Biodegradation Characteristics and Mechanism of Aflatoxin B1 by Bacillus amyloliquefaciens from Enzymatic and Multiomics Perspectives.

Aflatoxin B1 (AFB1), a mycotoxin and natural carcinogen, commonly contaminates cereals and animal feeds, posing serious health risks to human and animal. In this study, Bacillus amyloliquefaciens ZG08 isolated from kimchi could effectively remove 80.93% of AFB1 within 72 h at 37 °C and pH 7.0. Metabolome and transcriptome analysis showed that metabolic processes including glycerophospholipid metabolism and amino acid metabolism were most affected in B. amyloliquefaciens ZG08 exposed to AFB1. The adaptation mechanism likely involved activation of the thioredoxin system to restore intracellular redox equilibrium. The key genes, tpx and gldA, overexpressed in Escherichia coli BL21, achieved degradation rates of 60.15% and 47.16% for 100 µg/kg AFB1 under optimal conditions of 37 °C and pH 8.0 and 45 °C and pH 7.0, respectively. The degradation products, identified as AFD1, were less cytotoxic than AFB1 in HepG2 cells. These findings suggest potential strategies for utilizing probiotics and engineered enzymes in AFB1 detoxification.

The Similarities in Microbial and Chemical Patterns of Fermentation in Two Open Environments were Promoted by Using 150-Year-Old Nukadoko as Starters.

Nukadoko, a fermented rice bran employed in traditional Japanese pickling, uses lactic acid bacteria to ferment vegetables. Here, we report the microbial and chemical data of a mixture of matured 150-year-old nukadoko and commercially available rice bran placed in two open environments over 29 days. Across the two environments, Loigolactobacillus was identified as the dominant microbial genera in the later stages of fermentation in nukadoko. The period of increase in the relative abundance of Loigolactobacillus correlated with a decrease in pH and Oxidation-Reduction Potential (ORP) values. While the two environments showed a difference in the rate of change in microbial diversity, they shared the common process through which Loigolactobacillus outcompeted adventitious bacteria in nukadoko, as indicated by the alpha and beta diversity index. Thus, the similarities in microbial and chemical data across two open environments during fermentation using starters indicate that starters contribute to the stability of fermentation in open environments.

Species-level understanding of the bacterial community in Daqu based on full-length 16S rRNA gene sequences.

Daqu is used as the fermentation starter of Baijiu and contributes diversified functional microbes for saccharifying grains and converting sugars into ethanol and aroma components in Baijiu products. Daqu is mainly classified into three types, namely low (LTD), medium (MTD) and high (HTD) temperature Daqu, according to the highest temperatures reached in their fermentation processes. In this study, we used the PacBio small-molecule real-time (SMRT) sequencing technology to determine the full-length 16 S rRNA gene sequences from the metagenomes of 296 samples of different types of Daqu collected from ten provinces in China, and revealed the bacterial diversity at the species level in the Daqu samples. We totally identified 310 bacteria species, including 78 highly abundant species (with a relative abundance >0.1% each) which accounted for 91.90% of the reads from all the Daqu samples. We also recognized the differentially enriched bacterial species in different types of Daqu, and in the Daqu samples with the same type but from different provinces. Specifically, Lactobacillales, Enterobacterales and Bacillaceae were significantly enriched in the LTD, MTD and HTD groups, respectively. The potential co-existence and exclusion relationships among the bacteria species involved in all the Daqu samples and in the LTD, MTD and HTD samples from a specific region were also identified. These results provide a better understanding of the bacterial diversity in different types of Daqu at the species level.

Differential adaptation of the yeast Candida anglica to fermented food.

A single strain of Candida anglica, isolated from cider, is available in international yeast collections. We present here seven new strains isolated from French PDO cheeses. For one of the cheese strains, we achieved a high-quality genome assembly of 13.7 Mb with eight near-complete telomere-to-telomere chromosomes. The genomes of two additional cheese strains and of the cider strain were also assembled and annotated, resulting in a core genome of 5966 coding sequences. Phylogenetic analysis showed that the seven cheese strains clustered together, away from the cider strain. Mating-type locus analysis revealed the presence of a MATa locus in the cider strain but a MATalpha locus in all cheese strains. The presence of LINE retrotransposons at identical genome position in the cheese strains, and two different karyotypic profiles resulting from chromosomal rearrangements were observed. Together, these findings are consistent with clonal propagation of the cheese strains. Phenotypic trait variations were observed within the cheese population under stress conditions whereas the cider strain was found to have a much greater capacity for growth in all conditions tested.

A concise review of the nutritional profiles, microbial dynamics, and health impacts of fermented mushrooms.

Mushrooms have garnered significant attention for their nutritional composition and potential health benefits, including antioxidant, antihypertensive, and cholesterol-lowering properties. This review explores the nutritional composition of edible mushrooms, including their high protein content, essential amino acids, low fat, cholesterol levels, and bioactive compounds with medicinal value. Moreover, the study analyzes the microbiology of mushroom fermentation, focusing on the diverse microbial ecosystem involved in the transformation of raw mushrooms and the preservation methods employed to extend their shelf life. Special emphasis is placed on lactic acid fermentation as a cost-effective and efficient preservation technique. It involves controlling the growth of lactic acid bacteria to enhance the microbial stability and nutritional quality of mushrooms. Furthermore, the bioactivities of fermented mushrooms are elucidated, which are antioxidant, antimicrobial, anticancer, anti-glycemic, immune modulatory, and other biological activities. The mechanisms underlying these bioactivities are explored, emphasizing the role of fermented mushrooms in suppressing free radicals, enhancing antioxidant defenses, and modulating immune responses. Overall, this review provides comprehensive insights into the nutritional composition, microbiology, bioactivities, and underlying mechanisms of fermented mushrooms, highlighting their potential as functional foods with significant health-promoting properties.

Effects of community ecological network construction on physicochemical, microbial, and quality characteristics of inoculated northeast sauerkraut: A new insight in food fermentation processes.

The enhancement of the quality of northeast sauerkraut can be achieved by inoculation with lactic acid bacteria. However, a comprehensive ecological understanding of the intricate dynamic processes involved is currently lacking, which could yield valuable insights for regulating sauerkraut fermentation. This study compares spontaneously sauerkrauts with the sauerkrauts inoculated with autochthonous Lactiplantibacillus plantarum SC-MDJ and commercial L. plantarum, respectively. We examine their physicochemical properties, quality characteristics, bacterial community dynamics, and ecological network interactions. Inoculation with L. plantarum leads to reduced bacterial community richness and niche breadth, but an increase in robustness, interactions, and assembly processes. Notably, there appears to be a potential correlation between bacterial community structure and quality characteristics. Particularly, sauerkraut inoculated with L. plantarum SC-MDJ may produce a sourness more quickly, possibly attributed to the enhanced ecological role of L. plantarum SC-MDJ. This study establishes a foundation for the targeted regulation of sauerkraut fermentation.

Study of the effects of pasteurization and selected microbial starters on functional traits of fermented table olives.

Table olives are one of the most known fruit consumed as fermented food, being a fundamental component of the Mediterranean diet. Their production and consumption continue to increase globally and represent an important economic source for the producing countries. One of the most stimulating challenges for the future is the modernization of olive fermentation process. Besides the demand for more reproducible and safer production methods that could be able to reduce product losses and potential risks, producers and consumers are increasingly attracted by the final product characteristics and properties on human health. In this study, the contribution of microbial starters to table olives was fully described in terms of specific enzymatic and microbiological profiles, nutrient components, fermentation-derived compounds, and content of bioactive compounds. The use of microbial starters from different sources was tested considering their technological features and potential ability to improve the functional traits of fermented black table olives. For each fermentation assay, the effects of controlled temperature (kept at 20 °C constantly) versus not controlled environmental conditions (oscillating between 7 and 17 °C), as well as the consequences of the pasteurization treatment were tested on the final products. Starter-driven fermentation strategies seemed to increase both total phenolic content and total antioxidant activity. Herein, among all the tested microbial starters, we provide data indicating that two bacterial strains (Leuconostoc mesenteroides KT 5-1 and Lactiplantibacillus plantarum BC T3-35), and two yeast strains (Saccharomyces cerevisiae 10A and Debaryomyces hansenii A15-44) were the better ones related to enzyme activities, total phenolic content and antioxidant activity. We also demonstrated that the fermentation of black table olives under not controlled environmental temperature conditions was more promising than the controlled level of 20 °C constantly in terms of technological and functional properties considered in this study. Moreover, we confirmed that the pasteurization process had a role in enhancing the levels of antioxidant compounds.

Effects of seasonal harvest of kimchi cabbage on microbial and metabolic profiles of kimchi.

Kimchi cabbage, the key ingredient in kimchi, is cultivated year-round to meet high production demands. This study aimed to examine the effects of seasonal harvesting (spring, summer, fall, and winter) on the microbial and metabolic profiles of kimchi during 30 days of fermentation. Lactic acid bacteria distribution is notably influenced by seasonal variations, with Latilactobacillus dominant in fall-harvested kimchi group and Weissella prevailing in spring, summer, and winter. The microbial communities of spring and fall group exhibited similar profiles before fermentation, whereas the microbial communities and metabolic profiles of spring and summer group were similar after 30 days of fermentation. Seasonal disparities in metabolite concentrations, including glutamic acid, serine, and cytosine, persist throughout fermentation. This study provides a comprehensive understanding of the substantial impact of seasonal harvesting of kimchi cabbage on the microbial and metabolic characteristics of kimchi, providing valuable insights into producing kimchi with diverse qualities.

Lacto-fermented garlic handcrafted in the Lower Silesia Region (Poland): Microbial diversity, morpho-textural traits, and volatile compounds.

The aim of the present study was to provide a first characterization of lacto-fermented garlic manufactured by local small-scale artisanal producers in the Lower Silesia Region (Poland). The lacto-fermented garlic samples showed high nutritional features in terms of antioxidant activity. A total of 86 compounds, belonging to various chemical classes, were identified by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC/MS). Most of these compounds belonged to six main classes, being sulfur compounds, esters and acetates, oxygenated monoterpenes, monoterpene hydrocarbons, and alcohols. Aldehydes, acids, ketones, furans, and phenols were also identified. In the analyzed samples, counts up to 8 log cfu g-1 were observed for lactic acid bacteria. Metataxonomic analysis revealed the presence of Levilactobacillus, Lactiplantibacillus, Latilactobacillus, Secundilactobacillus, Weissella, Leuconostoc, Lactococcus, Pediococcus, and Lacticaseibacillus among the major taxa. These results were confirmed by the isolation and characterization of viable lactic acid bacteria. Indeed, the presence of the closest relatives to Lacticaseibacillus casei group, Pediococcus parvulus, Levilactobacillus brevis, Levilactobacillus parabrevis, and Lactiplantibacillus plantarum group was observed. A good acidification performance in salty garlic-based medium was observed for all the isolates that, between 8 and 15 days of fermentation, reached pH values comprised between 4 and 3.5, depending on the tested species. Of note, 15 out of the 37 lactic acid bacteria isolates (Levilactobacillus parabrevis, Pediococcus parvulus, Lactiplantibacillus plantarum group, and Lacticaseibacillus casei group) showed the presence of the hdcA gene of Gram-positive bacteria encoding for histidine decarboxylase. Furthermore, for 8 out of the 37 isolates the in-vitro exopolysaccharides production was observed. No isolate showed inhibitory activity against the three Listeria innocua strains used as surrogate for Listeria monocytogenes.

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.

Novel misos shape distinct microbial ecologies: opportunities for flavourful sustainable food innovation.

Fermentation is resurgent around the world as people seek healthier, more sustainable, and tasty food options. This study explores the microbial ecology of miso, a traditional Japanese fermented paste, made with novel regional substrates to develop new plant-based foods. Eight novel miso varieties were developed using different protein-rich substrates: yellow peas, Gotland lentils, and fava beans (each with two treatments: standard and nixtamalisation), as well as rye bread and soybeans. The misos were produced at Noma, a restaurant in Copenhagen, Denmark. Samples were analysed with biological and technical triplicates at the beginning and end of fermentation. We also incorporated in this study six samples of novel misos produced following the same recipe at Inua, a former affiliate restaurant of Noma in Tokyo, Japan. To analyse microbial community structure and diversity, metabarcoding (16S and ITS) and shotgun metagenomic analyses were performed. The misos contain a greater range of microbes than is currently described for miso in the literature. The composition of the novel yellow pea misos was notably similar to the traditional soybean ones, suggesting they are a good alternative, which supports our culinary collaborators' sensory conclusions. For bacteria, we found that overall substrate had the strongest effect, followed by time, treatment (nixtamalisation), and geography. For fungi, there was a slightly stronger effect of geography and a mild effect of substrate, and no significant effects for treatment or time. Based on an analysis of metagenome-assembled genomes (MAGs), strains of Staphylococccus epidermidis differentiated according to substrate. Carotenoid biosynthesis genes in these MAGs appeared in strains from Japan but not from Denmark, suggesting a possible gene-level geographical effect. The benign and possibly functional presence of S. epidermidis in these misos, a species typically associated with the human skin microbiome, suggests possible adaptation to the miso niche, and the flow of microbes between bodies and foods in certain fermentation as more common than is currently recognised. This study improves our understanding of miso ecology, highlights the potential for developing novel misos using diverse local ingredients, and suggests how fermentation innovation can contribute to studies of microbial ecology and evolution.

Synthetic microbial communities: Novel strategies to enhance the quality of traditional fermented foods.

Consumers are attracted to traditional fermented foods due to their unique flavor and nutritional value. However, the traditional fermentation technique can no longer accommodate the requirements of the food industry. Traditional fermented foods produce hazardous compounds, off-odor, and anti-nutritional factors, reducing product stability. The microbial system complexity of traditional fermented foods resulting from the open fermentation process has made it challenging to regulate these problems by modifying microbial behaviors. Synthetic microbial communities (SynComs) have been shown to simplify complex microbial communities and allow for the targeted design of microbial communities, which has been applied in processing traditional fermented foods. Herein, we describe the theoretical information of SynComs, particularly microbial physiological processes and their interactions. This paper discusses current approaches to creating SynComs, including designing, building, testing, and learning, with typical applications and fundamental techniques. Based on various traditional fermented food innovation demands, the potential and application of SynComs in enhancing the quality of traditional fermented foods are highlighted. SynComs showed superior performance in regulating the quality of traditional fermented foods using the interaction of core microorganisms to reduce the hazardous compounds of traditional fermented foods and improve flavor. Additionally, we presented the current status and future perspectives of SynComs for improving the quality of traditional fermented foods.

Multidrug resistance profile in Lactobacillus delbrueckii: a food industry species with probiotic properties.

Lactobacillus delbrueckii, a widely used lactic acid bacterium in the food industry, has been studied for its probiotic properties and reservoir of antibiotic-resistant genes, raising safety concerns for probiotic formulations and fermented products. This review consolidates findings from 60 articles published between 2012 and 2023, focusing on the global antibiotic resistance profile and associated genetic factors in L. delbrueckii strains. Resistance to aminoglycosides, particularly streptomycin, kanamycin, and gentamicin, as well as resistance to glycopeptides (vancomycin), fluoroquinolones (ciprofloxacin), and tetracyclines was predominant. Notably, although resistance genes have been identified, they have not been linked to mobile genetic elements, reducing the risk of dissemination. However, a significant limitation is the insufficient exploration of responsible genes or mobile elements in 80% of studies, hindering safety assessments. Additionally, most articles originated from Asian and Middle Eastern countries, with strains often isolated from fermented dairy foods. Therefore, these findings underscore the necessity for comprehensive analyses of new strains of L. delbrueckii for potential industrial and biotherapeutic applications and in combating the rise of antibiotic-resistant pathogens.

Microbial aromatic amino acid metabolism is modifiable in fermented food matrices to promote bioactivity.

Ingestion of fermented foods impacts human immune function, yet the bioactive food components underlying these effects are not understood. Here, we interrogated whether fermented food bioactivity relates to microbial metabolites derived from aromatic amino acids, termed aryl-lactates. Using targeted metabolomics, we established the presence of aryl-lactates in commercially available fermented foods. After pinpointing fermented food-associated lactic acid bacteria that produce high levels of aryl-lactates, we identified fermentation conditions to increase aryl-lactate production in food matrices up to 5 × 103 fold vs. standard fermentation conditions. Using ex vivo reporter assays, we found that food matrix conditions optimized for aryl-lactate production exhibited enhanced agonist activity for the human aryl-hydrocarbon receptor (AhR) as compared to standard fermentation conditions and commercial products. Reduced microbial-induced AhR activity has emerged as a hallmark of many chronic inflammatory diseases, thus we envision strategies to enhance AhR bioactivity of fermented foods to be leveraged to improve human health.

Functional roles and engineering strategies to improve the industrial functionalities of lactic acid bacteria during food fermentation.

In order to improve the flavor profiles, food security, probiotic effects and shorten the fermentation period of traditional fermented foods, lactic acid bacteria (LAB) were often considered as the ideal candidate to participate in the fermentation process. In general, LAB strains possessed the ability to develop flavor compounds via carbohydrate metabolism, protein hydrolysis and amino acid metabolism, lipid hydrolysis and fatty acid metabolism. Based on the functional properties to inhibit spoilage microbes, foodborne pathogens and fungi, those species could improve the safety properties and prolong the shelf life of fermented products. Meanwhile, influence of LAB on texture and functionality of fermented food were also involved in this review. As for the adverse effect carried by environmental challenges during fermentation process, engineering strategies based on exogenous addition, cross protection, and metabolic engineering to improve the robustness and of LAB were also discussed in this review. Besides, this review also summarized the potential strategies including microbial co-culture and metabolic engineering for improvement of fermentation performance in LAB strains. The authors hope this review could contribute to provide an understanding and insight into improving the industrial functionalities of LAB.

Starting with screening strains to construct synthetic microbial communities (SynComs) for traditional food fermentation.

With the elucidation of community structures and assembly mechanisms in various fermented foods, core communities that significantly influence or guide fermentation have been pinpointed and used for exogenous restructuring into synthetic microbial communities (SynComs). These SynComs simulate ecological systems or function as adjuncts or substitutes in starters, and their efficacy has been widely verified. However, screening and assembly are still the main limiting factors for implementing theoretic SynComs, as desired strains cannot be effectively obtained and integrated. To expand strain screening methods suitable for SynComs in food fermentation, this review summarizes the recent research trends in using SynComs to study community evolution or interaction and improve the quality of food fermentation, as well as the specific process of constructing synthetic communities. The potential for novel screening modalities based on genes, enzymes and metabolites in food microbial screening is discussed, along with the emphasis on strategies to optimize assembly for facilitating the development of synthetic communities.

Unveiling the synthesis of aromatic compounds in sauce-flavor Daqu from the functional microorganisms to enzymes.

Aromatic compounds serve as the primary source of floral and fruity aromas in sauce-flavor (Maotai flavor) baijiu, constituting the skeleton components of its flavor profile. Nevertheless, the formation mechanism of these compounds and key aroma-producing enzymes in sauce-flavor Daqu (fermentation agent, SFD) remain elusive. Here, we combined metagenomics, metaproteomics, metabolomics, and key enzyme activity to verify the biosynthesis pathway of aromatic compounds and to identify key enzymes, genes, and characteristic microorganisms in SFD. The results showed that the later period of fermentation was critical for the generation of aromatic compounds in SFD. In-situ verification was conducted on the potential key enzymes and profiles in various metabolites, providing comprehensive evidence for the main synthetic pathways of aromatic compounds in SFD. Notably, our results showed that primary amine oxidase (PrAO) and aldehyde dehydrogenase (ALDH) emerged as two key enzymes promoting aromatic compound synthesis. Additionally, two potential key functional genes regulating aromatics generation were identified during SFD fermentation through correlation analysis between proteins and relevant metabolites, coupled with in vitro amplification test. Furthermore, original functional strains (Aspergillus flavus-C10 and Aspergillus niger-IN2) exhibiting high PrAO and ALDH production were successfully isolated from SFD, thus validating the results of metagenomics and metaproteomics analyses. This study comprehensively elucidates the pathway of aromatic compound formation in SFD at the genetic, proteomic, enzymatic, and metabolomic levels, providing new ideas for the investigation of key flavor substances in baijiu. Additionally, these findings offer valuable insights into the regulatory mechanisms of aromatic compounds generation.

Whole genome sequencing of the poly-γ-glutamic acid-producing novel Bacillus subtilis Tamang strain, isolated from spontaneously fermented kinema.

Kinema, a traditional fermented soybean food from the Himalayas, is well-liked for its sticky texture and flavourful umami taste. Among 175 bacterial strains from spontaneously fermented kinema samples, Bacillus subtilis Tamang strain stood out for its high stickiness and viscosity. The strain's Poly-γ-glutamic acid (γ-PGA) contains various groups of glutamic acid and has a molecular weight of 660 kDa. It demonstrates the ability to solubilize iron, preserve ferritin in Caco-2 cells, and exhibit antibacterial properties. The genome of B. subtilis Tamang is devoid of plasmid elements but does feature nine insert elements. Noteworthy is the presence of unique secondary metabolites with potential antimicrobial effects, such as amyloliquecidin GF610, bogorol A, and thermoactinoamide A. A total of 132 carbohydrate-active enzymes (CAZy) were identified, hinting at possible prebiotic characteristics. The genome analysis revealed genes responsible for γ-PGA production via the capBCA complex. Furthermore, genes associated with fibrinolytic activity, taste enhancement, biopeptides, immunomodulators, and vitamins like B12 and K2 were found, along with probiotics and various health benefits. The genetic material for L-asparaginase production, known for its anti-cancer properties, was also detected, as well as CRISPR-Cas systems. The absence of virulence factors and antimicrobial resistance genes confirms the safety of consuming B. subtilis Tamang as a food-grade bacterium.

A comprehensive review on novel synthetic foods: Potential risk factors, detection strategies, and processing technologies.

Nowadays, the food industry is facing challenges due to the simultaneous rise in global warming, population, and food consumption. As the integration of synthetic biology and food science, novel synthetic foods have obtained high attention to address these issues. However, these novel foods may cause potential risks related to human health. Four types of novel synthetic foods, including plant-based foods, cultured meat, fermented foods, and microalgae-based foods, were reviewed in the study. The original food sources, consumer acceptance, advantages and disadvantages of these foods were discussed. Furthermore, potential risk factors, such as nutritional, biological, and chemical risk factors, associated with these foods were described and analyzed. Additionally, the current detection methods (e.g., enzyme-linked immunosorbent assay, biosensors, chromatography, polymerase chain reaction, isothermal amplification, and microfluidic technology) and processing technologies (e.g., microwave treatment, ohmic heating, steam explosion, high hydrostatic pressure, ultrasound, cold plasma, and supercritical carbon dioxide) were reviewed and discussed critically. Nonetheless, it is crucial to continue innovating and developing new detection and processing technologies to effectively evaluate these novel synthetic foods and ensure their safety. Finally, approaches to enhance the quality of these foods were briefly presented. It will provide insights into the development and management of novel synthetic foods for food industry.

Extraction of Proteins from Fermented Food.

The metaproteomic approach allows a deep microbiome characterization in different complex systems. Based on metaproteome data, microbial communities' composition, succession, and functional role in different environmental conditions can be established.The main challenge in metaproteomic studies is protein extraction, and although many protocols have been developed, a few are focused on the protein extraction of fermented foods. In this chapter, a reproducible and efficient method for the extraction of proteins from a traditionally fermented starchy food is described. The method can be applied to any fermented food and aims to enrich the extraction of proteins from microorganisms for their subsequent characterization.