Potential of Whey Protein-Fortified Blackberry Juice in Transporting and Protecting Lactic Acid Bacteria: A Proteolytic Profile Analysis and Antioxidant Activity

Abstract

This study investigates the potential of blackberry juice fortified with whey as a carrier for transporting and protecting lactic acid bacteria (LAB). The interactions between whey proteins and the juice were examined to assess their impact on probiotic stability and protection during storage and passage through the gastrointestinal tract. Additionally, the study explored how this combination influences the antioxidant properties of the product. The results indicated that the blackberry juice and whey protein mixture provided moderate protection to Lacticaseibacillus rhamnosus GG compared to the positive control (inulin), suggesting that whey proteins may enhance probiotic viability. Proteolytic analysis revealed progressive protein hydrolysis during fermentation, leading to the release of bioactive peptides, indicating the formation of compounds with potential functional benefits. Moreover, samples inoculated with LAB exhibited higher antioxidant activity than those without inoculum. This research demonstrates the promise of fermented blackberry juice fortified with whey proteins as an effective probiotic delivery system. It opens new possibilities for developing functional foods to promote intestinal health and overall well-being.

1. Introduction

In recent years, probiotics have garnered considerable attention for their potential health benefits, especially in relation to gut microbiota [1]. One of the most widely studied and utilized probiotics is Lacticaseibacillus rhamnosus GG, known for its positive effects on digestion, immune system regulation, and protection against gastrointestinal infections [2,3]. However, maintaining probiotic viability during storage and ensuring survival through the gastrointestinal tract poses a significant challenge due to factors like acidic pH and bile salts [4,5]. Various strategies have been explored to address this challenge and enhance probiotic stability [4,6].

One promising approach involves developing vehicles that serve as protective matrices, facilitating the controlled release of microorganisms at the optimal location, such as the small intestine [7]. Natural matrices like fruit juices have become an attractive option [8,9,10]. Specifically, blackberry juice, rich in bioactive compounds, including antioxidants, flavonoids, and dietary fiber, is an excellent candidate for formulating functional products [11,12]. This juice may also support probiotics’ survival and biological activity [13].

On the other hand, whey proteins, which are byproducts of the cheesemaking process, are known for their functional properties, including their ability to interact with microorganisms [14,15]. These proteins have shown potential in enhancing probiotic stability, acting as protective agents against harsh gastrointestinal conditions and, in some instances, assisting in the adhesion of probiotics to intestinal cells [16,17]. For instance, whey proteins fermented by Pediococcus acidilactici produce low-molecular-weight peptides (<7 kDa) with inhibitory activity against angiotensin 1-converting enzyme (ACE) [18]. Additionally, fermenting yellow whey with Lacticaseibacillus casei YQ336 for 48 h increased organic acids production and antioxidant activity [19]. L. casei has also shown potential antidiabetic activities measured by the inhibition of α-glucosidase and α-amylase in vitro [20]. Fortifying fruit juices with whey proteins might provide an effective delivery system for Lacticaseibacillus rhamnosus GG, boosting probiotic viability and efficacy [21].

Studies have reported the use of cheese whey in L. plantarum 67- and L. paracasei grx 701-fermented blueberry juice. Fourier-Transform Infrared Spectroscopy analysis revealed that anthocyanins interact with whey protein through electrostatic interactions, hydrogen bonding, and hydrophobic interactions, resulting in protein fluorescence quenching and structural changes. Adding whey protein to blueberry juice increased the survival rate compared to whey protein alone [22]. In addition, interactions between whey proteins and apple polyphenols enhanced polyphenol bioaccessibility and bioavailability in simulated gastrointestinal digestion and human Caco-2 cells [23].

Previously, we reported that using lactic acid bacteria (LAB) consortia to enhance biomass during fermentation in fruit juices can be beneficial, resulting in a 3-logarithmic increase in growth with whey addition. The increased survival was related to lactic acid production and antioxidant changes during fermentation and 28-day storage at 4 °C [24].

Therefore, the potential of whey protein-fortified blackberry juice as a carrier for transporting and protecting LAB was analyzed through the proteolytic profile of LAB consortia. We aim to understand how whey proteins interact with blackberry juice and how this combination affects the probiotic’s protection and stability during storage and transit through the gastrointestinal tract. Additionally, we will explore how the composition of this matrix impacts the final content of phenolic acids, flavonoids, and organic acid, bacteriocins production, and antioxidant properties, providing an added benefit to fortifying blackberry juice with bioactive compounds.

This study evaluates the effectiveness of this system as a probiotic delivery tool, presenting new opportunities for developing functional products promoting intestinal health and overall well-being.

2. Materials and Methods

2.1. Materials

The raw materials used to produce the beverages include blackberries (Rubus fruticosus) from Atotonilco el Grande, Hidalgo, Mexico. Whey protein (100% isolated) was purchased from HolixLab (Hilmar ingredients, Guadalajara, Jalisco, Mexico).

Bacterial cultures of Lactiplantibacillus plantarum, Pediococcus acidilactici, Levilactobacillus brevis, Lacticaseibacillus rhamnosus, and Lacticaseibacillus casei were provided from the strain collection of the Nutrition Department from the Universidad Autónoma del Estado de Hidalgo. The probiotic strain Lacticasebacillus rhamnosus GG was sourced from the strain collection of the Food Biotechnology Department at the Universidad Autónoma Metropolitana, Iztapalapa Campus, México.

(+)-Catechin, (−)-epicatechin, (−)-epigallocatechin gallate, 2,2′-azobis(2-methylpropionamidine) dihydrochloride (AAPH), 2,4,6-trinitrobenzenesulfonic acid (TNBS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), 4-hydroxybenzoic acid, acetic acid, benzoic acid, ellagic acid, butyric acid, caffeic acid, gallic acid, HPLC grade formic acid, iron trichloride (FeCl3), isovaleric acid, myricetin, p-coumaric acid, propionic acid, radical 2,2-diphenyl-1-picrylhydrazyl (DPPH∙), rutin, sodium fluorescein, trans-ferulic acid, Trolox, and vanillic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). HPLC grade methanol was obtained from JT Baker (Wayne, PA, USA).

2.2. Obtaining Thermoultrasonic Blackberry Juice

Blackberry juice (BJ) was extracted under aseptic conditions using colloidal silver (Microdyn^®, Ciudad de Mexico, CDMX, Mexico) as a disinfectant. Subsequently, the fruit was crushed, and seeds were separated. The juice was clarified by centrifugation (10,000 rpm for 30 min, 4 °C) followed by thermo-ultrasound treatment (80% amplitude for 17 min), according to the optimization conditions reported by Cervantes-Elizarrarás et al. [25].

2.3. Fermentation

A mixture of BJ and whey (WH) was prepared in equal concentrations (1:1), and the pH was adjusted to 6 with 1 M NaOH. The beverage was subdivided into 3 parts for inoculation (9 Log CFU/10 mL): consortium 1 (C1) was inoculated with L. plantarum, P. acidilactici, and L. brevis; consortium 2 (C2) was inoculated with L. rhamnosus and L. casei; and a sample without inoculum (C−). Fermentation was carried out for 16 h at 37 °C, with samples taken at 0, 8, and 16 h.

2.4. Determination of Prebiotic Activity

The method described by Jaime-Ordaz et al. [26] was applied with modifications to determine the prebiotic activity through in vitro digestion study simulating the small intestine. This method included MRS medium carbon-free, and the BJ and BJ + WH juices were used as the carbon source. Inulin (1 g/L solution) was used as the positive control of the prebiotic activity. All samples were inoculated with 1 × 10⁷ CFU/mL of L. rhamnosus GG. The flasks were kept at 37 °C for 48 h on an orbital shaker (at 150 rpm). The viable probiotic count was determined by seeding on MRS agar (Hilmar) and incubating at 37 °C under anaerobic conditions for 72 h. Carbon source consumption was determined by measuring the total carbohydrates using the DNS (3,5-dinitrosalicylic acid) method.

2.5. Proteolytic Profile Assessment

2.5.1. Determination of Free Amino Groups by the TNBS Technique

Free amino groups during fermentation were measured using the method described by Sebastian-Nicolás et al. [27]. In glass tubes shielded from light, 2 mL of a phosphate buffer (0.21 M, pH 8.2), 0.250 mL of the samples, and 2 mL of 0.10% TNBS were combined, stirred, and incubated for 60 min at 50 °C. The reaction was halted with 4 mL of 0.1 N HCl, and the absorbance was assessed in a spectrophotometer at 340 nm against a control of deionized water. A glycine calibration curve (0 to 0.25 mg/mL) was used.

2.5.2. Low-Molecular-Weight Peptides Separation by Tris–Tricine Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (Tris–Tricine SDS-PAGE)

The method suggested by Sebastián-Nicolás et al. [27] was applied with modifications. The protein concentration of the samples was determined using the nanodroplet. A total of 40 µL of lyophilized sample (20-fold concentrated) was mixed with 20 µL of running buffer (0.1 M Tris base–Tricine) and 3 µL of mercaptoethanol and incubated at 40 °C for 30 min. After incubation, 15 µL of the sample (C1, C2, and C−) was loaded. The polyacrylamide gel (15% T) was starting from a 30% T solution (acrylamide:bisacrylamide ratio 19:1 and 0.1 M Tris–Tricine). Electrophoresis was carried out at 30 V for 1 h, followed by 95 V for approximately seven hours. Image Lab^TM software from Gel-Doc^TM EZ system 1708270 (Bio-Rad, Hercules, CA, USA) was used to analyze the bands after the gels were stained with silver (Bio-Rad, Hercules, CA, USA).

2.6. Organic Acids Quantification

For the quantification of organic acids, a Gases Chromatography-Flame Ionization Detector (GC-FID) method was used [28]. The calibration curve was constructed with the following commercial compound standards: acetic acid, propionic acid, butyric acid, and isovaleric acid at concentrations of 0.05 to 0.13%.

One microliter of sample was injected into a PerkinElmer AutoSystem XL gas chromatograph (PerkinElmer, Springfield, IL, USA) equipped with a DB-FFAP capillary column (15 m × 0.53 mm × 0.50 µm; Agilent, Santa Clara, CA, USA), a flame ionization detector at 250° C, and a split/splitless injector (split ratio 30:1). Hydrogen was used as a carrier gas of 45 mL/min.

The oven temperature ramp for the analysis was as follows: the oven temperature was raised to 60 °C for 1 min; then, the temperature was increased to 165 °C at 20 °C/min and held for 1.25 min; finally, the temperature was increased to 200 °C at 10 °C/min and held for 4 min. The compounds were identified by comparing their retention time and absorption spectra with the following commercial standards: gallic acid, (+)-catechin, 4-hydroxybenzoic acid, (−)-epigallocatechin gallate, vanillic, (−)-epicatechin, caffeic acid, p-coumaric acid, trans-ferulic acid, benzoic acid, ellagic acid, rutin, and myricetin. Their quantification was carried out using calibration curves.

2.7. Quantification of Phenolic Compounds

Phenolic compounds were analyzed by HPLC using the methodology developed by García-Falcón et al. [29], with some modifications. A Zorbax Eclipse XDB-C18 column (5 µm particle size, 15 cm × 4.6 mm i.d.; Agilent, Santa Clara, CA, USA), was used. The mobile phase was set to 1.5 mL/min and comprised 1% formic acid in methanol (Phase A) and 1% formic acid in water (Phase B). During the analysis, the solvent gradient was programmed to increase from 10% to 100% B in A over 30 min. A sample volume of 20 μL was injected into the column. A diode array detector was utilized, programmed for four different wavelengths: 260, 280, 320, and 360 nm. The compounds were identified by comparing their retention times and absorption spectra with the following commercial standards: gallic acid, (+)-catechin, 4-hydroxybenzoic acid, (−)-epigallocatechin gallate, vanillic, (−)-epicatechin, caffeic acid, p-coumaric acid, trans-ferulic acid, benzoic acid, ellagic acid, rutin, and myricetin. Their quantification was carried out using calibration curves.

2.8. Antioxidant Capacity Assays

2.8.1. Oxygen Radical Absorbance Capacity (ORAC) Test

The ORAC test was determined according to the method described by Fernandez-Rojas et al. [30]. The AAPH compound was used as a peroxyl radical generator, and Trolox (0.5 to 10 µM) served as a standard. Briefly, 25 µL of diluted samples were mixed with 25 µL of 153 mM AAPH and 150 µL of 0.05 µM fluorescein in 96-well black microplates (Costar^®, Corning, NY, USA). Fluorescence was measured every minute for 90 min at 37 °C in a Synergy HT multimode microplate reader (BioTek Instruments, Winooski, VT, USA) using fluorescence filters with an excitation wavelength of 485 nm and an emission wavelength of 520 nm. Data were expressed as µM of Trolox equivalents.

2.8.2. Ferric Ion-Reducing Antioxidant Power (FRAP) Assay

This parameter was determined using the methodology described by Ramírez-Rodríguez et al. [31]. Briefly, 30 µL of the diluted samples and 300 µL of the FRAP solution (1.66 mM FeCl₃ and 0.83 mM TPTZ in 300 mM acetate buffer, pH 3.6) were mixed in a 96-well microplate. After 15 min of incubation at room temperature, the absorbance at 593 nm was measured. Data were expressed as mM of Trolox equivalents.

2.8.3. Antioxidant Activity Assay Against 2,2-Diphenyl-1-picrylhydrazyl Radical (DPPH^●)

This assay was created according to Koren et al. [25]. Briefly, 27 µL of the sample and 20 µL of 2 mM DPPH^● methanolic solution were added directly to the 96-well microplate and allowed to stand for 2 min. Subsequently, 285 µL of methanol were added, followed by another 2 min standing period, and the optical density at 517 nm was read. The results were expressed as the percentage of DPPH removed.

2.9. Extraction of Low-Molecular-Weight Peptides

The peptide extract was prepared following the method described by Gaspar et al. [32] with modifications. The pH of 50 mL of samples (C1 and C2) was adjusted to 6.5 using 1 M NaOH and stirred for 30 min at room temperature. Subsequently, the samples were heated in a water bath at 70 °C for 30 min. The cells were collected by centrifugation at 4500 rpm for 15 min at 4 °C and washed twice with a sodium phosphate buffer (5 mM at pH 6.5). The cells were collected again by centrifugation under the same conditions. The recovered cells were resuspended in 30 mL of NaCl solution (100 mM at pH 2, adjusted with phosphoric acid) and stirred for 1 h at 4 °C using a magnetic stirrer. The suspension was centrifuged at 4500 rpm for 15 min at 4 °C, and the supernatant was collected and adjusted to pH 6.5 with 1 M NaOH. The recovered supernatant was filtered through a cellulose acetate filter with a pore size of 0.22 µm, and finally, the extract was lyophilized for analysis. Each sample was analyzed by gel electrophoresis using the same technique described in Section 2.5.2.

2.10. Statistical Analysis

The results were analyzed using a one-way analysis of variance (ANOVA) test and a post hoc Tukey’s test at a significance level of p < 0.05. Data are presented as the mean and standard deviation (SD) of three independent experiments. Comparisons were made between each consortium at different fermentation times (0, 8, and 16 h) and differences between consortia at the same fermentation time. All analyses were carried out using the SPSS V.19 statistical package.

3. Results

3.1. Prebiotic Activity

The effect of treatments on prebiotic activity was assessed in terms of the survival of L. rhamnosus GG (Figure 1A) and the concentration of reducing sugars (Figure 1B) in BJ and its mixture with whey (BJ + WH). In Figure 1A, inulin, used as a positive control, significantly enhanced the survival of L. rhamnosus GG compared to BJ and BJ + WH (p < 0.05). The samples showed intermediate survival, indicating that whey components may offer some protective effects.

In panel B, the initial concentration of reducing sugars was higher in BJ than in BJ + WH and the inulin control. After 48 h of incubation at 37 °C, the concentration of reducing sugars decreased in BJ and the control inulin, reflecting their consumption by the microorganisms and suggesting a more efficient metabolism by L. rhamnosus GG, supporting its prebiotic effect.

3.2. Proteolytic Profile

The proteolytic profile of BJ mixed with whey (1:1) was evaluated for three samples of C1 (L. plantarum, P. acidilactici, and L. brevis); C2 (L. casei and L. rhamnosus); and C− (BJ + WH without inoculum). The protein hydrolysis was determined by measuring free amino groups and analyzing the molecular weights using SDS-PAGE electrophoresis, as shown in Figure 2.

The evolution of free amino groups during fermentation reflects the dynamics of protein hydrolysis and the subsequent release of peptides and amino acids [33]. The initial free amino group concentration was 1676.6 mg/L on average. During the first 8 h of fermentation, samples C1 and C2 showed significant decreases to 983.8 mg/L and 1140.3 mg/L, respectively, likely due to rapid assimilation by LAB for biomass synthesis and energy metabolism. At 16 h, free amino group concentrations were maintained in C1 (860.6 mg/L) and C2 (1128.7 mg/L) compared to C− (1296.8 mg/L). The action of proteases and peptidases can degrade proteins into lower molecular weight peptides and free amino acids [34].

Differences between C1 and C2 suggest moderated variability in proteolytic activity, with C1 showing greater accumulation of free amino groups, indicating more efficient protein degradation by the generation of low-molecular-weight peptides. The increased concentration of free amino groups during the final hours of fermentation indicates the possible presence of bioactive peptides in the fermented matrix.

The variation in free amino groups throughout the fermentation process demonstrates the differing proteolytic activity of the evaluated bacterial consortia [28]. The greater production of these compounds in C1 suggests their potential to generate peptides with bioactive activity, creating opportunities for developing fermented beverages with improved functional properties [35].

SDS-PAGE analysis revealed the gradual degradation of proteins during fermentation, illustrating the generation of lower molecular weight peptides over time (see Figure 3). Initially (time 0), high-molecular-weight bands diminished in intensity as the fermentation progressed, indicating the activity of bacterial proteases on the proteins in whey and blackberry juice.

After 8 h, partial fragmentation was observed, with lower molecular weight bands emerging, indicating ongoing hydrolysis. By 16 h, a significant accumulation of low-molecular-weight peptides and disappearance of larger protein bands were evident, reflecting the proteolytic activity of LAB, such as L. plantarum and P. acidilactici, which are known to produce enzymes that release bioactive peptides during fermentation. Notably, C1 showed increased smaller peptides (5 and 2 kDa) at 16 h. This is likely associated with the formation of compounds with bioactive potential, including those exhibiting antioxidant, antihypertensive, or immunomodulatory properties, highlighting the importance of this process in the development of functional foods [34].

3.3. Quantification of Organic and Phenolic Compounds During Fermentation

Table 1. Quantification of phenolic acids, flavonoids, and organic acids in fermented beverages (C1, C2, and C−) during fermentation (0, 8, and 16 h).

Fermentation	0 h			8 h			16 h
Sample	C1	C2	C−	C1	C2	C−	C1	C2	C−
Phenolic acids
Gallic acid	0.356 ± 0.007 A,a	0.394 ± 0.014 A,a	0.353 ± 0.031 A,a	0.144 ± 0.060 B,a	0.171 ± 0.017 B,a	0.349 ± 0.021 A,b	0.147 ± 0.004 B,a	0.163 ± 0.001 B,a	0.347 ± 0.016 A,b
Hydroxybenzoic	0.031 ± 0.001 A,a	0.029 ± 0.002 A,a	0.030 ± 0.001 A,a	0.017 ± 0.002 B,a	0.017 ± 0.004 B,a	0.028 ± 0.002 AB,b	0.013 ± 0.001 C,a	0.018 ± 0.001 B,b	0.026 ± 0.001 B,c
Vanillic acid	0.072 ± 0.004 A,a	0.046 ± 0.023 A,a	0.063 ± 0.010 A,a	0.042 ± 0.022 AB,ab	0.027 ± 0.013 A,a	0.078 ± 0.002 A,b	0.020 ± 0.014 B,a	0.015 ± 0.004 A,a	0.073 ± 0.005 A,c
Caffeic acid	0.069 ± 0.001 A,b	0.060 ± 0.004 A,a	0.064 ± 0.002 A,ab	0.062 ± 0.016 A,a	0.040 ± 0.009 B,a	0.038 ± 0.004 B,a	0.031 ± 0.006 B,a	0.024 ± 0.002 C,a	0.031 ± 0.012 B,a
Coumaric acid	0.157 ± 0.001 A,a	0.126 ± 0.015 A,a	0.157 ± 0.022 A,a	0.138 ± 0.028 A,a	0.023 ± 0.005 B,b	0.111 ± 0.001 B,b	0.046 ± 0.002 B,c	0.026 ± 0.001 B,a	0.037 ± 0.001 C,b
Ferulic acid	0.075 ± 0.005 A,b	0.023 ± 0.006 B,a	0.092 ± 0.004 A,c	0.030 ± 0.003 C,a	0.051 ± 0.008 A,b	0.086 ± 0.008 A,c	0.051 ± 0.002 B,a	0.056 ± 0.004 A,a	0.087 ± 0.004 A,b
Benzoic acid	1.064 ± 0.057 A,a	1.588 ± 0.174 A,b	1.742 ± 0.142 A,b	1.084 ± 0.268 A,a	1.252 ± 0.206 AB,a	1.407 ± 0.113 B,a	1.324 ± 0.032 A,a	1.126 ± 0.127 B,a	1.211 ± 0.074 B,a
Ellagic acid	0.063 ± 0.002 A,a	0.063 ± 0.005 A,a	0.056 ± 0.001 A,a	0.047 ± 0.010 AB,b	0.024 ± 0.002 B,a	0.062 ± 0.004 A,b	0.028 ± 0.008 B,a	0.033 ± 0.006 B,ab	0.059 ± 0.019 A,b
Flavonoids
Rutin	0.683 ± 0.029 A,a	1.172 ± 0.143 A,b	0.526 ± 0.031 B,a	0.686 ± 0.081 A,b	0.467 ± 0.052 B,a	0.852 ± 0.021 A,c	0.494 ± 0.019 B,a	0.511 ± 0.067 B,a	0.910 ± 0.079 A,b
Myricetin	0.062 ± 0.016 A,a	0.053 ± 0.004 A,a	0.070 ± 0.005 A,a	0.067 ± 0.005 A,b	0.031 ± 0.002 B,a	0.055 ± 0.016 A,b	0.021 ± 0.002 B,b	0.007 ± 0.001 C,a	0.053 ± 0.009 A,c
Epicatechin	0.407 ± 0.042 A,b	0.166 ± 0.009 B,a	0.333 ± 0.029 B,b	0.404 ± 0.027 A,b	0.343 ± 0.015 A,a	0.441 ± 0.021 A,b	0.364 ± 0.008 A,a	0.369 ± 0.018 A,a	0.474 ± 0.007 A,b
EGCG	<LOD	<LOD	<LOD	<LOD	0.132 ± 0.005 B,a	<LOD	0.0134 ± 0.005 b	0.142 ± 0.005 A,a	0.215 ± 0.008 A
Catechin	3.307 ± 0.271 AB,a	2.249 ± 0.911 A,a	2.882 ± 0.296 B,b	3.554 ± 0.220 A,b	2.882 ± 0.119 A,a	3.454 ± 0.052 A,b	2.848 ± 0.085 B,a	3.067 ± 0.063 A,b	3.418 ± 0.104 A,c
Organic acids
Acetic acid	52.4 ± 4.26 C,b	49.22 ± 4.77 B,b	64.71 ± 3.81 A,a	77.34 ± 7.65 B,a	64.75 ± 4.78 AB,ab	58.32 ± 6.15 A,b	93.91 ± 3.38 A,a	65.94 ± 6.00 A,b	53.00 ± 2.50 B,c

Note. Data are presented in mg/100 mL. C1 (L. plantarum, P. acidilactici, and L. brevis); C2 (L. casei and L. rhamnosus); and C− (without inoculum); EGCG: epigallocatechin gallate; LOD: Limit of Detection. Values represent the mean ± standard deviation of three experiments (n = 3). Different capital letters in subscript indicate significant differences between different hours for each sample. Different lowercase letters indicate significant differences between samples for each hour of fermentation.

displays the changes in phenolic acids, flavonoids, and organic acids in C1, C2, and C− samples at 0, 8, and 16 h of fermentation. A noteworthy decrease in certain phenolic acids, such as gallic acid and hydroxybenzoic acid, was observed in the inoculated samples (C1 and C2) and benzoic acid in C2, suggesting their potential metabolism by LAB. In contrast, the levels of these compounds remain stable in the non-inoculated sample (C−), indicating that microbial fermentation influences their transformation. Furthermore, catechin and epicatechin show fluctuating concentrations, likely due to flavonoid conversion or their interaction with proteins and peptides released during fermentation.

EGCG (epigallocatechin gallate), which was not detected in the three samples at 0 h, appears in C2 at 8 and 16 h after fermentation, potentially due to the conversion of other flavonoids. Benzoic acid, a microbial metabolite produced from phenolic acid degradation, showed varying concentrations, with higher levels observed in the control sample (C−), suggesting LAB fermentation may influence its production.

The rutin and myricetin levels decreased in inoculated samples (C1 and C2) during fermentation, suggesting degradation or conversion into other bioactive compounds. Myricetin showed the most significant reduction in C2, potentially due to higher enzymatic activity. Additionally, acetic acid, a marker of fermentation activity, increased over time in C1 and C2, confirming the metabolic activity of LAB in converting sugars and other precursors into organic acids. In contrast, acetic acid levels remained stable in the control sample (C−), highlighting the role of bacteria fermentation.

3.4. Antioxidant Capacity

Table 2. Antioxidant properties of fermented beverages during fermentation.

Samples	Fermentation (Hours)	ORAC *	FRAP *	DPPH (% of Elimination)
C1	0	2447.8 ± 48.5 A,b	2.7 ± 0.09 A,a	51.71 ± 5.79 A,a
	8	2106.5 ± 274.6 A,b	2.4 ± 0.28 AB,a	51.27 ± 1.04 A,a
	16	2061.5 ± 203.1 A,a	2.04 ± 0.10 B,a	43.36 ± 1.82 A,a
C2	0	2919.8 ± 290.7 A,a	2.75 ± 0.27 A,a	45.31 ± 2.83 A,a
	8	2291.7 ± 148.5 B,a	2.38 ± 0.17 AB,a	45.27 ± 2.32 A,b
	16	2982.6 ± 30.17 A,a	1.99 ± 0.11 B,a	46.27 ± 1.95 A,a
C−	0	2589.5 ± 90.3 A,ab	2.51 ± 0.22 A,a	44.39 ± 1.42 A,a
	8	2652.6 ± 92.9 A,a	2.61 ± 0.45 A,a	39.7 ± 2.51 A,c
	16	2164.1 ± 165.2 B,a	2.19 ± 0.2 A,a	44.78 ± 2.04 A,a

* Results of the ORAC and FRAP assays are presented as µM and mM Trolox equivalents, respectively. C1 (L. plantarum, P. acidilactici, and L. brevis); C2 (L. casei and L. rhamnosus); and C− (without inoculum). Values represent the mean ± standard deviation of three experiments (n = 3). Capital letters in supscripts indicate significant differences between different hours for each sample. Different lowercase letters indicate significant differences between samples for each hour of fermentation.

presents the antioxidant properties of fermented blackberry juice, evaluated through the ORAC, FRAP, and DPPH assays at 0, 8, and 16 h of fermentation, by evaluating three samples: C1, C2, and C−.

The overall antioxidant capacity, evaluated by ORAC, varied throughout fermentation, with C2 exhibiting a higher value at 16 h (2982.6 µM Trolox equivalents) after a temporary decrease at 8 h. In contrast, the antioxidant capacity of C1 and C− declined over the 16 h of fermentation. Sample C1 showed the lowest antioxidant capacity at 16 h (2061.5 µM Trolox equivalents), indicating that the presence of LAB influences this property [36].

The FRAP assay displays the reducing power of ferric ions by the antioxidants present in the samples. The results showed a decrease in ferric reduction ability through fermentation time. C1 and C2 showed a significant decrease from the initial values of 2.7 mM Trolox equivalents.

The DPPH assay showed a decreasing trend throughout fermentation, with C1’s scavenging capacity diminishing from 51.71% to 43.36% at 16 h. In contrast, C2 and C− maintained their DPPH scavenging ability.

In summary, the antioxidant properties of fermented blackberry juice are influenced by the presence of LAB, with C2 exhibiting more favorable behavior. The results suggest that the fermentation process gradually reduces antioxidant activity, as measured by the ORAC, FRAP, and DPPH assays, with variations between samples reflecting the specific interactions between LAB and the antioxidant compounds in blackberry juice [37].

3.5. Separation of Bacteriocins

Figure 4 shows electropherograms of the SDS-PAGE separation of bacteriocins in whey-fortified blackberry beverages throughout fermentation at 0, 8, and 16 h. Panels A, B, and C present the results obtained at 0, 8, and 16 h of fermentation.

In Figure 4A (0 h), several bands corresponding to the molecular weights of the bacteriocins in the initial samples are visible. In C1, the approximate molecular weights are 15.4 kDa, 7.9 kDa, and 5.7 kDa, while, in C2, two bands with molecular weights of 15.5 kDa and 5.6 kDa were detected. These bands indicate the presence of bacteriocins with varying molecular sizes at the beginning of fermentation. In Figure 4B (8 h), all three bacteriocins are still present in C1, suggesting that, although some degradation or modification has occurred, the bacteriocins remain intact. In contrast, C2 retains only the 5.5 kDa bacteriocin, implying that the higher molecular weight bacteriocins have been broken down or altered by the activity of LAB during fermentation.

Finally, in Figure 4C (16 h), all bacteriocins in both samples nearly disappear, indicating that, after 16 h of fermentation, the bacteria in the beverage have fully degraded or converted them into smaller or bioactive compounds. This pattern highlights the metabolic activity of LAB facilitating probiotic’s stabilization and viability through fermentation, which can break down bacteriocins and change the antimicrobial properties of the final product [38].

4. Discussion

This research demonstrated the effect of the addition of whey to a complex matrix (blackberry juice) on probiotic viability. This can be attributed to its protein and carbohydrates, which promote probiotic growth [15]. It has been reported that β-lactoglobulin and α-lactalbumin can interact with the cell membrane of L. rhamnosus GG [39], enhancing its stability under harsh conditions such as gastric acidity and bile salts [40]. This indicates that whey supplies essential nutrients and may act as a structural protector, supporting probiotic viability in fermented systems.

Our study shows an 80% survival rate of L. rhamnosus GG in BJ and BJ + WH, suggesting these matrices support probiotic viability and potentially serve as effective delivery systems. The decrease in reducing the sugar content in BJ after 48 h suggests active carbohydrate consumption by microorganisms, whereas BJ + WH showed no significant decrease, potentially due to L. rhamnosus proteolytic activity targeting whey proteins, as previously reported the liberation of free amino groups in whey after fermentation [41]. Without the addition of whey, L. rhamnosus and L. plantarum effectively metabolize simple and complex carbohydrates to produce organic acids and bioactive metabolites [42]. The significant decrease in reducing sugar in the inulin control confirms its high fermentability.

Protein hydrolysis during fermentation confirms the formation of low-molecular-weight peptides, aligning with previous studies on the proteolytic activity of LAB in dairy and plant matrices [35,43,44]. The greater protein reduction in C1 suggests increased enzymatic activity from L. plantarum, P. acidilactici, and L. brevis, promoting the release of peptides with functional properties [20].

Protein hydrolysis increased the free amino groups, indicating the formation of peptides with specific bioactive sequences. SDS-PAGE gel electrophoresis revealed a progressive breakdown of proteins during fermentation in C1 and C2, which suggests the formation of low-molecular-weight peptides with potential bioactive properties.

Several studies have demonstrated that peptides generated by LAB can exhibit antihypertensive, antioxidant, and antimicrobial effects, depending on their amino acid composition and structure [38]. The peptide profile observed in this study aligns with previous research identifying bioactive sequences from casein and β-lactoglobulin that exhibit angiotensin-converting enzyme inhibitory and antioxidant activities [44,45]. Specifically, the peptides found in sample C2 may contain sequences with strong antioxidant activity, suggesting their potential to influence metal binding activity in biological models.

The proteolytic activity during fermentation releases low-molecular-weight peptides with enhanced antioxidant potential due to the increased accessibility of specific amino acid residues like tyrosine, tryptophan, and histidine [41]. Rather than losing bioactivity, peptide degradation can result in the formation of new, functionally active sequences that contribute to the overall antioxidant capacity of the fermented matrix.

The fermentation of beverages C1 and C2 significantly increased the total acidity and acetic acid concentration while reducing the pH. These changes are associated with LAB metabolism, which generates organic acids and facilitates the enzymatic hydrolysis of serum proteins, releasing bioactive peptides [46]. Previous studies showed that fermentation-derived compounds, like phenolic metabolites and organic acids, can synergize with bioactive peptides, enhancing antimicrobial and antioxidant activities through mechanisms such as membrane destabilization and redox modulation [47,48,49]. This synergism is reflected in the fermented beverage’s functional properties, with indicators like the total acidity and acetic acid levels, serving as reliable markers of bioactivity [50]. Monitoring these parameters provides insights into fermentation’s contribution to the functional potential of the final product.

The protective effect of LAB is associated with the degradation of protein–peptide compounds, like bacteriocins, which produce peptides with biological and antioxidant properties through hydrolysis. Whey proteins provide a nitrogen source, supporting metabolism during fermentation without affecting bacteriocin stability. Probiotic microorganisms trigger various reactions, generating not only organic acids but also metabolites with biological activities. The whey protein-enriched matrix provides a favorable environment for maintaining probiotic activity [51,52,53].

The organic acid profile shows increased acetic acid production in C1 and C2, indicating active fermentation by LAB [54]. This finding aligns with previous studies emphasizing the ability of heterofermentative LAB, such as L. plantarum and L. brevis, to convert carbohydrates into organic acids like acetic and lactic acid [55]. These acids help modulate the medium’s acidity, enhancing the product’s microbiological and sensory stability [56]. The production of organic acids is closely tied to substrate availability and LAB enzymatic activity. The BJ + WH combination provided an environment rich in fermentable sugars and proteins, influencing bacterial metabolism. Recent research has shown that organic acids and bioactive peptides can work synergistically, enhancing stability and bioavailability in the gastrointestinal tract [57].

Regarding the conversion of phenolic compounds, a significant reduction in the concentration of phenolic acids, such as gallic acid and ferulic acid, was noted after fermentation, indicating their potential metabolism by lactic microbiota. Certain LABs are known to produce phenolases and esterases that can alter the chemical structure of phenols, leading to metabolites with improved biological activity [58,59]. Specifically, transforming ferulic acid into smaller phenolic compounds could enhance their solubility and antioxidant capacity, thus facilitating absorption in the small intestine [60,61].

The increase in EGCG content in all the samples (C1, C2, and C−) may be related to protein–phenol interactions and the specific LAB metabolism, although the evidence is limited. Similarly, fermented yogurt with Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus showed reduced protein–phenol affinity, facilitating the EGCG release [62].

The higher retention of phenolic compounds in consortium C1 compared to C2 and the control (C−) suggests variations in enzymatic activity among the tested microorganisms. This may indicate that certain strains have more effective mechanisms for bioconverting polyphenols into bioactive forms with improved antioxidant effects [60]. Previous studies have demonstrated that the biotransformation of phenolic compounds by LAB can enhance their antioxidant and anti-inflammatory properties, implying that the resulting fermented product could mitigate oxidative stress at the cellular level [61].

Lactobacillaceae microorganisms, including L. plantarum and L. brevis, are known for converting polyphenolic compounds. Esterases, reductases, and decarboxylases mediate the conversion of hydroxycinnamic and hydroxybenzoic acids [63]. This biotransformation is strain-specific, as reported by Montijo-Prieto et al., where L. plantarum 748T consumed p-coumaric acid through cinnamoyl esterase and decarboxylase activity, resulting in increased antioxidant activity [64].

Phenolic compounds are sensitive to pH and temperature changes during fermentation. Previous research showed that BJ + WH samples exhibit high variability in the total phenolic content at pH 6, whereas C1 and C2 were more stable at pH 3.5 [24]. Phenolic compounds have also been reported to be more stable during vegetable fermentation [65]. Additionally, they can remain stable during fermentation at 37 °C and storage at 4 °C [24,66,67].

Antioxidant activity, measured by ORAC, FRAP and DPPH, showed a slight decrease in some samples after 16 h of fermentation, but consortium C2 maintained its antioxidant capacity. This activity may be attributed to the production of antioxidant metabolites during fermentation, including transformed organic acids, phenolic compounds, and bioactive peptides released through whey protein hydrolysis [44,68]. Additionally, previous studies have indicated that peptides derived from dairy protein fermentation may exhibit antioxidant activity, primarily due to amino acid residues that effectively neutralize reactive oxygen species [43]. Specifically, residues such as tyrosine, histidine, and tryptophan have been associated with the antioxidant capacity of low-molecular-weight peptides [44].

In this context, the peptides released in sample C2 may contribute to the observed antioxidant activity, as their generation was more pronounced compared to the other samples. Several studies have shown that peptides with a high content of hydrophobic groups can interact with free radicals and help stabilize cell membranes, thereby preventing oxidative damage in biological systems [20,44].

Furthermore, the interaction between bioactive peptides and phenolic compounds in fermented systems has been recognized as enhancing antioxidant activity [69]. Some studies suggest that certain peptides can form insoluble complexes with polyphenols, shielding them from degradation and boosting their bioactivity in the gastrointestinal tract [70]. This synergy between peptides and phenolic compounds may account for the stable antioxidant activity observed in consortium C2, where a lesser decrease in antioxidant capacity was noted following fermentation.

As the antioxidant activity of peptides depends on their specific structure and amino acid sequence [34], future studies may concentrate on characterizing these peptides using mass spectrometry and evaluating their capacity to modulate antioxidant pathways in cellular and animal models. This strategy would allow for a more precise assessment of their potential in preventing diseases related to oxidative stress, including cardiovascular and neurodegenerative disorders.

The findings of bacteriocin degradation by LAB during fermentation are consistent with previous reports. Perez et al. [71] noted that LAB can release antimicrobial peptides during dairy products fermentation, indicating a similar mechanism for bacteriocins in the whey blackberry beverage [72]. This degradation process affects the stability of bacteriocins and improves the final product’s functional properties. The transformation of bacteriocins into smaller peptides that enhance bioavailability and absorption, as observed in this study, aligns with findings from other researchers, such as Abbasiliasi et al. [38], who discussed the fragmentation of high-molecular-weight proteins during fermentation, which facilitates the absorption of bioactive compounds in the intestinal tract, resulting in a product with greater therapeutic potential.

Finally, concerning the enhancement of organoleptic properties in the final product, the results of this study correspond with observations from other research, which indicates that fermentation not only enhances the functional properties of foods but also has a positive impact on their sensory characteristics [73]. In this context, Hu et al. [74] highlighted that the enzymatic activity of LAB during fermentation helps develop appealing flavors and textures, making the product more enticing to consumers. These findings strengthen the idea that manipulating bacteriocins during fermentation can positively influence both the health benefits and sensory quality of the final product.

5. Conclusions

The findings of this study highlight the potential of fermenting blackberry juice with whey as a strategy to improve probiotic viability and encourage the release of bioactive peptides. The use of specific LAB consortia promotes the generation of peptides, but the specific interactions between LAB could impact the results of the antioxidant activity. In this context, the use of C2 (L. casei and L. rhamnosus) consortia showed maintained antioxidant activity, suggesting that such products could play a valuable role in developing functional foods.

Future research should aim to identify and characterize the peptides produced during fermentation, particularly through mass spectrometry, and conduct in vivo studies to evaluate their bioavailability and biological activity. Furthermore, optimizing the LAB consortia and fermentation conditions could improve the functionality of the final product, paving the way for the development of fermented beverages with therapeutic potential and enhanced health benefits.