Hop acids, derived from spent brewer’s yeast, exhibit strong antimicrobial properties that could enhance aquaculture’s economic and environmental sustainability. A 56-day trial was conducted with Nile tilapia (Oreochromis niloticus) to evaluate the effects of hop acids (Humulone and Lupulone) on production metrics, lysozyme activity, and fillet characteristics. The results indicated no significant changes in overall production, except for the viscerosomatic index. Although the feed treatment with approximately 300mg hop acids/kg showed slightly higher average fish weight, there were no notable differences in color or aroma of the fillets compared to the control group. Importantly, the inclusion of hop acids did not adversely affect the fish. While the study did not demonstrate significant benefits from hop acid supplementation, the absence of negative effects suggests potential for using brewing byproducts in fish feed formulations.

1 INTRODUCTION

As aquaculture continues to be the main seafood producer, new practices for maximizing yield, such as high-density rearing and formulated feeds, are necessary to meet the growing demand for seafood while considering rising operational costs (FAO, 2024). In formulated feed, functional feed additives are vital for producers as they can potentially enhance feed efficiency, boost growth, and support immune health (Alemayehu et al., 2018). Additives such as pre- and probiotics, like dietary Saccharomyces cerevisiae in tilapia feed, can provide improvement in gut health to help enhance digestibility and weight gain (Abu-Elala et al., 2013). When added to tilapia feed, phytogenic substances such as Moringa oleifera (Gbadamosi et al., 2016) and Allium sativum (Metwally, 2009) have resulted in oxidative stress reduction and improved immune response. Therefore, feed additives can be used to support the production of healthier fish, including tilapia, and lead to better economic outcomes for producers and farmers.

Hop acids may offer a potential novel feed additive in aquaculture. Found in hops, Humulus lupulus, hop acids are well known for their antimicrobial activity against gram-positive bacteria (Shimwell, 1937). Effective at concentrations as low as 10–25 ppm (Cocuzza & Peifer, 2021), hops have traditionally been incorporated in beer production as a potent antimicrobial. Hop acids as a feed additive have been explored in cattle feed with shown improvements in ruminal fermentation (Wang et al., 2010) and reduce methane production from microbes (Blaxland et al., 2021). In addition, hop acids have also been associated with antioxidant, anticancer, and anti-inflammatory activities in humans (Olšovská et al., 2016; Puligundla et al., 2020; Van Cleemput et al., 2009) potentially offering additional benefits to aquaculture production. Utilizing hop acids as a natural antimicrobial may also be enticing to consumers, thereby presenting a useful feed additive for aquaculture producers.

Hop acids are a prominent component of spent brewer’s yeast (SBY), their utilization as a protein supplement in fish feed holds potential for enhancing both economic and environmental sustainability in aquaculture operations. Spent brewer’s yeast is a major byproduct of the brewing industry that is presently being researched as a novel protein supplement for aquaculture (Covert et al., 2025; Jaeger et al., 2020). Through the brewing process, hop acids are adsorbed onto yeast cells, therefore becoming a difficult-to-separate component of SBY. The concentration of hop acids found in SBY can vary widely across different beer styles, ranging from 60 to 3000 μg hop acid/gram of dry yeast (Bryant & Cohen, 2015).

While previous research has initially investigated the antimicrobial efficacy of HAs in aquaculture (Barnes et al., 2012; Lee et al., 2022), little attention has been paid to broader applications. In terrestrial systems, various animal models have explored the inclusion of hop acids in animal feeds, revealing increased growth rates in swine (Sbardella et al., 2016), improved growth rates in poultry (Bortoluzzi et al., 2014), reduced methane production in ruminants (Pszczolkowski et al., 2016), and reduced ammonia production in caprines (Flythe, 2009). The distinctive bitter flavor, potent aroma, and characteristic green pigment of hops raise concerns for incorporation into fish feed, as evidenced by previous research on feed additives affecting the color (Oehlenschläger & Ostermeyer, 2016) and aroma (Jones et al., 2016) of the final fillet.

Given the potential benefits and avenues hop acids may be incorporated into feed, the objective of this study was to comprehensively assess the impact of hop acid inclusion on various growth and health metrics during a controlled feed trial to gain a holistic understanding of their potential as a feed additive in aquaculture.

2. METHODS:

   2.1 Experimental Overview

The study involved a 56-day feeding trial to evaluate the impact of hop acid on Nile tilapia growth. Four different concentrations of hop acids were tested: approximately 0, 60, 300, and 1200 mg/kg of feed. Weekly measurements of fish weight were taken to assess growth, and the feed conversion ratio (FCR) was calculated to analyze growth patterns. Additional metrics, including the condition factor (CF), fillet yield (FY), and viscerosomatic index (VSI), were recorded at the trial’s conclusion to evaluate the internal composition of the fish. Health assessments were conducted by measuring the hepatosomatic index (HSI), while antimicrobial effects were evaluated through lysozyme activity in blood samples.

   2.2 Experimental Conditions

All animal procedures received approval from the University of Florida’s IACUC. The trial was conducted in a flow-through system at the Aquatic Laboratory, utilizing twelve 2082-L circular tanks. Water was sourced from underground aquifers and filtered before entering the tanks. Each tank was equipped with an aeration system to ensure optimal conditions. Ninety-six juvenile Nile tilapia were acclimated for seven weeks prior to the trial, during which they were fed commercial tilapia feed until reaching an average weight of approximately 36.03 g. The fish were sorted into tanks to ensure equal weight distribution before the trial commenced.

   2.3 Feed Design and Production

Four treatment diets with varying hop acid concentrations were formulated for the study. A basal diet was derived from previous experimental tilapia feed, with hop acids added as liquid extracts. The concentration of hop acids was calculated based on a 40% replacement of fish meal protein using spent brewer’s yeast (SBY). The treatments included low (60 mg/kg), medium (300 mg/kg), high (1200 mg/kg), and a control (no hop acids). The formulations aimed to mimic the profiles of hop acids found in different sources of SBY from both craft and multinational breweries, ensuring a comprehensive evaluation of hop acid effects on tilapia.

   2.4 Chemical analysis

The methods used for analyzing water quality parameters, which were adapted from established protocols by HACH and the American Public Health Association. Key metrics such as water temperature, dissolved oxygen concentration, pH, nitrite, and nitrate levels were monitored throughout the trial, ensuring consistent water quality in the flow-through tanks. The water temperature was maintained at 26.5 ± 1.2°C, while dissolved oxygen and pH levels were kept at 6.1 ± 0.4 ppm and 8.1 ± 0.2, respectively. Additionally, nitrite and nitrate levels were carefully controlled, demonstrating the rigorous approach taken to maintain optimal conditions for the tilapias.

Prior to the trial, a thorough proximate and mineral analysis of the treatment feeds was conducted by a third-party laboratory to ensure they met the nutritional requirements for tilapia growth. The analysis confirmed that the feed formulations adhered to the nutritional guidelines established by Nguyen et al. (2018), targeting a dry basis composition of approximately 7% fat and 38% protein. This careful formulation process was crucial for supporting the health and growth of the tilapias throughout the experimental period.

   2.5 Sampling

At the conclusion of the 56-day trial, eight fish were randomly selected from each treatment group (CTRL, LOW, MED, HIGH) based on the HA concentration in their feed. Fish from three tanks sharing the same treatment were collected together for dissection. Each fish was euthanized using a buffered tricaine methanesulfonate solution, after which their weight and length were recorded for condition factor measurement. Blood was collected from the caudal vessels using a syringe and needle, dispensed into EDTA-coated tubes to prevent clotting, and stored on ice for later lysozyme activity analysis. Additionally, viscera and liver samples were removed and weighed for visceral and hepatosomatic indices, while carcasses were filleted for further analysis, including aroma and color assessments.

   2.6 Lysozyme Activity

Post-sampling, blood samples were centrifuged to separate plasma, which was then stored at −80°C until analysis. The lysozyme activity in the plasma was measured using a commercial fluorescence-based assay kit. This assay detects lysozyme activity by assessing its effect on Micrococcus lysodeikticus cell walls, with a sensitivity down to 20 U/mL. The measurement of lysozyme activity was crucial for analyzing the antimicrobial effects of hop acids, as this enzyme plays a significant role in the innate immune response of the fish.

   2.7 Growth Performance of Fish

Fish production metrics, including survival rates, feed conversion ratio (FCR), and weight gain, were evaluated across the four treatment groups (CTRL, LOW, MED, HIGH HA). Daily assessments were made to monitor survival counts and any adverse effects leading to fish mortality. Weekly measurements of weight gain and FCR were recorded for each of the 12 tanks. At the end of the feeding trial, various health indices such as hepatosomatic index (HSI), visceral somatic index (VSI), condition factor (CF), and fillet yield (FY) were calculated from the harvested specimens of each treatment group to assess overall growth and nutritional performance.

   2.8 Sensory assessment

Skinned and deboned fillets from fish fed with  and different concentrations feeds were compared using a consumer panel. Fillets were baked at 180°C for 8 min to follow the preparation method of Turchini et al. (2003).

   2.9 Color assessment

Fillets from each treatment were trimmed of any bone/sinew, skinned, and sliced into 1 cm cubes before being placed in a clear plastic cup and scanned for their Hunter LAB color using a MiniScan XE (HunterLab, Reston, VA, USA). Because of the dark coloring of hop acids (alpha, beta, and iso-alpha), the LAB was recorded to determine whether a potential transfer of color from HA was found in the fish fillet.

   2.10 Statistical analysis

All data was assumed to be normally distributed and model residuals were heteroscedastic. One-way and two-way ANOVA followed by Dunnett’s test was performed using a combination of RStudio.

3. RESULTS

 The results  from this study investigating the effects of hop acids (HA) as a feed additive in tilapia aquacultureindicate that while hop acids did not significantly alter growth metrics or immune responses, they may still offer benefits without negatively impacting sensory qualities.

4. Implications for Sustainable Aquaculture

The study highlights the potential of hop acids as a sustainable feed additive, particularly when combined with spent brewer’s yeast. This aligns with consumer demand for environmentally friendly and minimally processed food production. However, the controlled laboratory conditions limit the applicability of the findings to commercial aquaculture settings, where water quality and microbial challenges vary. Future research should focus on large-scale trials and explore interactions between hop acids and other dietary components to optimize feed formulations.

5. Conclusion

 while hop acids did not significantly impact growth metrics or immune responses in tilapia, their inclusion in feed presents a promising avenue for sustainable aquaculture practices without compromising sensory quality.

Source : Lee, J. K., Farzad, R., Lee, T., Ying Chuah, S. X., Omidvar, R., Sims, C., Zhang, B., Ropicki, A., & MacIntosh, A. J. (2025). Effect of hop acid inclusion as a feed additive on Nile tilapia, Oreochromis niloticus, production, lysozyme activity, fillet color, and aroma. Journal of the World Aquaculture Society, 56(2), e70014. https://doi.org/10.1111/jwas.70014.