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Ontogenetic shifts in digestive enzyme kinetics drive age-dependent in vitro digestibility outcomes for fungal biomass and conventional ingredients in Oncorhynchus mykiss

This study demonstrates that the in vitro digestibility of fungal biomass compared to conventional ingredients in rainbow trout is highly age-dependent, driven by non-linear ontogenetic shifts in gastric and intestinal enzyme kinetics that result in fungal proteins being more efficiently hydrolyzed than fishmeal in older (2+) fish but less so in younger (0+ and 1+) fish.

Original authors: Mahtab Mohammadi, Enric Gisbert, Mohammad J. Taherzadeh, Amir Mahboubi Soufiani

Published 2026-10-05

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CC BY 4.0

Original authors: Mahtab Mohammadi, Enric Gisbert, Mohammad J. Taherzadeh, Amir Mahboubi Soufiani

Original paper licensed under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/). ✨ This is an AI-generated explanation of the paper below. It is not written or endorsed by the authors. For technical accuracy, refer to the original paper. Read full disclaimer

The world's aquaculture industry, which raises fish for human consumption, faces a growing dilemma. To feed billions of farmed fish, producers have long relied on fishmeal, a protein source made from wild-caught marine life. This practice strains ocean ecosystems, prompting a shift toward plant-based alternatives like soy and wheat. However, these terrestrial crops bring their own problems, from deforestation to competition with human food supplies, and they often lack the nutritional balance fish need to thrive. In response, scientists are turning to a different kind of protein: fungal biomass. Grown by fermenting organic matter, these microscopic fungi offer a sustainable, scalable source of nutrition. But before they can replace traditional ingredients, researchers must understand how well a fish's body can break them down and absorb their nutrients. This process is not static; a fish's digestive system changes dramatically as it grows from a tiny fry into a large adult, much like a human child's stomach differs from an adult's. If scientists test new feed ingredients using only one age group, they risk getting a misleading picture of how the food will perform in the real world.

A team of researchers set out to solve this puzzle by studying rainbow trout at three distinct stages of life: young fish weighing about 100 grams, medium-sized fish around 700 grams, and large adults nearing 3,000 grams. Instead of feeding the fish and waiting months to see if they grew, the team used a laboratory method that simulates the fish's digestive tract. They collected stomach and intestinal tissues from the fish, extracted the natural enzymes responsible for digestion, and tested how these enzymes interacted with six different protein sources. These sources included the standard fishmeal, soybean meal, and wheat gluten, alongside three types of fungal biomass grown in the lab. The researchers carefully measured the speed and efficiency of the enzymes, noting that the chemical behavior of these biological tools was not the same for every fish size. They found that the digestive enzymes in the youngest fish worked best at low concentrations of food, while the enzymes in the largest fish behaved differently, showing a tendency to slow down when faced with too much food at once.

When the team ran the simulated digestion, the results revealed a surprising reversal in performance depending on the age of the fish. For the young and medium-sized trout, the traditional fishmeal was the easiest to digest, with the enzymes breaking down more than half of its protein. The fungal proteins performed well, but they did not quite match the fishmeal, while the plant-based ingredients were the hardest to break down. However, the story changed completely for the large, adult fish. In these older specimens, the fishmeal became surprisingly difficult to digest, with the enzymes managing to break down only about 14 percent of its protein. In contrast, two of the fungal ingredients, specifically those from the species Neurospora intermedia and Aspergillus oryzae, became the most digestible options, with the enzymes releasing nearly 28 percent of their protein. The plant-based ingredients remained the least digestible for this age group as well.

The researchers traced this shift back to the changing nature of the fish's digestive enzymes as they matured. The young fish possessed enzymes that were highly efficient at grabbing onto and breaking down simple proteins quickly, which suited the easily digestible fishmeal. As the fish grew, their digestive strategy shifted. The large adults developed enzymes better suited for a slower, sustained breakdown of complex prey, a trait that inadvertently made the standard fishmeal harder to process within the short timeframe of the experiment. The fungal proteins, however, seemed to possess a unique structural advantage for these older fish. The study suggests that the adult fish's stomach enzymes may have a specific ability to weaken the tough outer walls of the fungi, allowing the internal proteins to be released and digested more effectively than the rigid structures found in plant meals. This finding highlights that the "best" feed ingredient is not a fixed choice but depends entirely on the life stage of the fish being fed.

The study also ruled out the idea that the fungi were simply breaking down on their own inside the stomach without help from the fish. While some of the fungal ingredients did show a slight ability to digest themselves, the vast majority of the protein breakdown was driven by the fish's own enzymes. Furthermore, the researchers noted that previous studies comparing these ingredients might have been misleading if they did not account for these age-related changes. For instance, a different study had suggested that one type of fungus was superior to another, but the current research indicates that such rankings can flip depending on whether the fish is young or old. The team concluded that to create truly effective and sustainable fish feed, nutritionists must stop treating all fish as if they have the same digestive needs. Instead, they must design diets that match the specific enzymatic capabilities of the fish at each stage of its life, ensuring that alternative proteins like fungal biomass are used where they will perform best.

Technical Summary: Ontogenetic Shifts in Digestive Enzyme Kinetics and Age-Dependent In Vitro Digestibility in Rainbow Trout

Problem Statement
The aquaculture industry faces a sustainability crisis driven by reliance on finite marine resources (fishmeal) and the ecological and nutritional limitations of terrestrial plant-based alternatives (e.g., deforestation, anti-nutritional factors). Fungal biomass (mycoprotein) has emerged as a promising alternative protein source; however, its nutritional validation traditionally relies on resource-intensive in vivo trials. Furthermore, existing in vitro digestibility models often fail to account for the ontogenetic development of the fish gastrointestinal tract (GIT). Current literature predominantly utilizes single life-stage models, potentially yielding misleading results because digestive physiology, enzyme profiles, and morphological maturation change significantly from juvenile to adult stages. This study addresses the gap in understanding how age-dependent enzymatic kinetics influence the in vitro digestibility of fungal biomass compared to conventional ingredients in rainbow trout (Oncorhynchus mykiss).

Methodology
The study employed a modified in vitro digestion protocol adapted from the INFOGEST framework, utilizing physiologically relevant enzyme extracts rather than commercial enzymes.

Subjects: Digestive tissues were harvested from three distinct age classes of rainbow trout: 0+ (~~100 g), 1+ (~~700 g), and 2+ (~3000 g).
Ingredients: Six protein sources were tested: three fungal strains (Neurospora intermedia, Aspergillus oryzae, Rhizopus oligosporus) and three conventional ingredients (fishmeal, roasted soybean meal, wheat gluten).
Enzyme Kinetics: Gastric and intestinal enzyme extracts were prepared and kinetically modeled against varying substrate concentrations. Gastric activity was assayed using hemoglobin at pH 2, and intestinal activity using casein at pH 8. Kinetic parameters ( $V_{max}$ , $K_m$ , and inhibition constants $K_i$ ) were determined using Michaelis-Menten, Hill, and substrate inhibition models.
Digestibility Assay: A two-phase digestion (2-hour gastric at pH 2, 4-hour intestinal at pH 8) was conducted at 13°C. Substrate concentrations were standardized based on the specific kinetic parameters of each age class (targeting $2 \times K_m$ for gastric, limited by intestinal $K_i$ ). Degree of Hydrolysis (DH%) was calculated based on the release of free primary amino groups relative to total amino acids.

Key Results

Ontogenetic Shifts in Enzyme Kinetics:
- Gastric Enzymes: Followed Michaelis-Menten kinetics across all age classes. $V_{max}$ values fluctuated non-linearly: 11.02 (0+), 7.85 (1+), and 15.71 $\mu$ mol mg $^{-1}$ min $^{-1}$ (2+). The $K_m$ increased with age (0.36% in 0+ to 1.21% in 2+), indicating a shift from high substrate affinity in juveniles to lower affinity in adults, with the 1+ stage representing a transitional minimum in catalytic capacity.
- Intestinal Enzymes: Exhibited substrate inhibition kinetics. The inhibition constant ( $K_i$ ) varied significantly: 0.55% (0+), 3.99% (1+), and 2.02% (2+). The 0+ stage showed the strongest inhibition, suggesting a regulatory mechanism to prevent autodigestion during rapid growth, while adults showed reduced sensitivity to substrate inhibition.
Age-Dependent Digestibility Outcomes:
- 0+ and 1+ Fish: Fishmeal demonstrated the highest hydrolysis (49–55%), followed by N. intermedia (39–43%) and A. oryzae (~40%). Plant-based ingredients (soybean meal, wheat gluten) and R. oligosporus showed the lowest hydrolysis (21–29%).
- 2+ Fish: A complete inversion of the hierarchy occurred. N. intermedia (27%) and A. oryzae (28%) became the most extensively hydrolyzed ingredients, significantly outperforming fishmeal (14%).
- Auto-hydrolysis: N. intermedia and A. oryzae exhibited significant endogenous proteolytic activity (auto-hydrolysis), which contributed to their higher DH values, particularly in the 2+ age class where fish enzymes alone provided a lower baseline.

Key Contributions

Kinetic Standardization: The study established a method to characterize precise enzymatic kinetics at different fish size classes to standardize in vitro assay parameters (substrate-to-enzyme ratios), moving beyond static models.
Dynamic Digestibility: It demonstrated that protein digestibility is not a static property of an ingredient but a dynamic variable fundamentally determined by the interaction between the fish's life stage and the ingredient's structural matrix.
Fungal Protein Evaluation: It provided comparative data showing that while fishmeal is superior for younger trout, specific fungal proteins (N. intermedia, A. oryzae) may offer superior or comparable digestibility in older trout, challenging the assumption that fishmeal is universally the most digestible protein source.

Significance and Claims
The paper claims that the nutritional evaluation of feed ingredients cannot rely on a single developmental stage. The findings highlight that ingredient ranking is not static; it depends on the specific interaction between feed structure and the fish's digestive profile at a given age.

Physiological Insight: The shift in enzyme kinetics (from high-affinity/low-capacity in juveniles to lower-affinity/high-capacity in adults) reflects a metabolic reorientation from rapid somatic growth to metabolic maintenance and reproduction.
Methodological Implication: Applying adult-derived in vitro models to juvenile diets (or vice versa) is scientifically unsound and risks producing false results. The study advocates for age-specific approaches in in vitro digestibility assessments to accurately inform aquafeed formulation.
Ingredient Potential: Fungal proteins, particularly Ascomycota strains, show promise as competitive alternatives, with their performance relative to fishmeal being highly dependent on the target age group of the fish. The study suggests that the structural complexity of fungal cell walls interacts differently with the evolving enzymatic capabilities of the trout, particularly in adult stages where gastric chitinase activity and fungal autolysis may play synergistic roles.

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