Palometa fish (Serrasalmus maculatus) meat: Amino acids profile by gas chromatography–mass spectrometry, pulping and application in salty cookies

Abstract

Palometa (Serrasalmus maculatus) is a predatory freshwater fish that is abundant in South America. Palometa is rarely consumed and has low commercial value because of its short storage time and low acceptability due to the large number of small bones. Despite being a good source of protein, the amino acid profile of this meat remains unknown. This study analyzed the amino acid profile of palometa meat using gas chromatography–mass spectrometry. The meat was also pulped and applied to salty cookies. The amino acid concentration range was 2.82 (glutamine) to 234.17 (glutamic acid) mg g-1 of protein. All essential amino acids were detected in the meat, pulp, and cookies, except for tryptophan, and lysine and leucine were prominent. The cookies with 10% pulp presented an increase in lysine, isoleucine, and leucine compared to the standard, and the chemical score demonstrated that palometa is a good source of threonine. The sensory attributes of the cookies showed an acceptance rate of over 70.0%. In conclusion, the development of salty cookies can be a way to incorporate palometa fish, with high protein quality, in food formulations, valuing this underutilized raw material and being a strategy to increase fish consumption in the population.

Key words
Lysine; Leucine; Threonine; Fish consumption; Protein quality; Gas chromatography–mass spectrometry

INTRODUCTION

Recent statistics from the Food and Agriculture Organization of the United Nations (FAO) show that global fish production reached 178.5 million tons in 2018. Of this total, 87.6% of the production was intended for human consumption (frozen, fresh, canned, or dry), and 12.4% was intended for other purposes (FAO 2020). In recent years, consumers have shown greater interest in fish meat, mainly because of its high nutrient content, high protein content, high-quality lipids, and low cholesterol levels (Tiwari et al. 2021, Souza et al. 2022). In addition, fish and their by-products are recognized not only as some of the healthiest foods on the planet but also as having the least impact on the natural environment (Viero et al. 2021).

As for nutritional aspects, fish and by-products are important components in the diet as a source of polyunsaturated fatty acids, minerals (calcium, phosphorus, iron, copper and selenium) and fat-soluble vitamins (A and D) (Viero et al. 2021, Souza et al. 2022). Furthermore, fish and their by-products are a nutritious source of high-quality proteins that are rich in essential amino acids. Altogether, 20 amino acids make up proteins, of which nine amino acids are not synthesized by the human body. These amino acids are called essential, and therefore must be introduced into the body through the diet for structural function and proper cell growth (Nollet & Toldrá 2012, Nelson & Cox 2021).

Nevertheless, Brazil has one of the lowest fish consumption rates worldwide. The Brazilian population consumes, on average, approximately 6.8 kg/inhabitant/year; however, the FAO recommends 12 kg/inhabitant/year, which is probably due, among other factors, to the lack of knowledge of the importance of this product in food (FAO 2020). In South America, there is an abundance of a predatory freshwater fish, popularly known as Palometa (Serrasalmus maculatus), which has an excellent protein percentage (Viero et al. 2021). Despite being abundant in nature, scientific studies on this raw material are extremely rare, and its amino acid profile remains unknown. This is an important gap to be addressed, as the amino acid profile is fundamental information for evaluating the protein quality of a meat product.

Palometa is rarely consumed and has low commercial value. Thus, there is good potential for its exploitation, both by the fishing sector and by research bodies, aiming at its addition in the preparation of food products, where the use of noble species is not required (Viero et al. 2021). However, it is necessary to create strategies to overcome the limitations in the commercialization of Palometa meat, which are due to the short storage time and low acceptability due to the large number of small bones. In this context, the transformation of meat into pulp is a promising alternative to overcome these barriers (Liu et al. 2023, Azambuja et al. 2025).

Cookies are among the most popular fast-food items and are consumed by people of all ages and economic groups. However, cookies are prepared with wheat flour (refined), sugar, and fat, along with other minor ingredients, and are characterized as high-sugar and high-fat products. The gluten protein in wheat flour is a basic structural component of cookies because it possesses unique characteristics that expand the air cells and provide rigidity after baking (Farheena et al. 2015). Nevertheless, wheat proteins are relatively low in protein quality due to the deficiency of some essential amino acids. Additionally, gluten proteins are partially resistant to proteolytic digestion and cause celiac disease. Consequently, the fortification of bakery products with quality proteins (especially animal proteins) has gained considerable interest in recent years (Sittichoke et al. 2019, Wabali et al. 2020, Hashem et al. 2023).

In this sense, considering the nutritional quality of fish and the need to improve fish consumption by the population, considering the natural abundance, underutilization, and scarcity of data on the amino acid profile of palometa, and considering that the pulping of fish for use in salty cookies is a viable and promising alternative for the valorization of these species and for the protein improvement of highly consumed products, the objective of this work was to evaluate the amino acid profile and perform the pulping of Palometa meat, applying the pulp to salty cookies, and evaluating its technological and sensorial characteristics.

MATERIALS AND METHODS

Raw material and pulping

The raw material used was palometa (Serrasalmus maculatus) from the Sanchuri Dam, located in the 5th district of the municipality of Uruguaiana/RS/Brazil, which was captured with the support of local fishermen. Soon after capture, the fish were washed with chlorinated water, eviscerated, and stored at -18°C until use. To obtain Palometa pulp, the procedure described by Centenaro et al. (2007) was used with modifications. The gutted fish were filleted and ground using a food processor. Subsequently, the minced meat was washed according to the methodology indicated by Furlan et al. (2009), using the following washing cycle: 1. Washed with NaHCO3 0.1%; 2 and 3. The samples were washed with distilled water; and 4. The samples were washed with a 0.3% NaCl solution at a 1:3 (m/v) ratio. The washing processes occurred under constant agitation in a shaft-propeller type agitator, model 711, Fisatom brand, washing solution with temperature between 5 and 7 °C and for a total extraction period of 6 minutes. At the end of the washes, the liquid phase was removed from the pulp using a Tyler 32 sieve (0.5 mm) to obtain a wet pulp. For the production of dry pulp, thin layers (0.4 mm) of wet pulp were distributed in containers and dried in an oven at 60°C for 4 h. After drying, the pulp was ground in an analytical knife mill and sieved to a 0.5 mm granulometry (Tyler 32) for better product uniformity and the yield was calculated as follow:

$$\text{Dry/Wet Pulp Weight (g)}/\text{Whole Fish Weight (g)}\times 100$$

Proximate composition

The proximal composition was determined according to the official methodology of the Association of Official Analytical Chemists (AOAC 2011), with some adaptations.

For moisture content, two grams (2 g) of each sample were weighed into a dry-cleaned crucible. The dish containing the sample was placed in a well-insulated oven at 103 °C for 3 h. The samples were then removed and transferred to desiccators at room temperature to cool. After cooling, the sample was weighed and placed in the oven for another 1 h. After this, the dish containing the sample was removed, cooled in a desiccator, and re-weighed. The procedure was repeated until a constant weight was achieved. The weight loss was reported as moisture content and calculated as follows:

$$\text{\% Moisture content}=(W2-W3/W3-W1)\times 100$$

Where:

W1 = weight of empty dish;

W2 = weight of empty dish + sample before drying (g);

W3 = weight of dish + sample dried to a constant weight (g).

For protein analysis, 0.2 g of the sample was mixed with 2 ml of concentrated H2SO4 in a digestion flask, and a tablet of selenium catalyst was added before heating under a fume cupboard until a clear solution was obtained (the digest). The digest was diluted to 20 ml in a volumetric flask and used for analysis. Then, 10 ml of the digest was mixed with an equal volume of 45 % NaOH solution in a Kjeldahl distillation flask containing 20 ml of 4 % boric acid and three drops of mixed indicator (bromocressol green). A total of 50 ml of distillate was collected and titrated against 0.1 N HCl from green to a deep red endpoint. A reagent blank was digested, distilled, and titrated. The nitrogen and protein contents were calculated using the following formula:

$$\text{\% Protein}=N2\times 6,25$$

$$N2=(100/10)\times (N\times 14)/1000\times (Vt/Va)xT-B$$

Where:

Vt = Total digest volume (20 ml);

Va = Volume of digest analyzed (10 ml);

T = Sample titre value;

B = Blank titre value.

For fat determination, ten grams (10 g) of the sample was carefully weighed into a thimble and placed in the sample holder of the Soxhlet extraction apparatus. A clean, dried, and weighed Soxhlet extraction flask was filled with 250 ml of petroleum ether, and the entire apparatus was assembled. The flask was placed on a heating mantle and heated at 60 °C for 6 h. After extraction, the solvent was evaporated by drying in an oven. Drying, cooling, and re-weighing of the sample were repeated until a constant weight was obtained. The percentage fat content was determined using the following equation:

$$\text{\% Lipids}=(X-Y)/Z\times 100$$

Where:

X = weight of fat + flask;

Y = weight of flask;

Z = weight of sample.

For ash content analysis, the crucible was weighed, dried in an oven, cooled in desiccators, and weighed again. Then, two grams (2 g) of the samples were weighed into an empty porcelain crucible respectively, which was placed in a muffle furnace and ignited at 550 °C for 45 min after which the crucible was transferred into the desiccators, cooled and weighed. The percentage ash content of the samples was determined using the following equation:

$$\text{\% Ash content}=[(W2-W1)/\text{Weigth of sample}]\times 100$$

Where:

W1 = weight of empty crucible;

W2 = weight of crucible + Ash.

The carbohydrate content of the samples was determined by difference as follows:

$$\text{\% Carbohydrates}=100-\mathrm{\%}(\text{Moisture}+\text{Proteins}+\text{Lipids}+\text{Ash})$$

Amino acid profile

The amino acid content of the proteins was determined according to the method described by Furlan et al. (2024). The samples were first subjected to acid hydrolysis to release amino acid monomers, followed by a derivatization procedure with N-tert-Butyldimethylsilyl-N-methyltrifluoroacetamide (MTBSTFA) and subsequent separation, identification and quantification using a gas chromatograph coupled to mass spectrometry (GC-MS) (Shimadzu Corporation QP2010-Plus, Kyoto, JP). Initially, 500 mg of sample was subjected to acid hydrolysis with hydrochloric acid (6 mol/L) containing 1 g L-1 of phenol as a catalyst at 110 °C for 24 h under vacuum, so that the protein amino acids were solubilized. The hydrolyzate was then subjected to a derivatization procedure. The derivatization process was applied to both standard amino acid solutions and hydrolyzed protein extracts to enhance analyte volatility. This procedure was adopted from the method described by Alves et al. (2020). The hydrolysates or standard solutions (100 μL) and internal standard (DL-norleucine:100 μL at 10 μg mL_-1) were evaporated at 60 °C under a nitrogen flow, and the residues were combined with 50 μL of acetonitrile and 50 μL of MTBSTFA. The mixture was then left to react for an additional 120 min at 100 °C. After derivatization, chromatographic analysis was performed according to the optimized conditions detailed by Ribas et al. (2022). A 1 μL sample was injected in splitless mode into the GC–MS system at 285 °C using a specific injection protocol (1 min with the split valve closed, followed by a 10:1 split). Helium (99.9995%) was used as the carrier gas at a constant linear velocity of 40 cm/s. Compounds were separated on an NST-5MS capillary column (30 m × 0.25 mm; 0.25 μm thickness film; Nano Separation Technologies, Brazil). The initial column temperature was set to 100 °C for 1 min, followed by a ramp to 220 °C at 20 °C/min. The temperature was then increased to 250 °C at 5 °C/min and subsequently raised to 260 °C at 2 °C/min, and held isothermal for 1 min; then, it was further raised to 265 °C at 2 °C/min. Subsequently, the temperature was increased to 285 °C at 5 °C/min and held isothermal for 1 min. Finally, it reached 300 °C at 15 °C/min and was held for 2 min, totaling 29.5 min. The GC–MS interface and electron impact ionization source (EI at 70 eV) temperatures were 280 °C and 210 °C, respectively. The quadrupole mass analyzer was operated in full-scan mode, enabling us to acquire the total ion chromatogram at a mass-to-charge ratio (m/z) ranging from 50 to 500 for the unequivocal identification of analytes. Selected ion monitoring was used for amino acid quantification. The analytes were positively identified by comparing their retention times and mass spectra with those of the 23 amino acid analytical standards. Quantification was performed by external calibration (0.2–60 μg mL-1) with areas previously normalized by the internal standard. The LOD and LOQ values ranged from 0.01 to 0.10 μg mL-1 and from 0.02 to 0.31 μg mL-1, respectively. The results were expressed as milligrams of amino acids per gram of protein. The experiments were performed in triplicate. The chemical score was calculated as the ratio between the content of each essential amino acid in the protein and the FAO (2013) reference standard as follows:

$\mathrm{Chemical score }=\mathrm{ }\frac{\left(\mathrm{mg of amino acids per g of protein in the sample}\right)}{\left(\mathrm{mg of the same amino acid per g of reference protein}\right)}$

Application of dry pulp of palometa in salty cookies

Based on preliminary tests using the formulation proposed by Haj-Isa et al. (2011), who enriched salty cookies with the addition of hake, three formulations of biscuits were defined, as shown in Table I: one standard formulation (with no addition of fish) and two others with the addition of 5 and 10% dried Palometa pulp.

Table I
Formulations, proximal composition and specific volume and expansion factor of salty cookies with dry pulp of Palometa fish.

Ingredients	Formulations (%)
	Standard	5.0%	10.0%
Wheat flour	49.5	44.5	39.5
Corn starch	17.5	17.5	17.5
Dry pulp	0.0	5.0	10.0
Margarine (60.0% lipids)	15.0	15.0	15.0
Sugar	2.0	2.0	2.0
Egg yolk (in natura)	2.0	2.0	2.0
Chemical baking powder	2.0	2.0	2.0
Salt	1.0	1.0	1.0
Dried condiments (garlic 0.5 g and basil 0.5 g dried)	1.0	1.0	1.0
Water	10.0	10.0	10.0
Component (g/100 g)
Moisture	1.41±0.13b	1.17±0.30b	2.73±0.22a
Proteins	9.34±0.03c	13.42±0.80b	18.27±0.41a
Lipids	12.27±0.10b	12.80±0.04a	13.01±0.10a
Ash	3.22±0.04c	3.46±0.01b	3.50±0.05a
Carbohydrates	73.76±0.13a	69.15±1.07b	62.49±0.71c
Calorific value (kcal/100 g)	442.86±0.66b	445.48±1.18a	440.13±1.10c
SV and EF
SV	1.74±0.07a	1.21±0.04b	1.31±0.07b
EF	18.36±1.11a	20.42±0.35a	18.80±0.74a

• Adapted from Haj-Isa et al. (2011). 5.0% = Salty cookie with 5.0 % of dry pulp of Palometa fish. 10.0% = Salty cookie with 10.0 % of dry pulp of Palometa fish. Mean values ± standard deviation (4 repetitions); different letters on the same line: there is a significant difference. SV: specific volume; EF: expansion factor.

To achieve better homogeneity of the dough, the production of biscuits started with the individual sieving of the dry ingredients (flour, corn starch, sugar, chemical yeast, salt, and condiments) in a granulometry of 0.5 mm (Tyler 32), as well as the dry pulp. First, all the dry ingredients were mixed using a mixer for 1 min, and then the sifted egg yolks and water were added.

Water was gradually added until the gluten was completely developed and a dough of homogeneous consistency was obtained. Standard cookies and cookies with 5 and 10% dry pulp received 115, 125, and 150 mL of water, respectively. After 15 min of rest, the dough was rolled into a 4 mm thickness and cut with a manual cookie cutter. After cutting, the cookies were transferred to greased molds and placed in an oven preheated to 200 °C for 10 min. In the next step, the biscuits were cooled to room temperature and evaluated for physical-chemical composition, amino acid profile, and technological and sensory characteristics. For cookies, the correction factor for protein determination was %N × 5.7 (AOAC 2011).

The energy (calorific value) of the cookies was determined using the value obtained from proteins, lipids, and carbohydrates:

$$\text{Energy value (Kcal/100 g)}=(\text{Proteins}\times 4.0)+(\text{Lipids}\times 9.0)+(\text{Carbohydrates}\times 3.75)\text{kiloJoule}=\text{Energy value (k/100 g)}\times \mathrm{4.184.}$$

Technological assessment

The specific volume (cm3/g) of the cookies was calculated as the ratio between the apparent volume (cm3) obtained by displacing millet seeds and the mass of the cookies after baking (g), as described by Silva et al. (1998). In summary, the analyzed cookies were placed in the center of an aluminum capsule below a glass funnel supported by a tripod. The millet seeds were poured through the funnel and collected below until they overflowed into the aluminum capsule, which was previously weighed with millet seeds. The capsule was then leveled with a ruler, and the volume of millet in the capsule was measured using a graduated cylinder without the biscuit. The specific volume was calculated using the following equation:

$$\text{Specific volume (cm3/g)}=\text{Biscuit volume (cm3)}/\text{biscuit weight (g)}$$

$$\text{Biscuit volume}=\text{Capsule volume (cm3)}-\text{Capsule volume with biscuit}$$

The expansion factor was evaluated using the ratio between the diameter and thickness of the cookies after baking, measured using a caliper (Sharma et al. 2013).

Sensory evaluation

The sensory characteristics of the cookies were evaluated 24 h after the end of baking, using a 9-point hedonic scale for different attributes (odor, color, crispness, flavor, and global evaluation), ranging from “9-I liked it extremely” to “1-I disliked it extremely.” Simultaneously, an attitude scale was applied to assess the purchase potential, analyzed using a structured 5-point scale (“1-disliked a lot/would not buy” to “5-liked a lot/would definitely buy”) (Dutcosky 2011).

Statistical analysis

The results were subjected to analysis of variance (ANOVA), and significant differences were identified by comparison test between means (Tukey Test) at a 5.0% significance level. Previously, the Kolmogorov-Smirnov and Cochran tests were performed to verify the normality of the data and whether their variances were equal, respectively (Triola 2008). Afterwards, the data were submitted to Multivariate Analysis of Principal Components (PCA) using the TIBCO STATISTICA software (version 14.0, StatSoft, Inc., Palo Alto, CA, U.S.).

RESULTS AND DISCUSSION

Pulping

A white, wet pulp with a fishy odor was obtained after pulping. The wet pulp yield obtained after the process was 56.7% based on the weight of the fish. After drying the wet pulp and sieving, we obtained a powdery product with the appearance of flour, whitish in color, and with a slight fishy odor, which was defined as dry pulp. The final yield of the dried, ground, and sieved pulp was 2.5% relative to the weight of the whole fish. Similar results were observed by Gonçalves (2014), who reported a yield of freeze-dried cobia pulp (Rachycentron canadum) of 3.3% relative to the total mass of fish. After filleting the palometa, we also observed a low yield of the meat obtained (40.9%) compared to the whole fish. This species is considered a small fish, and in the adult phase, it reaches an average size of 20 cm and a weight of 250 g, which are the characteristics that make it difficult to remove the maximum amount of meat. According to Liu et al. (2022), some machines for producing high-yielding fillets can handle short-bodied fish species. Therefore, modern filleting machines could achieve higher yields compared to traditional human-based filleting. As an alternative to improve yield, the use of industrial pulpers or filleting machines could increase the efficiency of the process, with a consequent increase in the yield of dry pulp.

Proximate composition of palometa fillet, wet and dry pulp, and cookies

Tables I and II present the proximal composition of Palometa fillets, wet pulp, dry pulp, and salty cookies. Regarding moisture, Table II shows that the moisture content of the fillet was approximately 79%, whereas that of the wet pulp was approximately 92%. Regarding the protein, lipid, and ash content in the fillet, wet, and dry pulp were in the range of 8–92 % (proteins), 0.46–5.54 % (lipids), and 0.16–1.96 % (ash). Regarding the proximal composition of the cookies, we found values in the range of 1.17 to 2.73% (moisture), 9.34 to 18.27% (proteins), 12.27 to 13.01 % (lipids), 3.22 to 3.50 % (ash), and 62.49 to 73.76 % (carbohydrates).

Table II
Proximal composition of fillet, wet pulp and dry pulp of Palometa fish.

Component (g/100 g)	Fillet	Wet pulp	Dry pulp
Moisture	79.44 ± 0.27	91.69 ± 0.31	-
Proteins	18.79 ± 1.35	7.65 ± 2.17	92.02 ± 2.17
Lipids	1.12 ± 0.09	0.46 ± 0.14	5.54 ± 0.14
Ash	0.98 ± 0.05	0.16 ± 0.09	1.96 ± 0.09

• Mean values ± standard deviation (4 repetitions).

Ogawa & Maia (1999) described fish as one of the main sources of animal protein, presenting variations in its nutritional composition of approximately 50.0-85.0% for water, 12.0-24.0% for protein, 0.1-22.0% for fat and 0.8-2.0% for minerals. Thus, we perceive that the values found in the present study are in accordance with those reported in the literature. We also observed a significant reduction in the concentrations of proteins, lipids, and ash in the washed wet pulp (washed) in relation to the Palometa fillets. This occurred due to the washing step, whose objectives are to clarify the pulp and remove the natural components of the fish meat, such as proteins soluble in water and weak saline solutions, such as sarcoplasmic proteins (myoglobin and hemoglobin), lipids, and other components that accelerate deterioration (lipid oxidation and microorganisms), improving the quality and maintaining the functional characteristics of the food (Ogawa & Maia 1999, Jesus et al. 2001). Furthermore, it is known that the washing process promotes an increase in the concentration of myofibrillar proteins, providing favorable conditions for good gelatinous and elastic formation (Belibagli et al. 2003).

This study also showed that after drying the wet pulp, the proteins, lipids, and ash were concentrated, as described by Centenaro et al. (2007). There was a reduction in moisture content when compared to the initial moisture content of Palometa fillet. According to Rebouças et al. (2012), this decrease in moisture is an advantage from the point of view of preserving the product, especially if microbiological changes are considered. The result of the dry pulp protein content (92.02%) was higher than that found by Centenaro et al. (2007) (82.2%). This appreciable amount of protein in the dry pulp is favorable, considering that the proposed objective is the protein enrichment of the salty cookies. As for the lipid content of the dry pulp, the value found was 5.54%, a value close to those described by Centenaro et al. (2007), for goat pulp (6.2%) and by Rebouças et al. (2012), for Nile Tilapia protein concentrate (8.2%). Therefore, it is observed that the dry pulp presented, in addition to the high protein content, a low lipid content, which is a favorable aspect, considering the health problems related to high lipid intake.

Codex Alimentarius (2018), emphasizing that fish proteins are considered superior from a nutritional point of view, due to its composition of essential amino acids.

The caloric value of the standard formulations, with 5.0 and 10.0% of dry pulp, developed in this study, ranged from 440 to 445 kcal/100 g. These values are close to those demonstrated by Goes et al. (2016), who produced cookies with the addition of Pacu and Tilapia and reached caloric values of 448.58 and 426.58 kcal/100 g, respectively.

Amino acid profile

Bearing in mind that the addition of dry pulp in the formulation of salty cookies promoted a significant increase in protein in the product and that proteins are formed by amino acids, the amino acid composition of the raw material and the elaborated formulations was verified. The amino acid concentration range was 2.82 (glutamine) to 234.17 (glutamic acid) mg g-1. All essential amino acids were detected in the meat, in the wet and dry pulp of palometa as well as in the cookies, except for tryptophan (Table III). Within the essential amino acids, in greater concentration, lysine and leucine stand out (Figure 1), agreeing with studies by other authors who also report that the main essential amino acids found in freshwater fish are lysine and leucine (Kaya & Kocatepe 2014).

Table III
Protein amino acids (mg g-1) of meat, wet pulp and dry pulp of Palometa fish, and salty cookies with dry pulp of Palometa fish.

	Meat	Wet Pulp	Dry Pulp	Standart Cookie	Cookie 5.0%	Cookie 10.0%	Standart FAO (2013)
	Amino acid (mg/ g protein)
Alanine	57.80 ± 2.22a	56.91 ± 2.40a	45.60 ± 1.72b	24.42 ± 1.08c	34.35 ± 2.53d	47.73 ± 1.85b
Glycine	32.11 ± 1.24a	39.60 ± 4.38c	11.82 ± 0.57d	33.79 ± 1.84a	23.90 ± 0.49b	21.95 ± 0.95b
Valine	48.17 ± 1.85a	46.55 ± 2.57ab	51.80 ± 0.03a	32.20 ± 2.20c	38.65 ± 0.47d	47.12 ± 0.04ab	40
Leucine	68.51 ± 2.63a	68.19 ± 3.17ab	82.20 ± 1.18c	45.89 ± 4.11d	62.61 ± 1.10ab	76.44 ± 0.94c	61
Isoleucine	40.68 ± 1.56a	40.92 ± 1.71ab	48.98 ± 0.60c	24.81 ± 2.69d	32.64 ± 0.23e	41.68 ± 0.57ab	30
Proline	56.75 ± 2.15a	56.15 ± 3.15ab	52.50 ± 1.30ab	186.55 ± 6.45c	147.00 ± 6.20d	104.65 ± 2.35e
Methionine	11.77 ± 0.45a	11.79 ± 1.60ab	23.40 ± 2.40e	13.51 ± 0.00abc	13.04 ± 0.75abcd	13.29 ± 0.01abcd	23
Serine	44.96 ± 1.73a	46.13 ± 0.72ab	58.55 ± 1.09c	38.18 ± 1.32d	45.07 ± 3.72ab	54.37 ± 0.04c
Threonine	52.45 ± 2.02a	50.77 ± 0.21ab	101.91 ± 4.01d	35.18 ± 1.68c	36.69 ± 0.20c	47.95 ± 0.95ab	25
Phenylalanine	40.68 ± 1.56a	38.57 ± 1.28a	43.91 ± 0.03ab	36.24 ± 4.30ac	38.59 ± 1.71a	40.60 ± 0.70a	41
Aspastic Acid	103.75 ± 4.32a	106.26 ± 6.81ab	104.72±0.07ab	32.56 ± 1.84c	64.59 ± 6.47d	81.76 ± 0.74e
Hydroxyproline	8.89 ± 0.37a	7.08 ± 0.59ab	17.45 ± 1.70c	7.99 ± 0.61ab	11.19 ± 1.11a	15.00 ± 1.00c
Cysteine	7.90 ± 0.33a	7.07 ± 0.58a	9.01 ± 0.01ab	18.65 ± 1.15d	9.87 ± 0.74bc	10.43 ± 0.43bc
Glutamic Acid	144.26 ± 6.01a	143.89 ± 8.10a	173.41 ± 4.62c	234.17 ± 4.17b	226.47 ± 8.23b	208.99 ± 1.01d
Asparagine	<LQ	<LQ	<LQ	<LQ	<LQ	<LQ
Lysine	97.82 ± 4.08a	98.47 ± 8.58a	20.55 ± 0.55b	24.38 ± 3.88b	46.97 ± 6.67c	48.48± 3.82c	48
Glutamine	3.95 ± 0.16a	2.82 ± 0.04b	3.38 ± 0.00c	5.21 ± 0.29d	3.15 ± 0.20bc	3.62 ± 0.00ac
Cystine	12.85 ± 0.54a	14.14 ± 1.16ab	14.07 ± 0.55ab	23.34 ± 1.23d	19.55 ± 1.66c	18.20 ± 1.30c
Arginine	76.08 ± 3.17a	107.72 ± 10.40c	42.07 ± 0.43d	77.04 ± 4.56ab	83.67 ± 3.21ab	61.05 ± 2.55e
Histidine	21.74 ± 0.91a	19.76 ± 0.29ab	20.38 ± 0.12abc	57.45 ± 0.95d	32.10 ± 4.03e	21.12 ± 0.62abc	16
Tyrosine	36.56 ± 1.52a	37.20 ± 1.05ab	47.86 ± 0.59c	19.04 ± 0.62d	29.91 ± 0.85e	35.04 ± 0.03ab
Tryptophan	n.d	n.d	n.d	n.d	n.d	n.d	6.6
ΣPA	967.70	999.99	973.57	970.61	999.99	999.47
ΣEssentials	381.82	375.02	393.12	269.67	301.28	336.69
ΣNon-essential	585.88	624.96	580.44	700.94	698.71	662.79

• Mean values ± standard deviation (3 repetitions); different letters on the same line, there is a significant difference (p< 0.05). 5.0% = cookie with 5.0% of dry pulp; 10.0% = cookie with 10.0% of dry pulp. ΣPA: Sum of protein amino acids. ΣEssential: Sum of essential amino acids; ΣNon-essential: Sum of non-essential amino acids; <LQ: Less than the limit of quantification; n.d: not detected.

Figure 1
Concentration of essential amino acids (mg g-1) in standard salty cookies, with 5.0 and 10.0% of dry pulp.

Positive and negative variations were observed in the amino acid composition of the meat in relation to the dry pulp, which may be due to the dehydration process after washing. The thermal treatment used may contribute to altering the distribution of amino acids, a fact observed in obtaining dry pulp from palometa meat. The significant reduction in the content of alanine, glycine, glutamine and arginine, however, does not completely compromise the nutritional quality of the dry pulp, since the concentration of essential amino acids, with the exception of lysine, meets the recommendations established by FAO (2013).

Twenty-one amino acids were detected in the cookies for all formulations, as described in Table III. The essential amino acids identified and quantified were valine, leucine, isoleucine, methionine, threonine, phenylalanine, lysine and histidine. In addition, thirteen non-essential amino acids were also found (alanine, glycine, proline, serine, aspartic acid, hydroxyproline, cysteine, glutamic acid, asparagine, glutamine, cystine, arginine and tyrosine). Among essential amino acids, comparing the standard cookies and those with 10% of dry pulp, the greatest increases occurred for lysine, isoleucine and leucine, followed by valine, threonine and phenylalanine, in that order. Among the non-essential amino acids, those that showed a significant increase when fish was added in greater quantity in the formulations were aspartic acid, alanine, hydroxyproline, tyrosine and serine. The significant increase in the amino acids lysine (98.8%) and threonine (36.3%) has nutritional importance, considering that they are limiting amino acids in cereals (Neiva et al. 2011). The fact that the cookies in question have a good source of essential amino acids becomes a nutritional advantage, since said amino acids must be obtained through a balanced diet, which meets the needs of each individual (Tirapegui et al. 2016).

Cookies with 10% of dry pulp had a higher content of essential amino acids (336.69 mg/g protein) compared to the other formulations. With the exception of histidine, 10% formulation presented all essential amino acids in amounts significantly greater than or equal to the standard cookie. Therefore, cookies fortified with 10% dry pulp of palometa appear as a product of good nutritional quality, a result that corroborates the work of Cercel et al. (2016), and that reports that the inclusion of fish protein concentrate in the formulation of breads with wheat flour improved the nutritional quality of the products due to the excellent source of amino acids. Thus, replacing wheat flour with dry pulp improved the protein content and nutritional quality of the cookies produced. The results of this study are in harmony with Abraha et al. (2018), where biscuits with 10% powdered sturgeon fillet (Acipeneser sinensis) were produced and had a higher content of essential amino acids compared to biscuits without addition or fortified with 5% fish.

The absence of tryptophan can be attributed to the effect of acid hydrolysis on this amino acid. According to the AOAC (2000), acid hydrolysis does not provide good recovery for the amino acid tryptophan, thus recommending the use of alkaline hydrolysis to determine tryptophan. Our research group, attempting to apply the technique suggested by the AOAC (2000), has dedicated itself in recent months to implementing and validating this method, which is a prospect for future studies. Furthermore, enzymatic hydrolysis is another possibility for future studies, but to date, it has not been proven capable of providing complete hydrolysis in food matrices.

Figure 2 presents the chemical score of the amino acids found in the meat, in the wet and dry pulps (Figure 2a) and in the cookies for this study (Figure 2b). Through the chemical score, it was possible to identify the nutritional value and determine the order of the limiting amino acids in this study. A protein with chemical scores greater than 1.0 for all essential amino acids is considered of high biological value, and a value below 1.0 means that the evaluated protein has at least one essential amino acid in insufficient quantity (Sartori et al. 2022). The results presented in Figure 2 show that for meat, wet pulp and for all cookies formulations, methionine and phenylalanine were considered the limiting amino acids, as they presented scores lower than 1.0. Sulfur-containing amino acids such as methionine are prone to oxidation (Nollet & Toldrá 2012). In this work, the samples for determination of amino acids were hydrolyzed in an acidic medium with high temperature and the reaction was carried out in sealed flasks under vacuum in order to minimize the possibility of degradation of some amino acids. Therefore, during the reaction, some amino acids such as methionine may have been oxidized, reducing their concentration in the biscuits. In contrast, this fish proved to be a good source of threonine, with scores of 4.08 and 1.92 for dry pulp and cookie with 10%, respectively. Considering that threonine is nutritionally important, the addition of dry pulp of palometa in foods is beneficial, as according to Nollet & Toldrá (2012) this amino acid produces antibodies and immunoglobulins, forms the connective tissue of tendons and skin and participates in lipid metabolism, preventing the accumulation of fat in liver cells.

Figure 2
Chemical score of essential amino acids in meat, wet and dry pulp (a) and in salty cookies (b). Amino acid score = (mg of amino acids per gram of protein in the sample) / (mg of the same amino acid per gram of reference protein) (FAO 2013).

Principal component analysis (PCA) was used to better visualize the effect of different treatments on the amino acid profile of the studied samples. The first two principal components explained 91.12% of the total variance of the results obtained, and can be used to verify similarities and differences within the sample set, as shown in Figure 3. It is clearly observed the formation of three distinct groups: the first, formed only by the meat and the wet pulp, the second by the standard cookies and with 5% of dry pulp and the third by the dry pulp itself and the cookies with 10% of the same. Thus, it became evident that the process used in each sample influences the separation of clusters along the PCI and PCII components, as shown in Figure 3. The compounds that provided greater variability in treatments were Valine, Leucine, Isoleucine, Proline, Aspartic Acid, Histidine and Tyrosine in the first principal component (PCI) and in the second principal component (PCII) were Glycine, Lysine and Arginine.

Figure 3
Graphical representation of the amino acid composition (a), for the different samples of fish, pulp and cookies (b) showing the main components Factor 1 (PCI) and Factor 2 (PCII).

Palometa meat and wet pulp showed similarity and the variables that contributed to this similarity were Alanine, Aspartic Acid and Lysine, that is, these compounds made these samples differ from the others, mainly due to the higher concentrations of these amino acids (Figure 2b). This demonstrates that washing the meat to obtain washed palometa pulp does not result in significant changes in the concentration of most amino acids.

The concentration of amino acids in the dry pulp and cookie with 10% of dry pulp also showed similarity and the variables that contributed to these samples differing from the others were Valine, Leucine, Isoleucine, Methionine, Serine, Threonine, Phenylalanine, Hydroxyproline and Tyrosine, suggesting that these samples have similar characteristics. Comparing the treatments that refer to the cookie formulation, the product added with 10% of dry pulp had a higher percentage of essential amino acids compared to standard cookies and with 5%. These, in turn, differed from the other samples due to the higher concentrations of the amino acids Proline, Cysteine, Glutamic acid, Glutamine, Cystine and Histidine.

Technological assessment

Table I shows the results of the specific volume and the expansion factor of the standard cookies and with different concentrations of Palometa dry pulp. The range of results for specific volume and expansion factor were 1.21 to 1.74 % and 8.36 to 20.42 %, respectively. It is possible to verify a decrease in the specific volume of cookies enriched with 5.0 and 10.0% of Palometa dry pulp in relation to the standard. The fact that the standard formulation has a higher amount of wheat flour, resulted in greater water retention due to the presence of wheat proteins (glutenins and gliadins), for better gluten development, which is a functional quality of wheat flour on the cookie not added dry pulp. Therefore, it is necessary that the mixture has the appropriate amount of these proteins, otherwise, the weak protein structure decreases gas retention in the dough and favors the formation of a compact and low-volume structure (Soares et al. 2018).

The expansion factor is an important parameter for determining the quality of baked goods. In general, cracker expansion decreases whenever the wheat flour substitution level increases (Chung et al. 2014). However, cookies that expand greatly when baked become difficult to pack in standardized packaging compared to those that expand little.

To improve these characteristics, emulsifiers (such as mono and diglycerides of fatty acids, sorbitan monostearate, for example) can be added to the cookie formulation. Recently, research has also shown that oleogels (a combination of oils and structuring agents) are alternative ingredients that can be added to cookies to reduce the saturated fat content, enhancing the structuring effect without affecting the dimensional properties volume and expansion factor (Barros 2022).

Sensory evaluation

The acceptance rate for the studied formulations can be seen in Figure 4a, and the acceptance range was approximately 70 to 95%. The formulation added with 10.0% Palometa dry pulp resulted in a cookie with the lowest acceptance rate. However, all attributes (odor, color, crispness, flavor and global evaluation) evaluated by the judges showed acceptance rates above 70.0% for the three formulations. According to Dutcosky (2011), when the results of the acceptability indexes show values above 70.0% among the judges, the product has the potential to be accepted in the market. Figure 4b presents the results corresponding to the purchase intention of the standard savory cookies with the addition of 5.0 and 10.0% Palometa dry pulp. The purchase intention test identified that none of the judges indicated as a response “I certainly would not buy” for the cookie enriched with 5.0% Palometa dry pulp. The biscuit with 5.0% dry pulp obtained the best purchase intention score compared to the product with 10.0%, which had the lowest grades and acceptance rate. Considering the future commercial potential of the product, additional research can be carried out to explore the storage stability of the enriched cookies by evaluating flavor and lipid stability, for example. Nevertheless, considering the increasing prevalence of food allergies which affect both children and adults, information about the inclusion of fish-based products in cookies should be provided to the consumer.

Figure 4
Acceptance index (a) and purchase intention (b) of standard salty cookies, with 5.0 and 10.0% of dry pulp.

CONCLUSIONS

The present work demonstrated the characterization of the amino acid profile of the fillet and pulp of palometa, highlighting the presence of all essential amino acids, except tryptophan.

Furthermore, we demonstrated the feasibility of applying dry palometa pulp to salty cookies, improving the protein quality of the cookies and providing sensory acceptance above 70%.

These results highlight the importance of developing technologies to utilize palometa pulp, a nutrient-rich raw material with low market value, allowing for better utilization of this product while simultaneously enriching the protein content of nutritionally poor foods. Future studies should seek to overcome the low yield of dehydrated palometa pulp and conduct allergenicity testing of the products studied, as well as focusing on stability studies.

Acknowledgements

This publication was partial funded by the Brazilian Federal Agencies: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Brazil – Finance Code 001, and by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil. The authors gratefully acknowledge Brazilian University where this study was developed, Federal University of Pampa.

• Data availability
  All the data used in the manuscript are available in the tables and figures. Code Availability Not applicable.

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Edited by

• Handling editor
  Gisele da Rocha

Data availability

All the data used in the manuscript are available in the tables and figures. Code Availability Not applicable.

Publication Dates

• Publication in this collection
  20 July 2026
• Date of issue
  2026

History

• Received
  9 Apr 2025
• Accepted
  29 Nov 2025