Investigating the Effect of Different Bovine Colostrum Concentrations Added to Ground Rabbit Patties on the Survival of Listeria monocytogenes and Meat Quality

Abstract

Bovine colostrum is naturally rich in antimicrobial and antioxidant compounds, making it a promising candidate for improving the safety and quality of fresh meat products. This study aimed to evaluate the effect of incorporating bovine colostrum at 1%, 3%, and 5% (w/w) into ground rabbit meat patties on the growth potential of Listeria monocytogenes and on meat quality during refrigerated storage at 4 ± 2 °C. Microbiological analyses revealed that bovine colostrum significantly reduced (p < 0.001) the growth potential of Listeria monocytogenes in a dose-dependent manner, with the 5% formulation showing the slowest growth rate (μ = 0.055 h⁻¹; doubling time = 12.5 h) compared with the control (μ = 0.063 h⁻¹; doubling time = 10.9 h). In parallel, physicochemical analyses demonstrated that patties containing bovine colostrum, particularly at 5%, had a lower peroxidability index (p < 0.05), reduced lipid oxidation (p < 0.001), and higher sulfhydryl group content (p < 0.001), indicating improved oxidative stability in fresh meat. These findings demonstrate that bovine colostrum, particularly at 5%, effectively inhibits microbial growth while preserving lipid and protein integrity. Overall, bovine colostrum shows strong potential as a natural antimicrobial and antioxidant ingredient in fresh meat, supporting its use in multi-hurdle preservation strategies to extend shelf life and improve consumer safety.

1. Introduction

Listeria monocytogenes is a psychrotrophic, Gram-positive foodborne pathogen of major concern in the European Union due to its high hospitalization and case-fatality rates. According to the most recent EFSA and ECDC reports, 2.770 confirmed human cases of listeriosis were recorded in the EU in 2022, with a hospitalization rate of approximately 82% and a mortality rate of about 9% [1,2]. Foods of animal origin, particularly Ready-to-Eat (RTE) products, are considered the main vehicles of transmission, as Listeria monocytogenes is able to survive and proliferate under refrigeration and tolerate a wide range of pH and water activity (a_w) conditions [3]. Consequently, the development of effective strategies to limit its growth in high-risk foods remains a critical priority for food safety [4].

In this context, functional natural ingredients have gained increasing attention as part of multi-hurdle preservation strategies. Plant extracts [5], essential oils [6], probiotics, and bioactive dairy fractions [7] have been widely studied for their antibacterial, antioxidant, and nutraceutical properties [8,9]. These natural compounds may inhibit microbial growth through multiple mechanisms, including disruption of cell membranes [10], interference with metabolic pathways [10], and modulation of oxidative processes [10]. In particular, several studies have highlighted their effectiveness against foodborne pathogens such as Listeria monocytogenes, supporting their application as natural biopreservatives [11]. In addition, their antioxidant activity can help preserve product quality by limiting lipid and protein oxidation during storage [12].

Bovine colostrum (BC), the first secretion of the mammary gland after calving, is a rich source of immunoglobulins, lactoferrin, lysozyme, growth factors, and bioactive peptides with documented antimicrobial, anti-inflammatory, and antioxidant activities [13,14,15]. Although traditionally employed in neonatal nutrition to support immune development and growth [15], BC and its derivatives have more recently been investigated as functional food ingredients in dairy matrices and beverages. These applications highlight their potential to enhance both the safety and technological stability of perishable foods. However, their direct use in fresh meat or RTE products remains largely underexplored, representing a promising opportunity for future research.

Previous studies [16,17] investigated the effects of bovine colostrum dietary supplementation in rabbits, demonstrating improvements in meat shelf life and quality. However, no studies have yet evaluated the direct incorporation of bovine colostrum into meat products to assess its antimicrobial activity against foodborne pathogens. The present study therefore addresses this gap by testing its effectiveness in ground rabbit meat patties through a challenge test with Listeria monocytogenes while simultaneously monitoring oxidative stability and quality parameters during storage.

Challenge tests are widely recognized as the gold standard for evaluating the antibacterial potential of natural functional ingredients. By artificially inoculating a known concentration of a target pathogen into the food matrix, these tests allow the quantification of microbial growth or inactivation under defined storage conditions, thus providing realistic evidence of an ingredient’s efficacy [18].

This study aimed to evaluate the effect of bovine colostrum, incorporated at varying concentrations into homemade ground rabbit meat patties, on the growth potential of Listeria monocytogenes during storage at 4 ± 2 °C. The selected temperature and storage duration were designed to replicate typical household refrigeration conditions throughout the product’s shelf life. A multifactorial statistical approach was applied to evaluate both the individual and interactive effects of bovine colostrum addition and storage temperature, while kinetic modeling was employed to characterize microbial growth dynamics. Furthermore, physicochemical stability parameters were monitored to assess potential quality changes during storage.

2. Materials and Methods

2.1. Inoculum Preparation

Lyophilized L. monocytogenes strain ATCC 13932 was rehydrated in Brain Heart Infusion (BHI) broth and incubated at 37 °C for 24 h to obtain an active culture. To acclimate the bacterial cells to low-temperature conditions representative of household refrigeration, the pre-adapted culture was transferred into fresh BHI broth and incubated at 4 ± 2 °C for 24 h. The pre-adapted suspension was subsequently diluted in sterile physiological saline solution (0.85% NaCl) to achieve a target concentration of approximately 10⁵ CFU/mL. Enumeration of viable cells was performed using decimal serial dilutions in sterile saline, followed by surface plating on Oxoid™ Brilliance Listeria Agar (OCLA-ISO formulation).

The plates were incubated at 37 ± 1 °C for 24 to 48 ± 2 h to allow colony development and enumeration. To minimize changes in the physicochemical characteristics of the rabbit meat patties, the volume of the bacterial inoculum was maintained below 1% of the total sample weight.

2.2. Preparation of Rabbit Meat Patties

Fresh New Zealand rabbit carcasses, 48 h post-mortem, were transported under refrigerated conditions (4 ± 1 °C) to the Food Inspection Laboratory at the Department of Veterinary Medicine and Animal Science, University of Milan (Lodi, Italy). Upon arrival, hind legs and Longissimus dorsi muscles were excised and ground into a homogeneous meat paste using a commercial meat grinder. Immediately after mincing, the rabbit meat was analyzed to confirm the absence of Listeria monocytogenes prior to inoculation. Subsequently, the minced meat was allocated to four experimental formulations: a control (CTR) group consisting of untreated minced rabbit meat, and three treatment groups supplemented with bovine colostrum at 1% (w/w; BC1%), 3% (w/w; BC3%), and 5% (w/w; BC5%), corresponding to low, medium, and high concentrations, respectively. The bovine colostrum used in the challenge test was commercially purchased and intended for human consumption, and its chemical–nutritional composition is reported in Table S1. For each group, three independent batches were prepared by placing 150 g of meat into sterile stomacher bags. Each batch was subsequently inoculated with L. monocytogenes strain ATCC 13932. After homogenization for 60 s in stomacher, treated and control groups were stored at 4 ± 2 °C. L. monocytogenes count according to ISO 11290-2:2017 [19] and ISO 11290-2:2017/DAmd 1 [20], was carried out at 0, 1, 2, 6, 8, 24 and 48 h of storage. The experimental design is summarized in

Table 1. Experimental design.

Group	Batch	Timepoints (h)	Parameters
			L. monocytogenes	pH, aw, and Total Viable Count	Proximate Composition, FA 1 Profile and PI 2	Color, TBARS, and Sulfhydryl
CTR	B1 B2 B3	0	✔ 3	✔ 4	✔ 4	✔ 4
		1	✔
		2	✔
		6	✔
		8	✔			✔ 4
		24	✔			✔ 4
		48	✔	✔ 4		✔ 4
		96				✔ 4
BC1%	B1 B2 B3	0	✔ 3	✔ 4	✔ 4	✔ 4
		1	✔
		2	✔
		6	✔
		8	✔			✔ 4
		24	✔			✔ 4
		48	✔	✔ 4		✔ 4
		96				✔ 4
BC3%	B1 B2 B3	0	✔ 3	✔ 4	✔ 4	✔ 4
		1	✔
		2	✔
		6	✔
		8	✔			✔ 4
		24	✔			✔ 4
		48	✔	✔ 4		✔ 4
		96				✔ 4
BC5%	B1 B2 B3	0	✔ 3	✔ 4	✔ 4	✔ 4
		1	✔
		2	✔
		6	✔
		8	✔			✔ 4
		24	✔			✔ 4
		48	✔	✔ 4		✔ 4
		96				✔ 4

¹ FA = fatty acids; ² PI = peroxidability index; ³ immediately after L. monocytogenes inoculation; ⁴ non-inoculated batches; ✔= analyses performed at the corresponding sampling time.

. An equivalent number of non-inoculated batches were analyzed at 0 and 48 h, corresponding to the beginning and end of the challenge test, to assess the total viable count (TVC) ISO 4833-1:2013/Amd 1:2022 [21], pH (pH meter HI 99163; Hanna Instruments, Villafranca Padovana, Italy), and a_w (Rotronic probe type HC2-AW-USB; Rotronic AG, Bassersdorf, Switzerland).

2.3. Microbiological Analysis

At each sampling timepoint, 10 g of minced rabbit meat from each experimental group was aseptically collected and blended with 90 mL of sterile buffered peptone water. The mixture was homogenized for 60 s at room temperature using a stomacher homogenizer.

Serial ten-fold dilutions were then prepared in sterile buffered peptone water, and 1 mL of each dilution was surface plated in duplicate onto 90 mm OCLA plates. Following incubation at 37 ± 1 °C for 48 ± 2 h, colonies were enumerated, and the results were expressed as Log CFU/g of minced rabbit meat.

2.4. Physicochemical Parameters

Physicochemical parameters were evaluated exclusively on non-inoculated samples at five timepoints: 0, 8, 24, 48, and 96 h of refrigeration storage. The proximate analysis and fatty acid profile were determined only at the initial timepoint (0 h). All measurements were carried out in triplicate.

2.4.1. Color Evaluation

The chrome index (C*; color saturation or intensity) and hue angle (H°) values were derived from the a* and b* coordinates according to Equations (1) and (2). In addition to complementing the L*, a*, and b* values, the C* and H° were calculated to provide a more comprehensive description of the color of rabbit meat; in particular, the hue angle is considered a sensitive indicator of pigment oxidation, reflecting the shift from oxymyoglobin to metmyoglobin.

Color measurements were carried out by exposing minced meat to air for 30 min to allow the blooming effect to develop. The color parameters were then recorded using the CIEL* a* b* system, assessing lightness (L*), redness (a*), and yellowness (b*) using a Konica Minolta CM-3600 D spectrophotometer (Sensing, Inc., Osaka, Japan). Measurements were performed under D illuminant conditions (6504 K, daylight) chosen because this illuminant represents a standardized reference light source in colorimetry, ensuring comparability of results with previous studies and alignment with internationally accepted protocols for meat quality evaluation. The instrument used employs a diffuse/8° geometry. This setting is particularly appropriate for fresh meat surfaces, as it minimizes the effect of gloss and surface irregularities, thereby improving reproducibility.

The chrome index (C*; color saturation or intensity) and hue angle (H°) values were derived from the a* and b* coordinates according to Equations (1) and (2):

$$Chrome\left(C^{*}\right)=\sqrt{a^2}+b^2$$

(1)

$$Hue(H°)={tang}^{-1}{b}^{*}/a^{*}$$

(2)

The difference in color between groups (ΔE) was calculated as follows in the Equation (3):

ΔE− = √(ΔE)² + (Δa*)² + (Δb*)²

(3)

where Δ represents the difference in the color parameter between the different times for each group. Differences in perceivable color can be analytically classified as very distinct (ΔE > 3), distinct (1.5 < ΔE ≤ 3), and small (ΔE ≤ 1.5) as reported in Pathare et al. [22]. Data were reported in Table S2.

2.4.2. Thiobarbituric Acid Reactive Substance Assay (TBARS) and Sulfhydryl Group Analysis

Lipid oxidation was assessed using the TBARS assay. Meat samples (2.5 g) were homogenized in water containing 2.8% ethanolic BHT. An aliquot (1 mL) of the homogenate was mixed with 1 mL of trichloroacetic acid (TCA) and then centrifuged, and the resulting supernatant was incubated with 0.28% TBA at 80 °C for 30 min. After cooling, 10 μL was analyzed by HPLC (Alliance 2695, Waters, Milford, MA, USA) using a C18 reverse-phase column (Kinetex 5 μm EVO, Phenomenex, Torrance, CA, USA). The results were expressed as mg MDA/kg meat.

Protein oxidation was assessed by measuring alterations in sulfhydryl compounds (–SH) also referred to as thiol groups, in rabbit minced meat samples. Briefly, for the determination of thiol groups, 1 g of meat was homogenized in 0.15 M potassium chloride solution. After a 1:50 dilution, two aliquots were prepared: one was mixed with 8 M urea in 100 mM phosphate buffer and 10 mM 2,2’-dithiobis(5-nitropyridine) (DTNP), while the other served as a blank and was mixed only with urea. The samples were incubated for one hour in the dark, and absorbance was recorded at 386 nm using a Lambda 25 spectrophotometer (PerkinElmer, Shelton, CT, USA). Thiol concentrations were expressed as nmol SH per mg of protein.

2.4.3. Proximate Composition, Fatty Acid Analysis, and Determination of Peroxidability Index

The proximate composition was determined following the official protocols outlined by the AOAC, specifically: method 934.01 for dry matter (calculating moisture as 100% dry matter), method 942.05 for ash content, and method 984.13 for crude protein (CP) quantification.

Lipids from the meat samples were extracted in duplicate following the Folch et al. [23] method. Subsequently, the extracted lipids were methylated according to the IUPAC protocol [24]. Quantification of fatty acid methyl esters (FAMEs) was carried out using a gas chromatograph (Agilent Technologies, Santa Clara, CA, USA) equipped with a flame ionization detector (FID). A CP-Sil88 fused silica capillary column (100 m length, 0.25 mm internal diameter, 0.2 µm film thickness; Agilent Technologies) was employed for the analysis. The procedures for fatty acid extraction, methylation, and chromatographic conditions were conducted as described by Failla et al. [25]. An internal standard (C19:0, nonadecanoate methyl ester) was added prior to extraction to assess recovery efficiency. Identification of FAMEs was achieved by comparing retention times with those of known standards, including Supelco Mix 37, CLA mix, 22:4-n-6 (docosatetraenoic acid methyl ester DTA), 22:5-n-3 (docosapentaenoic acid, DPA), and a branched-chain fatty acid mixture of BR2 and BR4. The FAME results and the different fatty acid classes—saturated (SFA), monounsaturated (MUFA), and polyunsaturated (PUFA)—were expressed as percentages of the total FAME content. Only the main classes of fatty acids, calculated as the sum of individual acidic components, were presented. In addition, the peroxidability index (PI) was determined following the method of Arakawa and Sagai [26], as shown in Equation (4).

$$\begin{array}{c}\mathit{P}\mathit{I}=\left(0.025 \times \mathit{\Sigma }\mathit{m}\mathit{o}\mathit{n}\mathit{o}\mathit{e}\mathit{n}\mathit{o}\mathit{i}\mathit{c} \mathit{a}\mathit{c}\mathit{i}\mathit{d}\mathit{s}\mathit{\%}\right)+\left(\mathit{\Sigma }\mathit{d}\mathit{i}\mathit{e}\mathit{n}\mathit{o}\mathit{i} \mathit{a}\mathit{c}\mathit{i}\mathit{d}\mathit{s}\mathit{\%}\right)+\left(2 \times \mathit{\Sigma }\mathit{t}\mathit{r}\mathit{i}\mathit{e}\mathit{n}\mathit{o}\mathit{i}\mathit{c} \mathit{a}\mathit{c}\mathit{i}\mathit{d}\mathit{s}\mathit{\%}\right)\\ +\left(4 \times \mathit{\Sigma }\mathit{t}\mathit{e}\mathit{t}\mathit{r}\mathit{a}\mathit{e}\mathit{n}\mathit{o}\mathit{i}\mathit{c} \mathit{a}\mathit{c}\mathit{i}\mathit{d}\mathit{s}\mathit{\%}\right)+\left(6 \times \mathit{\Sigma }\mathit{p}\mathit{e}\mathit{n}\mathit{t}\mathit{a}\mathit{e}\mathit{n}\mathit{o}\mathit{i}\mathit{c} \mathit{a}\mathit{c}\mathit{i}\mathit{d}\mathit{s}\mathit{\%}\right)+(8 \times \mathit{\Sigma }\mathit{h}\mathit{e}\mathit{x}\mathit{a}\mathit{e}\mathit{n}\mathit{o}\mathit{i}\mathit{c} \mathit{a}\mathit{c}\mathit{i}\mathit{d}\mathit{s}\mathit{\%})\end{array}$$

(4)

2.5. Materials

Lyophilized Listeria monocytogenes strain ATCC 13932 was obtained from the American Type Culture Collection, Cat. No. ATCC 13932, Manassas, VA, USA. The following materials were also obtained: Brain Heart Infusion (BHI) broth, Oxoid (Thermo Fisher Scientific, Waltham, MA, USA), Cat. No. CM1135B, Basingstoke, UK; Buffered Peptone Water, Oxoid (Thermo Fisher Scientific), Cat. No. OXCM0509B, Basingstoke, UK; and Oxoid™ Brilliance Listeria Agar (OCLA-ISO formulation), Oxoid (Thermo Fisher Scientific), Cat. No. PO1298A, Basingstoke, UK. Sterile physiological saline solution (0.85% NaCl) was prepared using analytical grade sodium chloride, Merck, Cat. No. S9888, Darmstadt, Germany. For microbiological quality assessment, Petrifilm^® Aerobic Count Plates were obtained from Neogen^®, Cat. No. 700002273, Lansing, MI, USA. Reagents used for lipid and protein oxidation analyses included 2-thiobarbituric acid (TBA), Merck, Cat. No. T5500, Darmstadt, Germany; butylated hydroxytoluene (BHT), Merck, Cat. No. B-1378, Darmstadt, Germany; trichloroacetic acid (TCA), Merck, Cat. No. T6399, Darmstadt, Germany; and 2,2′-dithiobis(5-nitropyridine) (DTNP), Merck, Cat. No. 1589194, Darmstadt, Germany. Malondialdehyde (MDA) reference standard, Sigma-Aldrich, Cat. No. 108383, St. Louis, MO, USA; internal standard nonadecanoate methyl ester (C19:0), Sigma-Aldrich, Cat. No. 74208, St. Louis, MO, USA; Supelco Mix 37, Sigma-Aldrich, Cat. No. CRM47885, St. Louis, MO, USA; CLA Mix, Sigma-Aldrich, Cat. No. L6031, St. Louis, MO, USA; docosatetraenoic acid methyl ester (22:4-n-6), Sigma-Aldrich, Cat. No. D3534, St. Louis, MO, USA; and docosapentaenoic acid methyl ester (22:5-n-3), Sigma-Aldrich, Cat. No. CRM47563, St. Louis, MO, USA. Branched-chain fatty acid mixtures BR2 and BR4 were obtained from Larodan, Cat. No. 90-1052, Solna, Sweden. All reagents and solvents were of analytical grade.

2.6. Statistical Analysis

2.6.1. Data Analysis

Microbiological, oxidative, and color parameters were analyzed using Linear Mixed Models (LMM) implemented with the PROC MIXED procedure of SAS software (version 9.4; SAS Institute Inc., Cary, NC, USA). The model included the fixed effects of Treatment (4 levels: CTR, BC1%, BC3%, and BC5%), Time (7 levels: 0, 1, 2, 6, 8, 24, and 48 h for L. monocytogenes; 2 levels: 0 and 48 for the TVC, pH, and a_w; and 5 levels: 0, 8, 24, 48, and 96 h for the others), and their interaction (Treatment × Time). Biological replicates (n = 3 per treatment group) were included as a random effect. Pairwise comparisons among least squares means were adjusted using the Bonferroni correction for multiple testing. Model assumptions were verified using diagnostic plots (normality of residuals, homoscedasticity, and detection of outliers). The significance level was set at p < 0.05.

2.6.2. Growth Curve Modeling

Survival curves were plotted as Log10 S(t), where S(t) represents the ratio between the bacterial count at a given time (Nt) and the initial bacterial count (N0). To describe the growth behavior, both linear and quadratic regression models were fitted to the experimental data. The linear model followed Equation (5):

$${Log}_{10}S\left(t\right)=kt+b$$

(5)

where k is the slope (Log₁₀ units/hour), representing the specific growth rate.

For interpretation in natural logarithmic terms, k was converted into the growth rate constant (μ) using the relation (Equation (6)):

$$\mu =k\times \mathrm{l}\mathrm{n}\left(10\right)$$

(6)

Subsequently, the doubling time (Td) was calculated as (Equation (7)):

$$Td={\displaystyle \frac{ln\left(2\right)}{\mu }}$$

(7)

The goodness of fit of each model was assessed using the coefficient of determination (R²), and the statistical significance was determined using the p value from the regression analysis (p < 0.05 was considered statistically significant).

3. Results

3.1. Chemical Composition

The inclusion of bovine colostrum in ground rabbit meat patties led to different modifications in the chemical composition and fatty acid profile of the samples (Table S2). In particular, regarding the lipid profile, saturated fatty acid (SFA) content was significantly higher in the BC5% group compared with the control. Similarly, branched-chain SFAs were significantly elevated in BC3% and BC5% compared with both the CTR and BC1% (p < 0.05). No significant differences were detected in MUFA and trans-MUFA contents. Conversely, a significant reduction in PUFA content was observed in all colostrum-enriched groups, both in the PUFA-n6 and PUFA-n3 fractions. In the BC5% group, PUFA-n6 levels were lower compared with the control (24.61 ± 0.66 vs. 26.10 ± 0.55, respectively), while PUFA-n3 values significantly declined in all BC groups compared with the control (p < 0.05).

As a result, the PI was significantly lower in the colostrum-treated groups than in the control.

3.2. pH, a_w, and Total Viable Count Results

pH and TVC values changed significantly between groups and over time (

Table 2. Results of pH, a_w, and total viable count in control (CTR) and bovine colostrum-treated rabbit meat patties (BC1%, BC3%, and BC5%) at 0 and 48 h.

				Parameters
Groups	pH			aw		Total Viable Count (Log CFU/g)			Δ (Log CFU/g)
	0	48		0	48	0	48
CTR	5.68 ± 0.009 c	5.36 ± 0.010 c	***	0.973 ± 0.020 ns	0.943 ± 0.024 ns	3.81 ± 0.021 b	4.70 ± 0.013 b	***	+0.89
BC1%	6.44 ± 0.030 b	5.94 ± 0.010 b	***	0.956 ± 0.015 ns	0.970 ± 0.017 ns	3.60 ± 0.012 c	4.08 ± 0.022 c	***	+0.48
BC3%	6.49 ± 0.022 ab	6.03 ± 0.019 a	***	0.964 ± 0.007 ns	0.948 ± 0.024 ns	3.95 ± 0.015 a	4.85 ± 0.014 a	***	+0.90
BC5%	6.53 ± 0.021 a	6.01 ± 0.027 a	***	0.962 ± 0.013 ns	0.964 ± 0.011 ns	3.57 ± 0.009 c	3.92 ± 0.022 d	***	+0.35
	<0.001	<0.001		ns	ns	<0.001	<0.001

a–d = values with different lowercase letters in the same column indicate significant differences between groups (p < 0.001); *** = asterisks indicate significant differences (p < 0.001) within the same group over time (0 h vs. 48 h) within the same group for each parameter; ns = non-significant; data are expressed as mean ± SD.

). At 0 h, pH values were higher in all BC groups compared with the control (p < 0.001), with BC5% showing the highest value (6.53 ± 0.021). Over 48 h, pH decreased significantly in all groups (p < 0.001) but remained consistently higher in the BC groups than in the CTR (p < 0.001).

No significant differences in a_w were observed either between groups or over time (p > 0.05), with all values remaining above 0.94. The total viable counts increased significantly during storage in all samples (p < 0.001).

Although statistically significant differences were observed among the groups (p < 0.001), the microbial growth remained below 1 Log CFU/g in all cases.

3.3. Growth Dynamics of Listeria monocytogenes: Effect of Bovine Colostrum Concentration

The evolution of Listeria monocytogenes populations during storage at 4 °C in this challenge test is reported in Figure 1. An overall increase in bacterial counts was observed in all groups over time (

Table 3. Changes in mean counts of L. monocytogenes (Log CFU/g) over storage time in control (CTR) and bovine colostrum-treated rabbit meat patties (BC1%, BC3%, and BC5%).

Groups	Timepoints (h)							p Value for Time Effect	Δ (Log CFU/g)
	0	1	2	6	8	24	48
CTR	3.98 ± 0.07 Ca	3.80 ± 0.45 Ca	3.69 ± 0.32 Ca	4.29 ± 0.24 BCa	4.32 ± 0.08 BCa	4.58 ± 0.12 Ba	5.21 ± 0.01 Aa	<0.05	+1.23
BC1%	3.93 ± 0.37 Ca	3.88 ± 0.34 Ca	3.85 ± 0.25 Ca	4.33 ± 0.10 BCa	4.38 ± 0.18 BCa	4.49 ± 0.03 Bab	5.11 ± 0.08 Aa	<0.01	+1.18
BC3%	3.86 ± 0.57 BCa	3.66 ± 0.38 Ca	3.58 ± 0.51 Ca	3.87 ± 0.43 BCb	3.98 ± 0.44 BCab	4.16 ± 0.29 Bab	5.10 ± 0.04 Aa	<0.001	+1.24
BC5%	3.81 ± 0.34 BCa	3.66 ± 0.23 Ca	3.60 ± 0.26 Ca	3.87 ± 0.42 BCb	3.90 ± 0.49 BCb	4.11 ± 0.86 Bb	4.88 ± 0.09 Aa	<0.05	+1.07
p Value for Group Effect	>0.05	>0.05	>0.05	<0.05	<0.05	<0.05	>0.05

A–C = values with different uppercase letters in the same row indicate significant differences over time within the same group (p < 0.05); a, b = values with different lowercase letters in the same column indicate significant differences among groups (p < 0.05); data are expressed as mean ± SD; Δ Log values represent the difference between counts at 48 h and 0 h.

), (p < 0.001).

However, samples enriched with bovine colostrum, particularly at 5%, exhibited a slower growth rate compared with the control. Moreover, from 6 to 24 h, significant differences among treatments were observed (h 6 = BC3% and BC5%; h 8 and 24 = BC5%; p < 0.05).

3.4. Kinetic Modeling of Listeria monocytogenes Growth: Effect of Bovine Colostrum Concentration

The survival kinetics of L. monocytogenes were modeled using linear and quadratic regressions based on log-transformed survival ratios (Log₁₀ S(t); Figure 2). Both models showed good fit, with R² values above 0.88 for all treatments. The quadratic model provided a slightly better fit than the linear model, with R² values >0.99 for the CTR and BC1% and ≥0.96 for BC3% and BC5%. The estimated specific growth rate (μ) decreased with increasing colostrum concentrations. The control group exhibited a growth rate of 0.063 h⁻¹ and a doubling time (Td) of 10.9 h, whereas BC5% samples showed the lowest μ value (0.055 h⁻¹) and the highest Td (12.5 h).

3.5. Colorimetric Parameters

The inclusion of bovine colostrum significantly affected the colorimetric parameters of rabbit meat patties during storage, with differences becoming more evident at later timepoints (Figure 3). L* values generally remained stable across all groups, except at 8 h where a slight but significant increase in lightness was observed compared with 0 h (

Table 4. Effect of storage time on physicochemical parameters of rabbit meat patties, irrespective of treatment.

Parameters	Timepoints (h)					p Value	RMSE
	0	8	24	48	96
L*	49.49 b	51.58 a	50.29 ab	48.72 b	48.64 b	<0.001	1.47
a*	−1.62 b	−1.42 b	−1.43 b	−1.46 b	−1.02 a	0.003	0.23
b*	6.92 b	7.25 b	7.84 ab	7.79 ab	8.15 a	<0.001	0.69
C*	7.15 b	7.43 b	8.00 a	7.94 ab	8.24 a	0.002	0.67
H°	76.11 c	78.10 bc	78.92 b	79.21 b	82.75 a	<0.001	2.28
TBARS (mg MDA/kg)	0.03 d	0.05 d	0.11 c	0.26 b	0.70 a	<0.001	0.02
Sulfhydryl (nmol SH/mg protein)	73.65 a	62.57 b	57.08 c	44.80 d	37.77 e	<0.001	3.31

RMSE = root mean square error; a–e = values with different lowercase letters in the same row indicate a significant difference during storage times (p > 0.05); data are expressed as mean.

). However, BC5% samples consistently maintained higher L* values than the control, with significant differences at 48 h and 96 h (p < 0.001, Figure 3). For a* values, all colostrum-enriched samples exhibited significantly lower values compared with the control at all timepoints (p < 0.001). Although values were relatively stable over time, a significant decrease was observed only at the end of refrigerated storage. Regarding b* values and the C*, a general increase was observed during storage. Nonetheless, the colostrum-treated rabbit meat patties showed significantly lower b* and C* values than the control from 0 h to 48 h (p < 0.001). Finally, the hue angle, representing perceived color tone, increased over time in all groups. However, it remained significantly lower in the colostrum-treated samples compared with the control at all timepoints (p < 0.001).

The ΔE results are reported in Table S3. In the control group, the ΔE exceeded 3 after 48 h and reached values above 4 at 96 h. In the 1% and 3% colostrum groups, the ΔE values already exceeded 3 at 24 h and continued to increase over time. In the 5% colostrum group, the ΔE values remained below 3 throughout the storage period, including at 96 h.

3.6. Oxidative Stability: TBARS and Thiol Content

Lipid oxidation increased progressively during storage in all groups (Figure 4). The CTR samples exhibited a marked increase starting from 24 h. In contrast, bovine colostrum-treated samples showed significantly lower TBARS levels from 8 h onward compared with the control (p < 0.001), with BC5% displaying the lowest values. Regarding protein oxidation, a time-dependent decrease in thiol content was observed across all treatments. The colostrum-enriched rabbit meat patties retained significantly higher thiol levels throughout storage (p < 0.001).

4. Discussion

The incorporation of bovine colostrum into ground rabbit meat patties significantly affected both microbial dynamics and chemical composition of the matrix, with relevant implications for food safety and oxidative stability. Colostrum supplementation induced a shift in the lipid profile toward a higher proportion of SFA, particularly branched-chain SFAs, which have been associated with improved oxidative stability in meat [17]. This shift was accompanied by a significant reduction in the PI, suggesting a protective effect of colostrum against lipid oxidation. The treated samples consistently showed lower TBARS values throughout storage compared with the control group.

Despite the increased fat content, a_w remained stable and above 0.94 in all treatment groups. This is noteworthy, as even slight reductions in a_w can inhibit microbial growth—thereby enhancing food safety—and slow down chemical degradation processes such as lipid and protein oxidation, both of which are known to be a_w dependent [27,28]. Although a consistent direct correlation between fat content and a_w has not been demonstrated, previous studies [29,30] suggest that fat may indirectly influence a_w by altering water mobility and distribution within the meat matrix. In particular, higher lipid levels have been linked to reduced availability of free water in certain meat systems, potentially lowering effective a_w [31].

The growth of L. monocytogenes was significantly influenced by colostrum concentration. The BC5% group exhibited a lower specific growth rate (μ = 0.055 h⁻¹) and a longer doubling time (12.5 h) compared with the control (μ = 0.063 h⁻¹; Td = 10.9 h). This inhibitory effect may be attributable to antimicrobial components naturally present in colostrum, such as lactoferrin, immunoglobulins, lysozyme, and bioactive oligosaccharides [14,30,32,33]. Interestingly, a dose-dependent effect was observed, as the BC3% group showed a slightly higher growth rate than the control, suggesting that moderate inclusion levels may provide nutrients (e.g., peptides) potentially supporting microbial proliferation. Although the antimicrobial potential of bovine colostrum has been documented in dairy and other food matrices (e.g., yogurt, fermented products) [34], its application in meat systems remains underexplored [17]. Our findings are consistent with those of Iram et al. [35], who reported similar inhibition patterns of Gram-positive pathogens in dairy matrices supplemented with colostrum.

One of the aims of this study was to evaluate the inhibitory effect of bovine colostrum on the growth of Listeria monocytogenes in ground rabbit meat. According to Regulation (EC) No. 2073/2005 [36], minced meat and meat products intended to be eaten cooked, such as rabbit meat patties, are not classified as ready-to-eat foods and are therefore not subject to specific microbiological safety criteria for L. monocytogenes. However, if these products were to be marketed in a pre-cooked format, they would fall under the RTE category. In such cases, the combination of physicochemical characteristics (e.g., pH and a_w) and shelf life determines whether the matrix can support the growth of L. monocytogenes. In this context, the upcoming revision of the European microbiological criteria (Reg. (EC) No. 2895/2024) [37], which will come into force in July 2026, places greater emphasis on ensuring product safety throughout the entire shelf life, particularly for RTE foods capable of supporting the growth of L. monocytogenes. In the absence of validated evidence demonstrating that bacterial levels will remain below the legal limit until the end of shelf life, stricter criteria will be applied. This evolving regulatory framework highlights the potential role of natural ingredients, such as bovine colostrum, in enhancing microbial control. Their inclusion may support food business operators in complying with safety requirements through scientifically validated approaches, including challenge tests and predictive microbiology.

Further studies involving the deliberate inoculation of pathogens into BC-enriched ground rabbit meat would provide additional insight into the antimicrobial effectiveness of BC, particularly against the Salmonella spp., which remains subject to an absence criterion (n = 5, c = 0, limit: not detected in 25 g).

Overall, although BC did not make the product microbiologically safe—since, as previously discussed, no specific legal limits for L. monocytogenes apply to this type of food product—it nonetheless reduced the pathogen’s growth potential in a dose-dependent manner and enhanced both physicochemical stability and overall meat quality.

In addition, BC supplementation improved color stability. The BC5% group was significantly lighter than the control at the last two sampling times, while the b* and C* coordinates showed lower values in all BC-enriched groups compared with the control up to 48 h. This trend is consistent with the previously reported lower TBARS values from 8 h onward and is further supported by the consistently higher thiol content measured from 0 h in the BC groups compared with the control. These findings suggest that BC limits lipid oxidation and protects protein sulfhydryl groups, thereby delaying oxidative pigment changes responsible for discoloration [27]. Overall, the combined preservation of meat color, lipid stability, and protein integrity supports the potential of BC as a multifunctional ingredient for improving the shelf life of fresh meat.

Moreover, the stability of meat color and the improved resistance to both lipid and protein oxidation further highlight the potential of BC as a natural preservative. These effects likely derive from bioactive compounds in colostrum—including vitamins A and E, enzymatic antioxidants like superoxide dismutase and catalase, and radical-scavenging peptides [13,38]. Such properties may make BC a promising ingredient for developing functional meat products that align with consumer demand for natural and health-promoting foods [39]. The ΔE results further support this perspective, as the 5% colostrum group maintained values below the perceptibility threshold of 3 throughout storage, suggesting a protective role in limiting discoloration. In contrast, both the control and the lower inclusion levels (1% and 3%) showed that the ΔE increases beyond this threshold, indicating distinct color changes. These findings highlight the potential of colostrum not only for its nutritional and bioactive value but also for its technological functionality in improving the visual quality and shelf life of meat products.

Given that consumer acceptability is a critical factor for industrial implementation, a second step of this research is currently underway, involving challenge tests on pre-cooked rabbit meat patties with the same levels of bovine colostrum inclusion. In parallel, sensory analyses (panel tests) are being conducted on non-inoculated samples to evaluate the effects of colostrum addition on taste, texture, and overall acceptability.

Bovine colostrum inclusion could therefore support a multi-hurdle preservation approach, particularly when combined with packaging strategies such as modified-atmosphere packaging (MAP) or active packaging [40]. Beyond its technological and microbiological benefits, BC, as a natural ingredient, also offers promising opportunities for sustainable resource management. Colostrum is often produced in quantities exceeding the needs of newborn calves, especially in high-yield dairy systems, and a considerable proportion is discarded or underutilized due to regulatory or logistical constraints [16]. When appropriately treated through thermal or high-pressure processing to ensure microbiological safety, colostrum not required for feeding newborn calves could be redirected into the human food chain as a functional ingredient. Its incorporation into meat products may therefore represent a circular economy strategy, reducing on-farm waste while improving the safety and technological quality of perishable foods. In this context, BC may be considered a high-value co-product capable of contributing to both food sustainability and consumer health when properly processed and integrated.

5. Conclusions

This study demonstrated that the inclusion of bovine colostrum, particularly at a 5% level, in ground rabbit meat patties significantly slowed the growth of Listeria monocytogenes during refrigerated storage and improved oxidative stability by reducing lipid oxidation and preserving protein thiol groups. Taken together, these results indicate that bovine colostrum can act as a natural multifunctional ingredient, simultaneously enhancing the microbial safety and physicochemical stability of fresh meat, and that its application could therefore be considered within multi-hurdle preservation strategies for perishable foods. In addition, the valorization of surplus colostrum represents a sustainable approach consistent with circular economy principles, providing added value to dairy by-products while supporting consumer health and safety. Future studies should further investigate the dose–response relationship of colostrum components, assess its effectiveness in different meat matrices and processing conditions, and evaluate sensory and regulatory implications for potential industrial applications.