Effect of Ice Storage on Freshness and Biochemical, Physical, Chemical, and Microbiological Quality of Leg Muscle Samples from Bullfrog (Lithobates catesbeianus)

Abstract

The present study evaluated the effect of ice storage on the freshness and quality of bullfrog (Lithobates catesbeianus) leg muscle. Biochemical, chemical, physical, and microbiological changes during 24 days of storage were analyzed. A rapid degradation of ATP into its intermediates (AMP, IMP, inosine, and hypoxanthine) was observed, with a significant increase in K-index (6.78% to 79.33%) and hypoxanthine concentration (3.93 ± 0.87 µmol/g), indicating a progressive reduction in freshness. The pH initially decreased due to post-mortem glycolysis but subsequently increased due to microbial activity and protein degradation. Volatile basic nitrogen (TVB-N) content increased significantly, reaching 27.36 mg/100 g, reflecting protein breakdown. A loss of texture was recorded, with a reduction in muscle firmness from 21.93 ± 1.36 Nw to 10.87 ± 1.08 Nw. Microbiological analyses showed an increase in bacterial load, with mesophiles and psychrophiles reaching 6.75 and 6.45 log CFU/g, respectively. These results indicate that the freshness and quality of bullfrog leg under ice storage remain within acceptable limits until day 18, but its quality and freshness decrease significantly toward the end of the study period.

1. Introduction

Aquaculture is one of the most important economic activities globally and considerably impacts human nutrition. For example, aquaculture accounts for 18% of the total output of fisheries in México [1]. Raniculture is a type of highly productive aquaculture activity that involves the cultivation and/or breeding of frogs in captivity [2] as well as the widespread export and import of frogs, primarily for their meat. Frog legs are the main edible portion of frogs alongside some other byproducts [3].

According to the current frog production statistics estimated by the Cultured Aquatic Species Information Program of the FAO, ~148 thousand tons of frogs are chiefly produced in countries such as China, Brazil, Ecuador, Guatemala, and México [4].

The bullfrog (Lithobates catesbeianus) is considered of great importance in México and is cultivated in the states of Sonora, Sinaloa, Jalisco, Michoacán, Tamaulipas, Veracruz, Tabasco, and Morelos. Taxonomically, L. catesbeianus belongs to the subphylum Vertebrata, class Amphibia, order Anura, family Ranidae, genus Lithobates, and species catesbeianus (Shaw, 1802). The nickname bullfrog comes from the sound emitted by the male bullfrog, which resembles that of a bull (laryngeal dimorphism), during the reproductive season. This species is native to North America and is characterized by its large size, as bullfrogs can reach a maximum length and weight of 43 cm and 2.5 kg, respectively. Frog legs are traditionally consumed in several countries worldwide [5,6].

Bullfrog production has several advantages that can be commercially exploited, such as their rapid life cycle and increased demand in international markets as well as the public preference for fresh over frozen products, which consequently results in higher prices and income associated with its sale during certain occasions [7].

Frog meat is considered a healthy food and is beneficial for resolving diet-associated issues such as obesity and malnutrition. Additionally, frog meat can aid in the alleviation of gastrointestinal disorders. Similarly to fish, frog meat is considered white meat and is lean, rich in essential amino acids, therapeutic for high cholesterol and hypertension, and easily digestible [8,9,10]. Furthermore, it has low carbohydrate and saturated fat content but high protein and water content [11], which is beneficial for consumer health.

Bullfrogs are considered a very important species in aquaculture, and similar to other aquatic products, their freshness and quality reduce with storage time. The loss of freshness and quality of fresh and frozen aquatic foods, as well as their deterioration pattern, is caused by postmortem changes, which occur in four stages: rigor mortis, dissolution of rigor, autolysis (loss of freshness), and bacterial spoilage. The duration of each stage depends on the species, physiological conditions, microbial contamination, and the storage temperature of the product [12,13].

Notably, quality indices based on nucleotide degradation have garnered special attention over the past few decades for freshness surveillance of aquatic products during manipulation and conversion. The concentration of the principal adenine nucleotides and their associated compounds in the postmortem muscle exhibits good correlation with the deterioration of the quality of a large variety of aquatic products. In this regard, the total molar concentration (TMC) of adenosine 5’ triphosphate (ATP) and its related metabolites in muscle tissue, along with the rate and pattern of its degradation at varying concentrations during storage, is influenced by both the species and the specific type of muscle tissue. Additionally, irrespective of species or muscle type, ATP undergoes rapid degradation within the first 24 h postmortem [14]. In aquatic and fishery organisms, ATP catabolism follows a sequential metabolic pathway: ATP → adenosine 5′-diphosphate (ADP) → adenosine 5′-monophosphate (AMP) → inosine 5′-monophosphate (IMP) → inosine (HxR) → hypoxanthine (Hx) [12,15]. The K-value, an established freshness indicator for aquatic and fishery products, is derived from the concentrations of ATP and its degradation products and serves as a key metric for assessing the rate of nucleotide degradation. This index is expressed as the percentage ratio of the sum of HxR and Hx concentrations to the total concentration of ATP and all its related catabolic intermediates [14,16]. Furthermore, the K-value has been shown to correlate strongly with storage duration, making it a reliable indicator of postmortem quality deterioration [14].

Previous studies have revealed that the postmortem changes that strongly affect the freshness and quality of aquatic products include the protein and ATP degradation, pH decrease, lipid oxidation, as well as the production of undesirable compounds such as trimethylamine (TMA-N) and total volatile bases of low molecular weight (TVB-N) as a result of bacterial action, which directly impacts the texture, water-holding capacity (WHC), and color of the muscle tissues [17,18,19]. Various methods that are currently used to evaluate the freshness and quality of the products of different aquaculture species are based on assessing changes in sensory, biochemical, chemical, and physical parameters as well as in microbiological composition [20]. Like other aquatic products, bullfrog meat is highly perishable due to microbial activity and handling conditions, highlighting the presence of psychrotrophic bacteria such as Pseudomonas spp., Shewanella spp., and Aeromonas spp. [21], which are responsible for spoilage. In addition, they may contain foodborne pathogenic microorganisms such as Salmonella spp. [22] and Escherichia coli O157:H7 [23]. While specific studies on the microbiota of bullfrog meat are limited, previous research has identified spoilage and pathogenic microorganisms in the skin, mouth, and intestine of different frog species, where the presence of Lactobacillus spp., Pediococcus spp., Micrococcus spp., Enterococcus faecalis, Enterococcus faecium, and members of the Enterobacteriaceae family, as well as Escherichia coli, Salmonella typhi, Vibrio cholerae, and Staphylococcus aureus have been reported [21,24]. This diversity in frog meat depends on environmental factors, habitat, management practices, etc.

The most important factors affecting the freshness and quality of bullfrog legs during ice storage are the same as those that apply to aquatic products in general. In fact, the freshness and quality of bullfrog leg muscle during ice storage are influenced by several factors, including physical, biochemical, microbiological, and sensory factors. However, these factors depend primarily on temperature control, in this case, on the ice-chilling process. To maintain constant cold conditions, frequent ice changes are essential, as this reduces the rate of microbial growth and slows down the chemical and enzymatic reactions responsible for product spoilage [13]. A key aspect for efficient temperature control is the constant replacement of ice because, over time, ice in contact with the muscle melts, reducing the cooling area and causing a slight increase in temperature. This can accelerate deterioration processes, favoring the appearance of undesirable changes in texture, color, odor, and microbial growth. Another determining factor in the quality and freshness of frog legs is storage time, since, as storage time increases, chemical, enzymatic, and microbiological reactions advance, affecting the stability of the product. Hence, the importance of precise temperature control [12]. In addition, proper handling during harvest and storage, together with good hygiene and packaging practices, can minimize initial contamination and prolong the shelf life of the product.

This study aims to evaluate the effect of ice storage on the freshness, biochemical, physical, chemical, and microbiological quality of leg muscle samples from the bullfrog (Lithobates catesbeianus). This is relevant particularly because of little to no previous research on these parameters, and the findings of this study will allow for the more efficient utilization of the species as well as improve its processing and marketing strategies for national and international consumption.

2. Materials and Methods

2.1. Collection of Bullfrogs

The bullfrogs (L. catesbeianus (Shaw 1802); n = 55) employed in the present study were obtained from the NutriFrog company (Mazatlán, Sinaloa, México) and included 28 females and 27 males with average body weights of 233.78 ± 22.43 and 260.62 ± 39.07 g, respectively, and an average age of 13 months. The bullfrogs were transported (for a duration of 4 h) in a container allowing air circulation to Caseros, S. de R. L. M. I (Navojoa, Sonora, México), where they were maintained with adequate food supply and allowed to rest for a period of 4 days to eliminate the stress of transport before their sacrifice.

The bullfrogs were sacrificed via desensitization via a dry blow to the anterior dorsal area, followed by evisceration, skin removal, and procurement of their legs [25]. The bullfrog legs so obtained were washed with potable water and individually placed in high-density polyethylene bags arranged in alternating beds of ground ice and bullfrog legs in a hermetic cooler for transport to the Food Research Laboratory of the University of Sonora (México). The time interval between the sacrifice of the bullfrogs and the receipt of the sample at the laboratory was no longer than 12 h.

2.2. Analysis of Samples During Storage on Ice

Following receipt at the laboratory, the bullfrog leg samples were repacked in polyethylene bags and again arranged in alternating beds of ground ice and bullfrog legs in a hermetic cooler. The frog legs subjected to storage on ice were analyzed over a duration of 24 days, with samples withdrawn on days 0, 3, 6, 9, 12, 15, 18, 21, and 24 to evaluate the biochemical, physical, and chemical changes in them, including ATP and degradation products, K-value, pH, total volatile bases (TVB-N), WHC, texture, and color (L*, a*, and b*). The total bacterial counts (mesophilic and psychrophilic counts) were determined on days 0, 6, 12, 18, and 24 of ice storage. Frog legs from four individual organisms for each day of sampling were employed for biochemical, physical, and chemical analyses, while frog legs from two individual organisms per day were employed for microbiological analyses. Finally, crushed ice was replaced as and when necessary during the storage of the bullfrog legs.

2.3. Analytical Determinations

2.3.1. ATP and Degradation Products

ATP, ADP, AMP, IMP, HxR, and Hx were quantified using high-performance liquid chromatography (HPLC), as detailed previously [26]. An extract was prepared by homogenizing bullfrog leg muscle (3 g) in 0.6 M perchloric acid (15 mL) at 0 °C using an Ultra-Turrax T18 Basic homogenizer (IKA Works Inc., Wilmington, NC, USA) at 18,000 rpm for 1 min. The homogenate was subsequently centrifuged at 5500× g for 10 min at 0 °C in a refrigerated Thermo Electron Model IEC-MULTI RF centrifuge (Thermo Fisher Scientific, Asheville, NC 28804, USA). Subsequently, a 7 mL aliquot of the supernatant was neutralized with 1 M KOH to attain a pH of 6.5–6.8 and allowed to rest for 30 min on ice. Potassium perchlorate was then removed via filtration using Whatman No. 4 paper. The supernatant was diluted to 15 mL with distilled water, and the samples were stored at −80 °C until further use.

The separation and quantification of the compounds via HPLC was performed by injecting the neutralized and diluted supernatant (20 µL) onto a Varian Prostar 240 chromatograph (Varian, Inc., Walnut Creek, CA, USA) that employs a Varian C18 Ultrasphere ODS reverse phase column (Beckman Instruments, Inc., Fullerton, CA, USA) with an inner diameter of 4.6 mm and a length of 250 mm. The mobile phase comprises phosphate buffer (0.04 M KH₂PO₄ and 0.06 M K₂HPO₄), at a flow rate of 1 mL/min. Nucleotides, nucleosides, and nitrogenous bases were detected at 254 nm using a UV-visible detector (Varian, Inc., Walnut Creek, CA, USA).

2.3.2. Calculation of K-Value

It is important to highlight that high-performance liquid chromatography (HPLC) is highly efficient for quantifying ATP and its degradation products due to its ability to separate molecules according to their polarity and partition coefficient. In reverse phase C18 columns, more polar compounds elute faster than less polar compounds, allowing accurate separation. In addition, UV detection at 254 nm ensures high sensitivity and specificity, since nucleotides have aromatic conjugated structures that absorb at this wavelength [26]. Compared to other methods such as UV-Vis spectrophotometry and enzymatic methods, to mention a few, HPLC allows quantifying each metabolite individually [27]. In addition, it combines sensitivity, reproducibility, and ease of application, being ideal for freshness and quality studies in biological and food products [14].

The concentrations of ATP, ADP, AMP, IMP, HxR, and Hx were employed for calculating the K-value using the equation described in a previous study [28], as follows:

K-value (%) = [(HxR + Hx)/(ATP + ADP + AMP + IMP + HxR + Hx)] × 100.

2.3.3. Determination of the pH of Frog Leg Muscle

The pH of the bullfrog leg muscle was determined according to a previously reported method [29]. For this purpose, bullfrog leg muscle (2 g) was homogenized in distilled water (18 mL) using the Ultra-Turrax T18 Basic homogenizer (IKA Works Inc., Wilmington, NC, USA) at 18,000 rpm for 1 min. The pH of the homogenate was determined by introducing the tip of the electrode of a potentiometer into it (Orion 420 A, Thermo Electron Corporation, Waltham, MA, USA). The equipment was calibrated daily with the standard pH buffer solutions.

2.3.4. Color

The color of bullfrog leg muscle was assessed using tristimulus colorimetry within the CIE Lab color space, employing a MiniScan HunterLab colorimeter (Reston, Virginia, VA, USA). The instrument operated in reflectance mode with a 0.5 cm reading port opening, capturing the L (lightness), a (red–green axis), and b (yellow–blue axis) values. Color measurements were conducted on both surfaces of the muscle tissue. Prior to data collection, the colorimeter was calibrated according to the manufacturer’s guidelines.

2.3.5. Texture

The texture of bullfrog leg muscle was evaluated using an EZ-S Shimadzu 346-54909-33 texture analyzer (Shimadzu Corp., Model EZ-S, Kyoto, Japan) equipped with a Warner–Bratzler shear cell. The maximum shear force (N) required to cut samples measuring 10 × 10 × 20 mm³ was recorded. A 50 kg compression cell was used, applying force at a head speed of 20 cm/min. The force was exerted perpendicular to the muscle fibers, and the required shear force was measured following the International System of Units (SI) standards.

2.3.6. Water Retention Capacity (WHC)

The water-holding capacity (WHC) of bullfrog leg muscle was determined according to a previously established method [30]. Two grams of muscle tissue were weighed and placed into a 50 mL centrifuge tube, followed by centrifugation in a refrigerated Thermo IEC-MULTI RF centrifuge (Thermo Fisher Scientific, Asheville, NC, USA) at 19,600× g for 60 min at 4 °C. The WHC was calculated as the percentage of water loss relative to the initial moisture content using the following formula:

WHC (%) = 100 − ([Wi − Wf]/Wi × 100),

where Wi and Wf correspond to the initial weight of fresh bullfrog meat and the final weight after centrifugation and surface drying, respectively.

2.3.7. Total Volatile Bases (TVB-N)

The determination of TVB-N content in the frog leg muscle was performed as detailed previously [29]. The bullfrog leg muscle (10 g) was weighed and transferred to a 1 L ball flask, followed by the addition of MgO (2 g) and distilled water (300 mL). The mixture was homogenized using an Ultra-Turrax T18 Basic homogenizer (IKA Works, Inc., Wilmington, NC, USA) for 1 min. Subsequently, edible vegetable oil (1-2-3^® brand, 20 drops) was added as a defoamer. The TVB-N content was obtained following distillation for 25 min and collected in 2% boric acid (15 mL). The distillate was titrated with 0.03 N H₂SO₄. A blank was distilled under similar conditions. Finally, the values obtained were expressed as mg N/100 g of the sample.

2.3.8. Aerobic Mesophilic and Psychrophilic Bacteria Count

The evaluation of aerobic mesophilic bacteria, “aerobic microorganism plate count”, in the bullfrog leg muscle samples was performed as previously reported [31]. For this purpose, the bullfrog leg muscle sample (10 g) was homogenized in sterile peptone water (1% w/v, 90 mL) using a previously sterilized conventional blender. Subsequently, the homogenized sample was subjected to five serial dilutions by mixing the original/diluted sample (1 mL) as the case may be with sterile peptone water (1% w/v, 9 mL), followed by manual mixing for 7 s with 25 movements in an arc of ~30 cm. Aliquots (1 mL) of each dilution were placed in a sterile Petri dish, and plate count agar (PCA, 20 mL) was added. The PCA assay was conducted in triplicate for each dilution. The mixtures of the agar and inoculum were homogenized as mentioned above, with six repetitions each of a circular motion from right to left, clockwise movements, movements from right to left, and movements from front to back. The plates were incubated in an inverted position at 35 °C ± 2 °C for 48 h for the mesophilic bacteria. The same procedure was employed for the psychrophilic bacteria; however, the plates were incubated at 5 °C ± 2 °C for 7–10 days. Finally, a colony count was performed at the dilution corresponding to the presence of 25–250 colony-forming units (CFU). The results were expressed as log CFU/g of bullfrog leg muscle.

2.4. Statistical Analyses

The collected data were analyzed using classical statistical methods, including the calculation of mean and standard deviation, analysis of variance (ANOVA), and Tukey’s multiple range test for post hoc comparisons. Statistical analyses were conducted using the NCSS software (Version 6.0.22; Kaysville, UT, USA). All analyses were performed with four replicates, except for the microbiological assays, where only two replicates were utilized. Additionally, a correlation matrix was generated to assess relationships among the measured parameters. A significance level of 5% (p < 0.05) was applied in all statistical evaluations.

3. Results

3.1. Estimation of the Concentrations of ATP and Its Degradation Products

One of the most important postmortem biochemical changes in the muscles of aquatic organisms and fishery products is associated with the degradation of ATP to ADP, AMP, IMP, HxR, and Hx, which is determined via the estimation of nucleotides, nucleosides, and nitrogenous bases. This process has been widely studied and utilized for monitoring the freshness and quality of the muscles of aquatic organisms [14]. Figure 1 shows the changes in the concentration of ATP and all its degradation products, including Hx, in the bullfrog leg muscles stored on ice for 24 days.

The results obtained in this study reveal that the ATP concentration in the bullfrog leg at the start of storage was 0.75 ± 0.12 µmol/g of muscle. The predominant nucleotides in the bullfrog leg at the start of storage included AMP and IMP, with initial concentrations of 2.53 ± 0.33 and 2.74 ± 0.37 µmol/g, respectively; these levels could be a consequence of the time elapsed from the sacrifice of the bullfrogs until measurement (~12 h). The high concentrations of AMP and IMP reported in the current study reflect the rapid degradation of ATP into AMP and IMP. In addition, a slight but significant (p < 0.05) decrease in the ATP and ADP concentrations was observed with increased storage time. Furthermore, the levels of AMP and IMP decreased in accordance with the equations y = −0.096x + 2.625 [r² value of 0.94 (p < 0.05)] and y = −0.098x + 2.814 [r² value of 0.89 (p < 0.05)], respectively. The average sum of the TMCs of the nucleotides, nucleosides, and nitrogenous bases was 7.11 ± 0.85 µmol/g. The concentration of HxR significantly increased (p < 0.05) till day 18 of storage (1.74 ± 0.03 µmol/g muscle), followed by a subsequent decrease until the end of the storage period, with concomitant production of Hx. Hx concentration in the frog leg muscle significantly increased at the end of the 24-day storage period on ice, attaining a final concentration of 3.93 ± 0.87 µM/g of muscle (y = 0.15x − 0.35, r² = 0.88 [p < 0.05]).

3.2. Estimation of K-Value

The K-value, or freshness index, is a mathematical measure used to assess the freshness of fish and other aquatic products. Its derivation is based on nucleotide quantification and measures the extent to which ATP degradation has progressed within the tissue. This freshness index is expressed as the content of the two final products of the ATP catabolic pathway (HrX and Hx) in percentage with respect to the total content of ATP and its degradation byproducts (ATP, ADP, AMP, IMP, HrX, and Hx). The lower the K-value, the more the freshness of the aquatic products. Moreover, this index has been recognized for several decades as the most effective and objective indicator of the freshness of several fish species and seafood products [32].

Figure 2 shows the variations in the K-value of the bullfrog leg muscle samples stored on ice for 24 days. A significant and linear increase (y = 3.10x + 0.60, r² = 0.98 [p < 0.05]) in the K-value of the bullfrog leg muscle samples from 6.78% ± 0.73% (day 0, not adjusted) to 79.33% ± 4.02% (day 24) was observed during the storage period.

3.3. Texture and WHC of the Frog Leg Muscle

Texture is a key parameter for evaluating the freshness of aquatic products [33], and its characteristics are species-dependent. The WHC refers to the amount of water retained per gram of protein or the ability of proteins to retain water against gravitational forces, influencing both texture and juiciness in food products [34]. A decrease in WHC is often indicative of protein denaturation through hydrolysis and/or aggregation of myofibrillar proteins during storage on ice [35], which is directly associated with the loss of muscle texture [36]. Figure 3 illustrates the postmortem changes in texture and WHC of bullfrog leg muscle stored on ice for 24 days. A significant decline (p < 0.05) in texture was observed, decreasing from 21.93 ± 1.36 Nw to 10.87 ± 1.08 Nw over the storage period. Conversely, WHC remained relatively stable, showing no significant differences (p > 0.05), with only a slight decrease from an initial value of 97.61% to 88.24% by day 24 of storage.

3.4. Estimation of pH and TVB-N

pH is an important indicator of the quality of aquatic organisms, as it can be used to assess the freshness, biochemical, physical, chemical, and microbiological quality of the aquatic and fishery products during storage [37,38]. Total volatile basic nitrogen (TVB-N) is often used as a biomarker of protein and amine degradation, and it is used to interpret meat freshness [39]. Figure 4 shows the changes in the pH of the bullfrog leg muscle samples during the 24-day storage period on ice.

Figure 4 reveals a significant decrease (p < 0.05) in the pH until reaching the minimum record of 6.2 ± 0.16 on day 12 of ice storage on ice, which was maintained until day 15 of storage, followed by an increase to 6.80 ± 0.24 on day 18 and finally, to 7.23 ± 0.06 on day 24. Meanwhile, the content of TVB-N started with a value of 14.82 ± 2.05 mg/100 g of muscle and increased until day 6, to decrease and maintain significant changes (p < 0.05) until day 15, to continue its increase and reach its maximum value (27.365 ± 2.20) until the end of the experiment.

3.5. Color of the Frog Leg Muscle

Color is one of the most important parameters employed for evaluating the quality of fishery products [30]. The surface color parameters of the bullfrog leg muscle are shown in Figure 5; the initial values corresponding to “L”, “a”, “b”, and ∆E were found to be 60.37 ± 1.36, 2.70 ± 1.36, 3.34 ± 1.36, and 0.00, respectively. At the end of storage in ice, a significant increase (p < 0.05) in the values of the parameters “L”, “b”, and ∆E but not in that of “a” (p > 0.05) was observed.

3.6. Effect of Storage on Microbiological Content

The microbiological analysis helps identify areas in aquatic organism handling and processing that need improvement to ensure hygiene quality or the requirement of any additional treatment for reducing the risk of diseases. The results of the analysis reveal the safety of the leg muscle from the bullfrog for human consumption. Figure 6 shows the total counts of mesophilic and psychrophilic bacteria in the leg muscles of the bullfrog during storage on ice for 24 days.

The initial counts of the mesophilic and psychrophilic bacteria were found to be 3.09 ± 0.12 and 2.70 ± 0.00 log CFU/g of muscle, respectively. The current study revealed a significant increase (p < 0.05) in the total counts of mesophilic and psychrophilic bacteria with storage time, resulting in counts of 6.75 ± 0.01 and 6.45 ± 0.02 log CFU/g, respectively, on day 24 of storage on ice.

3.7. Degree of Association Among the Different Quality Parameters

The correlation matrix between the quality parameters used to evaluate the postmortem changes in the bullfrog muscle is presented in

Table 1. Correlation matrix from the determinations.

	L	a	b	Texture	pH	WHC	TVB-N	ATP	ADP	AMP	IMP	HxR	Hx	KV	TPB	TMB
L	1.00000
a	0.99435	1.00000
b	0.90962	0.87377	1.00000
Texture	−0.94709	−0.95345	−0.91945	1.00000
pH	−0.46596	−0.49383	−0.59029	0.72525	1.00000
WHC	−0.98375	−0.96195	−0.96714	0.94319	0.49321	1.00000
TVB-N	0.99967	0.99319	0.90433	−0.93858	−0.44321	−0.98233	1.00000
ATP	−0.97815	−0.95773	−0.88456	0.86466	0.28706	0.96926	−0.98266	1.00000
ADP	−0.57261	−0.51384	−0.48395	0.29432	−0.41174	0.58389	−0.59187	0.73034	1.00000
AMP	−0.96383	−0.94565	−0.84048	0.82787	0.21549	0.94412	−0.97025	0.99612	0.76489	1.00000
IMP	−0.76715	−0.70665	−0.74268	0.55884	−0.09811	0.80144	−0.77986	0.88026	0.94502	0.88921	1.00000
HxR	0.97488	0.94984	0.90252	−0.86514	−0.29629	−0.97534	0.97905	−0.99865	−0.73374	−0.99158	−0.89076	1.00000
Hx	0.90611	0.86928	0.82125	−0.73489	−0.08565	−0.90885	0.91543	−0.97418	−0.86422	−0.98048	−0.96181	0.97591	1.00000
KV	0.89924	0.86165	0.81338	−0.72370	−0.06959	−0.90205	0.90894	−0.97048	−0.87231	−0.97781	−0.96518	0.97222	0.99987	1.00000
TPB	0.92171	0.88096	0.87693	−0.77701	−0.16951	−0.93953	0.92879	−0.97858	−0.82649	−0.97513	−0.95525	0.98474	0.99382	0.99252	1.00000
TMB	0.83052	0.76670	0.91503	−0.72357	−0.22267	−0.90171	0.83473	−0.89128	−0.77017	−0.86854	−0.93366	0.91275	0.92041	0.91898	0.95303	1.00000

Lab: Lab CIE or CIELAB chromatic model to describe colors; WHC: water retention capacity; TVB-N: total volatile bases; ATP: adenosine 5′ triphosphate; ADP: adenosine 5′ diphosphate; AMP: adenosine 5′ monophosphate; IMP: inosine 5′ monophosphate; HxR: inosine; Hx: hypoxanthine; KV: K-value; TPB: total psychrophilic bacteria; TMB: total mesophilic bacteria.

. The data indicated a high positive correlation between the ice storage time of the bullfrog leg with regard to the determination of K-Value, TVB-N, TMB, TPB, pH, HxR, and Hx. On the other hand, it was observed that the concentration of ADP, AMP, IMP, texture, and WHC had a negative correlation with the storage time.

3.8. Principal Component Analysis (PCA) Between the Different Quality Parameters

The Principal Component Analysis (PCA) allows us to visualize the grouping of variables and their impact on product quality over storage time. The results of the PCA plot are shown in Figure 7. It is noted the evaluation and relationship between freshness and quality parameters of frog legs stored in ice.

4. Discussion

4.1. Estimation of the Concentrations of ATP and Its Degradation Products

ATP is a molecule that provides energy for cellular processes in living organisms. It should be noted that there is a scarcity of previously published information that would allow the comparison of the TMCs of nucleotides, nucleosides, and the nitrogenous with those of other studies involving bullfrogs or other species of frogs. However, the result obtained herein is very similar to that of a previous study on cazón fish (Mustelus lunulatus; 6.93 µmol/g) [35], but lower than that reported for the seriola fish (Seriola quinqueradiata; 9.3 µmol/g) [40]. The TMC of ATP and related compounds in the muscle tissue as well as the rates and patterns of changes in these levels during storage are dependent on the species under consideration and also on the type of muscle tissue and storage conditions [41]. Variations in TMC have been associated with differences in species, seasons, physiological conditions, and feeding, among other variables [42].

The concentration of ATP obtained in the present study was less than that obtained in the frog semitendinosus muscle (9.9 µmol/g) in a previous study [43]. However, the low initial ATP concentration in the bullfrog leg reported in the current study can mainly be attributed to the time elapsed since slaughter (~12 h). In fact, ATP has been reported to be degraded within the first 24 h postmortem [44], and concentrations of ATP (0.15 and 0.10 µmol/g) similar to those obtained in the current study were previously reported in M. lunulatus [35] and rainbow trout (Oncorhynchus mykiss) [45], respectively. The degradation behavior of ATP similar results with respect to bullfrog leg muscle has hitherto not been reported. The degradation behavior observed herein parallels the degradation behavior reported in other aquatic species, which showed a good correlation with a reduction in freshness and sensory acceptability [46]. A previous study [47] reported that ATP is typically degraded to AMP and IMP leading to their relatively rapid accumulation in the fish muscles; AMP was found to accumulate in substantial quantities in the fish muscle followed by its deamination to IMP by AMP deaminase [15,48]. The concentrations of AMP and IMP are considered suitable indicators of the freshness of aquatic species in various studies; these compounds impart a sweet taste, which is considered a characteristic of fresh fish [27].

As with the other ATP degradation products, there is a lack of previously published research that would allow the comparison of the results obtained herein with those of other studies on bullfrogs; however, the formation and accumulation of HxR and Hx are reportedly associated with a bitter taste and, consequently, with a loss in freshness [16]. Concentrations of Hx similar to those observed herein have been previously reported for other aquatic species such as tilapia (Oreochromis niloticus) [49] after 18 days of storage on ice.

The postmortem formation of Hx in aquatic products indicates the onset of autolytic deterioration. Endogenous enzymes are responsible for the loss of freshness of aquatic products, and their action typically precedes and is independent of spoilage by bacteria [17]. The accumulation of Hx observed in the current study was a good indicator of the reduction in the freshness, which was linearly associated with ATP degradation and Hx formation [16]. In itself, aquatic products with high levels of ATP are of excellent freshness [27], while those with high concentrations of IMP are of lower freshness but with better flavor due to their contribution to the umami flavor, and when there are high concentrations of HxR and Hx, their quality will be reduced [50]. In summary, the degradation of ATP in the muscle of post-mortem bullfrog legs is a critical process that directly affects their shelf life and quality during storage on ice. The accumulation of degradation products, especially hypoxanthine, is associated with a decrease in the freshness and sensory quality of the product. Therefore, monitoring of these compounds is essential to ensure the quality and safety of the product during storage.

4.2. Estimation of K-Value

To the present knowledge, no study has been conducted to evaluate the K-value of the leg muscles of bullfrogs or any other species of frog. However, the initial K-values observed herein are lower than those reported in a previous study [49,51,52], where K-values of 11.35%, 21.23%, and 13.80% were obtained for the muscle samples of O. niloticus, sierra (Scomberomorus sierra), and Pacific salmon (Oncorhynchus nerka), respectively. However, the final K-value obtained in this study mirrored that obtained previously for the muscle sample of S. sierra muscle (80.6%) [51] but is higher than the corresponding values (69.61% and 58.39%) obtained for the muscle samples of O. niloticus [49] and M. lunulatus [35], respectively, after storage on ice for 18 days. Furthermore, the K-value obtained in this study is lower than that (90.0%) reported for turbot (Scophthalmus maximus) [53].

A system of classifying the freshness of fish products based on K-values was developed in a previous study [54]. According to this system, fish products with K-values of <20%, <50%, and >70% were considered very fresh, moderately fresh, and not fresh, respectively [27,55]. Based on these categories of K-value, the bullfrog leg muscle samples under the experimental conditions employed in the current study can be considered moderately fresh up to day 18 of storage on ice. Notably, this system of classification is dependent on the fish species (bullfrog is not a fish; however, it is considered an aquatic product [12,13]); thus, the K-value needs to be calculated for each type of aquatic organism.

4.3. Texture and WHC of the Frog Leg Muscle

Loss of texture during the storage of aquatic products has been previously reported [56], and several studies have indicated that the muscle tissue progressively loses its firmness after rigor mortis [57,58,59] a process mainly attributable to the enzymatic degradation of muscle proteins [60].

As per a previous study [61], the decrease in WHC is likely because of the reduction in the functionality of myofibrillar proteins, which are primarily involved in water retention in the muscle tissue [62].

It is well known that the properties of flesh change with a decrease in freshness, and the magnitude of the change depends on the original state of the muscle tissue [42]. The aquatic products typically exhibit an increase in toughness accompanying a progressive loss in fluids and reduction in WHC, possibly due to the loss of integrated myofibrillar protein during storage [61]. The results of this study indicate the probable denaturation (aggregation and/or hydrolysis) of the myofibrillar protein of the bullfrog leg muscle samples during the storage period, which affects the texture of the sample. Additional work is needed to determine the possible nature of protein conformational changes in the muscle tissue of this frog species during refrigerated storage on ice.

4.4. Estimation of pH and TVB-N

In the current study, a pH of 6.90 ± 0.08 was obtained on day 0, which was higher than the previously reported values (6.58, 6.43, and 6.24) for the leg muscle of the frog species Rana esculenta [7] and the muscles of M. lunulatus [35] and ray fish (Raja clavata) [63], respectively, at the start of storage on ice. However, a pH in the range of 6.7–7.0, which is similar to that obtained in the current study, was reported for fish products following capture [64]. The differences observed in the initial pH readings could be due to the species, season, diet, activity, or stress level during catching, and also to the type of muscle tissue [65].

The pH behavior of frog leg muscle during ice storage can be attributed to two consecutive biochemical processes: initially after slaughter, post-mortem glycolysis is activated under anaerobic conditions, in which glycogen is degraded to lactic acid, causing a decrease in pH due to the accumulation of this acid in the tissue caused by the cessation of blood flow. Conversely, as storage progresses, the activity of autolytic enzymes together with the proliferation of psychrotrophic bacteria favors the degradation of proteins and amino acids, generating basic compounds such as ammonia and various amines, resulting in a considerable increase in pH [65]. A previous study reported a significant increase in the pH of frog legs of the species R. esculenta [7] after 10 days of storage at 2 °C, attaining a pH of 7.11. These variations in pH may be due to the differences between the species of frogs and the temperatures at which the frog legs were stored.

TVB-N is very used to measuring the quality of aquatic products. The TVB-N content can also reflect the quality of bullfrogs, as it is closely related to the activities of bacteria and relative enzymes [66]. In light of the above information, the results of this study indicate that even after 24 days of storage on ice, the frog legs remained fit for human consumption as the maximum limit allowed for a product to be fit for human consumption was not exceeded. This increase in TVB-N content indicates a progressive deterioration of the muscle due to the action of microorganisms and endogenous enzymes. This increase is associated with the production of volatile compounds such as ammonia, trimethylamine (TMA), and dimethylamine (DMA), derived from the activity of psychrotrophic bacteria and from the breakdown of myofibrillar and sarcoplasmic proteins. However, further studies are warranted for establishing the duration of storage after which the frog leg muscle samples would exceed the maximum allowable limit of TVB-N in order to accurately determine the biochemical, physical, chemical, and microbiological quality of this product based on this quality index. In general, TVB-N is a useful indicator of the freshness and quality of frog leg muscle samples and can be employed to determine the degree of spoilage.

Both TVB-N and the spoilage degree are dependent on the species involved, and the TVB-N concentration cutoff value of 30 mg/100 g of muscle may not correspond to the organoleptic parameters of fish spoilage. Our results differ from those obtained in the bullfrog (Lithobates catesbeiana) [61], where an increase in TVB-N was reported from 6.65 to 43.81 mgN/100 g after 14 days of storage in refrigeration at 4 °C. These values are higher than those obtained in the present study carried out at 0 °C. These differences may be due to the storage temperature used, since at this temperature (4 °C), it favors enzymatic reactions and microbial growth, which accelerates the reduction in freshness and increases the rate of deterioration. Also, it has been reported that an increase in TVB-N can be caused by an autolytic process, which produces amino compounds and bacterial deterioration [67]. Therefore, the TVB-N concentrations at which a marine or aquatic product should be considered spoiled need to be established for each species.

4.5. Color of the Frog Leg Muscle

Color is a critical parameter for assessing the quality of food products. In this study, the initial values of L, a, and b positioned the bullfrog leg muscle samples within the yellow–red quadrant of the CIE color space, with a hue angle of 51.05, indicating an orange-toned coloration. At the end of the storage period on ice, a significant increase (p < 0.05) was observed in the L (lightness) and b (yellow–blue) values **, whereas *a (red–green) remained statistically unchanged (p > 0.05). The low a and b values suggest that the product exhibits an opaque appearance, as these values correspond to the grayish region of the chromatic sphere. A previous study [44] reported changes in the initial color of the aquatic and marine products during storage on ice, which affected their quality and impacted product acceptance by the consumers. There are no previous studies of color analysis in bullfrog legs that allow us to compare our results. In a study conducted on bullfrog (Lithobates catesbeiana) [66], a sensory analysis was carried out with trained judges, who assessed visible freshness indices, including texture, odor, color, and overall acceptability. The study reported that the shortest acceptable storage duration for bullfrog meat was eight days. Additionally, another study on the same species [68] examined muscle color and heme pigment content following electrical or thermal stunning, with or without bleeding procedures. The findings indicated that the myoglobin content in bullfrog meat was comparable to that found in avian white muscles.

4.6. Effect of Storage on Microbiological Content

The purpose of the microbiological analysis of food products is to evaluate the possible presence of bacteria and to assess the hygiene quality of the aquatic products owing to abuse of temperature and hygiene during handling and processing. Microbiological analysis is important to ensure food safety and prevent foodborne illnesses. The results of the initial counts of the mesophilic and psychrophilic bacteria are in alignment with the corresponding counts obtained previously for the frog leg samples of R. esculenta (3.83 ± 0.25 and 3.02 ± 0.09 log CFU/g, respectively) [7]. The current study revealed that the counts are lower than the corresponding counts (7.98 ± 0.10 and 7.71 ± 0.07 log CFU/g) previously determined for the frog legs of R. esculenta [7] following storage at 2 °C for 14 days.

Microbial processes play a key role in the deterioration of bullfrog (Lithobates catesbeianus) leg muscle during ice storage, as they promote protein degradation and the production of nitrogenous compounds such as ammonia and amines, among others, which promote microbial growth and loss of freshness. In addition, they negatively impact texture, reducing tissue firmness, which is attributed to the action of microbial proteolytic enzymes on muscle structural proteins. Together, bacterial growth and its metabolic effects accelerate product deterioration, reducing its shelf life. The Official Mexican Standard [26] and The International Committee for the Determination of Food Microbiological Properties [69] have set a limit of 7 log CFU/g or 10,000,000 CFU/g as the maximum permitted concentration of microorganisms in fresh and/or refrigerated fish for it to be fit for human consumption. Accordingly, the counts of mesophilic and psychrophilic bacteria in the bullfrog leg muscle samples were within permissible limits after 24 days of storage on ice, indicating their continued edibility. These results align with those described above with respect to the K-value, TVB-N concentration, pH, and the increase in hypoxanthine concentration on day 18 of storage. Biochemical, chemical, and physical changes in bullfrog legs stored in ice reflect a progressive loss of freshness and quality. The ATP degradation and increased K index are key indicators of spoilage, while increases in pH, TVB-N, and loss of texture indicate the progress of the decomposition process. These findings highlight the importance of controlling storage conditions to prolong product shelf life.

4.7. Degree of Association Among the Different Quality Parameters

The data obtained from the correlation matrix suggest that the decrease in the concentration of ADP, AMP, and IMP; texture; and WHC can be used in conjunction with the K index, TVB-N concentration, TMB, and TPB to monitor the freshness and quality of the muscle of bullfrog leg during ice storage [70]. During ice storage, frog legs undergo significant changes in their physicochemical, biochemical, and microbiological characteristics, which affect their quality and freshness. Luminosity (L) and color parameters a and b were observed to decrease progressively, correlating negatively with texture (−0.947, −0.953, and −0.919, respectively), indicating that loss of firmness is associated with changes in muscle coloration, possibly due to protein degradation and pigment oxidation [20]. Likewise, water holding capacity (WHC) decreases with time (−0.974), affecting the juiciness and sensory quality of the product, a common phenomenon in fish and shellfish stored on ice. In biochemical terms, ATP degradation (−0.996) follows the classical metabolic pathway to IMP, HxR, and Hx, which is reflected in an increase in K-value (0.972), a key freshness indicator widely used in postmortem quality assessment of aquatic products. The increase in TVB-N (0.972) and microbial load (TPB: 0.989, TMB: 0.926) confirms the progression of protein and microbiological deterioration as storage progresses, which is in agreement with previous studies that have shown that bacterial growth and the production of volatile nitrogenous compounds are directly related to protein degradation in refrigerated products. These results show that product quality is progressively compromised, suggesting that strict temperature control and continuous monitoring of indicators such as K-value, TVB-N, and WHC, which could be used as critical quality parameters, are necessary to maximize the freshness of frog legs [50].

4.8. Principal Component Analysis (PCA) Between the Different Quality Parameters

Finally, during ice storage, the quality of frog legs is affected by changes in physicochemical, biochemical, and microbiological parameters, as observed by Principal Component Analysis (PCA). Changes in color (L, a, b) are strongly related, suggesting a simultaneous alteration in visual appearance, which correlates negatively with texture, indicating that structural degradation of muscle occurs along with color deterioration [20]. Decreased water holding capacity (WHC), along with increased TVB-N, TPB, and TMB, reveals that microbial growth and production of volatile nitrogenous compounds contribute to the loss of sensory quality [70]. Furthermore, ATP degradation follows the classical pathway towards IMP, HxR, and Hx, showing a negative correlation with K-value, confirming its usefulness as an indicator of freshness. At the end, the positive relationship between TVB-N and Hx reinforces that microbial activity accelerates proteolysis and chemical deterioration of the product, highlighting the importance of temperature control and monitoring of these parameters to extend shelf life [50].

5. Conclusions

Ice storage significantly affects the freshness and quality of bullfrog leg muscle, as evidenced by ATP degradation, increased K-index, and hypoxanthine accumulation. Changes in pH, TVB-N, and texture reflect progressive biochemical and microbiological breakdown associated with bacterial growth and protein degradation. Although ice storage is an effective strategy to prolong initial freshness, the results suggest that optimal consumption is within the first 18 days, while freshness was reduced at day 24. The monitoring of biochemical and microbiological indicators is essential to establish better preservation strategies and to guarantee the quality and safety of this product in the aquaculture industry.