Nutrition & Food Science
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Item Three Clostridium species with Health Imparting Properties: In vitro Screening for Probiotic Potential(2024) Mochama, Victor Moronge; Obanda, Diana; Nutrition; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This research aimed to unlock the probiotic potential of the genus Clostridium, which is often overshadowed by the predominant focus on pathogenic species. The study specifically targeted three promising Clostridium species: C. disporicum, C. celatum, and C. vincentii, which have shown potential in mitigating diet-induced obesity. Despite the challenges presented by the anaerobic growth requirements of Clostridium bacteria, the study capitalized on their capacity to sporulate. This characteristic provides an avenue to use them as probiotics, with resilient and dormant spores capable of surviving food processing and harsh stomach conditions. The resilience of these spores was examined by exposing them to oxygen, heat, gastrointestinal juices, and bile salts. The spores survived oxygen exposure, exhibited resilience to both bile salts and gastric acids, and demonstrated a survival temperature of 70°C. When exposed to suitable germination conditions in vitro, the spores successfully germinated. The study assessed the colonization potential of the bacteria by evaluating their adhesion ability, and all bacteria were found to have the adhesion ability. Furthermore, a safety assessment was conducted by examining hemolytic activity and antibiotic susceptibility to selected antibiotics. The bacteria were found to be susceptible to the antibiotics and did not exhibit hemolytic activity. Bile salt hydrolase (BSH) activity and antibacterial activities were also assessed, and none of the bacteria exhibited BSH activity or antibacterial activity. Antioxidant tests revealed that C. vincentii had the highest antioxidant properties. Assessment of anti-inflammatory properties showed that C. celatum downregulated the gene expression of cytokine inflammation markers IL-6, IL-1, and iNOS while upregulating TGF-β expression. In summary all 3 bacterial species showed good probiotic potential from the in vitro tests. Particularly the formation of resistant spores that later germinated to vegetative cells that produced molecular patterns with antioxidant and anti-inflammatory properties. This necessitates further studies on their probiotic properties.Item EVALUATION OF THE COPPER HOMEOSTASIS AND SILVER RESISTANCE ISLAND AND ITS ROLE IN PERSISTENCE OF SALMONELLA ENTERICA(2023) Haendiges, Julie; Tikekar, Rohan; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Salmonella enterica is one of the leading bacterial cause of foodborne illness in the UnitedStates. Although there are many serovars, only a small subset causes human illness. Since Salmonella is ubiquitous in the environment, the Food and Drug Administration has established regulations for food processors to ensure that the products are free of contamination. Low-moisture foods are commonly ready-to-eat, and due to the low water activity do not promote growth of bacteria. However, Salmonella has been shown to persist in these foods. There havebeen two outbreaks and multiple recalls in the United States due to contaminated pistachios. Based on a retrospective study, results show that there is evidence of a contamination in the growing orchard and a significant number of Salmonella isolates from the environments contain the Copper Homeostasis and Silver Resistance Island (CHASRI) cassette. This raises several questions: what is the prevalence of CHASRI among different Salmonella isolates from food and environmental sources? Does presence of CHASRI enable Salmonella to survive better against copper stress? And whether presence of CHASRI provide cross-protection against other stresses such as desiccation and thermal treatment? This dissertation attempts to answer those questions. The prevalence of the CHASRI in Salmonella was determined by the use of publicly availablewhole genome sequencing data. The CHASRI was found in 61 different serovars and types of sources. The presence of the CHASRI in isolates from low-moisture foods that have caused previous outbreaks (peanut butter, nuts, spices) was interesting to note, and leads to future studies on correlations between this island and virulence. Based on results of phylogenetic analysis of CHASRI sequences from closed genomes, we determined there were four types of CHASRI found in Salmonella. Traditionally, the Salmonella Genomic Island-4 (SGI-4) is found but in addition the CHASRI can incorporate by itself, within a variant of SGI-4, or via a rare plasmid. Interestingly, the sequence of the CHASRI from SGI-4 and the variant SGI-4 were highly different. The high SNP differences in sequence along with the difference in the arsenic operon led to the conclusion that these variants arose independently. A Salmonella Senftenberg strain (CFSAN047523), isolated from pistachios, was used to createthree knockouts (∆cus, ∆pco, and ∆CHASRI). Previous studies have looked at the minimum inhibitory concentration (MIC) of strains with and without the CHASRI but have omitted the minimum bactericidal concentration (MBC). In this study, we used the knockouts to test for both MIC and MBC. While the MIC was similar for the strains, the MBC was greater in the wild type and partial CHASRI knockouts. Growth and inactivation kinetics were measured in different concentrations of copper sulfate. At higher levels of copper sulfate, the presence of the CHASRI made cells more resilient to inactivation by copper sulfate. Evidence shows that the stress response in Salmonella has the ability to crosstalk and provideprotection against multiple stresses. To investigate this phenomenon further, our isolates were tested against a multitude of stresses to evaluate for cross-protection that may be due to theCHASRI. Cells undergoing copper stress were better equipped to survive lethal copper concentrations and desiccation if the CHASRI was present. The presence of Salmonella in final pistachio products that have been fully processed identifies that some adaptation and stress response is occurring in the processing facility. Inoculated pistachios with the wild type and ∆CHASRI strain were thermally processed to test for survivors. This study showed that the presence of the CHASRI gave the isolate an advantage to survive thermal processing after desiccation. Overall, this study presents the prevalence of the CHASRI in Salmonella enterica as well as theimportant role it plays in copper tolerance. The evidence of cross-protection and tolerance to copper leads to future research regarding gene expression and virulence assessment.Item BACTERIAL GROWTH AND INACTIVATION IN HETEROGENEOUS ENVIRONMENT(2023) Tsai, Shawn; Tikekar, Rohan V.; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This dissertation explores the growth and inactivation of Salmonella enterica subsp. enterica Serovar Typhimurium in oil-in-water emulsion systems, with a particular focus on the effects of emulsifier type, oil fraction, temperature, molecular weight, and surface charge. First, investigation was conducted on the effects of emulsifier type (Tween 20, Tween 80, Triton X-100) and oil content (20%, 40%, 60%) on growth and thermal inactivation of S. Typhimurium in emulsions. The results indicated that while emulsifiers did not affect the bacterial growth rate or lag phase, the presence of high oil content (60%) prolonged the lag phase in emulsions. In addition, Tween 80 and Triton X-100 emulsifier solutions exhibited protective effects against thermal inactivation. Next chapter was focused on evaluating the impact of temperature on growth (7, 22, 37°C) and inactivation (55, 58, 60°C) kinetics of S. Typhimurium in emulsion systems with same emulsifier and oil fraction as previous chapter. The results indicated that temperature had a significant impact on bacterial kinetics, with increasing temperatures leading to faster growth and inactivation rates. Next, the effect of emulsifier molecular weight and surface charge on the growth and thermal inactivation of S. Typhimurium in emulsions was examined. To control the molecular weight with similar structure, whey protein was selected for experimentation. By adjusting the pH, it was possible to change the surface charge in whey protein. Results indicated that whey protein hydrolysate (WPH) with a lower molecular weight did not exhibit a lag phase in Salmonella growth. However, whey protein isolate (WPI) with higher molecular weight demonstrated no difference in the lag phase when compared to bacterial growth in TSB. Similar effects were observed with a positively charged emulsifier (WPI+). The findings suggest that the molecular weight of emulsifiers has a more significant impact on bacterial growth than their surface charge. Regarding the evaluation of inactivation, emulsifier solutions exhibited no significant difference compared to TSB, while emulsions stabilized by WPH and WPI+ showed some protective effects on S. Typhimurium. This observation can be attributed to the ability of positively charged emulsifiers to interact with the bacterial membrane, providing protective effects during thermal treatment. Lastly, to gain a comprehensive understanding of the mechanism concerned with the impact of emulsifier and oil inclusion on bacterial growth and inactivation behavior, S. Typhimurium was cultured in different emulsion-related environments and evaluated for nine stress-related genes (rpoE, rpoH, otsB, proV, fadA, fabA, dnaK, ibpA, ompC) after 20 hours of incubation at 37°C and after thermal treatment at 55°C for 45 min. It was found that ibpA was upregulated in all emulsifier environments, regardless of the presence of oil, indicating that IbpA was synthesized in emulsifier environments. Moreover, increased expression of fabA was also observed in Triton X-100 stabilized 60% emulsion, indicating poor heat resistance due to increased membrane fluidity. In the combination of gene expression data, our results showed that emulsifier solutions without oil exhibited a greater number of regulatory mechanisms compared to those containing oil, indicating that the presence of oil did not provide as much protection after thermal treatment. Based on these findings, the stress-related mechanism was constructed by the expression of those selected genes. Overall, this dissertation provides valuable insights into the factors influencing bacterial growth and inactivation in oil-in-water emulsion systems, as well as bacterial stress response in these systems. These findings provide important insights into the growth and inactivation behavior of S. Typhimurium in oil-in-water emulsion systems and the stress response mechanisms involved. Understanding these factors is crucial for developing effective control measures to ensure food safety and prevent foodborne illness outbreaks caused by this pathogen. This information can be used to optimize the formulation and processing of emulsion-based food products to minimize the risk of bacterial contamination and ensure their safety for consumption.Item Enhancement of thermal processing with food-grade antimicrobial compounds in low-moisture food matrix to improve food safety(2022) Ding, Qiao; Tikekar, Rohan V; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Heat resistant foodborne pathogens have been a concern in low-moisture foods and ingredients (LMFs). Increased thermal resistance of pathogens such as Escherichia coli O157:H7 and Salmonella Typhimurium at low water activity (aw) reduces the efficiency of thermal treatment in LMFs. Alternative methods are therefore needed to augment thermal processing and reduce food safety risk. This study investigated the enhancement of thermal treatment efficiency against pathogenic bacteria in LMF matrices at different aw by inclusion of food-grade antimicrobial compounds. Based on their similar target sites in cells, it is hypothesized that antimicrobial compounds may work synergistically with heat treatment in LMF matrices. The treatment effect may be dependent on both aw and matrix compositions. Physiological and transcriptional changes may take place within cells adapted to different environments and contribute to the varied bacterial resistance. A combination of Butylparaben (BP), a known antimicrobial and thermal treatment was tested to enhance inactivation rates of S. Typhimurium and E. coli O157:H7 in meat and bone meal (MBM) equilibrated to water activity (aw) of 0.4 and 0.7. Presence of BP significantly accelerated thermal inactivation of E. coli O157:H7 in MBM at both 0.4 and 0.7 aw. However, inactivation rate of S. Typhimurium was not affected by the combined treatment at either 0.4 or 0.7 aw. BP accelerated the inactivation of the thermal-resistant subpopulation in E. coli O157:H7 but not S. Typhimurium. Therefore, further studies were conducted to identify alternative antimicrobial compounds to enhance thermal treatment against S. Typhimurium in LMF matrices and investigate the resistance mechanisms. Trans-cinnamaldehyde (CA) and eugenol (EG) were selected from a screening study to assist thermal treatment against S. Typhimurium adapted to different aw in whey protein (WP), corn starch (CS) or peanut oil (PO). Although addition of CA or EG significantly accelerated thermal inactivation of S. Typhimurium in water and LMF components at 0.9 aw, similar effect was not observed in bacteria adapted to lower (0.4) aw in any of those matrices. The matrix effect on bacterial thermal resistance was observed at 0.9 aw and were ranked as WP > PO > CS. The combined heat treatments reduced bacterial metabolic activity, which was partially dependent on the food components as well. S. Typhimurium had lower membrane fluidity because of membrane fatty acid composition change when adapted to a lower aw, which contributed to resistance against the combined heat treatments. Based on the aw- and matrix- dependent bacterial resistance and physiological changes observed in the two previous studies, transcriptional analysis was conducted to further understand the molecular mechanism behind the bacterial resistance. Expression of nine stress-related genes in S. Typhimurium adapted to different aw in LMF components were analyzed with or without the CA-assisted heat treatment. The upregulation of rpoH and dnaK in S. Typhimurium was induced by the stress during bacterial adaptation to the low aw environments as well as the combined heat treatment, which contributed to the bacterial resistance to both desiccation and the combined treatment. Although its link to the desiccation response in bacteria is not fully understood, the downregulation of ompC during the combined treatment also partially contributed to the treatment resistance. The upregulation of rpoE, otsB, proV and fadA in S. Typhimurium was induced by the desiccation stress during incubation at the low aw environment but was not a major contributor to the resistance during the combined treatment. The observed upregulation of fabA and downregulation of ibpA could not be directly linked to either bacterial resistance to desiccation or the combined heat treatment. Differential expressions were also observed among different aw levels or in different matrices at the same aw. The inconsistencies between the expression profiles of dnaK and ompC and bacterial resistance during the combined treatment suggested the presence of additional stress-response pathways.In conclusion, the results have demonstrated the potential of using food-grade antimicrobial compounds to complement thermal treatment in LMF during processes that start with a relatively high aw (such as dehydration). However, their effectiveness may subside at low aw environments. Future works should focus on optimizing the parameters of the CA/ EG-assisted heat treatment for higher efficiency, studying the molecular mechanism behind the bacterial resistance at global scale, and exploring alternative non-thermal processing technologies to enhance thermal treatment efficiency in LMFs without raising thermal treatment intensity.Item Development of machine learning and advanced data analytical techniques to incorporate genomic data in predictive modeling for Salmonella enterica(2021) Karanth, Shraddha; Pradhan, Abani K; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The past few decades have seen a renaissance in the field of food safety, with the increasing usage of genomic data (e.g., whole genome sequencing (WGS)) in determining the cause of microbial foodborne illness, particularly for multi-serovar agents such as Salmonella enterica. However, utilizing such data in a preventative framework, specifically in the field of quantitative microbial risk assessment (QMRA) remains in its infancy, because incorporating such large-scale datasets in statistical models is hindered by the sheer number of variables/features introduced. Thus, the goal of this research is to introduce machine learning (ML)-based approaches to potentially incorporate WGS data in various stages of a risk assessment for Salmonella enterica. Specifically, we developed a machine learning-based workflow to obtain an association between gene presence/absence data from microbial whole genome sequences and severity of Salmonella-related health outcomes in host systems. A key contribution of this dissertation is assessing the applicability of Elastic Net model, a recursive feature selection technique, which resolves a well-known issue concerning WGS-based data analysis: variables/features outnumber the count of observations. Building on this finding, we developed a gene weighted Poisson regression method to incorporate genes into a dose-response framework for Salmonella enterica, thereby incorporating genetic variability directly into a risk assessment framework. Finally, we combined machine learning with count-based models to determine how significant genes interact with meteorological factors in impacting the severity of salmonellosis outbreaks. This dissertation uncovers some interesting findings. First, although commonly used classifiers (such as random forest) performed well in predicting disease severity, logistic regression, in conjunction with Elastic Net, performed significantly better. This finding is important, as the result of a logistic regression is generally more interpretable than that of other classifiers, easing its incorporation into predictive microbial modeling. Next, machine learning-supported count-based models, such as Poisson regression also proved to be a good fit for gene-informed dose-response modeling and determination of outbreak severity when combined with extrinsic factors such as atmospheric temperature and precipitation. Overall, this dissertation identified areas within a QMRA framework that could benefit from incorporating genetic information, and introduced ML models to incorporate such information.