Collagen Hydrolysate–Cranberry Mixture as a Functional Additive in Sausages

Abstract

Consumers increasingly seek clean-label meat products with improved nutrition and stability. We evaluated a collagen hydrolysate–cranberry mixture (CH-CR) as a functional additive in cooked sausages. Two formulations—control and CH-CR—were assessed for fatty acid profile; lipid and protein oxidation during storage; antioxidant capacity ferric-reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, and half-maximal inhibitory concentration (IC50); amino acid composition; and instrumental color. Relative to the control, CH-CR produced a more favorable lipid profile: lower saturated fatty acids (SFAs) 23.9% vs. 28.0%, higher monounsaturated fatty acids (MUFAs) 53.2% vs. 49.3%, slightly higher polyunsaturated fatty acids (PUFAs) 23.3% vs. 22.7%, a higher PUFA/SFA ratio of 0.97 vs. 0.81, and a lower omega-6/omega-3 (n-6/n-3) ratio of 13.5 vs. 27.1, driven by higher alpha-linolenic acid (ALA) 1.6% vs. 0.8%, with trans fats <0.1%. Storage studies showed attenuated oxidation in CH-CR: lower peroxide value (PV) at day 10 8.1 ± 0.4 vs. 9.8 ± 0.5 meq/kg and lower thiobarbituric acid-reactive substances (TBARS) at day 6 0.042 ± 0.004 vs. 0.055 ± 0.006 mg MDA/kg and day 10 0.156 ± 0.016 vs. 0.590 ± 0.041 mg MDA/kg); the acid value at day 10 was similar. Antioxidant capacity increased with CH-CR FRAP 30.5 mg gallic acid equivalents (GAE)/g vs. not detected; DPPH inhibition was 29.88% vs. 10.23%; IC50 56.22 vs. 149.51 µg/mL. The amino acid profile reflected collagen incorporation—higher glycine+proline+hydroxyproline 2.37 vs. 1.38 g/100 g and a modest rise in indispensable amino acids (IAAs) 5.72 vs. 5.42 g/100 g, increasing the IAA/total amino acid (TAA) ratio to 0.411 vs. 0.380. CH-CR samples were lighter and retained redness better under light, with comparable overall color stability. Overall, CH-CR is a natural strategy to improve fatty acid quality and oxidative/color stability in sausages.

1. Introduction

The growing consumer demand for functional and health-promoting foods has stimulated the search for novel natural ingredients that can improve both the nutritional and technological quality of meat products. Processed meat, particularly sausages, represents one of the most widely consumed food categories worldwide; yet its high fat content, susceptibility to lipid oxidation, and relatively low antioxidant capacity raise significant health and quality concerns [1,2,3]. Lipid oxidation not only reduces sensory acceptance due to rancidity and color deterioration but is also associated with the formation of potentially harmful compounds [4,5]. Therefore, there is an urgent need for strategies that enhance the oxidative stability and nutritional profile of sausages while maintaining consumer acceptability. Collagen hydrolysates have attracted attention as multifunctional ingredients due to their bioactive peptides, which exhibit antioxidant, antihypertensive, and anti-inflammatory properties [6,7,8]. These hydrolysates also improve protein digestibility and can modify the amino acid composition of meat products [9]. Several studies have demonstrated the use of protein hydrolysates from poultry, fish, and bovine sources as functional additives in meat systems, contributing to both texture and bioactivity [10,11,12]. However, the incorporation of collagen hydrolysates alone often requires complementary sources of natural antioxidants to maximize protection against lipid peroxidation. Cranberries (Vaccinium macrocarpon) are well recognized for their high content of phenolic compounds, flavonoids, and proanthocyanidins, which are powerful free-radical scavengers [13,14,15]. Their extracts have been successfully applied in diverse food systems, including beverage, bakery, and dairy products, demonstrating strong antioxidant and antimicrobial potential [16,17,18]. In meat products, cranberry phenolics have been reported to delay lipid oxidation, improve color stability, and contribute to an extended shelf life [19,20,21]. Nevertheless, there are diverging opinions regarding the impact of berry extracts on sensory acceptance due to their intense flavor and color, which may limit application levels [22,23]. The combination of collagen hydrolysate and cranberry is of particular interest because it unites the bioactive peptides from animal proteins with the phenolic antioxidants from plants, potentially leading to synergistic effects. Previous research has suggested that protein–polyphenol interactions may enhance radical scavenging activity, stabilize color pigments, and improve amino acid retention [24,25,26]. However, studies specifically investigating such combinations in sausages remain scarce, and the results are not always consistent across different experimental designs [27,28]. For instance, while some authors highlight improved oxidative stability and sensory acceptance, others report potential bitterness or changes in texture [29,30]. Here, we advance the field by combining collagen hydrolysate with cranberry polyphenols and positioning their interaction as a functional synergy rather than an additive sum. Collagen-derived peptides can donate electrons and chelate pro-oxidant metals, dampening radical propagation in both lipid and protein phases, while cranberry proanthocyanidins provide complementary radical-scavenging and metal-binding capacity. Beyond parallel actions, emerging evidence indicates protein–polyphenol interactions that can shield oxidation-sensitive residues, modulate interfacial properties in meat emulsions, and stabilize the heme pigment mechanisms that are expected to yield dual protection of lipids and proteins and improved color resilience. We therefore hypothesized that a collagen hydrolysate–cranberry (CH-CR) system would outperform either component alone by attenuating primary/secondary lipid oxidation and endpoint protein carbonylation, while maintaining desirable color and amino acid quality.

2. Materials and Methods

2.1. Materials

The sausages were produced using standard technological methods, including grinding the meat, mixing with additives, stuffing into casings, and subsequent thermal treatment (Figure 1). Poultry meat and premium-grade beef served as the main protein sources. Two formulations were developed: a control sample without collagen hydrolysate and cranberry powder, and an experimental sample enriched with these ingredients. In the control formulation, the mixture consisted of poultry meat 64%, beef 30%, hen egg 3%, and starch 3%, with no additional functional additives. In the experimental formulation, part of the poultry meat was replaced by 10% collagen hydrolysate and 3% cranberry powder (w/w, raw batter), yielding the following composition (%, w/w): poultry 51, beef 30, collagen hydrolysate 10, cranberry powder 3, hen egg 3, and starch 3; total = 100. Beef, egg, and starch levels were kept constant vs. the control; the 13% addition replaced poultry meat only.

The collagen hydrolysate used in the study was produced from bovine (Bos taurus) feet, including fetlock joint connective tissues (skin, tendons, and periarticular connective tissue) by enzymatic hydrolysis, followed by spray drying to obtain a fine powder (particle size ≤ 150 µm). Cranberry (Vaccinium macrocarpon) powder was prepared from freeze-dried berries ground to a homogeneous consistency with particle sizes below 150 µm. The chosen amounts (10% collagen hydrolysate and 3% cranberry powder) were based on preliminary trials that balanced biofunctional effects and sensory acceptability.

Thermal processing was carried out until the internal temperature of the sausages reached 72 °C. All samples were produced at the experimental facility of Almaty Technological University under standardized laboratory conditions to ensure reproducibility.

2.1.1. Storage Conditions for Oxidative Stability Testing

After thermal processing at a core of temperature 72 °C, the sausages were cooled to a room temperature of 20 ± 2 °C within 1 h, then vacuum-sealed in multilayer polyamide/polyethylene (PA/PE) pouches (oxygen permeability <40 cm³/m²·24 h·atm at 23 °C, 0% RH). Packaged samples were stored at 4 ± 1 °C in the dark, under a controlled chamber humidity of 65–70%, to simulate typical refrigerated shelf life conditions. Subsamples were taken and analyzed at days 0, 6, and 10. During storage, samples remained unopened until testing.

2.1.2. Collagen Hydrolysate and Cranberry Powder

Food-grade collagen hydrolysate was produced from bovine feet, including fetlock joint tissues (Bos taurus), which were purchased from the specialized supplier Agrofirma Kainar (TOO “AF Kainar”, Almaty, Kazakhstan). The raw material (skin, tendon, and periarticular connective tissue) was washed, trimmed, defatted, and demineralized in a mild acid solution, then neutralized to pH ~7.0 prior to enzymatic hydrolysis. The pretreated collagen 8% w/w solids were hydrated for 1 h at 50 °C, pH adjusted to 8.0 ± 0.1 with 2 M NaOH, and subjected to Alcalase 2.4 L (endoprotease; subtilisin) at 1.5% w/w per protein for 90 min at 55 ± 1 °C under pH-stat control. The slurry was then adjusted to pH 7.0 and treated with Flavourzyme 500 L (exopeptidase blend) at 1.0% w/w per protein for an additional 60 min at 50 ± 1 °C. Enzymes were inactivated at 90 °C for 10 min. Insoluble residues were removed by 5 µm filtration followed by 0.2 µm, and the clarified hydrolysate 20% solids were spray-dried at inlet 180 ± 5 °C, outlet 85 ± 5 °C, and sieved to ≤150 µm. The resulting hydrolysate had a degree of hydrolysis of 15–18% (OPA assay), moisture content < 5%, and ash < 5%.

Cranberry (Vaccinium macrocarpon) berries were also obtained from Agrofirma Kainar (TOO “AF Kainar”, Almaty, Kazakhstan). Fresh berries were washed, sorted, and frozen at −40 °C before freeze-drying in a laboratory lyophilizer −50 °C condenser, 0.04 mbar for 48 h, yielding a residual moisture < 5%. The dried berries were ground with a hammer mill, sieved to ≤150 µm, and stored in airtight containers at 4 °C, protected from light and humidity, until use in formulations.

2.2. Determination of Fatty Acid Composition

Total lipids were extracted by the Folch method using chloroform/methanol 2:1, v/v [31]. Lipids were trans-esterified to FAMEs with 14% boron trifluoride–methanol following the classical procedure [32]. FAMEs were analyzed by GC-FID on a polyethylene glycol capillary column HP-Innowax, 60 m × 0.32 mm × 0.5 µm under nitrogen, in line with ISO 12966 guidance for FAME preparation and capillary GC determination [33,34]. Fatty acids were identified against a 37-component FAME standard (Supelco 47885U, Merck KGaA, Darmstadt, Germany) and quantified by internal standardization; the results are expressed as % of total fatty acids.

2.3. Determination of Color Characteristics

Instrumental color was measured with a CR-400 chroma meter (Konica Minolta, Tokyo, Japan) following Iftikhar et al. [35] with minor adaptations. Before acquisition, the device was standardized using the manufacturer’s white reference tile and zero set. Readings were taken on freshly cut cross-sections at ambient laboratory temperature under controlled lighting. The results are reported in the CIE Lab* space (L*—lightness; a*—red–green; b*—yellow–blue). For each sample, three readings were collected at distinct locations and averaged to obtain the final value.

2.4. Protein Profiling (SDS-PAGE)

Sausage proteins were profiled by one-dimensional SDS–PAGE using the Laemmli system with minor modifications. Briefly, ~100 mg of homogenized sample was extracted on ice in a 500 µL lysis buffer of 4.5 M urea, 1% Triton X-100, 1% ampholytes pH 3–10, 2.5% β-mercaptoethanol for 30 min and centrifuged at 14,000× g for 20 min at 4 °C. Supernatants were mixed 1:1 with 2× Laemmli sample buffer of 125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue, and 10% β-mercaptoethanol, heated at 95 °C for 5 min, and briefly cooled. Equal protein amounts of 20 µg per lane; protein by BCA, and a prestained MW marker of 10–250 kDa were loaded onto 4% stacking/12% resolving polyacrylamide gels and electrophoresed at 120 V through the stacking gel, then 160 V through the resolving gel until the dye front reached the bottom [36].

Gels were stained with Coomassie brilliant blue G-250 following Neuhoff et al. [37], with minor modifications: staining in 0.05% CBB G-250 dissolved in 10% acetic acid/25% isopropanol for 60 min, then destaining in 10% acetic acid to a clear background. Gels were rinsed in water and imaged on a Bio-5000 Plus scanner (Serva, Heidelberg, Germany) at 600 ppi, 24-bit RGB. Band densitometry (background subtraction, peak integration, normalization to total lane intensity) was performed in ImageJ v1.53t [38]. The results are reported as mean ± SD from three independent extracts per treatment.

2.5. Sample Preparation for Oxidative Stability and Antioxidant Assays

General handling. Sausage subsamples were vacuum-packed and held at 4 °C ≤ 24 h before analysis or frozen at −20 °C for ≤7 days. Thawing was at 4 °C overnight. All extractions were protected from light, following common precautions for oxidation assays in meats [3].

2.5.1. Lipid Extraction for PV and AV

Lipids were extracted Folch from minced sausage 5.00 g with chloroform/methanol 2:1, v/v; 25 mL using an Ultra-Turrax homogenizer at 13,500 rpm, for 60 s [31]. After adding 5 mL 0.88% KCl, the mixture was vortexed, rested 10 min on ice, and centrifuged at 4000× g, for 10 min, at 4 °C. The lower chloroform phase was collected and brought to volume (typically 50 mL) with chloroform. This lipid extract was used immediately for PV and AV.

2.5.2. TBARS

A 10% (w/v) homogenate was prepared by blending sausage 10.0 g with 7.5% (w/v) trichloroacetic acid (TCA) containing 0.1% EDTA and 0.1% propyl gallate (90 s, Ultra-Turrax, on ice) and brought to 50 mL with the same TCA solution. After centrifugation at 5000× g, for 10 min, at 4 °C and filtration (Whatman No.1), 5 mL filtrate was mixed with 5 mL 0.02 M TBA in 90% acetic acid, heated at 95 °C for 45 min, cooled, and absorbance read at 532 nm (A532 corrected by A600). TBARS were calculated as mg malondialdehyde (MDA) per kg sample using ε = 1.56 × 10⁵ M⁻¹ cm⁻¹.

2.5.3. Protein Carbonyls

Sausage 2.0 g was homogenized in 20 mL of 20 mM phosphate buffer pH 6.5 with 0.6 M NaCl (90 s, on ice) and centrifuged at 10,000× g, for 15 min, at 4 °C. Protein in the supernatant was quantified by BCA. For carbonyls, 1.0 mL aliquots were reacted with 1.0 mL 10 mM DNPH in 2 M HCl (blanks received 2 M HCl without DNPH) for 1 h in the dark. Proteins were precipitated with 10% TCA, pellets washed 3× with ethanol/ethyl acetate 1:1 to remove free DNPH/lipids, then dissolved in 6 M guanidine-HCl in 20 mM phosphate buffer pH 6.5 at 37 °C for 30 min. Absorbance was read at 370 nm; carbonyls were calculated using ε = 22,000 M⁻¹ cm⁻¹ and expressed as nmol carbonyls per mg protein. Protein carbonyls were measured once at day 10 of storage in independent triplicate samples and expressed as mean ± SD.

2.5.4. Antioxidant Extracts for FRAP (Ferricyanide Reducing Power) and DPPH

For phenolic/antioxidant extraction, minced sausage 5.00 g was mixed with 80% methanol (v/v; 25 mL, pre-chilled), homogenized at 13,500 rpm, for 60 s, sonicated for 15 min in an ice bath, and centrifuged at 6000× g, for 10 min, at 4 °C. The residue was re-extracted with another 25 mL of 80% MeOH; supernatants were pooled to a final volume 50 mL, kept at −20 °C protected from light, and used the same day. An aliquot was evaporated under N₂ to constant mass to determine dry extract yield. Working solutions and basis. A stock solution was prepared at 10 mg/mL (dry extract basis) in methanol and serially diluted for assays. Unless stated, antioxidant results are expressed per g fresh weight (fw) of sausage; DPPH concentrations refer to the dry extract basis.

FRAP (ferricyanide reducing power). Reaction mixture: 0.5 mL extract, 2.5 mL 0.1 M phosphate buffer pH 6.6, 2.5 mL 1% (w/v) potassium ferricyanide; incubate at 50 °C for 20 min. Add 2.5 mL 10% TCA, centrifuge briefly; mix 2.5 mL supernatant with 2.5 mL water and 0.5 mL 0.1% FeCl₃; stand for 30 min; read A700. Calibrate with gallic acid (0–200 mg/L); report as mg gallic acid equivalents (GAE) per g fw [39].

DPPH radical scavenging and IC₅₀ determination. A 0.1 mg/mL DPPH solution in methanol was mixed 1:1 (v/v) with extract working solutions spanning 12.5–400 µg/mL (dry extract basis). After 30 min in the dark at room temperature, absorbance at 517 nm was recorded against a methanol control. Radical scavenging was calculated as % inhibition = (1 − A_sample/A_control) × 100. For descriptive comparison, % inhibition at 200 µg/mL is reported. The IC₅₀ was defined as the extract concentration µg/mL required to achieve 50% inhibition and was estimated by non-linear least squares fitting of a four-parameter logistic model to % inhibition vs. concentration; n = 3 independent extracts. Results are expressed as mean ± SD [40].

2.6. Amino Acid Composition Analysis

Approximately 200 mg of homogenized sausage sample was dried to a constant weight at 40 °C, in a vacuum. For total amino acids, samples were hydrolyzed with 6 M HCl containing 0.1% phenol under nitrogen at 110 °C for 24 h in sealed glass tubes. After cooling, hydrolysates were filtered, evaporated under vacuum, and reconstituted in 0.2 M sodium acetate buffer pH 6.8. L-norleucine 1.0 mM was added before hydrolysis as the internal standard.

For cysteine and methionine, samples were oxidized with performic acid formic acid/hydrogen peroxide 9:1, at 0–4 °C, for 16 h, neutralized with hydrobromic acid, and subjected to the same hydrolysis protocol. Tryptophan was determined separately after alkaline hydrolysis with 4.2 M NaOH, at 110 °C, for 16 h, under a nitrogen atmosphere, and neutralization with HCl.

All hydrolysates were derivatized using an AccQ-Tag Ultra kit (Waters Corp., Milford, MA, USA) according to the manufacturer’s protocol reaction at 55 °C for 10 min. Amino acids were analyzed on an Agilent 1260 Infinity HPLC system (Agilent Technologies, Inc., Santa Clara, CA, USA) with an AccQ-Tag Ultra C18 column 2.1 × 100 mm, 1.7 µm. A binary gradient of AccQ-Tag buffer (A) and acetonitrile (B) was applied at 0.7 mL/min, column temperature 40 °C, and detection at 260 nm. Identification and quantification were performed using a standard amino acid mixture (Sigma A9906, Sigma-Aldrich, St. Louis, MO, USA) and hydroxyproline standard for calibration of 0.5–250 µM, six points.

Quality control included blanks, recovery tests at 80, 100, 120%, and inter-day QC samples. Recoveries ranged from 95 to 105%, with repeatability RSD ≤ 5%; n = 3 independent hydrolyses per sample. LOD/LOQ values were approximately 0.01/0.03 g/100 g product. Final results were expressed as g/100 g product and used to calculate ∑IAA, ∑TAA, IAA/TAA, and collagen-associated residues (Gly + Pro + Hyp).

2.7. Statistical Analyses

The effects of collagen hydrolysate–cranberry mixture addition and storage time on the measured parameters were evaluated using the general linear model (GLM) procedure in SAS software, version 9.4 (SAS Institute Inc., Cary, NC, USA). Tukey’s multiple comparison test was applied in the GLM analyses to assess significant differences between treatments and storage periods. All experiments were conducted in triplicate, and the results were expressed as mean ± standard deviation. Statistical significance was established at p < 0.05.

3. Results and Discussion

3.1. Determination of Fatty Acid Composition

Table 1. Fatty acid profile (%) of sausages with and without the collagen hydrolysate–cranberry mixture.

Name of the Indicator	Unit of Measurement	Sausage Without Collagen Hydrolysate and Cranberry	Sausage with Collagen Hydrolysate and Cranberry
SFA
Butyric	C4:0	<0.1	<0.1
Caproic (Hexanoic)	C6:0	<0.1	<0.1
Caprylic (Octanoic)	C8:0	<0.1	<0.1
Capric (Decanoic)	C10:0	<0.1	<0.1
Undecanoic	C11:0	<0.1	<0.1
Lauric	C12:0	<0.1	<0.1
Tridecanoic	C13:0	<0.1	<0.1
Myristic	C14:0	<0.1	<0.1
Pentadecanoic	C15:0	0.2 ± 0.4	<0.1
Palmitic	C16:0	20.2 ± 2.1	17.0 ± 1.8
Margaric (Heptadecanoic)	C17:0	0.3 ± 0.4	0.4 ± 0.4
Stearic	C18:0	6.0 ± 2.1	5.0 ± 1.6
Arachidic	C20:0	0.3 ± 0.4	0.4 ± 0.4
Heneicosanoic	C21:0	<0.1	<0.1
Behenic	C22:0	0.4 ± 0.4	0.4 ± 0.4
Tricosanoic	C23:0	0.6 ± 0.4	0.6 ± 0.4
Lignoceric	C24:0	<0.1	<0.1
ΣSFA		28.0	23.9
MUFA
Myristoleic	C14:1	<0.1	<0.1
cis-10-Pentadecenoic	C15:1	<0.1	<0.1
Palmitoleic	C16:1	3.4 ± 0.4	4.0 ± 0.3
Heptadecenoic	C17:1	<0.1	<0.1
Oleic	C18:1	45.1 ± 2.1	48.5 ± 1.9
Elaidic (trans)	trans-C18:1	0.3 ± 0.4	<0.1
Gondoic	C20:1	0.5 ± 0.4	0.6 ± 0.4
Erucic	C22:1	<0.1	<0.1
Nervonic	C24:1	<0.1	<0.1
ΣMUFA		49.3	53.2
PUFA n-3
α-Linolenic (ALA)	C18:3 n-3	0.8 ± 0.4	1.6 ± 0.3
Eicosapentaenoic (EPA)	C20:5 n-3	<0.1	<0.1
Eicosatrienoic	C20:3 n-3	<0.1	<0.1
Docosahexaenoic (DHA)	C22:6 n-3	<0.1	<0.1
Σn-3 PUFA		0.81	1.61
PUFA n-6
Linoleic	C18:2 n-6	21.2 ± 2.1	21.0 ± 2.0
Linoelaidic (trans)	trans-C18:2 n-6	0.2 ± 0.4	<0.1
Dihomo-γ-linolenic (DGLA)	C20:3 n-6	<0.1	<0.1
Arachidonic	C20:4 n-6	0.5 ± 0.4	0.6 ± 0.4
Eicosadienoic	C20:2 n-6	<0.1	<0.1
Docosadienoic	C22:2 n-6	<0.1	<0.1
Σn-6 PUFA		21.89	21.69
ΣPUFA (n-3 + n-6)		22.70	23.30
Ratios
PUFA/SFA		0.81	0.97
n-6/n-3		27.1	13.5

summarizes the fatty acid profile (%) for the control formulation and the CH-CR formulation.

Compared with the control, the collagen hydrolysate–cranberry (CH-CR) formulation shifted the lipid profile toward a nutritionally and technologically more desirable configuration: lower ΣSFA 23.9% vs. 28.0%, higher ΣMUFA 53.2% vs. 49.3%, and a modest increase in ΣPUFA of 23.3% vs. 22.7%. These changes were mainly driven by a reduction in palmitic C16:0 and stearic C18:0 acids, an increase in oleic C18:1 and palmitoleic C16:1 acids, and a higher α-linolenic acid ALA, C18:3 n-3. The PUFA/SFA ratio improved from 0.81 (control) to 0.97 (CH-CR), and the n-6/n-3 ratio almost halved from 27.1 to 13.5, largely due to the ALA rise 0.8→1.6%. Such a pattern-less SFA, more MUFAs, and greater ω-3 have been repeatedly associated with improved healthfulness and favorable processing behavior in meat systems [1,2,3,4]. The reduction in C16:0 20.2→17.0% and C18:0 6.0→5.0% decreases the atherogenic SFA load, while the increase in C18:1 45.1→48.5% elevates MUFA, a class linked to beneficial plasma lipid responses and oxidative robustness relative to PUFA [1,2,5]. A slight rise in ΣPUFA—without increasing trans isomers—improves PUFA/SFA without incurring an undue oxidation penalty. This SFA↓/MUFA↑/PUFA profile is a common target in reformulation strategies for “healthier processed meats” [1,2,3,6]. The CH-CR sample showed higher ALA of 1.6% vs. 0.8%, reducing n-6/n-3 from 27.1 to ≈13.5. Lower n-6/n-3 ratios are broadly discussed as favorable for inflammatory and cardiometabolic endpoints, though the ideal range remains debated [2,3,4,7,22,41]. From a technological perspective, enriching ω-3 can raise oxidation susceptibility; however, the cranberry phenolics and collagen-derived peptides provide antioxidant buffering that helps preserve PUFA and stabilize color [3,4,10,28,42]. Berry phenolics can scavenge radicals and chelate pro-oxidant metals, curbing hydroperoxide formation and mitigating myoglobin oxidation, thus supporting color retention during storage [3,4,28,42]. Concurrently, protein–polyphenol interactions between collagen peptides and cranberry polyphenols may reduce the accessibility of oxidation-sensitive sites in both proteins and lipids and slightly alter interfacial properties in the emulsion matrix, reinforcing physical and chemical barriers to oxidation [13,14,15]. Together, these mechanisms help reconcile ω-3 enrichment with acceptable oxidative stability. The trans-MUFA elaidic and trans-PUFA linoelaidic fractions remained below detection <0.1% in the CH-CR treatment, aligning with clean-label and health-oriented positioning. Suppressing trans isomers while elevating MUFA and ω-3 is a desirable triad in current reformulation trends for processed meats [1,2,3,6,22]. Our results are in line with reports showing that (i) shifting lipid classes toward MUFA can provide a pragmatic compromise between nutrition and stability [1,2,6]; (ii) ω-3 enrichment is feasible in meat systems if accompanied by effective antioxidant strategies [3,4,28,42,43,44]; and (iii) collagen hydrolysates contribute bioactive peptides that may participate in radical quenching and metal ion binding [5,11,26,43,44]. The present data therefore support the dual functionality of the CH-CR mixture: nutritional modulation and technological resilience. For product development, the observed profile suggests that CH-CR can be used to (i) lower SFA and raise MUFA while (ii) modestly enhancing ω-3, keeping trans very low, and (iii) sustaining oxidative and color stability through inherent antioxidant mechanisms. Fine-tuning the dose, fat source, and process conditions can further optimize the health–quality balance [1,2,3,4,6,28,41,42].

3.2. Determination of Oxidative Stability During Storage

Table 2. Dynamics of lipid oxidation (PV) during storage and endpoint protein oxidation (protein carbonyls) in cooked sausages.

Indicator	Storage Time, Days	Sausage Without Collagen Hydrolysate and Cranberry	Sausage with Collagen Hydrolysate and Cranberry	p-Value, Treatment Within Storage Time
Peroxide value, PV (meq O2/kg fat)	0	4.1 ± 0.4	4.5 ± 0.5	0.343
	6	4.5 ± 0.5	5.0 ± 0.5	0.288
	10	9.8 ± 0.5	8.1 ± 0.4	0.011
Carbonyl compounds, nmol/mg of protein	10	106.13 ± 5.21	98.80 ± 4.87

Data are expressed as mean ± SD (n = 3 independent samples). PV was assessed at days 0, 6, and 10; p-values indicate treatment effects within each day (GLM, Tukey-adjusted). Protein carbonyls were measured once at day 10 as the endpoint marker of protein oxidation.

,

Table 3. Fat oxidation dynamics, with accumulation of thiobarbituric number in sausages during storage.

Thiobarbituric Number, Storage Time, Days	Sausage Without Collagen Hydrolysate and Cranberry	Sausage with Collagen Hydrolysate and Cranberry	p-Value, Treatment Within Storage Time
0	Below 0.039	Below 0.039
6	0.055 ± 0.006	0.042 ± 0.004	0.043
10	0.590 ± 0.041	0.156 ± 0.016	<0.001

and

Table 4. Fat oxidation dynamics, with accumulation of acid number in sausages during storage.

Acid Value, Storage Time, Days	Sausage Without Collagen Hydrolysate and Cranberry, mg KOH/g	Sausage with Collagen Hydrolysate and Cranberry, mg KOH/g	p-Value, Treatment Within Storage Time
0	2.5 ± 0.2	3.4 ± 0.2	0.005
6	3.2 ± 0.2	4.0 ± 0.3	0.024
10	4.4 ± 0.3	4.9 ± 0.3	0.111

present primary lipid oxidation (peroxide value, PV), secondary lipid oxidation (TBARS), hydrolytic fat degradation (acid value, AV), and endpoint protein oxidation (protein carbonyls) in boiled sausages at 0, 6, and 10 days of storage, comparing the control formulation with the collagen hydrolysate–cranberry treatment.

Across storage, the collagen hydrolysate–cranberry (CH-CR) formulation consistently mitigated lipid oxidation relative to the control. At day 10, the peroxide value was lower in CH-CR, 8.1 ± 0.4 vs. 9.8 ± 0.5 meq/kg; p = 0.011, indicating fewer primary peroxides accumulated (Table 2). Concomitantly, TBARS-a proxy for secondary carbonyls such as malondialdehyde were significantly reduced at both day 6, 0.042 ± 0.004 vs. 0.055 ± 0.006; p = 0.043, and day 10, 0.156 ± 0.016 vs. 0.590 ± 0.041; p < 0.001, corresponding to 24% and 74% decreases, respectively (Table 3). Although the acid value rose with time in both groups, between-treatment differences at day 10 were not significant, 4.9 ± 0.3 vs. 4.4 ± 0.3 mg KOH/g; p = 0.111, suggesting comparable endpoint hydrolysis under the applied conditions (Table 4). Protein oxidation—estimated by protein carbonyls—was numerically lower in CH-CR at the end of storage, 98.80 vs. 106.13 nmol/mg protein, in line with the lipid phase trends, though formal inference is limited without dispersion metrics (Table 2). The classical trajectory of lipid oxidation in cooked meat involves early accumulation of hydroperoxides, followed by their decomposition into aldehydes/ketones captured by TBARS and related assays [3,4,28,45]. In our data, the CH-CR treatment suppressed both the peak magnitude of primary oxidation and, more strikingly, the propagation/decomposition stage. Such dual-phase attenuation is consistent with an antioxidant system that both scavenges initiating radicals and retards hydroperoxide breakdown [45,46,47]. The outcome is technologically meaningful: lower TBARS is often associated with improved flavor stability and reduced rancid notes during chilled storage [3,48]. Cranberry supplies proanthocyanidins, flavanols, and phenolic acids capable of radical scavenging, metal chelation, and chain-breaking inhibition mechanisms repeatedly shown to lower PV/TBARS in meat systems [49,50,51]. In parallel, bioactive peptides present in collagen hydrolysate can contribute electron-donating and metal-binding activities, complementing phenolics and attenuating both lipid and protein oxidation pathways [5,52,53]. Moreover, protein–polyphenol interactions can shield reactive sites in proteins and at lipid–water interfaces, dampening cross-talk between lipid peroxyl radicals and amino acid side chains that lead to carbonyl formation [54,55]. Collectively, these effects explain the coherent reductions observed across PV, TBARS, and carbonyls. The slightly higher AV in CH-CR at early time points (significant at day 0 and 6) likely reflects matrix/assay effects rather than accelerated lipolysis: the titrimetric AV captures all titratable acids; so cranberry organic acids can elevate AV independently of free fatty acids. By day 10, the AV difference lost significance, while oxidative indices remained clearly improved—supporting the view that CH-CR primarily modulates oxidation, not hydrolytic rancidity. Similar dissociations between AV and TBARS have been reported when plant acids or phenolics are co-delivered in meat emulsions [28,50]. Here, we advance the field by combining collagen hydrolysate with cranberry polyphenols and positioning their interaction as a functional synergy rather than an additive sum. Collagen-derived peptides can donate electrons and chelate pro-oxidant metals, dampening radical propagation in both lipid and protein phases, while cranberry proanthocyanidins provide complementary radical-scavenging and metal-binding capacity. Beyond parallel actions, emerging evidence indicates that protein–polyphenol interactions that can shield oxidation-sensitive residues, modulate interfacial properties in meat emulsions, and stabilize heme pigment—mechanisms expected to yield dual protection of lipids and proteins and improved color resilience [24,25,26,43,44]. We therefore hypothesized that a collagen hydrolysate–cranberry (CH-CR) system would outperform either component alone by attenuating primary/secondary lipid oxidation and endpoint protein carbonylation, while maintaining desirable color and amino acid quality. For cooked sausages, the CH-CR mixture yielded lower primary and secondary lipid oxidation and a tendency toward lower protein carbonylation, without exacerbating final hydrolytic breakdown. These outcomes align with industry goals of clean-label antioxidant strategies that enhance shelf life and sensory stability while avoiding synthetic additives [49,50]. Optimization of dose, grind/particle size, and thermal profile may further leverage these benefits. The coherent reductions in PV and TBARS, together with lower endpoint protein carbonyls, are consistent with a synergistic peptide–polyphenol mechanism. Collagen peptides likely contribute electron donation and transition metal chelation, curbing initiation and propagation steps; cranberry phenolics furnish potent chain-breaking antioxidant activity. At the same time, non-covalent and covalent protein–polyphenol interactions may partially shield reactive amino acid side chains, reduce lipid–protein cross-talk, and stabilize myoglobin, explaining the concurrent mitigation of lipid and protein oxidation and the improved redness retention we observed. This synergy provides a mechanistic basis for the CH-CR mixture’s dual oxidative protection, distinguishing it from prior studies that evaluated collagen or berry extracts in isolation.

3.3. Determination of Amino Acid Composition

Table 5. Amino acid composition (g/100 g product).

Name of the Indicator	Sausage Without Collagen Hydrolysate and Cranberry, g/100 g Product	Sausage with Collagen Hydrolysate and Cranberry, g/100 g Product
Aspartic acid	1.31 ± 0.20	0.87 ± 0.13
Glutamic acid	2.31 ± 0.35	1.04 ± 0.16
Serine	0.53 ± 0.08	0.56 ± 0.08
Threonine	0.37 ± 0.06	0.47 ± 0.07
Glycine	0.78 ± 0.12	0.87 ± 0.13
Arginine	1.37 ± 0.21	1.37 ± 0.21
Alanine	1.45 ± 0.22	1.39 ± 0.21
Tyrosine	0.34 ± 0.05	0.41 ± 0.06
Cystine	0.14 ± 0.02	0.18 ± 0.03
Valine	0.73 ± 0.11	0.73 ± 0.11
Methionine	0.53 ± 0.08	0.28 ± 0.05
Isoleucine	0.58 ± 0.09	0.57 ± 0.09
Phenylalanine	0.48 ± 0.07	0.49 ± 0.07
Leucine	0.98 ± 0.15	1.37 ± 0.21
Proline	0.43 ± 0.06	0.89 ± 0.13
Lysine	0.86 ± 0.13	1.02 ± 0.15
Histidine	0.72 ± 0.11	0.61 ± 0.09
Tryptophan	0.17 ± 0.03	0.18 ± 0.03
Hydroxyproline	0.17 ± 0.02	0.61 ± 0.05

Values are expressed as g/100 g product and reported as mean ± SD (n = 3). Amino acids were quantified by HPLC after acid hydrolysis (6 M HCl, 110 °C, 24 h), with performic acid oxidation for Cys/Met and alkaline hydrolysis for Trp.

presents the amino acid composition (g/100 g product) of control sausages and sausages with the collagen hydrolysate–cranberry mixture, enabling comparison of essential amino acid supply and the collagen-associated signature relative to the control. The values shown in Table 5 were obtained by HPLC analysis after acid hydrolysis (6 M HCl, 110 °C, 24 h), performic acid oxidation for Cys/Met, and alkaline hydrolysis for Trp; IAA/TAA ratios were calculated on this basis.

The collagen hydrolysate–cranberry formulation remodeled the amino acid profile toward a collagen-associated signature while modestly improving indispensable amino acid (IAA) density. Summed IAA increased from 5.42 to 5.72 g/100 g of product, and the IAA/total amino acid (TAA) ratio rose from 0.380 to 0.411 despite slightly lower totals overall (14.25 vs. 13.91 g/100 g). Hallmark collagen residues increased, with glycine plus proline plus hydroxyproline rising from 1.38 to 2.37 g/100 g, driven by hydroxyproline from 0.17 to 0.61 g/100 g and proline from 0.43 to 0.89 g/100 g, consistent with incorporation of collagen peptides [56,57,58]. The branched-chain amino acids (BCAA; Leu + Ile + Val) sum increased from 2.29 to 2.67 g/100 g, with leucine rising from 0.98 to 1.37 g/100 g, supporting muscle protein synthesis relevance for older adults; lysine increased from 0.86 to 1.02 g/100 g, potentially improving the indispensable amino acid balance [59,60,61,62,63,64,65]. A trade-off was the decline in sulfur amino acids, with methionine plus cystine decreasing from 0.67 to 0.46 g/100 g, a known characteristic of collagen; phenylalanine and tyrosine were essentially maintained, indicating preservation of the aromatic amino acid supply at the current replacement level, and any sulfur amino acid deficit could be mitigated by blending with sulfur-rich proteins such as egg, dairy, or soy isolates [56,57,66,67]. Aspartic and glutamic acids decreased from 1.31 to 0.87 and from 2.31 to 1.04 g/100 g, respectively, in line with partial replacement of myofibrillar proteins; arginine remained at 1.37 g/100 g; threonine increased from 0.37 to 0.47 g/100 g; tryptophan was essentially unchanged at ~0.17–0.18 g/100 g. Overall, the data indicate that moderate replacement with the collagen hydrolysate–cranberry mixture enhances IAA density and leucine supply while conferring collagen-specific functionalities, with lower methionine best addressed through complementary protein blending.

3.4. Determination of Color Characteristics and Light Stability

Table 6. The dynamics of fat oxidation, with the accumulation of acid number in sausages during storage.

Samples	Color Characteristics Before Exposure to Light			Color Characteristics After Exposure to Light			Color Stability, %
	L-lightness	a-Redness	b-Yellowness	L-Lightness	a-Redness	b-Yellowness
Sausage without collagen hydrolysate and cranberry	61.14 ± 0.50	17.82 ± 0.15	13.70 ± 0.24	60.04 ± 0.42	16.46 ± 0.45	14.80 ± 0.61	94.20 ± 1.50
Sausage with collagen hydrolysate and cranberry	63.91 ± 0.97	13.82 ± 0.24	12.27 ± 0.17	62.62 ± 0.61	13.75 ± 0.76	13.98 ± 0.39	93.58 ± 1.76

presents instrumental color parameters (CIE L*, a*, b*) measured before and after light exposure, along with color stability (% retention), comparing control sausages and sausages formulated with the collagen hydrolysate–cranberry mixture.

Under light exposure, the CH-CR formulation produced a lighter product (L*) both before and after exposure of 63.91 vs. 61.14 and 62.62 vs. 60.04, respectively, while showing lower absolute redness (a*) at baseline of 13.82 vs. 17.82 and after exposure of 13.75 vs. 16.46, consistent with myoglobin dilution due to partial replacement of myofibrillar proteins by collagen [68,69]. Importantly, redness retention under light was superior with CH-CR: a* declined by only about 0.5% from 13.82 to 13.75; about 99.5% retention compared with about 7.6% in the control from 17.82 to 16.46; about 92.4% retention, indicating reduced metmyoglobin formation and better chromophore protection [3]. The b* coordinate increased during exposure in both groups, reflecting typical oxidative/yellowing trajectories; the increase was 1.10 units in the control from 13.70 to 14.80, about 8.0% and 1.71 units in CH-CR from 12.27 to 13.98, about 13.9%. This slightly larger b* rise with CH-CR may derive from phenolic–protein interactions and light-driven quinone chemistry that can shift the hue toward yellow-brown, even as phenolics slow heme oxidation and stabilize a* [3,51,70]. Despite these hue shifts, the overall color stability index was comparable at 94.20% in the control and 93.58% in CH-CR, suggesting that CH-CR maintains color integrity under light while trading some initial redness for improved redness resilience during display. Taken together, the data support a mechanism in which cranberry phenolics and peptide–polyphenol associations mitigate pigment oxidation, while collagen addition explains the lower starting a* via reduced myoglobin concentration [71].

3.5. Determination of Antioxidant Capacity

Table 7. Ferric-reducing antioxidant power (FRAP) and antioxidant activity (DPPH).

Indicator	Results		p-Value
	Sausage Without Collagen Hydrolysate and Cranberry	Sausage with Collagen Hydrolysate and Cranberry
Ferric-reducing antioxidant power (FRAP), mg GAE/g	Not detected	30.5 ± 0.04	<0.0001
DPPH radical-scavenging activity, %	10.23 ± 0.004	29.88 ± 0.01	<0.0001
IC50 of DPPH radical-scavenging activity, µg/mL	149.51 ± 12.23	56.22 ± 4.02	0.002

presents ferric-reducing antioxidant power (FRAP, mg GAE/g), DPPH radical-scavenging activity (%), and DPPH IC₅₀ (µg/mL) for control sausages and sausages with the collagen hydrolysate–cranberry mixture, where higher FRAP and DPPH values and a lower IC₅₀ indicate greater antioxidant capacity.

The collagen hydrolysate–cranberry formulation markedly enhanced antioxidant capacity relative to the control across all assays. Ferric-reducing antioxidant power was not detected in the control but reached 30.5 mg GAE/g in CH-CR, indicating a substantial increase in the electron-donating/reducing ability of the extractable fraction, consistent with the presence of cranberry phenolics and antioxidant peptides from collagen hydrolysate that can reduce Fe(III) complexes in the ferricyanide-based assay system [39,72]. DPPH radical-scavenging activity increased from 10.23% (control) to 29.88% (CH-CR) at the test concentration, a 2.9-fold improvement, while the IC₅₀ for DPPH decreased from 149.51 ± 12.23 to 56.22 ± 4.02 µg/mL, denoting a substantially higher radical-quenching potency. These outcomes align with established antioxidant mechanisms of berry polyphenol direct radical scavenging, hydrogen atom transfer, single-electron transfer, and metal chelation—and with reported bioactivities of collagen-derived peptides that can donate electrons/protons and bind pro-oxidant metals [28]. Beyond chemical assays, the stronger reducing and radical-scavenging capacity supports the lower PV and TBARS observed in storage (reported elsewhere), as higher FRAP/DPPH capacity typically translates into slower initiation/propagation of lipid oxidation and improved pigment stability in meat matrices [3]. The next steps will (i) run sensory tests to establish acceptable use levels; (ii) perform microbiological shelf life and challenge studies under vacuum/MAP; (iii) optimize dose and CH:CR ratio (incl. different collagen degrees of hydrolysis); (iv) broaden endpoints (ABTS/ORAC, protein thiols/carbonyls, GC–MS volatiles, myoglobin redox) and assess techno-functional traits (texture, WHC, emulsion stability); and (v) evaluate bioaccessibility via in vitro digestion and validate performance under retail display light and scale-up conditions.

4. Conclusions

Adding the collagen hydrolysate–cranberry mixture improved both the nutrition and stability of cooked sausages. The lipid profile shifted to lower ΣSFA 23.9% vs. 28.0% and higher ΣMUFA 53.2% vs. 49.3% with a slight rise in ΣPUFA 23.3% vs. 22.7%; PUFA/SFA increased 0.97 vs. 0.81 and n-6/n-3 nearly halved to 13.5 vs. 27.1 due to higher ALA of 1.6% vs. 0.8%, while trans isomers stayed <0.1%. Oxidative markers improved across storage: PV day 10 8.1 ± 0.4 vs. 9.8 ± 0.5 meq/kg, TBARS day 6 0.042 ± 0.004 vs. 0.055 ± 0.006; and day 10 0.156 ± 0.016 vs. 0.590 ± 0.041; AV day 10 was similar at 4.9 ± 0.3 vs. 4.4 ± 0.3 mg KOH/g. Antioxidant capacity rose sharply to FRAP 30.5 mg GAE/g vs. not detected; DPPH 29.88% vs. 10.23%; IC₅₀ 56.22 vs. 149.51 µg/mL. The amino acid profile gained a collagen signature Gly + Pro + Hyp 2.37 vs. 1.38 g/100 g and higher indispensable AA density of IAA 5.72 vs. 5.42 g/100 g; IAA/TAA 0.411 vs. 0.380, with increases in leucine 1.37 vs. 0.98 and lysine 1.02 vs. 0.86 g/100 g. Under light, the CH-CR samples had a lighter L of 63.91 vs. 61.14* and showed superior a* retention of 99.5% vs. 92.4% with comparable overall color stability of 93.58% vs. 94.20%. Collectively, CH-CR functions as a clean-label additive that improves fatty acid quality, boosts antioxidant protection, maintains color, and enriches key amino acids without increasing trans fats.