Comparison of Pretreatment Methods for Obtaining Collagen Hydrolysates from the Swim Bladder of Totoaba macdonaldi and Their Negative Impact on Cancer Cells

Abstract

The search for therapeutic bioactive peptides has led to the utilization of marine byproducts as collagen sources. This study evaluated the effect of collagen hydrolysates (CH) obtained from the swim bladder (SB) of Totoaba macdonaldi on breast (MCF-7) and colorectal (Caco-2) adenocarcinoma cells and on human dermal fibroblasts (CRL-1474), considering the need for less invasive and less toxic treatment alternatives. Two pretreatment methods for the SB were compared: (1) NaOH and butanol (SBPT), and (2) hexane (SBDF). The pretreated tissues underwent direct enzymatic hydrolysis using bromelain. The resulting hydrolysates were characterized by SDS-PAGE, Raman spectroscopy, and chromatographic profiling. Both pretreatments preserved the structure of type I collagen. Bromelain hydrolysis was efficient, yielding peptides with molecular weights below 20 kDa for CH-SBPT and below 10 kDa for CH-SBDF. CH of Totoaba macdonaldi significantly reduced MCF-7 and Caco-2 cells viability, particularly at 20 mg/mL. In CRL-1474 fibroblasts, CH-SBDF stimulated cell proliferation, while CH-SBPT had neutral effects. Hexane pretreatment is a viable alternative to NaOH, reducing processing steps without compromising yield or bioactivity. CH derived from Totoaba macdonaldi exhibit promising anticancer and regenerative properties, suggesting potential biomedical applications. Further research is needed to isolate specifically active peptides and elucidate their mechanisms of action.

1. Introduction

Collagen is the most abundant structural protein in the human body. It is made up of approximately 1000 amino acids, which may be released either individually or in the form of peptides through enzymatic hydrolysis. These compounds exhibit a diverse range of biological activities, including antioxidant, anti-inflammatory, and anticancer properties [1]. Collagen hydrolysates (CH) or collagen peptides have been produced from various sources, including bovine hides and bones, porcine skin, eggshells, and other byproducts from terrestrial animals. However, in recent years, there has been an increasing interest in producing collagen and CH from marine sources due to their health benefits as well as the increasing availability of commercial products for specific market segments [2,3]. In this regard, fish farming offers significant advantages as a source of raw materials for collagen extraction by providing a steady supply and consistent quality parameters. This positions marine sources as a promising area of focus in collagen research and industry development.

Among the species currently being farmed in México, Totoaba macdonaldi has gained importance in the last few years. This species is endemic to the Gulf of California in México and is considered an endangered species according to CITES (Convention on International Trade in Endangered Species of Wild Fauna and Flora) [4]. While Totoaba was historically overfished for its swim bladder (SB), some farms have successfully produced enough Totoaba to supply the domestic market.

Commercial production of marine CH involves three stages: 1. Sample pretreatment to remove protein, fats, pigments, and other undesirable components; 2. Extraction, separation, and purification of collagen; and 3. Enzymatic hydrolysis to obtain the peptides, which are then sterilized and dried. These steps make the process lengthy and energy intensive. In the hydrolysis stage, several enzymes have been used; however, bromelain exhibits the best results for antioxidant and functional properties in industrial applications [5]. Clearly, the choice of processing treatments markedly affects the amino acid sequences, molecular weights, and bioactive properties of the final peptides [6,7].

Numerous studies have investigated the bioactivities of collagen peptides. These compounds are associated with improved overall health and a reduced risk of chronic diseases, including cancer, diabetes, and cardiovascular disease. The high prevalence of these conditions has prompted extensive research into the potential of peptides as antioxidants, antihypertensives, anti-inflammatory, antidiabetics, and anticancer agents. Additional biological activities of interest include antimicrobial, anticoagulant, wound-healing, and anti-aging [6,8,9].

Previous reports have demonstrated the effects of protein hydrolysates on various cancer cell lines, including breast cancer cells [10]. Recently Çevik et al. [11] reported that proline-rich peptides, such as VPP (valine–proline–proline), induced cell death and inhibit proliferation and migration in breast cancer cells, indicating their potential as therapeutic agents [11]. Furthermore, previous studies have shown that samples of hydrolyzed fish protein and collagen exert negative effects on Caco-2 colorectal adenocarcinoma cell proliferation when tested at high concentrations [12]. More recently, research has indicated that doubly hydrolyzed collagen Type I and III, sourced from calves, inhibits the proliferation of HCT116 colorectal cancer cells by reducing cell growth and migration, modulating oxidative stress, and lowering tumor marker levels [13]. Additionally, CH have been reported to have low toxicity to normal cells and produce no significant harmful side effects [14,15]. Beyond these anticancer properties, CH have been shown to have several beneficial effects on human dermal fibroblasts (HDFs), which are crucial for skin health and regeneration [16].

Bioactive peptides derived from food proteins or byproducts represent an economical and sustainable alternative for developing functional foods and nutraceuticals. They also have multiple applications in the pharmaceutical and cosmetic industries. However, further studies are needed to investigate their safety and efficacy in more detail [6,17].

This study compared two pretreatment methods for the swim bladder (SB) of Totoaba macdonaldi. The first method involved sodium hydroxide (NaOH) pretreatment followed by defatting with butanol, while the second method utilized defatting with hexane. Following these pretreatments, the tissues underwent direct enzymatic hydrolysis with bromelain. Figure 1 shows the general procedure for producing CH. The biological activity of the resulting hydrolyzed collagen was evaluated on breast and colorectal adenocarcinoma cells, and on human dermal fibroblasts (HDFs).

2. Materials and Methods

2.1. Chemical Reagents

Thiazolyl Blue tetrazolium bromide (MTT), Bromelain (B4882), phosphate buffered saline (PBS), Dimethyl sulfoxide (DMSO), Ammonium Persulfate, β-Mercaptoethanol (β-Me), Tris base, glycine, Sodium dodecyl sulfate (SDS) RIPA buffer, RMPI-1640, Medio Esencial Mínimo de Eagle (EMEM), Dulbecco’s modified Eagle’s medium low glucose (DMEM), streptomycin–penicillin, trypsin 0.25% with EDTA, all were obtained from Sigma-Aldrich Inc., St. Louis, MO, USA. Coomassie blue G-250, 30% Acrylamide/Bis Solution 19:1 were purchased from BioRad Laboratories, Inc., Hercules, CA, USA. Fetal bovine serum (FBS) were obtained from Biowest USA Inc., Bradenton, FL, USA. All other reagents and solvents used were of analytical grade or better.

2.2. Swim Bladders Samples

Totoaba SB samples were provided by the Cygnus Ocean Farms. This farm is located at the “La Manga” bay, San Carlos Nuevo Guaymas, Sonora, México (Latitude 27.9420° N, Longitude 111.0617° W). The facility operates under approval permit DGVS-UMA-IN-1821-SON/17, issued on 17 January 2017 (SGPA/DGVS/00268/17), and 17 September 2018 (SGPA/DGVS/009141/18) [18]. Thus, it is an authorized and regulated aquaculture facility operating under current permits from SEMARNAT (Ministry of the Environment and Natural Resources) and CONAPESCA (National Commission for Aquaculture and Fisheries). No specimens were collected from the wild.

By sourcing from a certified aquaculture unit, the project supports sustainable practices that reduce pressure on wild populations and help create a legal, traceable supply chain. The use of byproducts, such as the swim bladder, also aligns with maximizing resource use and minimizing waste. The material was obtained from specimens that had already been harvested, so no animals were sacrificed solely for the purpose of this study.

The SBs were extracted from the fish under aseptic conditions, packed in polyethylene bags, and frozen and stored at −18 °C until sent to the laboratory, where they were kept frozen until processed. Before the pretreatment, the SB samples were cleaned manually to remove the blood vessels and fat and cut into small pieces (approximately 1 × 1 cm).

2.3. Extraction of Swim Bladder Total Protein Using RIPA Buffer

Ten milligrams of SB tissue were suspended in 500 μL of RIPA and sonicated for 15 min. RIPA total protein extraction lysis buffer is a traditional lysate for extracting proteins from cells and tissues.

2.4. Pretreatment with NaOH and Defatting with Butanol

NaOH treatment is commonly used to remove non-collagenous proteins from raw material. The pretreatment was performed following the methodology described by Cruz-López et al. [19] with slight modifications. 800 g of SB were immersed in a 0.1 M NaOH solution, kept at a 1:20 ratio (w/v). The mixture was continuously stirred at 4 °C for 12 h, and the alkali solution was changed every 4 h. The treated SB was then washed with distilled water until all the alkaline solution was eliminated. Subsequently, the tissue was defatted with a 15% butanol solution in H₂O at a 1:20 ratio (w/v) for 12 h a 4 °C, with butanol replenished every 4 h. The defatted sample was washed several times with distilled water and later submerged in distilled water overnight. Finally, the sample was freeze-dried and ground. This sample, labeled as SBPT, was further used to prepare hydrolysates.

2.5. Straight Defatting with Hexane

A total of 500 g of SB was freeze-dried and then submerged in hexane for defatting, using a 1:3 ratio (w/v). The mixture was continuously stirred for 24 h at 22 °C, and every 8 h, the hexane was replenished. Subsequently, the extracted SB was filtered to remove excess hexane, and the residual hexane was removed by evaporation. Finally, the sample was labeled as SBDF and used to prepare hydrolysates.

The following equation was used to determine dry weight yields of the SB after pretreatment:

$$\mathit{Yield} (\%)={\displaystyle \frac{\mathit{Weight} \mathit{of} \mathit{the} \mathit{swim} \mathit{bladder} \mathit{after} \mathit{pretreatment} (\mathit{final} \mathit{weight}, g)}{\mathit{Weight} \mathit{of} \mathit{the} \mathit{swim} \mathit{bladder} \mathit{tissue} (\mathit{initial} \mathit{weight}, g)}}\times 100\%$$

(1)

2.6. Electrophoretic Analysis

The molecular weight and type of collagen were determined by using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoretic patterns were determined according to Laemmli [20] with slight modifications, using 8% separating gel and 4% stacking gel for SB tissue in RIPA and SB sample pretreatment (SBPT and SBDF); and using 15% separating gel and 6% stacking gel for CH-SBPT and CH-SBDF. The samples (10 mL) were mixed with the sample loading buffer at a 1:1 ratio (v/v) in the presence of β-Me and incubated at 95 °C for 5 min. After electrophoresis, the gel was stained with Coomassie blue G-250 (BioRad Laboratories, Inc., Hercules, CA, USA). A protein ladder marker ranging from 10 kDa to 210 kDa (PageRuler^TM, Thermo Fisher Scientific Inc., Waltham, MA, USA) was used to estimate the molecular weight of collagen. The protein bands were visualized using ImageLab 5.0 Software (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

2.7. Direct Hydrolysis with Bromelain

The enzymatic hydrolysis of SBPT and SBDF was prepared by mixing samples in ultrapure water at a 1:20 ratio (w/v) and adding 100 mg of bromalin enzyme for every 100 mL of solution. The initial pH values of the mixture were measured: 6.59 for SBPT and 5.38 for SBDF. The mixtures were thoroughly homogenized and subjected to hydrolysis in a water bath using ultrasound at 50 °C for 3 h. After hydrolysis, the enzyme was inactivated by heating the mixture at 95 °C for 10 min. The samples were then allowed to cool and centrifuged at 5752× g for 20 min at 4 °C. This hydrolysis process followed Cruz-López with a slight modification [19]. The resulting supernatant was a hydrolysate. Finally, the samples were freeze-dried and labeled as CH-SBPT and CH-SBDF, respectively.

The following Equation (2) was used to determine yields of hydrolyzed collagens:

$$\mathit{Yield} (\%)={\displaystyle \frac{\mathit{Weight} \mathit{of} \mathit{the} \mathit{hydrolyzed} \mathit{collagens} \left(g\right)}{\mathit{Weight} \mathit{of} \mathit{the} \mathit{pretreated} \mathit{swim} \mathit{bladder} \left(g\right)}}\times 100\%$$

(2)

2.8. Chromatographic Profile of Hydrolyzed Collagens

The chromatographic profile of CH was performed using an ACQUITY Ultra Performance LC (Liquid chromatography) system with TUV Detector (Waters Corp., Milford, MA, USA). Briefly, 10 μL of each CH (10 mg/mL) were directly injected into a C18 Column (1.6 μm, 2.1 × 100 mm). CH were eluted using water as mobile phase A and acetonitrile as mobile phase B. An elution gradient was performed as follows: 0–5 min, 100% to 95% B; 5–10 min, 95% to 90% B; 10–15 min, 90% to 85% B; 15–20 min, 85% to 80% B; 20–25 min, 80% to 75% B; 25–30 min, 75% to 70% B; 30–35 min, 70% to 100% B; 36 min, 100% B. Total run time was 36 min with a 0.3 mL/min flow rate. Column temperature was set at 37 °C and UV detection performed at 215 nm.

2.9. Raman Analysis

Raman measurements were obtained in the range of 90–3100 cm⁻¹, using a laser with a wavelength of 1064 nm and an output power of 800 mW as the excitation source (Ocean Optic Inc., Dunedin, FL, USA).

2.10. Cellular Viability Assay

The MCF-7 cells (human breast adenocarcinoma, HTB-22) were cultured in RMPI-1640 supplemented with 10% heat-inactivated FBS and 0.5% streptomycin-penicillin. The Caco-2 cells (human colon adenocarcinoma, HTB-37) were cultured in EMEM supplemented with 10% heat-inactivated FBS and 0.5% streptomycin-penicillin. CRL-1474 cells (normal human dermal fibroblast, CCD-25Sk) were cultured in DMEM–low glucose supplemented with 10% heat-inactivated FBS and 0.5% streptomycin-penicillin. All cell lines were from the American Type Culture Collection (ATCC), Manassas, VA, USA. and were cultured in their growth media at 37 °C in a 5% CO₂ atmosphere. Cell viability assay was evaluated using MTT assay with slight modifications [21]. The cells were seeded into 96-well plates at a density of 10⁴ cells per well containing 100 μL and incubated for 24 h to allow cell adherence. Then, the medium was removed and substituted with 200 μL of different concentrations (20, 10 y 5 mg/mL) of CH-SBPT and CH-SBDF, which were prepared in medium without FBS. Cells were exposed for 48 h to the hydrolysates. Controls consisted of untreated cells and incubated using the same conditions as treated cells. 1% DMSO was used only as a cell death control. Then, 20 μL/well of MTT at 5 mg/mL were added, and the plates were returned to the incubation for 3.5 h. Afterwards, the 150 μL MTT solution was removed and 150 μL of DMSO: isopropanol was added. The absorbance was measured at 570 nm together with a reference of 690 nm using a microplate reader (Cytation 3 CYT3M, BioTek Instruments, Inc., Winooski, VT, USA). All experiments were carried out in triplicates and the cells were observed and photographed under an inverted microscope with a 5× objective lens (Leica, Wetzlar, Germany, DMi1).

2.11. Statistical Analysis

The software Graphpad Prism 9.0.1 (Boston, MA, USA) was used for statistical analysis applying Two-way ANOVA and Tukey’s post-hoc test for multiple comparison. Data were presented as means ± SEM and the statistical significance level was set at p < 0.05.

3. Results and Discussion

3.1. Pretreatment of Swim Bladders

The usual method to remove non-collagenous proteins is the use of NaOH, whose effectiveness depends on time, temperature, and concentration [22]. Additionally, the removal of fats and pigments was achieved using butanol or ethanol. Pretreatment usually involves several washes with NaOH, followed by the extraction with butanol. However, this process can take anywhere from a few days to several weeks and requires multiple water washes to neutralize the pH of the sample at each stage. To address these limitations, in this work, the SB was directly defatted using hexane and later hydrolyzed. This approach is less expensive and requires less processing time while producing a similar yield. The results are presented in the following

Table 1. Dry weight yields of SB after pretreatment with NaOH and Butanol (SBPT); and directly defatted (SBDF).

	Initial Weight (Wet Weight)	Final Weight (Dry Weight)	% Yield
SBPT	800 g	236 g	30
SBDF	500 g	166 g	33

.

3.2. Electrophoretic Analysis of Pretreated Samples and Swim Bladder

The SDS-PAGE analysis showed that the SB of Totoaba samples subjected to the two extraction conditions displayed at least two different α bands and a β dimer (α₁, α₂, β), which suggests that it is a Type 1 collagen, a heterotrimer containing two identical α₁-chains and one α₂-chain in the molecular form of [α₁(I)]₂α₂(I). The two bands of the α₁ and α₂ chains occur in a 2:1 ratio, with molecular weights of approximately 118 kDa and 108 kDa, respectively. Figure 2 shows the electrophoretic analysis of the two pretreated SB samples (SBPT and SBDF, Gel B) and a total protein extract in RIPA buffer of the SB tissue before pretreatment (Gel A). The patterns of the two α chains and one β dimer of SB with different pretreatment and SB before pretreatment were similar. Therefore, the conditions of pretreatment did not affect the native collagen structure. A previous study demonstrated that pepsin-soluble collagen from SB of Totoaba was a type I collagen, comprising α₁ and α₂ chains and a β dimer, with molecular weight of approximately 126 kDa and 116 kDa for the α₁ and α₂ chains, respectively [19]. These differences may be attributed to the growth conditions of the totoabas. In the study conducted by Cruz-López et al. [19], the authors used SB from 3-year-old totoaba grown under controlled conditions at the Wildlife Conservation Management Units (UMA, Unidad de Manejo Ambiental) of the Facultad de Ciencias Marinas, Universidad Autónoma de Baja California, Mexico. On the other hand, the totoabas used in this study were cultivated in the open sea in anchored cages.

3.3. Collagen Hydrolysates Yield

Following pretreatment of SB samples with NaOH-butanol and hexane, we proceeded directly to the enzymatic hydrolysis using bromelain, thus bypassing the collagen extraction stage. As previously noted, the conventional method to prepare hydrolyzed collagen involves three steps: 1. Pretreatment to remove protein, fats, pigments, and other undesirable components; 2. Extraction, separation, and purification of collagen; and 3. Enzymatic hydrolysis. Collagen is typically extracted using acetic acid, a process that requires several days and results in low yields [23].

In this work, we observed the electrophoretic profile of the samples (Figure 2) following both pretreatments, which revealed collagen as the most abundant protein. Based on these profiles, we selected direct enzymatic hydrolysis using bromelain, which enables a faster process and resulted in a high yield of hydrolysates. Initially, hydrolysis of SBPT was tested with pepsin, papain, and bromelain. Since bromelain produced smaller peptides, subsequent experiments focused exclusively on this enzyme. Supplementary Figure S1 presents the electrophoretic profile of hydrolysates obtained with papain and pepsin. Bromelain, a plant protease with high specificity, is widely used in the food industry and has been effective in the hydrolysis of collagen and producing peptides with high biological activity [5,24]. In this work, hydrolysis yields of 81% were achieved for both CH-SBPT and CH-SBDF, indicating that the type of pretreatment had no significant impact on the yield.

3.4. Electrophoretic Analysis of Collagen Hydrolysates

The CH-SBPT yielded molecular weights below 20 kDa, while the CH-SBDF had lower than 10 kDa (Figure 3). In both treatments, the same temperature and time combinations were used; however, there was a small difference in the pH of the initial solution. In the case of the SBPT, the pH was 6.59, while in the SBDF, it was 5.38. According to the scientific literature, bromelain is active at a pH range from 3 to 8, with an optimum value at 5 [24,25]. In this work, it was decided to work with the resulting pHs in the SB-water mixture, as they fell within the range of high enzyme activity. Forgoing additional washing to achieve pH neutralization is justified considering process efficiency. Maintaining a pH near 5 preserves optimal bromelain activity, which facilitates rapid and effective enzymatic hydrolysis in subsequent stages. This strategy also reduces water consumption while sustaining product quality and performance.

It is worth noting that the differences in the final pHs in the SBPT and SBDF mixtures could be attributed to the pretreatment process used. As described in the methods section, in the first case, NaOH/butanol was used, while for the SBDF, only hexane was used for defatting. In the first case, traces of NaOH may have contributed to the slight pH increase. As noted above, the CH-SBDF had a lower molecular weight than those of the CH-SBPT. This could be because the pH was closer to the optimum level for bromelain enzyme activity (pH value of 5) [24]. The pH value significantly influences not only enzyme activity but also the stability of collagen molecules. This relationship, in turn, impacts the susceptibility of peptide bonds to enzymatic cleavage [26,27].

3.5. Chromatographic Profile of Collagen Hydrolysates

As observed from the electrophoretic pattern of the CH, there is a different chromatographic profile for the two CH samples. Specifically, the chromatographic profile of the CH produced from SB showed that CH-SBDF has a higher number of peaks eluted from 0 to 10 min than those from CH-SBPT. This indicates that the peptides from CH-SBDF are more hydrophilic than those from CH-SBPT (Figure 4). Hydrolysis increased solubility by exposing ionizable residues that facilitate hydrogen bonding with water [28].

3.6. Raman Analysis

Raman spectroscopy was used to identify chemical changes between pretreated bladders before and after hydrolysis, as it provides valuable insights without the need for complex sample processing [29]. Figure 5 shows the Raman spectra of the SBPT, SBDF, CH-SBPT, and CH-SBDF, highlighting the observed Raman band signals. In the protein spectra, the C–C and C–N vibrational bands are associated with the polypeptide backbone. These bands are primarily attributed to the functional groups COO⁻, NH₃⁺, and OH⁻ present in the protein structure.

Generally, the stretch vibrations of amide I, amide III, and C-C from collagens are in the regions of 1620–1680, 1235–1280, and 764–980 cm⁻¹, respectively [29,30,31]. For example, the peaks at 850, 873, and 919 cm⁻¹ are attributed to the presence of proline (Pro) and hydroxyproline (Hyp) from collagen. Additionally, the band of 1256 cm⁻¹ may be assigned to secondary structures of the collagen α-helix. Meanwhile, the peak at 1460 cm⁻¹ is often due to the movement of the protein CH₂ group. The positions of the Raman bands in Amide I, Amide III, and C-C regions are similar for SF, but there are some differences in the ratio of the band intensities. A gradual loss of secondary structure of collagen leads to a broadening and weakening in the intensity of the bands [32]. The vibrations of amide I and amide III are most sensitive to changes in the conformation of the secondary structures of collagen. The vibrations related to these molecular groups appear in the spectral range from 1200 to 1750 cm⁻¹. In this sense, the most intense and defined bands can be found in the SBDF sample (1256, 1460, 1479, and 1653, 1671 cm⁻¹), suggesting that pretreatment with hexane has a lesser effect on the secondary structure of collagen compared with NaOH pretreatment (SBPT). Therefore, hexane pretreatment is a viable alternative to NaOH, reducing processing. Furthermore, for the CH of the SB in the Amide III region, a slight change was observed in the 1256 cm⁻¹ peak, as a shoulder appears at 1277 cm⁻¹; this bands include C-N stretching and N-H in-plane bending of the peptide bond, along with contributions from C-C stretching and C=O in-plane bending. Bands in the 1260–1300 cm⁻¹ range are typically associated with the α-helix conformation, while those between 1240 and 1250 cm⁻¹ are linked to both random coil and β-sheet structures [32]. This may reflect conformational changes in the collagen secondary structure caused by the hydrolysis of the collagen peptide bonds. It is important to note that, despite the evidence provided by Raman spectroscopy and SDS-PAGE analysis, some non-collagenic compounds may have been present, as no extraction method is 100% efficient. However, it is challenging to detect the presence of other compounds that are in low concentrations using Raman spectroscopy, as their bands can overlap with those of peaks, complicating the assignment of specific bands.

3.7. Cellular Viability Assay

There is great interest in the study of bioactive peptides isolated from various marine sources (sponges, tunicates, ascidians, mollusks, fish) with potential anticancer activity [33]. Previous reports have demonstrated the effects of protein hydrolysates on various cancer cell lines, including U-937 human lymphoma [34]; HT1080 human fibroid sarcoma, HeLa human cervix adenocarcinoma [35], MCF-7 and MDA-MB-231 breast cancer cells [10], among others. Furthermore, it has also been reported that gelatin peptides from other animal species, such as bovine, display activity against SKOV-3 human ovarian carcinoma cells [15]. In addition, marinated collagens have been shown to inhibit cell proliferation in Caco-2 colorectal adenocarcinoma cells, and doubly hydrolyzed collagens from calves reduce cell growth in HCT116 colorectal cancer cells [12,13].

In this work, we report on the effect of CH from the Totoaba macdonaldi SB on breast and colorectal adenocarcinoma cells. We also evaluate their biocompatibility on non-malignant HDFs. The hydrolysates were assessed against the MCF-7 and Caco-2 cell lines at concentrations of 5, 10, and 20 mg/mL. Both hydrolysates reduced the viability of MCF-7 and Caco-2 cells compared to the untreated control (0 mg/mL). On MCF-7 cells, CH-SBDF showed a more consistent dose–response. At 20 mg/mL, CH-SBDF inhibited cell viability more than CH-SBPT, with cell viabilities of 43% and 52%, respectively. In contrast, for Caco-2 cells, CH-SBPT exhibited a more consistent dose–response relationship. However, at 20 mg/mL, both CH-SBPT and CH-SBDF exhibited similar inhibition, with cell viability of 46% and 49%, respectively (Figure 6). Micrographic images at the end of the treatment demonstrate a lower cell density for the CH-SBDF treatment at 20 mg/mL compared to the untreated control (0 mg/mL) on MCF-7 cells. Figure 7 illustrates the morphological changes in untreated MCF-7 cells and MCF-7 cells treated with CH. MCF-7 cells exposed to CH exhibited distinct morphological changes that varied depending on the specific treatment administered. MCF-7 cells treated with CH-SBPT exhibited a reduction in cell density and a distortion in cell morphology, whereas treatment with CH-SBDF resulted in changes in cells size and more compact cell clusters. In both treatments, this may be indicative of cell death or apoptosis. In contrast, the untreated MCF-7 cells exhibit a normal, polygonal shape with a defined cell membrane boundary. Previously, it has been reported that marine collagen peptides promote apoptosis in breast cancer lines primarily by activating apoptotic pathways or interfering with survival signaling pathways. Moreover, studies indicate that collagen peptides modulate reactive oxygen species (ROS) levels, thereby facilitating apoptosis, necrosis, and autophagy in tumor cells [36].

In Caco-2 cells, both treatments resulted in a comparable decrease in cell density at 20 mg/mL, with very similar morphological changes in cell size and more compact cell clusters for both treatments (Figure 8). This suggests that CH may similarly inhibit the viability of MCF-7 and Caco-2 cancer cells. Further studies are needed to identify active fractions and determine specific peptide sequences with relevant activity, and clarify the molecular mechanisms involved.

In contrast, a different effect was observed in fibroblast cells in this study. CH-SBPT presented only a slight inhibition of cell viability, which was not significantly different from the control. On the other hand, the CH-SBDF displayed an increase in cell proliferation, particularly at a concentration of 5 mg/mL. For the remaining concentrations, a slight increase in cell viability was observed; however, this increase was not statistically significant compared to the control (Figure 6). In the micrographs, an increase in cell density is observed in relation to the control, which is more evident for the CH-SBDF treatment (Figure 9). Collagen peptides have well-documented beneficial effects on normal HDFs by upregulating the expression of genes encoding for collagen type I, elastin, and other extracellular matrix proteins. This increased gene expression promotes collagen synthesis, improves fibroblast proliferation, and migration [23,37]. Such an increase can accelerate the process of epithelial regeneration. Previous studies have reported that marine collagen peptides exhibit a range of bioactivities, making them promising candidates for various health and biomedical applications [38,39].

4. Conclusions

This investigation demonstrates that the pretreatment of the Totoaba macdonaldi SB with hexane can yield equal amounts of collagen-derived peptides, thus avoiding the protein extraction step with NaOH. Additionally, no differences were observed in the electrophoretic patterns of the native collagen structure between the two pretreatment methods. The enzymatic hydrolysis process was conducted directly using bromelain. The hydrolysis was verified through the SDS-PAGE, Raman spectroscopy, and chromatographic profile. CH from both pretreatments exhibited significant bioactivity against the MCF-7 breast adenocarcinoma and Caco-2 colorectal adenocarcinoma cell line, with a concentration of 20 mg/mL.

On the other hand, peptides from both pretreatments stimulated the growth and proliferation of HDFs, thus showing their beneficial effect on normal human cells. Further research is needed to isolate and characterize the specific peptides responsible for such promising effects as well as to identify the molecular mechanisms involved.