate (EC) hydrolase as a model food enzyme and conducted molecular dynamics (MD) simulations under varying temperature and pressure levels to elucidate its structure–function relationship. By systematically analyzing the dynamic changes in root mean square deviation (RMSD), root mean square fluctuation (RMSF), radius of gyration (Rg), solvent accessible surface area (SASA), hydrogen bonding, catalytic pocket conformation, and packing density under different temperature and pressure conditions, we revealed the structural adaptability of EC hydrolase. Furthermore, we analyzed the characteristics of EC hydrolase using molecular dynamics simulations with temperature and pressure levels, as well as conformational bias-based computer-aided engineering, providing both theoretical and experimental foundation for the adaptability mechanisms of enzymes under extreme conditions. Full article

Malolactic fermentation (MLF), an essential enological process for wine deacidification and aroma development, is predominantly mediated by Oenococcus oeni (O. oeni). This investigation characterized 170 indigenous O. oeni isolates from two principal Chinese viticultural regions (Yinchuan, Ningxia, and Changli, Hebei) through polyphasic analysis. Forty-nine strains demonstrating genetic potential for efficient malate metabolism and biosafety compliance (absence of ethyl carbamate and biogenic amines genes) were subjected to adaptive laboratory evolution under enologically relevant stress conditions. Comparative evaluation with the superior indigenous strain SD-2a revealed eight stress-adapted isolates exhibiting superior MLF kinetics, completing L-malic acid degradation in Marselan wine. Solid-phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS) profiling identified three isolates’ (3-31, 9-10, and 9-50) significant enhancement of key fermentation aromas in experimental fermentations. These oenologically adapted indigenous strains demonstrate promising potential as regional-specific starter cultures, providing a scientific foundation for developing terroir-expressive winemaking practices and optimizing microbial resources in China’s wine industry. Full article

Ethyl carbamate (EC), known as urethane, is a naturally occurring potentially carcinogenic metabolite that is widely found in alcoholic beverages and other food-related fermented products. The concern related to the presence of the EC and its toxicity in regularly consumed fermented alcoholic beverages raises global interest in assessing the possible risks to human health. EC mitigation approaches, such as molecular imprinting technology (MIT), have been proposed to target EC while preserving the sensory quality of fermented alcoholic beverages. This review explores the principles of MIT, the advantages and disadvantages of the most common polymerisation approach for molecularly imprinted polymer (MIP) synthesis, the analytical techniques used for MIP characterisation, and the strategies used to mitigate EC in fermented alcoholic beverages, with studies reporting removal efficiencies of up to 84%. Additionally, it highlights the novelty and potential of MIPs, offering practical insights into their integration within the production of fermented alcoholic beverages, highlighting their scalability and cost-effectiveness compared to traditional EC mitigation strategies. Full article

Background/Objectives: Natural cytokinins are a promising group of anti-tumor agents. In this work, we hypothesized that modification of the ethylenediurea moiety with alkyl and oxygen-containing groups could be a way to enhance the anti-proliferative properties of the molecule. Methods: Ten new analogs of ethylenediurea with these substitutions were tested for anti-proliferative activity in the human cancer cell lines MDA-MB-231 (breast cancer), A-375 (melanoma), and U-87 MG (glioblastoma) during 72 h of incubation using resazurin test and evaluated the substances receptor using molecular docking. Results: The compound with the carbamate link and ethylene substituent on the phenyl ring inhibited proliferation in these models by 70–90% without cytotoxic effects. The compound did not affect the viability of the immortalized fibroblast cell line Bj-5ta. The compound was also able to enhance the action of doxorubicin and temozolomide by about 20%. According to the molecular modeling data, the probable receptor target for the synthesized compound was the A2AR adenosine receptor. Conclusions: The results obtained on the ethylenediurea analogs with ethyl substituent in the aromatic ring are promising for the development of novel anticancer therapeutics. Full article

Lipid-like nanoparticles (LLNPs) have been shown to be an effective encapsulation and delivery tool for therapeutic molecules. While the preclinical development of lipid nanoparticle formulations has been of paramount importance, next-generation LLNPs present an opportunity of enhanced biocompatibility. With the change in amido functionality as part of the core backbone, our target, carbamate functionality within the LLNP core scaffold, was realized upon reaction of a protected amino alcohol onto the isocyanate generated in situ via a Curtius rearrangement. The single-step assembly of carbamate functionality starting from cyclohexane carboxylic acid in the presence of diphenylphosphoryl azide (DPPA) exceeds the metrics set forth for the rapid installment and enhanced biodegradability of next-generation lipid-like nanoparticles. Full article

In winemaking, malolactic fermentation (MLF), which converts L-malic acid to L-lactic acid, is often applied after the alcoholic fermentation stage to improve the sensory properties of the wine and its microbiological stability. MLF is usually performed by lactic acid bacteria, which, however, are sensitive to the conditions of alcoholic fermentation. Therefore, the development of wine yeast strains capable of both alcoholic fermentation and MLF is an important task. Using genome editing, we engineered a modified variant of the triploid wine yeast strain Saccharomyces cerevisiae I-328, in which the CAR1 arginase gene was replaced by the malate permease gene from Schizosaccharomyces pombe and the malolactic enzyme gene from Oenococcus oeni. Genome-wide transcriptional profiling confirmed the expression of the introduced genes and revealed a limited effect of the modification on global gene expression. Winemaking experiments show that genome editing did not affect fermentation activity and ethanol production, while use of the modified strain resulted in a tenfold reduction in malate content with simultaneous formation of lactate. The resulting wines had a softer and more harmonious taste compared to wine obtained using the parental strain. Inactivation of arginase, which forms urea and L-ornithine through the breakdown of arginine, also resulted in a twofold decrease in the content of urea and the carcinogenic ethyl carbamate in wine. Thus, the new strain with the replacement of the arginase gene with the MLF gene cassette is promising for use in winemaking. Full article

Enzymatic degradation of the carcinogen ethyl carbamate (EC) and its precursor urea is a promising method for controlling EC levels in alcoholic beverages. However, limited enzymes with EC-hydrolyzing activity and low ethanol or acid tolerance hinder their practical application. Here, a new urease with urea- and EC-hydrolyzing activities from Providencia sp. LBBE was characterized. The enzyme displayed considerable ethanol tolerance, retaining 42.4% activity after 1 h of incubation with 30% (v/v) ethanol at 37 °C. It exhibited broad pH tolerance (pH 3.0–8.0), with optimal pH 7.0 for EC and 7.5 for urea. After treatment at pH 4.5 and 5.0, it retained 41.3% and 59.4% activity, respectively. The K_m and V_max for EC and urea at pH 4.5 were 515.6 mM, 33.9 µmol/(min⸱mg) and 32.0 mM, 263.6 µmol/(min⸱mg), respectively. Using 6000 U/L purified enzyme at 30 °C for 9 h, 49.8% and 81.6% of urea was removed from rice wine (pH 4.5 and 7.0), respectively. No appreciable reduction in EC was observed under identical conditions, which may be ascribed to the minimal EC affinity. This study contributes to the future realization of the effective control of EC content in alcoholic beverages. Full article

Fermented foods have gained global attention for their unique flavor and immense health benefits. These flavor compounds and nutrients result from the metabolic activities of microorganism during fermentation. However, some unpleasant sensory characteristics and biohazard substances could also be generated in fermentation process. These quality and safety issues in fermented foods could be addressed by endogenous enzymes. In this review, the applications of enzymes in quality control of fermented foods, including texture improvement, appearance stability, aroma enhancement, and debittering, are discussed. Furthermore, the enzymes employed in eliminating biohazard compounds such as ethyl carbamate, biogenic amines, and nitrites, formed during fermentation, are reviewed. Advanced biological methods used for enhancing the enzymatic activity and stability are also summarized. This review focused on the applications and future prospects of enzymes in the improvement quality and safety qualities of fermented foods. Full article

In the present study, changes in the physicochemical indices, ethyl carbamate (EC) precursor and EC contents, and microbial communities of fermented grains under different fermentation patterns during strong-aroma Baijiu (SAB) fermentation and changes in EC precursor and EC contents during distillation were investigated to study EC formation during these processes. In detail, the amounts of sorghum added in protocols C and D were half those added in protocols A and B (the normal SAB-producing technology). When fermented for about 30 to 35 days, the fermented grains of protocols A and C were, respectively, remixed with Daqu and second-distilled SAB (so-called “Huijiu jiaqu”, HJJQ) and fermented for about 30 to 40 days. The results showed that the acidities of the final fermented grains of protocols A (2.43 ± 0.09 mmol/10 g) and C (3.18 ± 0.08 mmol/10 g) were lower than those of protocols B (3.71 ± 0.07 mmol/10 g) and D (4.66 ± 0.10 mmol/10 g), while the alcohol contents in the final fermented grains of protocols A (18.33 ± 0.76%) and C (15.33 ± 1.08%) were higher than those of protocols B (5.10 ± 0.85%) and D (1.85 ± 0.62%). No significant differences were observed in the other physicochemical indices among the samples. The HJJQ operation significantly increases the alcohol content and reduces the acidity of the fermented grains but has little influence on the other physicochemical indices during SAB fermentation. Excluding the influence of the HJJQ operation and a half input of sorghum on the EC precursor and EC contents for the fermented grains of protocol B, the linear relationships between the EC content and alcohol (R²: 0.4465), citrulline (R²: 0.6962), urea (R²: 0.4705), and HCN (R²: 0.6324) contents were good (all the confidence levels were at 0.05), indicating that these compounds were the dominant EC precursors during SAB fermentation. HJJQ also facilitated the reaction between alcohol and other EC precursors, decreasing EC precursor content and increasing the EC content. KEGG pathway analysis demonstrated that EC precursors were mainly synthesized by alcohol and arginine metabolism. HCN (R²: 0.3875 to 0.8198) and alcohol (R²: 0.4642 to 0.8423) were the dominant EC precursors during SAB distillation. Overall, the HJJQ operation, especially in protocol C, could significantly reduce the content of EC in base SAB, and the base SAB obtained was of good quality. This, therefore, may be an alternative and effective way to reduce the EC content in base Baijiu. Full article

Chemometric evaluation of potentially harmful volatile compound and toxic metal(loid) distribution patterns in fruit spirits relating to distinct fruit classes most commonly used in spirit production highlighted the potential of several volatiles as candidates for differentiation markers while dismissing toxic metal(loid)s. Pome fruit and grape pomace spirits were mostly characterized by a lower abundance of n-propanol, methanol, ethyl acetate and acetaldehyde, while stone fruit spirits contained lower amounts of isoamyl alcohol and isobutanol. Chemometric analysis of the fruit spirit composition of aromatics identified additional potential markers characteristic for certain fruits—benzoic acid ethyl ester, benzyl alcohol, benzaldehyde, butanoic acid 3-methyl-ethyl ester, butanoic acid 2-methyl-ethyl ester and furfural. This study explored the variability in the risk potential of the investigated spirits, considering that some chemicals known to be detected in spirits are potent health hazards. Ethyl carbamate in combination with acetaldehyde showed a higher potential risk in stone fruit spirits, methanol in stone and pome fruit spirits and acetaldehyde in grape pomace spirits. It is of great interest to evaluate to what extent consumers’ preference for spirits of distinct fruit types affects health risks. Consumers of stone fruit spirits are potentially at higher risk than those consuming pome fruit or grape pomace spirits. Full article

Climate change poses several challenges in the wine industry, including increasing risks related to chemical food contaminants such as biogenic amines and ethyl carbamate (EC). In this work, we focused on urea removal in red wines by immobilized acid urease aiming at limiting EC formation during wine storage. By considering separable kinetics of catalyst deactivation and urea hydrolysis, it was possible to model the time course of urea removal in repeated uses in stirred batch reactors. Treatments based on immobilized urease of red wine enriched with 30 mg/L of urea allowed the reduction in the contaminant concentration to <5 mg/L. After 28.5 h of treatment, the observed urea level was reduced to about 0.5 mg/L, corresponding to a decrease in the potential ethyl carbamate (PEC) from 1662 μg/L to 93 μg/L, below the level of the non-enriched wine (187 μg/L). As a comparison, when treating the same wine with the free enzyme at maximum doses allowed by the EU law, urea and PEC levels decreased to only 12 mg/L and 415 μg/L respectively, after 600 h of treatment. These results show that, for red wines, urease immobilization is an effective strategy for urea removal and, thus, effective reduction in ethyl carbamate as a process contaminant. This study provides the scientific background for the future scaling-up of the process at an industrial level. Full article

Lacticaseibacillus spp. are genetically close lactic acid bacteria species widely used in fermented products for their technological properties as well as their proven beneficial effects on human and animal health. This study, the first to include such a large collection of heterogeneous isolates (121) obtained from international collections belonging to Lacticaseibacillus paracasei, aimed to characterize the safety traits and technological properties of this important probiotic species, also making comparisons with other genetically related species, such as Lacticaseibacillus casei and Lacticaseibacillus zeae. These strains were isolated from a variety of heterogeneous sources, including dairy products, sourdoughs, wine, must, and human body excreta. After a preliminary molecular characterization using repetitive element palindromic PCR (Rep-PCR), Random Amplification of Polymorphic DNA (RAPD), and Sau-PCR, particular attention was paid to safety traits, evaluating antibiotic resistance profiles, biogenic amine (BA) production, the presence of genes related to the production of ethyl carbamate and diaminobenzidine (DAB), and multicopper oxidase activity (MCO). The technological characteristics of the strains, such as the capability to grow at different NaCl and ethanol concentrations and different pH values, were also investigated, as well as the production of bacteriocins. From the obtained results, it was observed that strains isolated from the same type of matrix often shared similar genetic characteristics. However, phenotypic traits were strain-specific. This underscored the vast potential of the different strains to be used for various purposes, from probiotics to bioprotective and starter cultures for food and feed production, highlighting the importance of conducting comprehensive evaluations to identify the most suitable strain for each purpose with the final aim of promoting human health. Full article

Baijiu is a renowned Chinese distilled liquor, notable for its distinctive flavor profile and intricate production process, which prominently involves fermentation and distillation. Ethyl carbamate (EC), a probable human carcinogen, can be potentially formed during these procedures, thus prompting significant health concerns. Consequently, the contamination of EC during Baijiu production has become an increasingly pressing issue. In this study, we developed a rapid and easily operable immunoassay for determining EC in the fermented materials used in Baijiu production. The development of a high-quality antibody specific to EC facilitated a streamlined analytical procedure and heightened method sensitivity. Furthermore, we systematically evaluated other essential parameters. Following optimization, the method achieved an IC₅₀ value of 11.83 μg/kg, with negligible cross-reactivity against EC analogs. The recovery study demonstrated the method’s good accuracy and precision, with mean recovery rates ranging from 86.0% to 105.5% and coefficients of variation all below 10%. To validate the feasibility of the technique, we collected and analyzed 39 samples simultaneously using both the proposed immunoassay and confirmatory gas chromatography–mass spectrometry (GC-MS). A robust correlation was observed between the results obtained from the two methods (R² > 0.99). The detected EC levels ranged from 2.36 μg/kg to 7.08 μg/kg, indicating an increase during the fermentation process. Full article

Ethyl carbamate (EC) is a natural by-product in the production of fermented food and alcoholic beverages and is carcinogenic and genotoxic, posing a significant food safety concern. In this study, Clavispora lusitaniae Cl-p with a strong EC degradation ability was isolated from Daqu rich in microorganisms by using EC as the sole nitrogen source. When 2.5 g/L of EC was added to the fermentation medium, the strain decomposed 47.69% of ethyl carbamate after five days of fermentation. It was unexpectedly found that the strain had the ability to produce aroma and ester, and the esterification power reached 30.78 mg/(g·100 h). When the strain was added to rice wine fermentation, compared with the control group, the EC content decreased by 41.82%, and flavor substances such as ethyl acetate and β-phenylethanol were added. The EC degradation rate of the immobilized crude enzyme in the finished yellow rice wine reached 31.01%, and the flavor substances of yellow rice wine were not affected. The strain is expected to be used in the fermented food industry to reduce EC residue and improve the safety of fermented food. Full article

Ethyl carbamate (EC) is a 2A classified carcinogen in Chinese liquor that has raised many problems regarding food safety. Applying microorganisms to control the content of EC precursors in fermented grains has been proven as an effective method to reduce EC in alcoholic beverages. However, the utilization of microorganisms to decrease the precursors of EC (urea and cyanide) is still incomplete in regard to Chinese liquor. Thus, it is necessary to isolate strains with the degradative activities of urea and cyanide. Herein, Bacillus sonorensis F3 and Bacillus licheniformis YA2 strains were isolated from the fermented grains through multiple rounds of high-throughput screening, and the degradative abilities in urea and cyanide reached 95.72% and 75.48%, respectively. In addition, the urease from the B. sonorensis F3 strain and the carbon nitrogen hydrolase from the B. licheniformis YA2 strain were identified by the heterogeneous expression in Escherichia coli. Then, both F3 and YA2 strains were combined at a ratio of 5:1 and applied to eliminate the EC in the simulated fermentation of Chinese liquor; as a result, 51.10% of EC was reduced without affecting the main composition of flavor substances. The obtained strains have great potential in terms of the improvement of quality and safety of Chinese liquor. Full article