This study presents an approach that utilizes low-value agro-industrial by-products as culture media for producing high-value proteolytic enzymes. The objective was to assess the impact of six agro-industrial by-products as culture media on the production of proteolytic enzymes. Bacillus subtilis strains, confirmed through comprehensive biochemical, morphological, and molecular analyses, were isolated and identified. Enzymatic activity was evaluated using azocasein and casein substrates, and the molecular sizes of the purified extract components were determined. The results demonstrated that the isolated bacteria exhibited higher metabolic and enzymatic activity when cultured in media containing 1 % soybean oil cake or feather meal. Furthermore, higher concentrations of the culture media were found to hinder the production of protease. Optimal protease synthesis on soybean oil cake and feather meal media was achieved after 4 days, using both the azocasein and casein methods. Semi-purification of the enzymatic extract obtained from Bacillus subtilis in feather meal and soybean oil cake resulted in a significant increase in azocaseinolytic and caseinolytic activities. Gel electrophoresis analysis revealed multiple bands in the fractions with the highest enzymatic activity in soybean oil cake, indicating the presence of various enzymes with varying molecular sizes. These findings highlight the potential of utilizing low-value agro-industrial by-products as efficient culture media for the sustainable and economically viable production of proteolytic enzymes with promising applications in various industries.

This study investigates the valorization potential of yellowfin tuna (Thunnus albacares) tails to produce high-value commercial products. Firstly, the tuna tails were placed in a perforated stainless-steel cylinder, and hydraulic pressure was applied to separate the skin from the muscle in the tails. The extracted muscle was then utilized as a nitrogen source for the growth of the proteolytic enzyme producer Bacillus subtilis, while the skins were employed for gelatin extraction. The proteases from B. subtilis were partially purified and used to produce antioxidant peptides from the obtained gelatin. The gelatin formed a gel upon cooling, with gelling and melting temperatures of 16 °C and 22 °C, respectively, and a Bloom strength of approximately 160. Response Surface Methodology (RSM) was employed to determine the optimal hydrolysis conditions to achieve the highest antioxidant activity (35.96% measured as DPPH radical scavenging activity), which were 50 °C and 6.5 IU of enzyme. The findings emphasize the importance of an integrated approach to maximize the value of tuna by-products, promoting sustainability within the framework of a circular bioeconomy. Overall, these results contribute to the efficient utilization of tuna by-products, waste reduction, and enhanced economic viability of the tuna industry.

The growing demand for efficient and cost-effective industrial proteases has intensified bioprospecting efforts in diverse ecosystems as a strategy to identify microorganisms with enhanced enzymatic performance. This study aimed to isolate, identify, and evaluate aerobic protease-producing bacteria from animal-protein matrices and water sources collected across the three continental regions of Ecuador, and to assess their suitability for industrial enzyme production A total of 34 bacterial strains were isolated and taxonomically assigned to the orders Enterobacterales, Pseudomonadales, and Bacillales. Proteolytic activity was evaluated using azocasein and casein assays after cultivation in an optimized medium containing 1% soybean paste as an inducer at 37 °C and 120 rpm for 72 h. Enterobacter cloacae (BC, pork), Bacillus paramycoides (P2, snook), and Pseudomonas aeruginosa (CH1, chontacuro) were identified as the most active protease producers from the Andean (Sierra), coastal (Costa), and Amazon regions, respectively. Production kinetics revealed marked strain-dependent differences. BC and P2 reached maximum proteolytic activity on day 4 followed by a decline, whereas CH1 peaked on day 2 and maintained stable activity over time, indicating superior enzymatic stability. Partial purification by gel-filtration chromatography (Sephadex G-100) yielded fractions with enhanced proteolytic activity, while SDS-PAGE analysis confirmed successful enrichment of protease-containing fractions. Overall, the results demonstrate that ecological origin strongly influences protease production and stability, and identify Pseudomonas aeruginosa CH1 as a particularly promising candidate for industrial applications requiring robust and sustained proteolytic activity.

Yellowfin tuna (Thunnus albacares) processing by-products are underutilized resources rich in high-quality proteins that can be valorized into bioactive ingredients. In this study, proteins from yellowfin tuna tail bones were hydrolyzed using either an endogenous protease extract recovered from tuna viscera or commercial trypsin. The endogenous extract was characterized and exhibited optimal proteolytic activity at pH 8 and 40 °C. Comparative bioactivity assessment showed that trypsin-derived hydrolysates exhibited significantly higher antioxidant capacity, with ABTS and DPPH values of 11.8 ± 0.04 and 13.3 ± 0.40 mg Trolox/g, respectively, compared with 10.1 ± 0.12 and 7.3 ± 0.21 mg Trolox/g for hydrolysates produced with endogenous enzymes. Trypsin hydrolysates also showed stronger dipeptidyl peptidase-IV (DPP-IV) inhibition (IC₅₀ = 0.83 ± 0.37 mg/mL) than endogenous-enzyme hydrolysates (IC₅₀ = 1.60 ± 0.47 mg/mL). Remarkably, hydrolysates generated with the endogenous enzymatic consortium exhibited exceptionally potent angiotensin-converting enzyme (ACE) inhibitory activity (IC₅₀ = 0.008 ± 0.004 mg/mL), outperforming trypsin hydrolysates (IC₅₀ = 0.01 ± 0.007 mg/mL). Neither hydrolysate showed evidence of acute toxicity in the Artemia salina model at the tested concentrations, supporting their favorable performance in this preliminary toxicity screening assay. Considering that nearly two-thirds of total fish biomass is discarded during processing, this integrated bioprocess demonstrates a sustainable strategy for the simultaneous valorization of skeletal and visceral waste streams. By employing endogenous enzymes as biocatalysts, this approach reduces dependence on commercial proteases, mitigates environmental burdens, and supports the development of a circular marine bioeconomy through the production of highly potent, peptide-rich functional ingredients with promising cardiovascular health applications. Further toxicological evaluation is required to confirm their safety for food and nutraceutical applications.

This study investigates the valorization potential of yellowfin tuna tails for the production of high-value commercial products. Muscle and skins were firstly mechanically separated by using a specifically designed device. The muscle was then used as a nitrogen source for the growth of the proteolytic enzymes-producer Bacillus subtilis, whereas the skins were employed for gelatin extrac-tion. The proteases from Bacillus subtilis were partially purified and used to produce antioxidant peptides from gelatin obtained. The gelatin obtained formed a gel upon cooling, with gelling and melting temperatures at 16 ºC and 22 ºC, respectively, and Bloom Strength of ≈ 160. RSM was used to determine the optimal hydrolysis conditions to obtain the highest antioxidant activity (35.96%, measured as DPPH radical scavenging activity), which were 50°C / 6.5 IU of enzyme. The findings emphasize the importance of an integrated approach to maximize the value of tuna by-products, promoting sustainability in the frame of circular bioeconomy. Overall, these results contribute to the efficient utilization of tuna by-products, waste reduction, and enhance economic viability of the tuna industry.