Scholarly Works, Food Science and Technology

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Characterization of Diarrheagenic Escherichia coli and Salmonella enterica from Produce in the Chobe District of Botswana
Bywater, Auja; Dintwe, Galaletsang; Alexander, Kathleen A.; Ponder, Monica A. (Elsevier, 2024-08-24)
Diarrheal disease is a leading cause of death in children in low- and moderate-income countries. Fresh produce, including fruits and vegetables, may harbor diarrheal disease-causing bacteria including strains of Salmonella enterica and Escherichia coli. This study aimed to determine the prevalence and antibiotic resistance profiles of S. enterica and E. coli isolated from produce samples (n = 207) obtained from retail markets in northern Botswana in Chobe District of Botswana in 2022. Samples were enriched in the appropriate selective media: Brilliant Green Bile Broth for E. coli and Rappaport Vassiliadis Broth for S. enterica. E. coli were confirmed by PCR detecting the phoA gene, and classified as potentially pathogenic through screening for the eae, stx, and stx2 and estIb genes. S. enterica isolates were confirmed using invA primers. Isolates were evaluated for resistance to ampicillin, amoxicillin-clavulanic acid, chloramphenicol, cefotaxime, doxycycline, streptomycin, sulfamethoxazole, and tetracycline antibiotic using the Kirby-Bauer Disk Diffusion method. E. coli was isolated from 15.5% of produce samples (n = 207). The gene eae was detected from 1.5% of samples, while stx1, stx2, and estIb were not detected. Resistance to one or more antibiotics was common (72%) with the majority of the resistant E. coli (n = 32) isolated from fruits (22%) and greens (18%) compared to other types of vegetables. Multidrug resistance (MDR, resistant to 3 or more antibiotics) was identified in 18% of samples. S. enterica was isolated from 3.4% of produce samples (7, n = 207). Resistance was uncommon among the S. enterica isolates (1/7). Overall prevalence of diarrheagenic S. enterica and E. coli was low; however, their presence and that of MDR E. coli in foods commonly consumed raw increases the risk to vulnerable populations. Strategies to reduce contamination of fresh produce and public education on washing and cooking some types of produce may be useful to reduce disease.
Antibiotic Resistance Profiles of Escherichia coli isolated from Chobe River water in the Northern Botswana
Fleming, Madison; Yi, Saehah; Ponder, Monica A.; Patino, Lena; Alexander, Kathleen A. (2024-04-26)
Antibiotic Resistance Profiles of Escherichia coli Isolated from Elephants in the Chobe Region of Botswana
Haddadin, Maisoon; Patino, Lena; Ponder, Monica A.; Alexander, Kathleen A. (2024-04-26)
Characterization of Escherichia coli isolated from poultry in the Chobe region of Botswana by molecular methods
Yi, Saehah; Bywater, Auja; Dintwe, Galaletsang; Haidl, Thomas; Cameron, Andrew; Ponder, Monica A.; Alexander, Kathleen A. (2024-07-15)
Introduction: Diarrheal illness incidence is high in rural dryland environments, including the Chobe region of Botswana, where peaks are often associated with high rainfall events. Antibiotic resistance (ABR) is also a growing problem in the region. Contributions of poultry to diarrheal illness and ABR in the region are not well understood. Purpose: Characterize the virulence potential and ABR of E.coli isolated from chicken purchased from local vendors in Botswana using molecular techniques, including whole genome sequencing. Methods: Poultry was purchased in Kasane, Botswana and enriched within 24h. E.coli was isolated using MacConkey and EMB agar. Isolates were shipped to Virginia Tech for molecular and antibiotic resistance characterization. Multiplex PCR was used to detect presence of genes phoA (all E.coli), and diarrheagenic E.coli (eae, and est1b). Phenotypic ABR was determined using CLSI disk diffusion methods. Whole genome sequencing was performed using an Illumina MiSeq system and genome annotated using BV-BRC. Results: E.coli was isolated from 62% (38/61) of poultry samples. Five isolates were classified as diarrheagenic based on eae, none were positive for est1LB. Multidrug resistance (3+) was seen in 26 isolates and resistance to 1 or more antibiotics in 41 E.coli isolates. Resistance amongst chicken isolates was as follows: tetracycline (29), doxycycline (21), sulfamethoxazole (20), streptomycin (15), ampicillin (15), chloramphenicol (9), ciprofloxacin (6), gentamicin (4), c ceftriaxone (4), amoxicillin-clavulanic acid (3) and azithromycin (1). Comprehensive analysis of the genomes from chicken intestinal isolates revealed large numbers of virulence genes (125-252) common amongst diarrheagenic E.coli. Antibiotic resistance genes (65-85) were also present including those encoding resistance to ampicillin, ceftriaxone, doxycycline, streptomycin, and tetracycline. Significance: While prevalence of diarrheagenic E.coli was low, antibiotic resistant E.coli were frequently isolated. E.coli may be an important sentinel species to evaluate ABR emergence and effectiveness of control strategies by Botswana poultry producers.
Characterization of key aroma compounds in microgreens and mature plants of hydroponic fennel (Foeniculum vulgare Mill.).
Liu, Jingsi; Li, Song; O'Keefe, Sean F.; Hurley, Ken; Rutto, Laban; Eriksen, Renee; Yin, Yun (Elsevier, 2024-10-23)
Fennel is a popular culinary herb known for its unique flavor. In this study, we identified key aroma-active compounds in fresh fennel (Foeniculum vulgare Mill.) and its microgreens. Fennel was cultivated hydroponically with soilless substrates. Upon harvesting, samples were ground with liquid nitrogen, and headspace solid phase microextraction (SPME) gas chromatography-mass-spectrometry-olfactometry (GC-MS-O) was used for volatile analysis. Thirty-two and 28 key aroma-active compounds were identified in fennel microgreens and mature leaves, respectively. Phenylpropenes, especially (E)-anethole, were the predominant aroma-active compound in all samples (36.3–83.4 %). Quantitation results showed that fennel microgreens contained a significantly higher amount of monoterpenes, showing an 81.4–98.1 % increase when compared to mature fennel. Principal component analysis (PCA) of identified volatiles indicated a distinctive difference in the overall aroma profile between microgreens and mature leaf. The changes in aroma contents over different growth stages revealed the underlying volatile biosynthesis discrepancy. This study provided baseline information for understanding the aroma evolution from microgreen to mature fennel herbs.
Evidence of horizontal gene transfer and environmental selection impacting antibiotic resistance evolution in soil-dwelling Listeria
Goh, Ying-Xian; Anupoju, Sai Manohar Balu; Nguyen, Anthony; Zhang, Hailong; Ponder, Monica A.; Krometis, Leigh-Anne H.; Pruden, Amy; Liao, Jingqiu (Nature Research, 2024-11-19)
Soil is an important reservoir of antibiotic resistance genes (ARGs) and understanding how corresponding environmental changes influence their emergence, evolution, and spread is crucial. The soil-dwelling bacterial genus Listeria, including L. monocytogenes, the causative agent of listeriosis, serves as a keymodel for establishing this understanding. Here, we characterize ARGs in 594 genomes representing 19 Listeria species that we previously isolated from soils in natural environments across the United States. Among the five putatively functional ARGs identified, lin,which confers resistance to lincomycin, is the most prevalent, followed by mprF, sul, fosX, and norB. ARGs are predominantly found in Listeria sensu stricto species, with those more closely related to L. monocytogenes tending to harbor more ARGs. Notably, phylogenetic and recombination analyses provide evidence of recent horizontal gene transfer (HGT) in all five ARGs within and/or across species, likelymediated by transformation rather than conjugation and transduction. In addition, the richness and genetic divergence of ARGs are associated with environmental conditions, particularly soil properties (e.g., aluminum and magnesium) and surrounding land use patterns (e.g., forest coverage). Collectively, our data suggest that recent HGT and environmental selection play a vital role in the acquisition and diversification of bacterial ARGs in natural environments.
Physical Cell Disruption Technologies for Intracellular Compound Extraction from Microorganisms
Zhao, Fujunzhu; Wang, Zhiwu; Huang, Haibo (MDPI, 2024-09-24)
This review focuses on the physical disruption techniques in extracting intracellular compounds, a critical step that significantly impacts yield and purity. Traditional chemical extraction methods, though long-established, face challenges related to cost and environmental sustainability. In response to these limitations, this paper highlights the growing shift towards physical disruption methods—high-pressure homogenization, ultrasonication, milling, and pulsed electric fields—as promising alternatives. These methods are applicable across various cell types, including bacteria, yeast, and algae. Physical disruption techniques achieve relatively high yields without degrading the bioactivity of the compounds. These techniques, utilizing physical forces to break cell membranes, offer promising extraction efficiency, with reduced environmental impacts, making them attractive options for sustainable and effective intracellular compound extraction. High-pressure homogenization is particularly effective for large-scale extracting of bioactive compounds from cultivated microbial cells. Ultrasonication is well-suited for small to medium-scale applications, especially for extracting heat-sensitive compounds. Milling is advantageous for tough-walled cells, while pulsed electric field offers gentle, non-thermal, and highly selective extraction. This review compares the advantages and limitations of each method, emphasizing its potential for recovering various intracellular compounds. Additionally, it identifies key research challenges that need to be addressed to advance the field of physical extractions.
The Preparation of W/O/W High-Internal-Phase Emulsions as Coagulants for Tofu: The Effect of the Addition of Soy Protein Isolate in the Internal Water Phase
Wang, Yongquan; Liu, Xuanbo; Zhang, Qiang (MDPI, 2024-08-29)
Tofu quality is determined by a controlled coagulation process using a W/O/W emulsion coagulant. The impact of adding soy protein isolate (SPI) to the inner water phase on the stability of W/O/W high-internal-phase emulsions (HIPEs) and its application as a coagulant for tofu was assessed. No creaming occurred during 7-day storage with SPI concentrations up to 0.3%, while the emulsion droplets aggregated with 0.5% and 0.7% SPI. Emulsions containing 0.3% SPI maintained a constant mean droplet size after 21 days of storage and exhibited the lowest TURBISCAN stability index value. HIPE stability against freeze–thaw cycles improved after heating. HIPEs with SPI concentrations above 0.3% demonstrated an elastic gel-like behavior. The increased viscosity and aggregation of the protein around droplets indicated that the interaction among emulsion droplets could enhance stability. W/O/W HIPE coagulants significantly increased tofu yield, reduced hardness, and produced a more homogenous tofu gel compared to a MgCl₂ solution. The HIPE with 0.3% SPI was found to be optimal for use as a coagulant for tofu.
Dietary Phosphorus Levels Influence Protein-Derived Uremic Toxin Production in Nephrectomized Male Rats
Cladis, Dennis P.; Burstad, Kendal M.; Biruete, Annabel; Jannasch, Amber H.; Cooper, Bruce R.; Hill Gallant, Kathleen M. (MDPI, 2024-06-08)
Gut microbiota-derived uremic toxins (UT) accumulate in patients with chronic kidney disease (CKD). Dietary phosphorus and protein restriction are common in CKD treatment, but the relationship between dietary phosphorus, a key nutrient for the gut microbiota, and protein-derived UT is poorly studied. Thus, we explored the relationship between dietary phosphorus and serum UT in CKD rats. For this exploratory study, we used serum samples from a larger study on the effects of dietary phosphorus on intestinal phosphorus absorption in nephrectomized (Nx, n = 22) or sham-operated (sham, n = 18) male Sprague Dawley rats. Rats were randomized to diet treatment groups of low or high phosphorus (0.1% or 1.2% w/w, respectively) for 1 week, with serum trimethylamine oxide (TMAO), indoxyl sulfate (IS), and p-cresol sulfate (pCS) analyzed by LC-MS. Nx rats had significantly higher levels of serum TMAO, IS, and pCS compared to sham rats (all p < 0.0001). IS showed a significant interaction between diet and CKD status, where serum IS was higher with the high-phosphorus diet in both Nx and sham rats, but to a greater extent in the Nx rats. Serum TMAO (p = 0.24) and pCS (p = 0.34) were not affected by dietary phosphorus levels. High dietary phosphorus intake for 1 week results in higher serum IS in both Nx and sham rats. The results of this exploratory study indicate that reducing dietary phosphorus intake in CKD may have beneficial effects on UT accumulation.
Flavor language in expert reviews versus consumer preferences: An application to expensive American whiskeys
Hamilton, Leah M.; Neill, Clinton L.; Lahne, Jacob (Elsevier, 2023-07)
Treating natural language flavor descriptions as data that can explain or “predict” consumer or market responses to a product, a process called Natural Language Processing or Text Mining, is increasingly common in food research. Text data has high variation in vocabulary usage and which features writers attend to, necessitating large datasets which tend to be from unblinded tastings with limited types of supplemental data. In this study, a random forest model trained on 4300 full-text whiskey reviews identified terms commonly describing higher- or lower-priced whiskeys. Ten terms were selected for a survey of American whiskey consumers. Professional whiskey reviewers commonly describe expensive whiskeys as tasting of “sultanas”, “oak”, “leather”, and “chocolate”. “Corn” and “grassy” are used commonly for inexpensive whiskeys. In contrast, US consumers are more likely to purchase whiskeys with “chocolate” and “caramel” flavor, ranking “corn” near the middle of the 10 terms tested and “tobacco”, “leather”, and “grass” the lowest. This study shows that the flavor terms reviewers use for expensive whiskeys aren’t necessarily most important to consumers, possibly due to bias from unblinded tastings or differences between reviewers and consumers. Predictions based on reviews can also overestimate the negative impact of common or expected flavors (like “corn” or “caramel” in whiskeys). Large correlational studies using convenient text corpora can effectively generate hypotheses or identify vocabulary and follow up surveys or controlled sensory experiments using the population of interest can provide additional insights about the product category and the groups of people interacting with it.
A taste of cell-cultured meat: a scoping review
To, K. V.; Comer, C. Cozette; O'Keefe, Sean F.; Lahne, Jacob (Frontiers, 2024-01-23)
Cell-cultured meat (CM) is a novel meat product grown in vitro from animal cells, widely framed as equivalent to conventional meat but presented as produced in a more sustainable way. Despite its limited availability for human consumption, consumer acceptance of CM (e.g., willingness to purchase and consume) has been extensively investigated. A key but under-investigated assumption of these studies is that CM’s sensory qualities are comparable to conventional, equivalent meat products. Therefore, the current review aims to clarify what is actually known about the sensory characteristics of CM and their potential impact on consumer acceptance. To this end, a structured scoping review of existing, peer-reviewed literature on the sensory evaluation of CM was conducted according to the PRISMA-ScR and Joanna Briggs Institute guidelines. Among the included studies (N = 26), only 5 conducted research activities that could be termed “sensory evaluation,” with only 4 of those 5 studies evaluating actual CM products in some form. The remaining 21 studies based their conclusions on the sensory characteristics of CM and consequent consumer acceptance to a set of hypothetical CM products and consumption experiences, often with explicitly positive information framing. In addition, many consumer acceptance studies in the literature have the explicit goal to increase the acceptance of CM, with some authors (researchers) acting as direct CM industry affiliates; this may be a source of bias on the level of consumer acceptance toward these products. By separating what is known about CM sensory characteristics and consumer acceptance from what is merely speculated, the current review reported realistic expectations of CM’s sensory characteristics within the promissory narratives of CM proponents.
Characterization of Diarrheagenic Escherichia coli Isolated from Food, Food Waste, and Water from the Chobe Region of Botswana
Bywater, Auja L.; Ponder, Monica A.; Alexander, Kathleen A. (2023-04-21)
Antibiotic Susceptibility of non-pathogenic Escherichia coli from meat and produce available in the Chobe region of Botswana
Saunders, Rachel; Bywater, Auja L.; Fleming, Madison; Kelly, Christine; Nuckolls, Evan; Alexander, Kathleen A.; Ponder, Monica A. (2023-04-21)
Food Waste from Campus Dining Hall as a Potential Feedstock for 2,3-Butanediol Production via Non-Sterilized Fermentation
Caldwell, Alicia; Su, Xueqian; Jin, Qing; Hemphill, Phyllicia; Jaha, Doaa; Nard, Sonecia; Tiriveedhi, Venkataswarup; Huang, Haibo; OHair, Joshua (MDPI, 2024-01-31)
Food waste is a major issue that is increasingly affecting our environment. More than one-third of food is wasted, resulting in over $400 billion in losses to the U.S. economy. While composting and other small recycling practices are encouraged from person-to-person, it is not enough to balance the net loss of 80 million tons per year. Currently, one of the most promising routes for reducing food waste is through microbial fermentation, which can convert the waste into valuable bioproducts. Among the compounds produced from fermentation, 2,3-butanediol (2,3-BDO) has gained interest recently due to its molecular structure as a building block for many other derivatives used in perfumes, synthetic rubber, fumigants, antifreeze agents, fuel additives, and pharmaceuticals. Waste feedstocks, such as food waste, are a potential source of renewable energy due to their lack of cost and availability. Food waste also possesses microbial requirements for growth such as carbohydrates, proteins, fats, and more. However, food waste is highly inconsistent and the variability in composition may hinder its ability to be a stable source for bioproducts such as 2,3-BDO. This current study focuses specifically on post-consumer food waste and how 2,3-BDO can be produced through a non-model organism, Bacillus licheniformis YNP5-TSU during non-sterile fermentation. From the dining hall at Tennessee State University, 13 food waste samples were collected over a 6-month period and the compositional analysis was performed. On average, these samples consisted of fat (19.7%), protein (18.7%), ash (4.8%), fiber (3.4%), starch (27.1%), and soluble sugars (20.9%) on a dry basis with an average moisture content of 34.7%. Food waste samples were also assessed for their potential production of 2,3-BDO during non-sterile thermophilic fermentation, resulting in a max titer of 12.12 g/L and a 33% g/g yield of 2,3-BDO/carbohydrates. These findings are promising and can lead to the better understanding of food waste as a defined feedstock for 2,3-BDO and other fermentation end-products.
Editorial: Antimicrobial resistant pathogens in sustainable food systems: how to prevent, detect, or control contamination?
Concha-Meyer, Anibal; Toro, Magaly; Eifert, Joseph D. (Frontiers, 2023-06-09)
Systematic literature review identifying bacterial constituents in the core intestinal microbiome of rainbow trout (Oncorhynchus mykiss)
Hines, Ian S.; Marshall, Maggie A.; Smith, Stephen A.; Kuhn, David D.; Stevens, Ann M. (Wiley, 2023-08-11)
Fish aquaculture has become the fastest growing sector in global food production. Thus, ensuring the sustainability of aquaculture practices is of the utmost importance. Studies in higher vertebrates (i.e. mammals) have demonstrated the role of the host microbiome in physiological processes from nutrient acquisition to pathogen protection. Therefore, analysis of fish microbiomes is an important factor to consider with regard to overall animal health and welfare. Rainbow trout (Oncorhynchus mykiss) are an economically valued fish cultured worldwide. Several studies have identified microbial constituents inhabiting the intestinal tract of rainbow trout. To better elucidate some of the core constituents of the rainbow trout intestinal microbiome, this systematic literature review analysed the relative abundance results from 25 articles published on the rainbow trout intestinal microbiome from 2017 to 2021. Bacteria classified within the phyla Firmicutes and Proteobacteria were observed in every study. At the family level, Lactobacillaceae was consistently observed. Additionally, bacteria in the Actinobacteria, Bacteroides, and Tenericutes phyla were identified in at least 50% of the studies. Interestingly, Mycoplasma spp. were occasionally the most dominant organisms present in the microbiome. Overall, the results here identify bacteria that are commonly found members of the rainbow trout intestinal microbiome.
Revitalizing the Future of Food Safety Extension
Arnold, Nicole L.; Schonberger, H. Lester; Ferelli, Angela; Murphy, Sarah I.; Yang, Lily L. (International Association for Food Protection, 2022-05-02)
Originally established to address agricultural needs by applying research and education in U.S. communities, the Cooperative Extension System (CES) has become increasingly involved in food safety through the supply chain. CES plays an integral role in food safety through consumer education, food employee training, regulatory guidance, and agricultural education for youth and students. CES food safety efforts have evolved to respond to current events and evolving public needs; subsequently, CES personnel communicated a myriad of challenges, including overextension within their roles, dwindling financial support, and pedagogical shifts. As a result, CES personnel have opted for creative, innovative, and timely solutions that can be harnessed by others with ties to CES. This article is based on a roundtable with Extension experts on “Revitalizing the Future of Food Safety Extension,” held at the 2019 International Association for Food Protection Annual Meeting detailing this imperative. This article serves as (i) a consolidated framework resource for educational purposes, (ii) an invitation to collaborate with food safety CES personnel, and (iii) a call for support and advocacy for CES and those within it. It also highlights the value and impact CES has, and will continue to have, in making food safer and more equitable.
The Impact of Marine Resource-Free Diets on Quality Attributes of Atlantic Salmon
McLean, Ewen; Campbell, Kelly B.; Kuhn, David D.; Tlusty, Michael F.; Barrows, Frederick T. (MDPI, 2024-01-17)
The influence of feeding Atlantic salmon for 90 days on diets that excluded fishmeal (FM) and fish oil (FO) was examined for influence on various quality traits. In addition, the effect of adding krill meal (KM; 0%, 2.5%, and 5%), as a putative feed palatant was also examined. Total replacement of FM/FO had a limited effect on production characteristics, affecting percentage yields of headed and gutted control fish and their standard length (p < 0.05). Variances between dietary groups were observed for pigmentation, and plant protein-based KM-free-fed fish returned deeper hues across their belly, NQC (Norwegian Quality Cut), and back portions (p < 0.03). No differences were measured for relative fin condition. δ¹³C and δ¹⁵N concentrations were lower and higher, respectively (p < 0.05) for fish fed the FM/FO-based diet. δ¹³C:δ¹⁵N likewise differed between treatments with FM/FO-fed salmon expressing higher ratios. Fillet mechanical characteristics varied with fish fed on animal protein-based diets, without KM expressing higher springiness and resilience (p < 0.05). Fish fed plant-based diets were generally preferred by younger taste testers. The results from this trial illustrate that FM/FO can be completely removed from salmon diets without problematic effects on quality and palatability attributes.
Quantitative texture analysis comparison of three legumes
Miller, Rebekah; Duncan, Susan; Yin, Yun; Zhang, Bo; Lahne, Jacob (Frontiers, 2023-06-19)
A validated texture-analysis method to evaluate product quality in frozen or cooked legumes is needed to support high-quality vegetable production but is not currently established in the literature. Peas, lima beans, and edamame were investigated in this study due to similar market use as well as growth in plant-based protein consumption in the United States. These three legumes were evaluated after three different processing treatments (blanch/freeze/thaw (BFT); BFT+microwave heat (BFT+M); BF+stove-top cooking (BF+C)), using both compression and puncture analysis following an American Society of Agricultural and Biological Engineers (ASABE) texture analysis method and moisture testing following an American Society for Testing and Materials (ASTM) standard method. Texture analysis results showed differences between legumes and processing methods. Compression analysis identified more differences between treatments within product type than puncture for both edamame and lima beans indicating compression might be more sensitive to texture changes in these products. Implementation of a standard texture method for legume vegetables for growers and producers would provide a consistent quality check to support efficient production of high-quality legumes. Due to the sensitivity obtained from the compression texture method in this work, compression should be considered for future research into a robust method to evaluate edamame and lima bean textures throughout the growing and production processes.
Analyzing larger sample sets with rapid methods: Incomplete-block designs with free-sorting and free-linking tasks
Ac-Pangan, Marlon; Tejedor-Romero, Marino; Swatko, Kyra; Orden, David; Lahne, Jacob (Elsevier, 2024-04)
As rapid, holistic methods for similarity and description—such as sorting and projective mapping—have grown in popularity, a limiting factor is the number of samples that can be presented to subjects: more than 25 food samples decreases the quality and stability of results. While incomplete-block designs could address this, their use has not been developed for these holistic methods. In this paper we present an empirical investigation into the use of incomplete-block designs with free sorting and the newer free linking. We compare these two methods because while their results are comparable, the cognitive tasks are different, and thus their suitability for incomplete-block designs may differ. We evaluated the effects of incomplete-block designs in two studies. In Study 1, 20 subjects evaluated 6/10 chocolate bars by free linking in an incomplete-block design, with each subject completing 2 blocks; results were compared to a complete-block evaluation of the 10 bars by free sorting and free linking. In Study 2, a total of 90 subjects evaluated 62 terms from a chocolate flavor-wheel in 3 conditions (between subjects): free sorting with complete blocks (N = 30, all 62 terms) and free sorting (N = 30) or free linking (N = 30) with 3 incomplete blocks of 16/62 terms. We introduce a novel method to evaluate stability for the incomplete-block designs that we call “pairwise simulation.” From Study 1, we find that pairwise simulation provides adequate stability estimates and that, with sufficient pairwise cooccurrences, free linking with incomplete blocks produces results that are comparable to free sorting or linking with complete blocks. From Study 2, we demonstrate that free linking with incomplete blocks can produce high quality results from a large sample set, maintaining the increased discrimination capacity that marks free linking in general, and that with incomplete blocks, free linking is likely to be more stable than free sorting. This research demonstrates that incomplete-block designs can be used with free linking, and also provides a new, effective method through pairwise simulation for evaluating stability with incomplete-block designs, which cannot be resampled using standard bootstrapping approaches.

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