Development of a cell line from skeletal trunk muscle of the fish Labeo rohita.

Labeo rohita is a widely cultivated tropical freshwater carp and found in rivers of South Asian region. A new cell line, designated LRM, has been developed from the muscle tissue of L. rohita. Muscle cells were subcultured up to 38 passages in a Leibovitz's-15 (L-15) supplemented with 10% FBS (Fetal Bovine Serum) and 10 ng/ml bFGF. The LRM cells exhibited fibroblastic morphology with a doubling time of 28 h, and a plating efficiency of 17%. A maximum growth rate was observed for LRM cells at 28 °C, 10% FBS and 10 ng/ml bFGF. A cytochrome C oxidase subunit I (COI) gene sequence was used to authenticate the developed cell line. Chromosome analysis revealed 50 diploid chromosomes. The fibroblastic characteristics of the of the LRM cells was confirmed by immunocytochemistry. The expression of MyoD gene in LRM cells was analyzed by quantitative PCR in comparison with passages 3, 18 and 32. The expression of MyoD was higher at passage 18 compared to the passages 3 and 32. The LRM cells attached properly onto the 2D scaffold and the expression of the F-actin filament protein was confirmed by phalloidin staining followed by counter staining with DAPI to observe the distribution of the muscle cell nuclei and the cytoskeleton protein. A revival rate of 70-80% was achieved when the LRM cells were cryopreserved at - 196 °C using liquid nitrogen. This study would further contribute to understanding the in vitro myogenesis and progress toward cultivated fish meat production.

Toward sustainable culture media: Using artificial intelligence to optimize reduced-serum formulations for cultivated meat.

When considering options for future foods, cell culture approaches are at the fore, however, culture media to support the process has been identified as a significant contributor to the overall global warming potential (GWP) and cost of cultivated meat production. To address this issue, an artificial intelligence-based approach was applied to simultaneously optimize the GWP, cost, and cell growth rate of a reduced-serum culture media formulation for a zebrafish (ZEM2S cell line) cultivated meat production system. Response surface methodology (RSM) was used to design the experiments, with seven components - IGF, FGF, TGF, PDGF, selenium, ascorbic acid, and serum - selected as independent variables, given their influence on culture media performance. Radial basis function (RBF) neural networks and genetic algorithm (GA) were applied for prediction of dependent variables, and optimization of the culture media formulation, respectively. The results indicated that the developed RBF could accurately predict the GWP, cost and growth rate, with a model efficiency of 0.98. Subsequently, the three developed RBF neural networks predictive models were used as the inputs for a multi-objective genetic algorithm, and the optimal quantities of the independent variables were determined using a multi-objective optimization algorithm. The suggested RSM + RBF + GA framework in this study could be applied to sustainably optimize serum-free media development, identifying the combination of media ingredients that balances yield, environmental impact, and cost for various cultivated meat cell lines.

Spatial Versus Nonspatial Variance in Fecal Indicator Bacteria Differs Within and Between Ponds.

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.

Inhibition kinetics, molecular docking, and stability studies of the effect of papain-generated peptides from palm kernel cake proteins on angiotensin-converting enzyme (ACE).

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.

Evaluating the Potential of Marine Invertebrate and Insect Protein Hydrolysates to Reduce Fetal Bovine Serum in Cell Culture Media for Cultivated Fish Production.

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.

Nutritional Evaluation of Black Soldier Fly Frass as an Ingredient in Florida Pompano (Trachinotus carolinus L.) Diets.

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.

Cellular Aquaculture: Prospects and Challenges.

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.

SARS-CoV-2 Remains Infectious on Refrigerated Deli Food, Meats, and Fresh Produce for up to 21 Days.

SARS-CoV-2, the virus that causes COVID-19, has been detected on foods and food packaging and the virus can infect oral cavity and intestinal cells, suggesting that infection could potentially occur following ingestion of virus-contaminated foods. To determine the relative risk of infection from different types of foods, we assessed survival of SARS-CoV-2 on refrigerated ready-to-eat deli items, fresh produce, and meats (including seafood). Deli items and meats with high protein, fat, and moisture maintained infectivity of SARS-CoV-2 for up to 21 days. However, processed meat, such as salami, and some fresh produce exhibited antiviral effects. SARS-CoV-2 also remained infectious in ground beef cooked rare or medium, but not well-done. Although infectious SARS-CoV-2 was inactivated on the foods over time, viral RNA was not degraded in similar trends, regardless of food type; thus, PCR-based assays for detection of pathogens on foods only indicate the presence of viral RNA, but do not correlate with presence or quantity of infectious virus. The survival and high recovery of SARS-CoV-2 on certain foods support the possibility that food contaminated with SARS-CoV-2 could potentially be a source of infection, highlighting the importance of proper food handling and cooking to inactivate any contaminating virus prior to consumption.

Prevalence of Microplastics in the Eastern Oyster Crassostrea virginica in the Chesapeake Bay: The Impact of Different Digestion Methods on Microplastic Properties.

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.

Assessing the Enzymatic Hydrolysis of Salmon Frame Proteins through Different By-Product/Water Ratios and pH Regimes.

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.

The Effect Ultrasound and Surfactants on Nanobubbles Efficacy against Listeria innocua and Escherichia coli O157:H7, in Cell Suspension and on Fresh Produce Surfaces.

Removing foodborne pathogens from food surfaces and inactivating them in wash water are critical steps for reducing the number of foodborne illnesses. In this study we evaluated the impact of surfactants on enhancing nanobubbles' efficacy on Escherichia coli O157:H7, and Listeria innocua removal from spinach leaves. We evaluated the synergistic impact of nanobubbles and ultrasound on these two pathogens inactivation in the cell suspension. The results indicated that nanobubbles or ultrasound alone could not significantly reduce bacteria in cell suspension after 15 min. However, a combination of nanobubbles and ultrasonication caused more than 6 log cfu/mL reduction after 15 min, and 7 log cfu/mL reduction after 10 min of L. innocua and E. coli, respectively. Nanobubbles also enhanced bacterial removal from spinach surface in combination with ultrasonication. Nanobubbles with ultrasound removed more than 2 and 4 log cfu/cm2 of L. innocua and E. coli, respectively, while ultrasound alone caused 0.5 and 1 log cfu/cm2 of L. innocua and E. coli reduction, respectively. No reduction was observed in the solutions with PBS and nanobubbles. Adding food-grade surfactants (0.1% Sodium dodecyl sulfate-SDS, and 0.1% Tween 20), did not significantly enhance nanobubbles efficacy on bacterial removal from spinach surface.

Survival of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Herpes Simplex Virus 1 (HSV-1) on Foods Stored at Refrigerated Temperature.

Outbreaks of coronavirus infectious disease 2019 (COVID-19) in meat processing plants and media reports of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on foods have raised concerns of a public health risk from contaminated foods. We used herpes simplex virus 1, a non-Biosafety Level 3 (non-BSL3) enveloped virus, as a surrogate to develop and validate methods before assessing the survival of infectious SARS-CoV-2 on foods. Several food types, including chicken, seafood, and produce, were held at 4 °C and assessed for infectious virus survival (herpes simplex virus 1 (HSV-1) and SARS-CoV-2) at 0 h, 1 h, and 24 h post-inoculation (hpi) by plaque assay. At all three time points, recovery of SARS-CoV-2 was similar from chicken, salmon, shrimp, and spinach, ranging from 3.4 to 4.3 log PFU/mL. However, initial (0 h) virus recovery from apples and mushrooms was significantly lower than that from poultry and seafood, and infectious virus decreased over time, with recovery from mushrooms becoming undetectable by 24 hpi. Comparing infectious virus titers with viral genome copies confirmed that PCR-based tests only indicate presence of viral nucleic acid, which does not necessarily correlate with the quantity of infectious virus. The survival and high recovery of SARS-CoV-2 on certain foods highlight the importance of safe food handling practices in mitigating any public health concerns related to potentially contaminated foods.

Effects of Enzymatic Hydrolysis on the Functional Properties, Antioxidant Activity and Protein Structure of Black Soldier Fly (Hermetia illucens) Protein.

The effects of chemical protein extraction, and enzymatic hydrolysis with Alcalase, papain and pepsin, on the functional properties, antioxidant activity, amino acid composition and protein structure of black soldier fly (H. illucens) larval protein were examined. Alcalase hydrolysates had the highest degree of hydrolysis (p < 0.05), with the highest hydrolysate and oil fraction yield (p < 0.05). Pepsin hydrolysates showed the lowest oil holding capacity (p < 0.05), whereas no significant differences were observed among other enzymes and protein concentrates (p > 0.05). The emulsifying stability and foam capacity were significantly lower in protein hydrolysates than protein concentrate (p < 0.05). The antioxidant activity of protein hydrolysates from protein concentrate and Alcalase was higher than that with papain and pepsin (p < 0.05), owing to the higher hydrophobic amino acid content. Raman spectroscopy indicated structural changes in protein α-helices and ß-sheets after enzymatic hydrolysis.

Inactivation of Aeromonas hydrophila and Vibrio parahaemolyticus by Curcumin-Mediated Photosensitization and Nanobubble-Ultrasonication Approaches.

The antimicrobial efficacy of novel photodynamic inactivation and nanobubble technologies was evaluated against Vibrio parahaemolyticus and Aeromonas hydrophila as two important aquatic microbial pathogens. Photodynamic inactivation results showed that LED (470 nm) and UV-A (400 nm)-activated curcumin caused a complete reduction in V. parahaemolyticus at 4 and 22 °C, and a greater than 2 log cfu/mL reduction in A. hydrophila, which was curcumin concentration-dependent (p < 0.05). Furthermore, the photodynamic approach caused a greater than 6 log cfu/mL V. parahaemolyticus reduction and more than 4 log cfu/mL of A. hydrophila reduction in aquaponic water samples (p < 0.05). Our results with the nanobubble technology showed that the nanobubbles alone did not significantly reduce bacteria (p > 0.05). However, a greater than 6 log cfu/mL A. hydrophila reduction and a greater than 3 log cfu/mL of V. parahaemolyticus reduction were achieved when nanobubble technology was combined with ultrasound (p < 0.05). The findings described in this study illustrate the potential of applying photodynamic inactivation and nanobubble-ultrasound antimicrobial approaches as alternative novel methods for inactivating fish and shellfish pathogens.