Analía Verónica Fernández-Gimenez’s research while affiliated with National University of Mar del Plata and other places

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Publications (18)


From waste to value: protein hydrolysates from byproducts of the Argentine hake (Merluccius hubbsi) processing using endogenous enzymes and Alcalase® 2.4L
  • Article
  • Full-text available

January 2025

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13 Reads

Marine and Fishery Sciences (MAFIS)

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Analia Fernández-Gimenez

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Maria Florencia Fangio

The valorization of fishery byproducts is essential to reduce waste and create high-value products. Waste from Argentine hake (Merluccius hubbsi) could enhance its functional and antioxidant properties through hydrolysis, releasing peptides with bioactive properties. Protein hydrolysates of Argentine hake were produced through autolysis (Aut) and enzymatic hydrolysis using Alcalase® 2.4L at concentrations of 0.24% and 2% (v/v) (Alc-0.24 and Alc-2), respectively, over 150 min. Alkaline peptidase activity, degree of hydrolysis, and antioxidant activity were assessed using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2’-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) radical ABTS·+ scavenging assays. All hydrolysates retained alkaline peptidase activity throughout the process. Alcalase-treated hydrolysates exhibited significantly higher peptidase activity and hydrolysis degree compared to autolysis. At 60 min, Alc-0.24 reached peptidase activity levels similar to Alc-2, and by 30 min, both had comparable degrees of hydrolysis. ABTS·+ scavenging activity increased over time for Alc-0.24, with both Alcalase® 2.4L concentrations outperforming autolysis. No significant differences were found between Alc-0.24 and Alc-2. Although all hydrolysates showed DPPH scavenging activity, no significant differences were detected between treatments or reaction times. These findings highlight the potential for producing value-added protein hydrolysates from Argentine hake waste.

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Zymogram of alkaline proteinases from M. hubbsi, P. brasiliensis, U. brasiliensis, and C. guatucupa. MWM, molecular weight standards. The table below shows the molecular weights with the band number in the super index. The presence of multiple bands in the electrophoretic separation of digestive proteinases is due to the different molecular weights of several enzymes, such as trypsin, chymotrypsin, collagenase, and pepsin, which are present in intestine crude extracts and conform the alkaline protease group.
Effect of several metal ions on the residual proteolytic activity (%) of the crude extracts of M. hubbsi, P. brasiliensis, U. brasiliensis, and C. guatucupa. Values indicate the means and standard error from three replicates. Different lowercase letters indicate significant differences between different metal ions for the same species. Different symbols (#, ‡, §) indicate significant differences among different species (generalized linear mixed model, Tukey test, p < 0.05).
Residual proteinase activity (%) of crude extracts from M. hubbsi, P. brasiliensis, U. brasiliensis, and C. guatucupa in the presence of several organic solvents. Values indicate the mean and standard error from three replicates. Different lowercase letters indicate significant differences between different organic solvents for the same species. Different symbols (#, ‡, §) indicate significant differences among different species (generalized linear mixed model, Tukey test, p < 0.05).
Stability of the alkaline proteases from M. hubbsi, P. brasiliensis, U. brasiliensis, and C. guatucupa in the presence of various commercial laundry detergents. Values indicate the means and standard error from three replicates. Different lowercase letters indicate significant differences between different detergents for the same species. Different symbols (#, ‡, §) indicate significant differences among different species (generalized linear mixed model, Tukey test, p < 0.05).
Recovery of alkaline proteinases from fisheries wastes: biochemical characterization and applications

Fish visceral waste, which is normally discarded, is considered one of the richest sources of proteinases with potential biotechnological applications. For this reason, alkaline proteinases from viscera of Argentine hake Merluccius hubbsi, Brazilian flathead Percophis brasiliensis, Brazilian codling Urophycis brasiliensis, and stripped weakfish Cynoscion guatucupa were characterized. Individuals were caught by a commercial fleet off the coast of the Argentinean Sea. The intestine and pyloric caeca were dissected out and then minced and triturated with distilled water. The proteinase activity of P. brasiliensis extracts was enhanced by all the ions tested (Mn²⁺, K⁺, Na⁺, Ca⁺²) while the enzymes of the other species were stable in the presence of those ions, retaining more than 60% of their enzymatic activity. Alkaline proteinases of all species showed extreme stability to 5% v/v surfactants at 60 min (Sodium dodecyl sulfate, Triton X‐100, Tween 20, Tween 80), and relative stability toward an 6% v/v oxidizing agent (H2O2) and organic solvents 80% (acetone, isopropanol, methanol, ethanol). The enzyme extracts were incubated for 60 min with these compounds. Interestingly, alkaline proteinases from all species were compatible with the commercial detergents (Ala, Skip, and Ace). These results demonstrate that proteinases recovered from a no‐cost sample such as fishery residues can be used for industrial applications, such as detergent formulations.


Effects of a Dietary Multienzyme Extract on Isotope Biokinetics in Red Claw Crayfish Cherax quadricarinatus Juveniles

November 2024

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50 Reads

Understanding the nutritional and metabolic physiology of aquatic organisms is essential for optimizing aquaculture practices and informing ecological models. We investigated the influence of dietary composition, specifically the incorporation of multienzyme extract derived from Pleoticus muelleri waste, on the growth and metabolic processes of juvenile Cherax quadricarinatus. We investigated how these dietary changes influence dietary assimilation and tissue turnover using stable isotope δ¹³C and δ¹⁵N dynamics, in both the pleon muscle and hepatopancreas. Juvenile crayfish were subjected to two isocaloric diets for a 90-day period: a control diet (diet C) and a diet supplemented with a multienzyme extract (diet E) from red shrimp waste. Despite comparable growth rates, isotopic analysis (δ¹³C and δ¹⁵N) of the pleon muscle and hepatopancreas revealed distinct metabolic patterns between both dietary treatments. The hepatopancreas exhibited accelerated isotopic turnover relative to muscle tissue, irrespective of diet, suggesting a more dynamic metabolic role. Diet E further accelerated turnover rates in both tissues, indicative of enhanced nutrient assimilation and utilization. Consistent isotopic disparities between the hepatopancreas and muscle tissues highlighted tissue-specific metabolic functions, with the hepatopancreas serving as a metabolic hub. Molting-induced shifts in isotopic patterns underscored the dynamic interplay between metabolic processes and nutrient mobilization. Isotopic equilibrium was reached earlier for δ¹³C than δ¹⁵N, with lower discrimination factors in the hepatopancreas. While δ¹³C primarily supported metabolic processes, δ¹⁵N contributed substantially to growth, especially in muscle. These findings illuminate the complex interplay of dietary composition, isotopic fractionation, and physiological regulation in C. quadricarinatus. The metabolic enhancements induced by the diet supplemented with the extract warrant further investigation to optimize nutrient utilization and growth performance in aquaculture settings.




Kinetic characterization of digestive proteinases extracted from the processing waste of South Atlantic fish

July 2023

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19 Reads

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1 Citation

Bioresource Technology Reports

In Argentina, the fishing industry is highly developed, producing large quantities of waste products from fish processing which contain digestive enzymes with potential biotechnological uses. Among these enzymes are proteinases that hydrolyze proteins and can convert them into functional ingredients. To use these value-added compounds in industrial processes, kinetic characterization of these proteinases under specific conditions, such as contact time, pH, and temperature, must first be carried out. The objective of the present work was to perform a kinetic characterization of crude extracts of alkaline proteinases from intestine-ceca of Merluccius hubbsi, Percophis brasiliensis, Urophycis brasiliensis, and Cynoscion guatucupa. Results showed that the reached maximum values of A440 depended on both the species analyzed and the initial concentration of azocasein. The maximum concentration of hydrolysis products obtained at long times was proportional to the initial concentration of the target protein. P. brasiliensis was the most suitable tested species to obtain high hydrolysis rates at high azocasein concentrations, and exhibited the highest proteinase activities, so it can be considered a candidate species for future biotechnological applications.



State of knowledge about biotechnological uses of digestive enzymes of marine fishery resources: A worldwide systematic review

February 2023

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55 Reads

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13 Citations

Aquaculture and Fisheries

The characterization of digestive enzymes presents in fish and their potential biotechnological uses is a well-developed and studied field. However, there has not been a systematic review that analyzes the state of knowledge of these enzymes at a global level. Therefore, a systematic literature search on three platforms was carried out to review and analyze existing knowledge about digestive enzymes of marine fishes from fisheries and aquaculture and their potential application in industrial processes. Using the PRISMA method for selecting journal manuscripts, we found 112 scientific articles published between 1984 and 2020 studying different digestive enzymes from 87 fish species. Most studies were carried out in Tunisia and Mexico and only 6 articles were published in South American countries. The most studied digestive enzymes were alkaline proteases, mainly trypsin, and the proposed uses for these enzymes were mainly as additives in commercial detergents. There is a vacancy in the characterization of other digestive enzymes as pepsins, lipases and amylases and the study of species that are distributed in the Southwest Atlantic Ocean. It is necessary to expand the knowledge about other digestive enzymes and to carry out new studies in regions with an important fishery development.


Table 1
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Amino acid prole of extruded pea meal and shrimp protein hydrolysate.
Potential of agricultural and fishery wastes as sustainable feed ingredients for Nile tilapia (Oreochromis niloticus)

December 2022

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20 Reads

Soybean meal (SBM) is the most widely employed vegetable ingredient in aquafeeds, but its rising price and the negative environmental impacts of its cultivation open research efforts for its replacement. More sustainable aquaculture may be achieved by utilizing agricultural and fishery by-products; thus, this study aims to incorporate pea meal (PM) and shrimp protein hydrolysate (SH) as protein ingredients in tilapia juveniles’ feed. The presence of protease inhibitors and the in vitro digestion were evaluated for raw (PM0) and extruded pea meals (PM150), while SH characteristics were studied as well. The nutritional quality of alternative ingredients was assessed to formulate a diet that meets tilapia juveniles’ requirements, and its effects on growth were evaluated. Extrusion significantly reduced the PM inhibitory effect on tilapia proteases (from 5.4–1.9%). In vitro protein digestion of PM150 (7.8 ± 1.35%) did not differ from the control treatment with fish meal (FM; 11.8 ± 1.94%). Additionally, SH presented 41.6 ± 2.91% of DPPH radical scavenging activity. Tilapias fed with a diet where SBM was totally replaced by the dietary inclusion of 25% PM150 and 10% SH, presented comparable growth indicators to those exposed to the control diet. Extrusion reduced PM proteolytic inhibitory effect and improved protein digestibility. SH presented antioxidant effect, as well as, provided flavor enhancers and essential amino acids to the diet. It is feasible to achieve a sustainable dietary formulation for juvenile tilapia using the evaluated by-products without affecting its growth performance.


Table 1
Amino acid prole of extruded pea meal and shrimp protein hydrolysate.
Potential of agricultural and fishery wastes as sustainable feed ingredients for Nile tilapia (Oreochromis niloticus)

December 2022

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36 Reads

Soybean meal (SBM) is the most widely employed vegetable ingredient in aquafeeds, but its rising price and the negative environmental impacts of its cultivation open research efforts for its replacement. More sustainable aquaculture may be achieved by utilizing agricultural and fishery by-products; thus, this study aims to incorporate pea meal (PM) and shrimp protein hydrolysate (SH) as protein ingredients in tilapia juveniles’ feed. The presence of protease inhibitors and the in vitro digestion were evaluated for raw (PM0) and extruded pea meals (PM150), while SH characteristics were studied as well. The nutritional quality of alternative ingredients was assessed to formulate a diet that meets tilapia juveniles’ requirements, and its effects on growth were evaluated. Extrusion significantly reduced the PM inhibitory effect on tilapia proteases (from 5.4–1.9%). In vitro protein digestion of PM150 (7.8 ± 1.35%) did not differ from the control treatment with fish meal (FM; 11.8 ± 1.94%). Additionally, SH presented 41.6 ± 2.91% of DPPH radical scavenging activity. Tilapias fed with a diet where SBM was totally replaced by the dietary inclusion of 25% PM150 and 10% SH, presented comparable growth indicators to those exposed to the control diet. Extrusion reduced PM proteolytic inhibitory effect and improved protein digestibility. SH presented antioxidant effect, as well as, provided flavor enhancers and essential amino acids to the diet. It is feasible to achieve a sustainable dietary formulation for juvenile tilapia using the evaluated by-products without affecting its growth performance.


Citations (6)


... Moreover, it is essential to study the biochemical characteristics of the enzymes from these new sources to obtain an efficient and economical application on a commercial scale. These enzymes are generally incorporated as crude extracts since it is a more economical method than the production of purified enzymes, which implies a protein engineering focus and therefore a higher economic cost (Friedman & Fernández-Gimenez, 2024). ...

Reference:

Recovery of alkaline proteinases from fisheries wastes: biochemical characterization and applications
State of knowledge about biotechnological uses of digestive enzymes of marine fishery resources: A worldwide systematic review
  • Citing Article
  • February 2023

Aquaculture and Fisheries

... In zebrafish, the dorsal and ventral buds fuse at the time of hatching [9]. While the post-hatching morphogenic features of the pancreas have been investigated in various fish species including Nile tilapia (Oreochromis niloticus) [10] and zebrafish (Danio rerio) [11], the early development of the pancreas and its differentiation in grass carp have not been fully studied. The present study aimed to characterize the morphogenic steps in the pancreas of grass carp (C. ...

Morphological changes, peptidase activity, and effects of exogenous enzymes in the early ontogeny of Nile tilapia, Oreochromis niloticus

... Fishery residues are composed mainly of viscera. These digestive organs are evidently a great source of digestive enzymes, as has been already reported for decapods (Bonadero et al. 2022) and cephalopods (Boucaud-Camou and Roper 1995;Kim et al. 2014), among many other taxa. Therefore, the extracts obtained here from the cephalothorax of P. muelleri, the midgut gland of L. santolla and the digestive gland of I. argentinus (which contain soluble proteins), are rich in digestive enzymes, such as proteases, lipases, and glycosidase. ...

Marine sources for biotechnology: Preliminary digestive enzymes assessment of seven decapod species from the southwest Atlantic
  • Citing Article
  • July 2022

Regional Studies in Marine Science

... Globally, our multivariate analysis supports that fish digestion plasticity regarding environmental temperature largely relies on trypsin and chymotrypsin activities, being chymotrypsin the most responsive enzyme, though lipid digestion seems also to be under temperature control. Most of the digestive enzymes of greater amberjack share optimal conditions with those from other fish species (Kishimura et al., 2006;de la Parra et al., 2007;Candiotto et al., 2018) and crustaceans (Perera et al., 2008;Chavez-Rodrıǵuez et al, 2020;Muhlia-Almazań and Fernańdez-Gimenez, 2022), pointing out the relative high thermal optimum of alkaline proteases, although a previous study described lower thermal optimum for trypsin and chymotrypsin of greater amberjack (Oliveira et al., 2017). Also, the enzymes studied are all susceptible to acidic conditions, with lipase being the most sensitive enzyme, probably as a result of a more complex tridimensional architecture and structural requirements for activity as reported for other lipases (Perera et al., 2008). ...

Understanding the Digestive Peptidases from Crustaceans: from Their Biochemical Basis and Classical Perspective to the Biotechnological Approach

Marine Biotechnology

... World fisheries and aquaculture production of aquatic animals is expected to grow further reaching 205 million tonnes (live weight equivalent) in 2032, and an additional 19 million tonnes or 10% increase relative to 2022 per capita apparent consumption of aquatic animal foods is projected to reach 21.3 kg in 2032 (FAO 2024). However, approximately 70% of fish used for industrial processing ends up as waste (Olsen, Toppe, and Karunasagar 2014), including fish viscera that constitute 12-18% of the total fish weight (Friedman et al. 2022;Sahu et al. 2016). At present, the comprehensive utilization of fish viscera is limited, with a significant proportion being discarded directly. ...

Digestive proteinases from the marine fish processing wastes of the South‐West Atlantic Ocean: Their partial characterization and comparison

... In addition, SPW can be used as a potential source of proteases, which can participate in the hydrolysis process. Pereira et al. (2022) isolated endogenous alkaline proteases from Pleoticus muelleri waste and further used them for hydrolysis of P. muelleri cephalothorax to obtain PH. The resultant PH had DH of about 72% when prepared using 2% (v/v) of enzyme extract for 90 min and showed DPPH radical scavenging activity. ...

Characterization of liquid protein hydrolysates shrimp industry waste: Analysis of antioxidant and microbiological activity, and shelf life of final product