Introduction
The yellow mealworm (Tenebrio molitor), an emerging alternative ingredient for aquafeeds with potential to influence the immune and oxidative status of fish [1]. Modifying the rearing substrate used in mealworm production is a promising strategy to enhance productivity, which may modulate not only the nutritional value, but also the functional properties of the end-product [2, 3]. Spent mushroom substrate (SMS), the main waster of mushroom production, presents a sustainable alternative to conventional cereal-based rearing substrates traditionally used in mealworm farming [4, 5]. This study aimed to evaluate the impact of larval rearing substrate composition on the bioactive and functional properties of mealworm meal (MM). Additionally, the effects of dietary inclusion of both MM types on the immune and oxidative status of juvenile rainbow trout (Oncorhynchus mykiss) were evaluated.
Materials and methods
Two different MM were used, a commercial MM (CC-MM, crude protein 51.9%, crude lipids: 32.8%) and a teste MM (SMS-MM, crude protein 53.1%, crude lipids: 24.2%), produced using a substrate composed by 80% of commercial rearing subtrate and 20% of SMS. The bioactive and functional properties of CC-MM and SMS-CC were assessed in an in vitro assay. Functional compounds were extracted from both MM using three different extraction methods: distilled water, 1:1 acetone and methanol mixture, or dichloromethane. The antimicrobial activity of the crude extracts was tested against six fish-relevant reference microorganisms (Aeromonas hydrophila DSM 3018, Yersinia ruckeri ATCC 29473, Listonella anguillarum ATCC 19264, Lactococcus garvieae DSM 20684, Tenacibaculum maritimum ATCC 43397 and Edwardsiella tarda DSM 30052) using the agar-based disk diffusion method [6]. A growth trial was then conducted to determine the effect of the dietary inclusion of both MM types on the immune and oxidative status of rainbow trout. Five isoproteic (45%) and isolipidic (22%) experimental diets were formulated: a control diet (CTRL) similar to a traditional commercial trout diet and four test diets based on the CTRL diet, including 12.5% or 25% of CC-MM or SMS-MM, replacing either 50% or 100% of fishmeal, respectively (CC12.5, CC25, SMS12.5, SMS25 diets). Fifteen groups of 20 juvenile rainbow trout (11.4 ± 1.5g) were randomly distributed over 15 fiberglass tanks (120L) within a recirculating aquaculture system (RAS). Fish were fed twice daily, ad libitum, for eight weeks. Haematologic parameters, plasma metabolites, innate immune parameters, and hepatic and intestinal oxidative stress indicators were analysed.
Results
The in vitro antimicrobial assays showed that extracts from CC-MM could inhibit the growth of Lactococcus garvieae and Tenacibaculum maritimum, while no inhibitory effect was observed in SMS-MM extracts. At the end of the growth trial, the increased inclusion level of MM from 12.5 to 25%, regardless of MM type, reduced lactate levels and alternative complement pathway activity and increased cholesterol levels in the plasma of juvenile rainbow trout. No significant effect was observed on plasma glucose or triglyceride levels, nor on lysozyme, peroxidase, protease, or anti-protease activities. Fish fed CC25 showed significantly higher hemoglobin levels compared to those fed the CTRL diet, while hematocrit values remained unaffected by dietary treatment. Hepatic and intestinal lipid peroxidation was also unchanged across experimental diets. Hepatic catalase activity was higher in fish fed 12.5% MM inclusion diets compared to those fed 25% inclusion diets, regardless of the MM type. Notably, fish fed the SMS diets demonstrated increased intestinal glutathione S-transferase activity. Fish fed the CTRL diet showed a significantly higher intestinal glutathione reductase activity than all other experimental treatments.
Conclusion
In the present study, CC-MM extracts showed antimicrobial activity, while the SMS-MM extracts showed no detectable effect. However, when included in aquafeeds, both CC-MM and SMS-MM had a similar impact on rainbow trout juveniles’ immune and hepatic and intestinal oxidative status. These findings indicate that producing MM with a circular approach, using SMS, does not impair fish health. Further studies, including bacterial challenge trials, are recommended to assess the impact on disease resistance.
Acknowledgements
This work was funded by the SAFE project (SmartAqua4FuturE) within the European Union’s Horizon Europe programme under grant agreement no. 101084549. This research was also supported by national funds through FCT (Foundation for Science and Technology), within the scope of UIDB/04423/2020, UIDP/04423/2020 and D. Amaral doctoral grant (2023.03476.BD).
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