Recipient Organization
ATLANTIC FISH CORPORATION
3740 W ST NW
WASHINGTON,DC 20007
Performing Department
(N/A)
Non Technical Summary
Despite being the most widely consumed animal protein in the world, our current seafood production system is not environmentally sustainable. Cellular agriculture is a transformative technology with the potential to substantially reduce the environmental impact of our current system. This process involves the expansion of animal cells in a bioreactor and subsequent differentiation into the tissue types found in meat, followed by assembly into a final food product. We have developed an immortalized, myogenic fish cell line from black sea bass and associated bioprocesses to expand these cells and differentiate them into muscle. In this project, we propose to develop a hybrid fish nugget consisting of cultivated fish muscle tissue and plant-based ingredients. We will first characterize the molecular composition of our cultivated fish muscle and how it affects the organoleptic properties of the hybrid nugget. We will use this information to refine our cultivation process, improving the quality of our hybrid nugget until it reaches parity with traditional fish nuggets. This project will enable us to develop an entirely novel product, gain market traction, and benefit from early cycles of learning. In the long-term, the knowledge generated regarding the specific molecular composition of our cultivated muscle tissue and how it relates to the organoleptic properties of our fish nuggets will form a proprietary competitive moat that will enable us to develop more advanced products, including hybrid products with a striated muscle structure and fully cell-based filets, while reducing the environmental impact of our seafood production system.
Animal Health Component
40%
Research Effort Categories
Basic
10%
Applied
40%
Developmental
50%
Goals / Objectives
Our goal for this project is to rationally develop a hybrid fish nugget consisting of cultivated fish muscle tissue and plant-based ingredients. In the short-term, this project will enable us to develop a product which does not currently exist, giving us market traction and allowing us to benefit from early cycles of learning in a nascent industry. In the long-term, the knowledge generated regarding the specific molecular composition of our cultivated muscle tissue, how it relates to the organoleptic properties of our fish nugget, and how different production processes can improve this will form a proprietary competitive moat that will enable us to develop more advanced products, including hybrid products with a striated muscle structure and fully cell-based products. It will be accomplished via the following specific objectives:Objective 1: Produce and characterize cultivated fish muscle tissue.The outcome of this Objective is a thorough, quantitative dataset describing the molecular composition of our cultivated fish muscle tissue and traditional fish muscle.Objective 2: Develop and characterize a prototype hybrid fish nugget.The outcome of this Objective is both an edible prototype hybrid fish nugget and a quantitative understanding of how cultivated fish muscle affects the prototype compared to traditional fish muscle.Objective 3: Develop rational process improvements for our cultivated fish muscle tissue.The outcome of this Objective is an improved, commercially competitive hybrid fish nugget with less than 10% deviation from those produced using traditional fish muscle in instrumented and sensory panel testing.
Project Methods
Objective 1: Produce and characterize cultivated fish muscle tissueExpansion of C. striata muscle progenitor cells: We will expand our immortalized muscle progenitor cell line (BSB509) on edible microcarriers in a 2L stirred tank bioreactor. Note that the microcarriers will not be part of the final product, but we are using an edible formulation to alleviate any food safety concerns. We will use magnetic stir flasks to create a seed train, expanding cellsuntil we have > 50M cells. The bioreactor will be inoculated with a starting volume of 500mL at 100k cells/mL and 3.6g of microcarriers and run in fed batch mode with a preset time profile for feeding until cells reach confluency on the microcarriers in 1.5L of media (working volume). At this point, cells and microcarriers will be harvested via centrifugation and used in the next step. Our cells will be maintained at 25C, pH 7.4 under atmospheric CO2.Differentiation into muscle tissue: Confluent cells on microcarrierswill becultured in differentiation medium for 7 days as the cells differentiate into muscle tissue. Next, the tissue will be harvested by dissociating the cells from the microcarriers, dissolving the microcarriers via enzymatic digest, andcentrifugation to collect the final cell mass. This will be transferred into appropriate food-safe containers prior to flash freezing for storage or transport to NCFIL in the following objectives. We will repeat expansion and differentiation until we have collected ~250g of tissue. We expect that cell mass will increase slightly during differentiation but have budgeted 5x bioreactor runs (~70 days) in case there are any losses. A sample from each experiment will be stained by immunolabeling for skeletal muscle-specific myosin heavy chain to verify differentiation (>70% fusion index, defined as % of nuclei in MF20+ myotubes).Characterization of cultivated fish muscle: For all characterization approaches, we will also analyze muscle tissue harvested from C. striata to be used as a future benchmark (Objective 3). Proximate analysis will be performed by first grinding the tissue using a laboratory mill to a powder (~150-200 µm) and tested for moisture, fat, ash, and carbohydrate (by difference) using the CEM Oracle and Sprint instrumentation suite. Aroma analysis will be performed using automated headspace solid-phase microextraction - gas chromatography-mass spectrometry. Extraction time/temperature, sample concentration/volume, and fiber material will be optimized to achieve the highest sensitivity. Samples will be spiked with an internal standard of 2-methyl-3-heptanone and methyl nonanoate in HPLC-grade methanol. Mass spectra will be analyzed using commercially available software. Quantification of compound odor activity values will be performed by comparison of comparison of chromatographic peaks to the internal standard, followed by adjustment by the odor threshold value for each identified compound. To determine the equivalent umami concentration, we will first measure free amino acids and nucleotides by liquid chromatography- tandem mass spectrometry (LC-MS-MS).Umami flavor (g monosodium glutamate equivalent /100g tissue) will be calculated from the amino acids Asp and Glu and the nucleotides IMP, GMP, and AMP.Microbial analysis will be performed using the following AOAC test methods: 2015.13 (enumeration of aerobic bacteria in food), 991.14 (coliforms and E. coli in foods), and 997.02 (yeast and molds in foods).Objective 2: Develop and characterize a prototype hybrid fish nuggetSensory evaluation of fish nuggets: To perform sensory evaluation, we will use a trained panel of experts in accordance with ISO 8586:2023 and all other relevant ISO standards under the supervision of NCFIL. We will obtain IRB approval and informed consent from all participants. Products will be tested for contamination prior to tasting (see above for details).Development of plant-based nugget: NCFIL will perform an initial ingredient screen based on emulsifying and water binding/holding properties to select plant-based proteins, texturizers, binding agents, and flavoring compounds. They will create 3 experimental formulations that represent promising combinations based on initial gross subjective characterization by an experienced food scientist. These will be used to prepare entirely plant-based fish nuggets. To prepare the nuggets, NCFIL will combine the ingredients together in 20 g portions and form them into nuggets. The nuggets will be flash frozen and then battered with a tempura-style batter. They will then be deep fried in neutral oil and frozen and packaged. For sensory evaluation these nuggets will be re-heated via air fryer and organoleptically evaluated by our expert panel in comparison to commercially available premium fish nuggets.Development of hybrid fish nugget: Nuggets will be formulated using the plant-based ingredients selected above and either (a) cultivated fish muscle tissue or (b) C. striata muscle homogenate. To prepare the C. striata muscle homogenate, we will finely mince C. striata muscle tissue, wash with saline solution, and enzymatically digest using food-grade trypsin. We will wash the resulting suspension with saline to remove soluble compounds and excess trypsin and create a homogenate that has similar gross physical properties to our cultivated fish muscle tissue. Fish nuggets will be fabricated with 5, 10, 15, and 20% cultivated muscle or homogenate by weight for a total of 10 different groups (4x cultivated, 4x homogenate, plant only control, and traditional fish nugget control). The nuggets will be prepared and evaluated as described above. We will select (a) the cultivated hybrid nugget and (b) the homogenate hybrid nugget with scores closest to (or better than) traditional fish nuggets for further characterization.Characterization of hybrid fish nuggets:Proximate analysis will be performed as described above for both raw and cooked nuggets. To determine how each formulation responds to cooking, we will compare moisture and fat for raw and cooked nuggets. To quantify the relevant material properties of each formulation, we will perform texture profile analysis.Briefly, the product will be compressed 3x using a 35mm diameter plexiglass cylinder to 50% strain with a contact force of 5 g and force threshold of 20 g. Hardness (peak force during first compression), springiness (ratio of height at beginning of second indentation to original height), cohesiveness (ratio of area under second compression curve to area under first compression curve) and chewiness (hardness * cohesiveness * springiness) will be calculated to quantify relevant organoleptic characteristics. Shear force analysis will be performed by first cutting cooked fish nuggets into 2 x1 cm strips and shearing straight through with a shear blade at 5 mm/s. The corresponding force-displacement curves will be used to determine shear force value (maximal force).Objective 3: Develop rational process improvements for our cultivated fish muscle tissueWe will start with the categories that our results from Objective 2 identify as the largest differences between the cultivated and homogenate nuggets. To compare quantitative results, we will use appropriate tests (e.g., ANOVA) to determine if the results are statistically different. Based on this information, we will identify the compounds from Objective 1 that are likely culprits. We will then implement these changes, produce a test batch (~10g, using shake flasks) of cultivated muscle, and characterize the muscle to determine if the change was successful. After a successful result, we will send the updated cultivated muscle to NCFIL for formulation and sensory/instrumented testing. We will continue this iterative test loop until we observe a less than 10% difference from those produced using traditional fish muscle.