Browsing by Author "Ovissipour, Reza"

Now showing 1 - 13 of 13
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    Assessing the Enzymatic Hydrolysis of Salmon Frame Proteins through Different By-Product/Water Ratios and pH Regimes
    Valencia, Pedro; Valdivia, Silvana; Nuñez, Suleivys; Ovissipour, Reza; Pinto, Marlene; Ramirez, Cristian; Perez, Alvaro; Ruz, Manuel; Garcia, Paula; Jimenez, Paula; Almonacid, Sergio (MDPI, 2021-12-08)
    The enzymatic hydrolysis of fish by-product proteins is traditionally carried out by mixing ground by-products with water. In addition, pH control is used to avoid pH drops. Higher costs are involved due to the use of pH control systems and the consequent energy cost in the drying stage. This work aimed to evaluate the effect of these conditions on the hydrolysis of salmon frame (SF) proteins, including the SF hydrolysis without added water. SF hydrolysis by subtilisin at 50, 75, and 100% SF under different pH regimes were evaluated by released α-amino (α-NH) groups, total nitrogen, degree of hydrolysis, and estimated peptide chain length (PCL) at 55 °C. The concentration of released α-NH groups was higher in the conditions with less added water. However, the nitrogen recovery decreased from 50 to 24% at 50 and 100% SF, respectively. Changing the SF/water ratio had a more significant effect than changing the pH regime. Estimated PCL changed from 5–7 to 7–9 at 50 and 100% SF, respectively. The operating conditions affected the hydrolysis performance and the molecular characteristics of the hydrolysate.
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    Cellular Aquaculture: Prospects and Challenges
    Goswami, Mukunda; Belathur Shambhugowda, Yashwanth; Sathiyanarayanan, Arjunan; Pinto, Nevil; Duscher, Alexandrea; Ovissipour, Reza; Lakra, Wazir Singh; Chandragiri Nagarajarao, Ravishankar (MDPI, 2022-05-26)
    Aquaculture plays an important role as one of the fastest-growing food-producing sectors in global food and nutritional security. Demand for animal protein in the form of fish has been increasing tremendously. Aquaculture faces many challenges to produce quality fish for the burgeoning world population. Cellular aquaculture can provide an alternative, climate-resilient food production system to produce quality fish. Potential applications of fish muscle cell lines in cellular aquaculture have raised the importance of developing and characterizing these cell lines. In vitro models, such as the mouse C2C12 cell line, have been extremely useful for expanding knowledge about molecular mechanisms of muscle growth and differentiation in mammals. Such studies are in an infancy stage in teleost due to the unavailability of equivalent permanent muscle cell lines, except a few fish muscle cell lines that have not yet been used for cellular aquaculture. The Prospect of cell-based aquaculture relies on the development of appropriate muscle cells, optimization of cell conditions, and mass production of cells in bioreactors. Hence, it is required to develop and characterize fish muscle cell lines along with their cryopreservation in cell line repositories and production of ideal mass cells in suitably designed bioreactors to overcome current cellular aquaculture challenges.
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    Developing cultivated mollusks through establishing primary cell culture methods of Eastern Oyster, Crassostrea virginica, as a model bivalve
    Aung, Thet Me Me (Virginia Tech, 2022-08-17)
    Cultivated seafood is a potential alternative protein source that can address the rising global food demand with exponentially rising human population growth. Cultivated seafood is made by growing animal cells in vitro using stem cells for edible food, eliminating the need to raise the entire animal. A crucial first step in developing cultivated seafood is creating a well-characterized cell line that can continuously grow and differentiate into desired cell types. Due to difficulties in determining optimal primary cell culture conditions, no continuous cell lines of food-relevant mollusks have been established so far. This study used the adult Eastern Oyster, Crassostrea virginica, as a model bivalve to study the decontamination, cell dissociation, and culture conditions suited for mollusk adductor muscle cells. Oyster adductor (OAD) cells were obtained via tissue explant, mechanical and enzymatic digestion. The cells were routinely monitored using an inverted microscope for phase-contrast and fluorescence imaging. Culture vessels were coated with surface proteins such as fibronectin, laminin, matrigel, and poly-d-lysine to promote cell attachment. The tissue decontamination with Penicillin-Streptomycin (100 µg/mL), Amphotericin B (0.25 µg/ml), and algaecide solution (0.03%) was effective in controlling microbial growth. OAD cells grew best at lower nutrient levels in a one-to-one ratio of Lebovitz L-15 media and artificial seawater. Lower fetal bovine serum levels, 1-5%, provided a high number of cell attachments and consistent growth in combination with 1% adult oyster whole-body or larvae extract. The tissue explant method resulted in the optimal cell dissociation from the three methods, and proceeding cultures had attached cells surviving for up to 10 days. All the plate coatings promoted cell attachment, but fibronectin provided optimal cell attachment of OAD cells. Fibroblast-like, neuron-like, epithelial-like, and rounded cells were observed. Fluorescence cell staining confirmed the presence of cytoskeleton and nuclei in the OAD cell cultures. These advances in primary cell culture methods of OAD cells may be beneficial for establishing mollusk cell lines for cultivated seafood production.
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    Developing Serum-Free Media Via Bioprocessing For Cultivated Seafood Products
    Batish, Inayat (Virginia Tech, 2022-09-08)
    Global food production management has become a challenge with an anticipated population of 10 billion people by 2050 and the ongoing COVID-19 epidemic. Seafood is a vital food source due to its widespread consumption, excellent nutrient profile, and low feed conversion ratio, rendering its sustainable production quintessential. Cellular agriculture or cultured meat can increase seafood production; however, the conventional use of Fetal Bovine Serum (FBS) in culture media restricts its utilization at an industrial level. FBS is effective but has many limitations: unethical animal extraction, high demand and low supply, poorly defined ingredients, variable performance, and high cost that impedes the feasibility and commercial viability of cellular agriculture. Thus, employing serum-free media becomes a quintessential need for cellular agriculture. This project aims to replace or reduce the typical 10% serum usage in Zebrafish embryonic stem cell (ESC) production media with protein hydrolysates derived from low-cost natural sources with high protein content. Enzymatic hydrolysis was performed on nine sources: insects (black army fly and cricket), plants (pea), fungi (mushroom and yeast), algae, and marine invertebrates (oyster, mussel, and lugworm). The resulting hydrolysates were evaluated for serum replacement in zebrafish ESCs. All hydrolysates were used at five different concentrations (10, 1 0.1, 0.01 and 0.001 mg/mL) in serum concentrations of 10%, 5%, and 0% with four biological replicates. The best hydrolysate sources and concentrations were selected for further testing at 2.5% and 1% serum concentrations. All hydrolysates, except for cricket, could restore or significantly increase cell growth with 50% less serum at a concentration of 0.1-0.001mg/mL. Protein hydrolysate concentration of 10 and 1mg/mL was toxic for cells. Additionally, the eight hydrolysates could reduce serum concentrations up to 75–90%. However, no protein hydrolysate could completely replace serum, as cells using only protein hydrolysates exhibited morphological aberrations and decreased growth. Replacing serum with protein hydrolysates lowers cellular agriculture's overall cost, thus enabling the commercialization of cultured meat and the development of a sustainable food system. In the future, blending various protein hydrolysate sources with or without the addition of conventional growth factors could be done to create the ideal serum-free media.
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    Enhancing Seed Quality and Application of Soybean through High Test Weigh and Low Trypsin Inhibitors
    Shea, Zachary (Virginia Tech, 2023-02-09)
    Soybean is an integral oilseed crop that is used all over the world. Despite this, soybean has been experiencing a decrease in seed quality seen by the decreasing trend in test weight, and is limited in its uses due to the antinutritional factor, trypsin inhibitor (TI). This project first worked to identify single nucleotide polymorphisms (SNPs) associated with high test weight through a genome-wide association study (GWAS) as well as determine the relationship between important seed composition traits and test weight. Additionally, it focused on identifying and knocking out KTI genes specifically expressed in soybean seed tissue through CRISPR/Cas9. Lastly, this project investigated further use of soybean in aquaculture by evaluating the performance of Rainbow trout fed diets with low-TI, 'VT Barrack' soy meal. Nine SNPs on chromosome 15 were found to be significantly associated with high test weight as and eight potential gene candidates were identified. Test weight was found to be significantly, and negatively related to seed oil content and had inconsistent correlations with protein and sugar content. The KTI genes KTI1, Glyma01g095000, and KTI3, Glyma08g341500, were found to be only expressed in seed tissues and multiple KTI knock-out soybean plants with decreased seed TI content and activity were developed. Lastly, we found that our low-TI soymeal could replace 30% of fish meal in aquafeeds with no negative effects on trout growth and health.
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    Evaluating the Potential of Marine Invertebrate and Insect Protein Hydrolysates to Reduce Fetal Bovine Serum in Cell Culture Media for Cultivated Fish Production
    Batish, Inayat; Zarei, Mohammad; Nitin, Nitin; Ovissipour, Reza (MDPI, 2022-11-16)
    The use of fetal bovine serum (FBS) and the price of cell culture media are the key constraints for developing serum-free cost-effective media. This study aims to replace or reduce the typical 10% serum application in fish cell culture media by applying protein hydrolysates from insects and marine invertebrate species for the growth of Zebrafish embryonic stem cells (ESC) as the model organism. Protein hydrolysates were produced from black soldier flies (BSF), crickets, oysters, mussels, and lugworms with a high protein content, suitable functional properties, and adequate amino-acid composition, with the degree of hydrolysis from 18.24 to 33.52%. Protein hydrolysates at low concentrations from 0.001 to 0.1 mg/mL in combination with 1 and 2.5% serums significantly increased cell growth compared to the control groups (5 and 10% serums) (p < 0.05). All protein hydrolysates with concentrations of 1 and 10 mg/mL were found to be toxic to cells and significantly reduced cell growth and performance (p < 0.05). However, except for crickets, all the hydrolysates were able to restore or significantly increase cell growth and viability with 50% less serum at concentrations of 0.001, 0.01, and 0.1 mg/mL. Although cell growth was enhanced at lower concentrations of protein hydrolysates, the cell morphology was altered due to the lack of serum. The lactate dehydrogenase (LDH) activity results indicated that BSF and lugworm hydrolysates did not alter the cell membrane. In addition, light and fluorescence imaging revealed that the cell morphological features were comparable to those of the 10% serum control group. Overall, lugworm and BSF hydrolysates reduced the serum by up to 90% while preserving excellent cell health.
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    Factors Influencing The Ecology and Epidemiology of Microbial Indicators and Foodborne Pathogens In Surface Waters and Development of Risk Mitigations
    Murphy, Claire Margaret (Virginia Tech, 2023-04-25)
    Foodborne outbreaks have continued to be associated with produce contamination originating from on-farm sources, such as soil or agricultural water. Additionally, the heterogeneity of the pre-harvest environment complicates the development of universal strategies for managing produce safety risks. Understanding the ecology and epidemiology of foodborne pathogens and fecal indicator bacteria (FIB) by growing regions, sample types, scale of analysis, and detection method is essential for developing targeted mitigation strategies. This dissertation utilized quantitative research methods and statistical modeling to examine the impact of sampling method, spatial, temporal, meteorological, and physicochemical factors on pathogen prevalence and FIB levels. Key findings highlight that the drivers of prevalence differ between pathogens and were influenced by sample type, scale, and region.. The variations in associations emphasize that risk varies by space and time. Therefore, results support regional and scale-dependent food safety standards and guidance documents for controlling hazards to minimize risk. Additionally, the method used for pathogen detection influences prevalence highlighting the need for standard methods since methodological differences confound comparisons across studies. Furthermore, since agricultural water quality is an important food safety priority, this dissertation aimed to determine the efficacy of chemical antimicrobial sanitizers against Salmonella in pre-harvest agricultural water. Results demonstrated that certain sanitizer treatments and conditions can significantly reduce Salmonella populations in preharvest agricultural water sources and thus may serve as a risk reduction option when used correctly.
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    Inhibition kinetics, molecular docking, and stability studies of the effect of papain-generated peptides from palm kernel cake proteins on angiotensin-converting enzyme (ACE)
    Zarei, Mohammad; Ghanbari, Raheleh; Zainal, Najib; Ovissipour, Reza; Saari, Nazamid (Elsevier, 2022-12-30)
    Three novel peptide sequences YGIKVGYAIP, GGIF, and GIFE from papain-generated protein hydrolysate of palm kernel cake proteins were used for stability study against ACE, ACE-inhibition kinetics, and molecular docking studies. Results showed that peptide YGIKVGYAIP was degraded, and its ACE-inhibitory activity decreased after 3 h pre-incubation with ACE, while peptides GGIF and GIFE were resistant. However, although the ACE-inhibitory activity of GIFE increased during this time, the ACE inhibitory activity of GGIF decreased after pre-incubation with ACE, indicating that peptide. YGIKVGYAIP and GGIF are substrate-type, whereas GIFE is a true-inhibitor type. Peptide YGIKVGYAIP showed the lowest Ki (0.054 mM) in the inhibition kinetics study compared to GGIF and GIFE, with Ki of 1.27 m M and 18 mM, respectively. In addition, YGIKVGYAIP revealed the lowest Km and Vmax and higher CE in different peptide concentrations, implying that the enzyme catalysis decreased, and peptides had some binding affinity to the enzyme in lower concentrations, which led to reduced catalytic ability. Furthermore, YGIKVGYAIP showed the lowest docking score of −14.733 and 21 interactions with tACE, while GGIF revealed the higher docking score of −8.006 with 15 interactions with tACE.
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    Nutritional Evaluation of Black Soldier Fly Frass as an Ingredient in Florida Pompano (Trachinotus carolinus L.) Diets
    Banavar, Amiti; Amirkolaei, Samad Keramat; Duscher, Lexi; Khairunisa, Bela Haifa; Mukhopadhyay, Biswarup; Schwarz, Michael; Urick, Steve; Ovissipour, Reza (MDPI, 2022-09-14)
    The aquaculture industry is in need of sustainable fish feed to reduce the use of expensive and environmentally invasive wild-caught fish currently fed to many carnivorous species. The black soldier fly (BSF) has become a popular sustainable alternative protein source; however, the nutritional waste byproduct of BSF, frass, has not been extensively studied as a feed replacement in carnivorous species. This study evaluates the potential of BSF frass on the growth, body composition, and intestinal microbiome of the Florida pompano, Trachinotus carolinus. Four experimental diets were formulated containing different levels of frass, replacing plant-based carbohydrate sources. As a result of this study, the frass did not improve the growth performance, resulting in a lower specific growth rate and higher feed conversion rate. While the frass diets did not alter the body composition, the visceral somatic index (VSI) significantly increased compared to the control diet and the hepatosomatic index (HIS) was lowered. The microbiome analysis showed high variation among the diets, with the control diet having the most distinct consortia, which may have been driven by the increased levels of starch compared to frass diets. This study indicates that BSF frass may not be a suitable feed replacement for carnivorous pompano; however, frass could still potentially be a replacement feed for herbivore or detritivore fish and should be further studied.
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    Prevalence of Microplastics in the Eastern Oyster Crassostrea virginica in the Chesapeake Bay: The Impact of Different Digestion Methods on Microplastic Properties
    Aung, Thet; Batish, Inayat; Ovissipour, Reza (MDPI, 2022-01-10)
    This study aimed to determine the microplastic prevalence in eastern oysters (C. virginica) in three sites in the Chesapeake Bay in Virginia and optimize the digestion methods. The digestion results illustrate that the lowest recovery rate and digestion recovery were related to enzymatic, enzymatic + hydrogen peroxide (H2O2), and HCl 5% treatments, while the highest digestion recovery and recovery rate were observed in H2O2 and basic (KOH) treatments. Nitric acid digestion resulted in satisfying digestion recovery (100%), while no blue polyethylene microplastics were observed due to the poor recovery rate. In addition, nitric acid altered the color, changed the Raman spectrum intensity, and melted polypropylene (PP) and polyethylene terephthalate (PET). In order to determine the number of microplastics, 144 oysters with an approximately similar size and weight from three sites, including the James River, York River, and Eastern Shore, were evaluated. Fragments were the most abundant microplastics among the different microplastics, followed by fibers and beads, in the three sites. A significantly higher number of fragments were found in the James River, probably due to the greater amount of human activities. The number of microplastics per gram of oyster tissue was higher in the James River, with 7 MPs/g tissue, than in the York River and Eastern Shore, with 6.7 and 5.6 MPs/g tissue.
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    Serum-free media development using black soldier fly protein isolate and hydrolysate for cultivated meat
    Garg, Palak (Virginia Tech, 2024-01-03)
    The global demand for animal proteins is projected to rise by 14% by 2030, amplifying the environmental toll of conventional animal-based protein production. Cultivated meat technology can alleviate the growing demand for protein and address the environmental and ethical concerns associated with conventional livestock farming. However, it faces a critical challenge: the high cost of cell culture media, primarily due to the use of Fetal Bovine Serum (FBS). Substituting serum with protein hydrolysates reduces the production expense of cultivated meat products and promotes establishing a sustainable food system. This study explores black soldier fly larvae (Hermetia illucens) as an emerging ethical and cost-effective alternative protein source to replace serum in media, particularly for cultivated meat production. The development of BSFL protein isolate involved defatting the larva, followed by protein extraction. The protein isolate was then hydrolyzed using an enzyme to produce BSFL hydrolysates. The goal was to supplement the protein isolate and hydrolysates with a serum-free media (B8) and determine their efficacy in replacing the 20% serum requirement for the cell culture of Bovine Satellite Cells. The BSFL protein isolate developed had a crude protein content of 80.42% and an amino acid composition conducive to cell proliferation. Experimental concentrations, ranging from 0.006 mg/ml for hydrolysate to 0.06 mg/ml for protein isolate, exhibited enhanced cell growth. Data from dsDNA quantification revealed no significant difference in growth between cells fed serum-containing growth media (BSC-GM) and BSFL protein hydrolysate (BSFLH_1h) over a short-term study. Results from the multi-passage growth study revealed that BSFLH_1h significantly improved cell growth compared to B8 over 4 passages. However, its doubling time was slower than BSC-GM. Additionally, it was observed that the protein isolate and hydrolysate were cytotoxic at higher concentrations. In the future, identifying and removing the cytotoxic compounds can further optimize the media composition. Immunostaining using Pax7 and DAPI identified supplemented media-maintained satellite cell identity of Bovine satellite cells, offering crucial insights into cellular proliferation. Furthermore, since each cell type requires varying serum and nutrients, testing these isolates and hydrolysates on different cell lines can provide better insight into creating a universal serum-free media.
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    Sorghum as a Potential Valuable Aquafeed Ingredient: Nutritional Quality and Digestibility
    Zarei, Mohammad; Amirkolaei, Abdolsamad K.; Trushenski, Jesse T.; Sealey, Wendy M.; Schwarz, Michael H.; Ovissipour, Reza (MDPI, 2022-05-06)
    The world population is increasing, and our current agricultural practices are not sustainable enough to address the concerns. Alternative proteins including plant-based proteins would provide a more sustainable source of food and feed ingredients. Among food systems, the aquaculture industry is rapidly growing, while still depending on marine sources as a main source of protein. Thus, using alternative plant-based proteins as a source for developing aquafeed would make this industry more viable. Sorghum is a valuable grain with high protein contents, proper mineral and fatty acids balance, and is available all around the world. However, sorghum has not been used widely for aquafeed development. In this review article, we cover sorghum production, composition, sorghum as a protein source for aquafeed development, and bioprocessing methods for enhancing the quality of sorghum.
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    Spatial Versus Nonspatial Variance in Fecal Indicator Bacteria Differs Within and Between Ponds
    Murphy, Claire M.; Weller, Daniel L.; Ovissipour, Reza; Boyer, Renee R.; Strawn, Laura K. (Elsevier, 2023-03)
    Surface water environments are inherently heterogenous, and little is known about variation in microbial water quality between locations. This study sought to understand how microbial water quality differs within and between Virginia ponds. Grab samples were collected twice per week from 30 sampling sites across nine Virginia ponds (n = 600). Samples (100 mL) were enumerated for total coliform (TC) and Escherichia coli (EC) levels, and physicochemical, weather, and environmental data were collected. Bayesian models of coregionalization were used to quantify the variance in TC and EC levels attributable to spatial (e.g., site, pond) versus nonspatial (e.g., date, pH) sources. Mixed-effects Bayesian regressions and conditional inference trees were used to characterize relationships between data and TC or EC levels. Analyses were performed separately for each pond with ≥3 sampling sites (5 intrapond) while one interpond model was developed using data from all sampling sites and all ponds. More variance in TC levels were attributable to spatial opposed to nonspatial sources for the interpond model (variance ratio [VR] = 1.55) while intrapond models were pond dependent (VR: 0.65–18.89). For EC levels, more variance was attributable to spatial sources in the interpond model (VR = 1.62), compared to all intrapond models (VR < 1.0) suggesting that more variance is attributable to nonspatial factors within individual ponds and spatial factors when multiple ponds are considered. Within each pond, TC and EC levels were spatially independent for sites 56–87 m apart, indicating that different sites within the same pond represent different water quality for risk management. Rainfall was positively and pH negatively associated with TC and EC levels in both inter- and intrapond models. For all other factors, the direction and strength of associations varied. Factors driving microbial dynamics in ponds appear to be pond-specific and differ depending on the spatial scale considered.