A Novel Wild-Type Lacticaseibacillus paracasei Strain Suitable for the Production of Functional Yoghurt and Ayran Products

Abstract

Raw goat and ewe’s milk samples were used for the isolation of seven lactic acid bacteria new strains. After testing hemolytic activity and resistance to antibiotics, specific functional properties were evaluated; Lactococcus lactis subsp. lactis FBM_1321 and Lacticaseibacillus paracasei FBM_1327 strains resulted in the highest cholesterol assimilation percentages ranging from 28.78 to 30.56%. In addition, strong adhesion capacity to differentiated Caco-2 cells (1.77–21.04%) was mapped, and the lactobacilli strains exhibited strong antagonistic activity against foodborne pathogens compared to lactococci. The strains were able to grow at low pH and high NaCl concentrations, conditions that prevail in food systems (cell counts ranged from 1.77 to 8.48 log CFU/mL after exposure to pH 3 and from 5.66 to 9.52 log CFU/mL after exposure to NaCl concentrations up to 8%). As a next step, freeze-dried immobilized Lc. paracasei FBM_1327 cells on oat flakes were used for the preparation of functional yoghurt and ayran products. Cell loads of the functional strain remained high and stable in both products (7.69 log CFU/g in yoghurt and 8.56 log CFU/g in ayran after 30 days of storage at 4 °C) throughout their shelf life. No significant changes in the volatile profile were noticed, and the new products were accepted by the panel during the sensory evaluation.

1. Introduction

Consumers increasingly adopt dietary habits aimed at improving physical and mental well-being. In this vein, probiotic [1] products have become an integral part of modern diets leading to a notable rise in probiotic industrial production, fueled by the continuous introduction of new products to the global market [2]. Additionally, consumers progressively seek less industrialized products and favor traditional, artisanal, or natural options, i.e., products made by smaller producers following traditional recipes, resulting in a unique sensory character [3]. Thus, the isolation of novel probiotic strains from fermented products, with potential applications in the fermentation of dairy products, juices, and other foods, offers new opportunities in the market. The challenge for microbial biomass producers is to maximize production efficiency and maintain cell viability over long periods (stability) [3].

Recently, there has been a global increase in obesity rates and associated diseases (diabetes, hypercholesterolemia, etc.) [4]. Scientific evidence supports the potential of functional cultures as effective supplements for the prevention and/or treatment of hypercholesterolemia [5,6], and thus, they have been tested as dietary supplements for patients with moderate to severe hypercholesterolemia, significantly reducing the risk of cardiovascular disease (CVD), which is a significant public health issue with a high mortality rate. Dyslipidemia is one of the factors linked to the onset of various CVD-related conditions. Numerous clinical studies have demonstrated a correlation between elevated cholesterol levels, particularly low-density lipoprotein cholesterol (LDL-c), and the progression of CVD. Probiotics have garnered considerable attention for their various health benefits, notably their ability to lower blood cholesterol in humans [7].

For the isolation and subsequent evaluation of presumptive beneficial strains, several issues must be explored, concerning the following aspects: (1) precise strain identification using up-to-date methods; (2) safety assessment; (3) evaluation of specific health-promoting attributes; and (4) stability control to assess cell viability throughout the product’s shelf-life [8]. Apart from a clinically proven health-promoting effect, high survival rates of the strains during the manufacturing process, handling, distribution, and storage until consumption should be achieved. It has been documented that cell immobilization in natural food ingredients, defined as a natural occurring process limiting cells to a solid matrix separated from the main liquid phase [9,10], may enhance the viability of the cultures during production, freeze-drying, and storage [10], as well as during digestion [11]. The formulation (either pill-type or food-type) process is a crucial factor; hence, physicochemical parameters may influence cell viability and functionality during production and storage. Features, such as pH, salt, oxygen concentration, water, and sugar content, and factors like the food matrix and the fermentation conditions significantly affect cell proliferation.

Immobilization of lactic acid bacteria (LAB) on cereals, such as wheat grains [12,13,14,15,16,17,18], oat flakes [11,19,20], zea [10], and wheat flakes [11], has been previously reported in the literature. Combining functional strains with a food component that contains prebiotic dietary fibers (e.g., beta-glucans) is of great importance for developing suitable delivery vehicles of health-promoting cultures. Of note, daily consumption of oat flakes that contain prebiotic [21] beta-glucans has been associated with the management of CVD by improving total cholesterol and LDL-c [22,23]. In Europe, in 2010, beta-glucans received a health claim stating that “Oat beta-glucan has been shown to lower/reduce blood cholesterol. Blood cholesterol lowering may reduce the risk of (coronary) heart disease” [24]; the daily intake is set at least 3 g of oats/day.

Several studies explored the incorporation of newly isolated probiotic strains into yogurt and other dairy products. Fermented milks and especially yogurt have been characterized as “the most popular probiotic food carrier” [25], leading to numerous studies focusing on the incorporation of probiotics in such products [20,26,27,28].

Our study aimed at isolating wild-type lactic acid bacteria (LAB) with potential functional properties from raw goat and ewe’s milk, exploring their use as adjunct cultures. After cell immobilization of a selected Lc. paracasei strain on oat flakes, production of functional fermented yoghurt and ayran products was tested. Data suggesting the suitability of the immobilized cells as a functional ingredient are presented.

2. Materials and Methods

2.1. Isolation and Identification of New LAB from Goat and Ewe’s Milk

2.1.1. Isolation of LAB

Samples of goat and ewe’s milk (freshly harvested from farmers of the Eastern Macedonia and Thrace Region in Greece) were collected and stored at 4 °C. Five milliliters of each milk sample were aseptically introduced into a sterile (121 °C for 15 min) De Man−Rogosa−Sharpe (MRS) (Condalab, Madrid, Spain) or M17 broth (Condalab), and incubated at 37 °C for 48–72 h. After incubation, approximately 1 mL of each sample was transferred to test tubes containing sterile PPS (Peptone Physiological Solution, 0.1% peptone, and 0.85% NaCl in deionized water). The samples were homogenized by gentle agitation, serially diluted and plated on MRS agar (Condalab) and M17 agar (Condalab). Plates were incubated at 37 °C under anaerobic conditions (2.5 L anaerobic jar; Sachets Merck Millipore, Darmstadt, Germany) (for lactobacilli) or 30 °C (for lactococci) for 72 h. Colonies were recorded based on color, size, texture, and shape, and individual colonies were randomly selected. These colonies were spread on MRS and M17 agar and re-incubated under the same conditions until clear single colonies were observed. Each distinct colony was collected, inoculated into tubes containing MRS or M17 broth supplemented with 20% (v/v) glycerol and stored at −20 °C. Initial characterization of the novel strains was carried out using the API50CH biochemical system (BioMèrieux, La Balme-les-Grottes, France).

2.1.2. Molecular Identification Using Multiplex PCR Assays

Bacterial DNA extraction was performed using the NucleoSpin™ Tissue kit (Macherey-Nagel GmbH & Co. KG, Düren, Germany) following the manufacturer’s instructions. Identification of lactobacilli was based on species-specific multiplex PCR targeting the following groups: (a) the Lacticaseibacillus spp. group (species Lacticaseibacillus casei, Lacticaseibacillus paracasei, and Lacticaseibacillus rhamnosus) [29]; and (b) the Lactiplantibacillus spp. group (species Lactiplantibacillus plantarum, Lactiplantibacillus paraplantarum, and Lactiplantibacillus pentosus) [30]. Likewise, lactococci were identified using species-specific multiplex PCR targeting Lactococcus spp. [31]. PCRs were performed using an Eppendorf 5333 MasterCycler Thermal Cycler (Hamburg, Germany) and the PCR KAPA SYBR^® FAST qPCR Master Mix (2×) universal kit (Kapa Biosystems Inc., Wilmington, MA, USA) in a 20 μL reaction volume. PCR products were visualized after electrophoresis on a 2% w/v agarose gel stained with ethidium bromide.

For species identification of the Lacticaseibacillus spp. group, the mutL genetic loci were targeted using the following primers: CZ (5′-CAGCGCTGGTGGAAGACTTG-3′), PC2a (5′-GGATTGGGTTTTGCGTGATGGTCGC-3′), and RHfor (5′-GACTTCTCAACCAGCAGCGCAGA-3′) for Lc. casei, Lc. paracasei, and Lc. rhamnosus, respectively, and the reverse primer CPRrev (5′-TGCATTTCCCCGCTTTCATGACT-3′) that resulted in the production of specific products: 666 bp for Lc. casei, 253 bp for Lc. paracasei, and 801 bp for Lc. rhamnosus [29]. Similarly, for identification of the Lactiplantibacillus strains, the genetic loci recA were targeted using the primers: planF (5′-CCGTTTATGCGGAACACCTA-3′), paraF (5′-GTCACAGGCATTACGAAAAC-3′), and pentF (5′-CAGTGGCGCGGTTGATATC-3′) for Lp. plantarum, Lp. pentosus, and Lp. paraplantarum, respectively, and the reverse primer pREV (5′-TCGGGATTACCAAACATCAC-3′). These combinations produced PCR products as follows: 318 bp for Lp. plantarum, 218 bp for Lp. pentosus, and 107 bp for Lp. paraplantarum [30]. Finally, for Lactococcus spp. species identification was accomplished using the primers LcLspp-F (5′-GTTGTATTAGCTAGTTGGTGAGGTAAA-3′), Lc-R (5′-GTTGAGCCACTGCCTTTTAC-3′), LcCr-F (5′-TGCTTGCACCAATTTGAAGAG-3′), and Lc-R (5′-GTTGAGCCACTGCCTTTTAC-3′) that targeted a specific region of the 16s rRNA gene, producing the following products: 387 bp for Lactococcus lactis subsp. lactis and 551 bp for Lactococcus lactis subsp. lactis cremoris [31].

2.2. Microbial Cultures and Culture Conditions

All lactobacilli isolates, as well as Lc. rhamnosus ATCC 53103 (GG), Lp. paracasei DSM 20008, and Lp. plantarum 20174, which were used as reference strains, were cultured in MRS broth and incubated at 37 °C for 24 h. All lactococci isolates were cultured in M17 broth and incubated at 30 °C for 24 h.

To evaluate the antagonistic activity of isolates against foodborne pathogens, the following strains were used: Salmonella enterica subsp. enterica ser. Enteritidis PT4, Listeria monocytogenes NCTC 10527 serotype 4b, Clostridioides difficile, and Escherichia coli ATCC 25922. The strains were grown in Brain Heart Infusion (BHI) broth (Condalab) and incubated at 37 °C for 24 h, except C. difficile, which was incubated at 37 °C for 48 h.

For the manufacturing of yogurt and ayran products, the YC-381 (Novenesis, Lyngby, Denmark) starter culture, consisting of Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, was used.

2.3. In Vitro Safety Assessment

2.3.1. Hemolytic Activity

The hemolytic activity of the isolates was evaluated following the method described by Nelios et al. [32]. The presence of surrounding zones (α- and β-hemolysis) was recorded after incubation at 37 °C for 48 h.

2.3.2. Antibiotic Susceptibility

The antibiotic susceptibility of the isolates was assessed following the broth microdilution method [32]. Phenotypic antibiotic resistance was evaluated based on cut-off values for ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, clindamycin, tetracycline, and chloramphenicol reported by the European Food Safety Authority (EFSA) for Lactobacillus spp. and Lactococcus spp. [33].

2.4. In Vitro Evaluation of Probiotic Properties

2.4.1. Cell Surface Hydrophobicity and Auto-Aggregation

The bacterial adhesion to hydrocarbons test (BATH), described by Collado et al. [34], was used to assess the hydrophobicity of the isolates, using xylene as a solvent. Hydrophobicity (%) was calculated following the formula:

H (%) = [(A₀ − A)/A₀] × 100,

where A₀ and A refer to the optical density before and after the incubation with xylene, respectively.

The auto-aggregation ability of LAB strains was assessed according to the method described by Li et al. [35]. The bacterial suspensions were incubated at 37 °C (lactobacilli) or 30 °C (lactococci), and the absorbance at 600 nm was measured at 0 and 5 h. The auto-aggregation (%) was calculated using the formula:

A (%) = [(A₀ − A)/A₀] × 100,

where A₀ and A refer to the optical density before and after the incubation, respectively.

2.4.2. Adhesion to Differentiated Caco-2 Cells

The adhesion ability of the isolates was further investigated using the human colon cancer cell line Caco-2 (ATCC, Manassas, VA, USA) as a model surface, following the method of Lappa et al. [36]. The cell loads of the strains were calculated by microbiological analysis. The bacterial adhesion (%) to the differentiated Caco-2 cell layer was estimated as the ratio of the number of bacterial cells that remained attached to the total number of bacterial cells initially added to the Caco-2 monolayer.

2.4.3. Bile Salt Hydrolase (BSH) Activity

The bile salt hydrolase (BSH) activity of the isolates was determined using the methodology described by Bosch et al. [37]. Sterile paper disks impregnated with a freshly grown culture of each isolate were placed on MRS (for lactobacilli) or M17 agar plates (for lactococci) supplemented with 5 g/L of sodium salt of taurodeoxycholic acid (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and 0.37 g/L CaCl₂. Plates were incubated at 37 °C anaerobically for lactobacilli and at 30 °C for lactococci, and the presence of precipitation zones was reported.

2.4.4. In Vitro Cholesterol Assimilation

BSH-positive isolates were evaluated for cholesterol removal, following the method of Lappa et al. [36]. A standard curve was prepared using the following concentrations: 50, 100, 125, 166.6, 250, and 500 μg/mL Cholesterol-PEG 600 (Sigma-Aldrich) in MRS broth (R² = 0.99), and cholesterol assimilation was determined using the following equation:

Cholesterol assimilation (%) = [(C₀ − C₁)/C₀] × 100,

where C₀ and C₁ represent the uninoculated and inoculated MRS or M17 broth—cholesterol-PEG 600, respectively.

2.4.5. Antagonistic Activity of Isolates Against Foodborne Pathogens Using a Co-Culture Assay

The antagonistic activity of the isolates against pathogens was evaluated using a co-culture assay described by Wittman et al. [38]. After co-cultivation of the isolates with each pathogen separately, the cell viability was determined by 10-fold serial dilution and plate counting on MRS or M17 agar for the isolates, Listeria agar base Palcam ISO (Condalab) supplemented with Palcam Listeria Selective Supplement (Condalab) for Lis. monocytogenes, MacConkey Agar EP/USP/ISO (Condalab) for E. coli and S. Enteritidis, and Tryptose Sulfite Cycloserine Agar (Condalab) supplemented with Egg yolk Tellurite (Condalab) and D-cycloserine (Condalab) under anaerobic conditions for C. difficile. Cell viability was expressed as Log CFU/mL.

2.4.6. Survival After In Vitro Digestion

To evaluate the survival of the isolates in conditions that simulated the digestive system, a static in vitro digestion model was performed according to Nelios et al. [32]. Cell loads of the isolates were determined at the beginning and at the end of every simulated phase by microbiological analysis, and the survival rates of the isolates were calculated using the following formula:

Survival rate (%) = [Log CFU(a)/Log CFU(b)] × 100,

where Log CFU(a) and Log CFU(b) refer to cell loads after incubation at a specified digestion phase and before the beginning of the simulated digestion, respectively.

2.5. In Vitro Evaluation of Technological Properties

2.5.1. pH Tolerance

The low pH tolerance of the wild-type strains was tested following Ng et al. [39]. In brief, the grown culture of each strain was centrifuged (8500× g, 10 min, and 4 °C), and the cells were resuspended in a sterile PBS solution. Then, pH 2.0 and 3.0 PBS solutions were inoculated with cultures (initial concentration of 10⁸ CFU/mL), while a pH 7.0 PBS solution was used as a positive control. Cell viability of the cultures was determined after 0, 1, 2, and 3 h of incubation at 37 °C (for lactobacilli) under anaerobic conditions and 30 °C (for lactococci) after microbiological analysis. The results are expressed as Log CFU/mL.

2.5.2. NaCl Tolerance

The LAB strains were tested for resistance to osmotic stress using the protocol previously described by Ng et al. [39]. Briefly, an MRS or M17 broth medium (for lactobacilli under anaerobic conditions or lactococci, respectively) supplemented with various concentrations of NaCl (1, 4, and 8% w/v) was inoculated with grown cultures of each strain (10⁷ CFU/mL initial inoculum). At the same time, MRS or M17 broths without NaCl served as controls. After 24 h incubation at 37 or 30 °C (for lactobacilli under anaerobic conditions or lactococci, respectively), cell viability was determined, and the results were expressed as Log CFU/mL.

2.6. Immobilization of Lc. paracasei FBM_1327 Cells on Oat Flakes Followed by Freeze-Drying

Prior to the production of fermented dairy products, freeze-dried immobilized Lc. paracasei FBM_1327 cells on oat flakes were prepared, as previously described [10,11]. In brief, Lc. paracasei FBM_1327 was grown in a sterile (food grade) medium developed by our research group (unpublished data) with the following composition at 37 °C for 24 h: glucose (20 g/L), yeast extract (25 g/L), KH₂PO₄ (2 g/L), CH₃COONa (6 g/L), MgSO₄ (0.3 g/L), and MnSO₄ (0.005 g/L). The initial pH was set to 6.5 using baking soda. Following centrifugation (8500× g, 10 min, and 4 °C), the cells were washed with a ¼ Ringer’s solution.

Before immobilization, the oat flakes (used as the immobilization support) were pre-heated at 140 °C for 30 min to eliminate microbial load. The cell biomass was dissolved in a ¼ Ringer’s solution (9.32 Log CFU/mL), combined with the oat flakes (50% w/v) and incubated at room temperature for 30 min. The wet immobilized Lc. paracasei FBM_1327 cells on oat flakes were then strained and washed with a ¼ Ringer’s solution to remove non-immobilized cells. In the following step, the wet immobilized cells were frozen at −80 °C for 18 h and freeze-dried (30–35 Pa, −101 °C) for 36 h in a BenchTop Pro (Virtis, SP Scientific, Warminster, PA, USA) freeze-dryer. For comparison purposes, wet and freeze-dried free (non-immobilized) cells were also produced.

2.7. Production of Dairy Products with Lc. paracasei FBM_1327 as Adjunct Cultures

2.7.1. Yoghurt Production

Yoghurt products were prepared according to Dimitrellou et al. [26]. Yoghurts containing wet or freeze-dried immobilized Lc. paracasei FBM_1327 cells on oat flakes were prepared (Y_WIO and Y_FDIO). For comparison purposes, products with the YC-381 culture alone (YC) or with the YC-381 culture supplemented with oat flakes (without immobilized cells) (YC_O), and products with free Lc. paracasei FBM_1327 cells (wet: Y_WF; freeze-dried: Y_FDF) were also produced. In brief, fresh full fat pasteurized milk (Evrofarma S.A., Didymoteicho, Greece) (3.6 g protein, 3.5 g fat, and 4.5 g carbohydrate per 100 mL) was heated to 42 °C, and free or immobilized Lc. paracasei FBM_1327 cells on oat flakes were added to achieve a concentration of 10 Log CFU/100 mL of milk (2 g of wet immobilized cells (5 × 10⁹ CFU/g)/100mL, or 13 g of freeze-dried immobilized cells (7.60 × 10⁸ CFU/g)/100 mL, or 0.59 g of wet free cells (1.7 × 10¹⁰ CFU/g)/100 mL, or 0.052 g of freeze-dried free cells (1.9 × 10¹¹ CFU/g)/100 mL were added). After 20 min, the mixture was inoculated with a YC-381 commercial culture (0.05% w/v), followed by fermentation at 42 °C to a final pH value of ~4.5 (approximately 3–3.5 h). Then, the products were transferred to 4 °C and stored at a refrigerated temperature (4 °C) for 30 days in sterile plastic cups [26].

2.7.2. Ayran Production

Ayran products were prepared according to Akal et al. [40]. Ayran containing wet and freeze-dried cells of Lc. paracasei FBM_1327 immobilized on oat flakes was prepared (A_WIO and A_FDIO). For comparison purposes, products with the YC-381 culture alone (AC), or with the YC-381 culture supplemented with oat flakes (without immobilized cells) (AC_O), and products with free cells of Lc. paracasei FBM_1327 (wet: A_WF; freeze-dried: A_FDF) were also produced. Briefly, fresh pasteurized, semi-skimmed cow’s milk (Farm Koukakis SA, Kilkis, Greece) (3.3 g protein, 1.5 g fat, and 4.7 g carbohydrate per 100 mL) was heated to 42 °C, and free or immobilized Lc. paracasei FBM_1327 cells on oat flakes were added (10 Log CFU per 100 mL of milk, as stated above). After 20 min, the samples were inoculated with a YC-381 culture (0.05% w/v), followed by fermentation at 42 °C to a final pH value of ~4.5 (approximately 3–3.5 h). After incubation, the products were diluted with sterile water (1:1) and mixed well, and NaCl (0.1% w/v) was added [41]. The products were then transferred to 4 °C and stored for 30 days in sterile glass bottles.

2.8. Analyses

2.8.1. Scanning Electron Microscopy

Cell immobilization of Lc. paracasei FBM_1327 on oat flakes was confirmed through scanning electron microscopy (SEM) as described previously [11,42].

2.8.2. Physicochemical Analyses

Determination of pH

The pH of yoghurt and ayran products was determined using a pH-300i pH-meter (WTW GmbH, Weilheim, Germany), according to the manufacturer’s instructions.

Titratable Acidity (TA)

The titratable acidity (TA) of the yogurt and ayran products was determined according to the method described by Matela et al. [43] and expressed as lactic acid percentage (% w/w) using the following equation:

TA (%) = [(volume of NaOH solution (mL) × 1N NaOH × equivalent weight of lactic acid)/weight of original sample (g)] × 100.

Results were reported as % lactic acid [44].

Quantification of Residual Sugars and Organic Acids by HPLC

The sugar and organic acid content in yoghurt and ayran products were quantified using High Performance Liquid Chromatography (HPLC), as described by Leite et al. [45] and Nikolaou et al. [46]. Samples were acidified and homogenized (10 mL product in 10 mL H₂SO₄ 0.15 mM), centrifuged (9000× g, 4 °C, and 20 min) and treated with of trichloroacetic acid (1.2 mL of 85% w/v trichloroacetic acid in 10 mL of sample). After cooling on ice for 45 min, protein precipitation was completed after centrifugation (17,000× g, 20 °C, and 20 min). The supernatant was filtered twice through a 0.22 µm nylon filter, and the samples were stored at −20 °C until analysis.

Quantification of sugars (glucose, lactose, and galactose) and organic acids (lactic acid and citric acid) was performed using a Shimadzu system (Shimadzu Corp., Duisburg, Germany), equipped with a Nucleogel ION 300 OA column (Macherey-Nagel GmbH & Co. KG), a degassing unit DGU-20A5R, an LC-20AD pump, a CTO-20AC oven at 85 °C, and an RID-10A refractive index detector. Briefly, a 0.049 g/L H₂SO₄ solution was used as the mobile phase at a flow rate of 0.3 mL/min, and 20 µL of each sample was injected directly onto the column. Cell temperature was set at 60 °C. Concentrations were calculated using a standard curve constructed from commercial standards (R² ≥ 0.99).

Minor Volatile Analysis by HS-SPME GC/MS

Identification and semi-quantification of the minor volatiles of the yoghurt and ayran samples collected after 20 days of storage was performed on an HS-SPME GC/MS system (6890N GC, 5973 Networked MS MSD, HP-5MS column (30 m, 0.25 mm i.d., and 0.25 μm film thickness); Agilent Technologies, Santa Clara, CA, USA) using 4-methyl-2-pentanol as an internal standard. The analysis was performed as described elsewhere [47].

2.8.3. Microbiological Analyses

Cell enumeration of the isolates throughout the in vitro assays was determined by 10-fold serial dilutions and plate counting on MRS agar at 37 °C under anaerobic conditions for lactobacilli and on M17 agar at 30 °C for 72 h for lactococci.

Cell levels of wet and freeze-dried, free, or immobilized Lc. paracasei FBM_1327 cells were also determined after 10-fold serial dilutions and plating on MRS agar followed by incubation at 37 °C for 72 h in anaerobic conditions.

The microbiological analyses of yoghurt and ayran were carried out one day post-production (day 1) and during storage at various intervals (days 10, 20, and 30). Samples (10 g) were homogenized in 90 mL of a sterile ¼ Ringer’s solution followed by serial decimal dilutions. The population of Lc. paracasei FBM_1327 cells was determined by plating on MRS Agar (Condalab) and incubation at 37 °C for 72 h under anaerobic conditions. S. thermophilus loads were quantified by plating on M17 agar (Condalab) at 37 °C for 48 h, and L. delbrueckii subsp. bulgaricus loads were quantified by plating on modified MRS agar (pH adjusted to 4.3) at 37 °C for 48 h in anaerobic conditions.

To assess possible microbial contaminations during storage, the levels of the following species were also determined:

(a) Total aerobic counts in Plate Count Agar (Condalab) after incubation at 30 °C for 96 h; (b) Enterobacteriaceae on Violet Red Bile Glucose Agar (Condalab) after incubation at 37 °C for 24 h; (c) coliforms on Violet Red Bile Agar (Condalab) after incubation at 37 °C for 24 h; (d) staphylococci in Baird Parker Medium Base (Condalab) after incubation at 37 °C for 48 h; (e) yeasts/fungi in Malt Agar (Condalab) after incubation at 30 °C for 96 h; (f) Pseudomonas spp. on Pseudomonas CFC selective agar (Condalab) after incubation at 30 °C for 72 h; and (g) Salmonella spp. in Xylose-Lysine-Deoxycholate Agar (VWR) after incubation at 37 °C for 48 h.

In all cases, cell concentration was expressed as Log CFU/g or Log CFU/mL.

2.8.4. Sensory Evaluation

The new products were evaluated for their sensory characteristics by 13 untrained participants (7 women and 6 men aged 21–50 years), familiar with the consumption of dairy products. The organoleptic evaluation was conducted on the 1st day of storage, with yoghurt samples offered in plastic containers and ayran products in 50 mL glass bottles. All samples were coded with random three-digit numbers and served in random order to each participant. During the testing, the participants were asked to cleanse their palate with unsalted crackers and tap water. The products were assessed for their aroma, taste, consistency, color, and overall quality on a 0–5 point scale, ranging from 0 (unacceptable) to 5 (very good).

2.9. Statistical Analyses

A schematic flowchart of the experimental design is presented in Figure 1.

All experiments were repeated at least in triplicates. Results were analyzed by Analysis of Variance (ANOVA), and significant differences were determined by Tukey’s post-hoc test (ANOVA tables and significance levels (p < 0.05) were calculated by Statistica v.12 software). XLSTAT 2024.3.0.1423 was used to create the principal component analysis (PCA).

3. Results and Discussion

The aim of the present study was to test the suitability of a functional food ingredient consisting of immobilized cells of a newly isolated wild-type strain, previously tested for potential functional properties, ensuring cell loads over the minimum required levels for conferring a beneficial effect throughout the products’ self-life.

3.1. Isolation and Molecular Identification of Isolated Strains

Milk samples were used as sources to isolate LAB. Multiple strains were isolated and kept in an initial stock. However, considering their potential use as adjuncts or functional cultures in fermented milk products, seven strains were selected for further evaluation. The seven strains isolated from goat and ewe’s milk were Gram-positive bacteria and catalase-negative. According to the multiplex PCR verification, the isolates were identified as Lactococcus lactis subsp. lactis (FBM_1321 and FBM_1331), Lc. paracasei (FBM_1327), Lp. plantarum (FBM_1422), and Lc. rhamnosus (FBM_1423, FBM_1433, and FBM_1434) (Supplementary Figure S1).

3.2. In Vitro Safety Assessment of Hemolysis and Antibiotic Susceptibility

In order to use a new isolate as candidate strain in the food industry, it should be firstly assessed for its safety. One important safety criterion is its hemolytic activity. According to the results, the newly isolated strains were considered as γ-hemolytic, as no zones on blood agar indicating α- or β-hemolysis were observed.

The next step concerned the evaluation of antibiotic susceptibility of the wild-type isolates, according to the recommendations of EFSA [33]. More specifically, potential resistance to vancomycin, gentamicin, kanamycin, streptomycin, tetracycline, erythromycin, clindamycin, chloramphenicol, and ampicillin was assessed, and the minimum inhibitory concentration (MIC) of the antibiotics against cell growth was determined (

Table 1. Minimum inhibitory concentration (MIC) values of antibiotics (μg/mL) against cell growth of wild-type strains.

Isolate	AM	CN	Chl	E	G	S	T	V	K
Lactococcus lactis FBM_1321	0.5	0.25	4	1	8	8	0.5	1	32
Lc p.aracasei FBM_1327	1	1	8 R	1	16	8	1	N.R. 1	32
Lactococcus lactis FBM_1331	0.5	0.125	8	0.5	8	32	0.5	1	32
Lp. plantarum FBM_1422	0.5	4 R	8	2 R	64 R	N.R. 1	16	N.R. 1	512 R
Lc. rhamnosus FBM_1423	2	2 R	16 R	1	32 R	32	1	N.R. 1	256 R
Lc. rhamnosus FBM_1433	1	2 R	8 R	1	32 R	16	1	N.R. 1	128 R
Lc. rhamnosus FBM_1434	1	2 R	8 R	1	128 R	128 R	1	N.R. 1	256 R

¹ Not required [33]. AM, ampicillin; CN, clindamycin; Chl, chloramphenicol; E, erythromycin; G, gentamicin; S, streptomycin; T, tetracycline; V, vancomycin; K, kanamycin; ^R, resistant to antibiotics according to the cut-off values by EFSA [33].

).

According to the results, all strains of the genus Lactococcus, isolated from goat milk, were characterized as susceptible to all antibiotics assessed. Lp. plantarum FBM_1422 was resistant to clindamycin, erythromycin, gentamicin, and kanamycin. The strain Lc. paracasei FBM_1327 was resistant to chloramphenicol, while the strains Lc. rhamnosus FBM_1423, Lc. rhamnosus FBM_1433, and Lc. rhamnosus FBM_1434, originating from ewe’s milk, were resistant to clindamycin, chloramphenicol, gentamicin, and kanamycin. Lc. rhamnosus FBM_1434 was also characterized as resistant to streptomycin. Resistance to aminoglycosides, such as gentamicin, kanamycin, and streptomycin, is due to the lack of cytochrome-mediated electron transfer and is, therefore, considered an intrinsic property [48]. In addition, several studies have reported resistance to chloramphenicol by Lp. plantarum and Lc. rhamnosus strains, which can be attributed to the presence of specific genes, such as the cat gene, but may also result from reduced expression of several genes, including genes related to oxidative stress, as well as genes encoding outer membrane proteins [48]. Nevertheless, antibiotic resistance is a major selection parameter, since resistance genes located on plasmids or transposable genetic loci have a high risk of transfer to the normal human microbial flora or to potentially pathogenic microbes that colonize the gut, resulting in horizontal spread of antibiotic resistance genes [33,49].

3.3. In Vitro Evaluation of Functional Properties

Resistance to gastric conditions, adhesion ability, and antagonistic activity are considered significant attributes in association with potential beneficial effects on health, such as reduction of cholesterol levels in blood [50], which can be evaluated in vitro.

3.3.1. Cell Surface Hydrophobicity and Auto-Aggregation

The adhesion of bacteria to intestinal epithelial cells is a complex process and is considered a significant property for beneficial bacteria, promoting colonization and proliferation in the gut, in order to exert their functional properties. The bacterial adhesion to hydrocarbons test has been extensively used as a tool to evaluate this trait, as adhesion ability is thought to be associated with the composition of the cell surface. It has been suggested that the presence of (glycol) proteinaceous material in cell surfaces is linked to higher hydrophobicity, whereas the presence of polysaccharides leads to more hydrophilic surfaces [34,51]. At the same time, cell aggregation of the same strain (auto-aggregation) is of considerable importance for the colonization of bacteria in the gut. In this vein, the hydrophobicity and auto-aggregation ability of the newly isolated strains was assessed, and the results are presented in

Table 2. Species identification, adhesion properties (hydrophobicity, auto-aggregation, and adhesion to cell line of Caco-2), BSH activity, and cholesterol assimilation dynamic of the newly isolated wild-type strains.

Isolates Code	Bacterial Species (after Multiplex PCR)	Source of Isolation	Hydrophobicity (%)	Auto-Aggregation (%)	Adhesion to Caco-2 (%)	BSH Activity *	Cholesterol Removal (%)
FBM_1321	Lactococcus lactis	Goat milk	38.70 ± 0.21	8.25 ± 0.04	20.76 ± 2.12	+	30.56 ± 0.11
FBM_1327	Lc. paracasei	Goat milk	13.81 ± 0.10	22.31 ± 0.15	6.87 ± 0.13	+	28.78 ± 0.15
FBM_1331	Lactococcus lactis	Goat milk	68.31 ± 0.45	11.22 ± 0.08	21.04 ± 0.80	-	-
FBM_1422	Lp. plantarum	Εwe milk	28.22 ± 0.15	17.04 ± 0.05	9.86 ± 0.71	+	1.20 ± 0.02
FBM_1423	Lc. rhamnosus	Εwe milk	3.65 ± 0.04	9.88 ± 0.03	1.77 ± 0.77	+	0.17 ± 0.01
FBM_1433	Lc. rhamnosus	Εwe milk	77.84 ± 0.22	28.91 ± 0.04	17.10 ± 0.64	+	4.67 ± 0.02
FBM_1434	Lc. rhamnosus	Εwe milk	4.22 ± 0.08	10.11 ± 0.06	5.45 ± 0.92	+	2.22 ± 0.01

* “+” suggests the presence of a precipitation zone indicating the ability of the isolates to secrete BSH.

.

A great heterogeneity was noticed among the strains in the hydrophobicity percentages. The highest (p < 0.05) percentage of hydrophobicity was observed by the strain Lc. rhamnosus FBM_1433 (77.84 ± 0.22%), while the lowest was exhibited by Lc. rhamnosus FBM_1423 and Lc. rhamnosus FBM_1434 strains (3.65 ± 0.04 and 4.22 ± 0.08%, respectively). Similar hydrophobicity rates were reported in similar studies, evaluating wild-type bacteria [34,35,51]. Indicatively, high rates of hydrophobicity (>50%) have been recorded for Lc. rhamnosus species [52]. The results concerning the auto-aggregation ability of isolated demonstrated variability in accordance with hydrophobicity levels observed. The auto-aggregation percentage of the strains ranged from 8.25 to 28.91%. The highest (p < 0.05) auto-aggregation capacity was recorded by Lc. rhamnosus FBM_1433 (28.91 ± 0.04%), while the lowest (p < 0.05) percentage was noted by Lactococcus lactis subsp. lactis FBM_1321 (8.25 ± 0.04%). The above results are in agreement with previous studies evaluating the ability of LAB to form aggregates [53,54].

3.3.2. Adhesion to Differentiated Caco-2 Cells

It is suggested that functional cultures should be capable of at least temporary colonization on the gut mucosa. In this vein, adhesion to the Caco-2 cell line was used to further evaluate the interaction between the wild-type strains and the surface of human colorectal adenocarcinoma cells of intestinal mucosal origin. The results are shown in Table 2.

Adhesion levels of the bacterial strains studied ranged from 1.77 to 21.04%. The highest adherence rates (p < 0.05) were recorded by Lactococcus lactis subsp. lactis FBM_1331 (21.04 ± 0.80) and Lactococcus lactis subsp. lactis FBM_1321 (20.76 ± 2.12), while the lowest levels (p < 0.05) were observed in the Lc. rhamnosus FBM_1423 strain (1.77 ± 0.77). The adhesion capacity of the strains varied and was not species-related, also verified by other studies [55,56].

3.3.3. Bile Salt Hydrolase (BSH) Activity

The secretion of the BSH enzyme by LAB cells is associated with a reduction in cholesterol levels in the intestinal lumen. Therefore, the isolated strains were assessed for their BSH activity, and the results are presented at Table 2.

3.3.4. In Vitro Cholesterol Assimilation

The development of coronary heart disease and colon cancer has been associated with high cholesterol levels in blood and a diet rich in fat. Lately, increasing attention on the cholesterol-lowering potential of LAB has been witnessed [57], since medications prescribed for cholesterol lowering could have side effects for patients. For this reason, the isolated strains were evaluated in vitro for their ability to reduce cholesterol levels, and the results are shown in Table 2.

Τhe percentage of cholesterol removal ranged from 0.17 to 30.56%. The greatest (p < 0.05) reduction in cholesterol levels was recorded by the strain Lactococcus lactis subsp. lactis FBM_1321 (30.56 ± 0.11%), isolated from goat milk. A cholesterol lowering capacity of 28.78 ± 0.15% was recorded for Lc. paracasei FBM_1327 strain, while the lowest (p < 0.05) percentage of cholesterol reduction was observed by the isolates Lc. rhamnosus FBM_1423 and Lp. plantarum FBM_1422 (0.17 ± 0.01 and 1.20 ± 0.02, respectively). These results are in agreement with similar studies on the in vitro ability of LAB to reduce cholesterol levels [50,58,59,60]. In particular, a high cholesterol-lowering capacity (43.70%) has been recorded by Lactococcus lactis strains isolated from a traditional drink produced from cereals [58], as well as by Lc. paracasei strains (>50%) [50].

3.3.5. Antagonistic Activity of Wild-Type Isolates Against Spoilage and Foodborne Pathogens

LAB have been receiving increasing attention for their ability to inhibit the growth of spoilage and foodborne pathogenic bacteria, a key characteristic with many prospects for their use as alternative biopreservative agents.

According to the results of the co-culture assay (

Table 3. Cell loads (Log CFU/mL) of foodborne pathogens after co-cultivation with the newly isolated wild-type strains.

Isolate	C. difficile	Lis. monocytogenes	S. Enteritidis	E. coli
Lactococcus lactis FBM_1321	6.67 ± 0.03	8.48 ± 0.01	8.76 ± 0.04	8.94 ± 0.01
Lc. paracasei FBM_1327	5.67 ± 0.05	5.53 ± 0.05	6.46 ± 0.03	5.32 ± 0.05
Lactococcus lactis FBM_1331	5.92 ± 0.04	8.23 ± 0.04	8.68 ± 0.05	8.85 ± 0.03
Lp. plantarum FBM_1422	4.16 ± 0.08	5.04 ± 0.02	4.61 ± 0.02	6.59 ± 0.02
Lc. rhamnosus FBM_1423	7.56 ± 0.07	5.37 ± 0.01	7.22 ± 0.06	7.25 ± 0.05
Lc. rhamnosus FBM_1433	4.42 ± 0.01	5.57 ± 0.04	4.61 ± 0.03	7.56 ± 0.01
Lc. rhamnosus FBM_1434	5.88 ± 0.04	6.40 ± 0.03	5.37 ± 0.01	7.59 ± 0.01
Growth control (of pathogen)	9.09 ± 0.01	9.08 ± 0.01	8.79 ± 0.02	9.05 ± 0.11

), all strains exhibited a significant (p < 0.05) antagonistic effect on the growth of C. difficile. Strong inhibitory activity of the majority of the isolates against Lis. monocytogenes was noted, as co-cultivation of the pathogen with each isolate separately led to significant reduction (p < 0.05) of cell levels (5.04–6.40 Log CFU/mL), except from Lactococcus lactis subsp. lactis FBM_1321 and Lactococcus lactis FBM_1331, which had no significant (p > 0.05) effect on pathogen growth. In the case of Gram-negative pathogens tested, only the isolates of the genus Lactobacillus exhibited strong (p < 0.05) antagonistic activity. More specifically, the final cell levels of E. coli and S. Enteritidis (after cultivation) ranged from 5.32 to 7.59 Log CFU/mL and from 4.61 to 7.22 Log CFU/mL, respectively, after incubation with the presence of lactobacilli. The newly isolated lactococci had no effect (p > 0.05) on the viability of these pathogens. Regarding the nature of the antimicrobial and antagonistic activity of LAB, these results could be attributed to the production of antimicrobial compounds, such as organic acids, hydrogen peroxide, and bacteriocins. Indeed, lactococci are well known for their ability to produce bacteriocin-like compounds [61]. However, it has been suggested that these antimicrobial agents are more effective on Gram-positive bacteria, while some Gram-negative bacteria seem to be resistant to such compounds [62,63].

3.3.6. Survival After In Vitro Digestion

As the conditions that are prevailing during the passage through the gastrointestinal (GI) tract may affect cell viability, but at the same time, the colonization of functional microbes in the gut is a prerequisite to exert their beneficial effects, a static in vitro digestion assay was followed to mimic digestion phases and evaluate the survival potential of four isolates with higher potential, according to the results demonstrated above. The results are presented in Figure 2.

Exposure to simulated salivary fluid (SSF), which consisted of 50 U/mL lysozyme, 75 U/mL α-amylase, and H₂O₂, had no significant effect (p > 0.05) on the viability of the tested isolates, as survival rates up to 99% were recorded. However, the incubation of strains in a solution that simulated the gastric juice (SGF) led to a reduction in cell loads, and the survival rates ranged from 70.97 to 90.84%. Further exposure to simulated intestinal fluid (SIF) resulted in even lower (p < 0.05) survival rates (41.63–55.56%). Lc. rhamnosus FBM_1433 exhibited the highest survival rates through all digestion phases, while Lactococcus lactis subsp. lactis FBM_1321 was more sensitive to acid environments and at the presence of bile salts. Similar results have been demonstrated by several studies evaluating the tolerance of LAB to conditions that mimic the passage through the GI tract [64,65]. In particular, Feng et al. [65] reported that the simulated oral solution had no effect on the viability of the tested isolates, while Nelios et al. [32] demonstrated that, for Lc. rhamnosus strains, a survival rate of <40% was observed after incubation in a gut-simulating solution.

3.4. In Vitro Evaluation of Technological Properties

The ability of a presumptive functional strain to survive under the harsh conditions of a specific food system, such as low pH and high salt concentrations, is considered an essential criterion for its suitability as a starter or adjunct culture.

3.4.1. pH Tolerance

As the ability of LAB to survive under low pH conditions is often used as a primary criterion for selecting suitable strains for dairy products, the wild-type strains were evaluated for their ability to survive in acidic conditions (

Table 4. Cell levels (Log CFU/mL) of newly isolated wild-type strains after incubation at different pH values; orin a medium supplemented with different NaCl concentrations.

Isolate	pH	Incubation Time (h)				NaCl Concentration (% w/v)
		0	1	2	3	0%	1%	4%	8%
Lactococcus lactis FBM_1321	2.0	8.48 ± 0.07	4.55 ± 0.01	2.94 ± 0.07	N.D.	9.42 ± 0.05	9.40 ± 0.11	8.66 ± 0.08	5.66 ± 0.07
	3.0		8.48 ± 0.03	4.55 ± 0.02	2.94 ± 0.07
	7.0		8.49 ± 0.01	8.51 ± 0.02	8.52 ± 0.08
Lc. paracasei FBM_1327	2.0	8.16 ± 0.03	3.62 ± 0.07	N.D.	N.D.	9.36 ± 0.02	9.33 ± 0.12	9.21 ± 0.14	8.28 ± 0.11
	3.0		5.17 ± 0.03	3.96 ± 0.07	1.77 ± 0.01
	7.0		8.17 ± 0.02	8.21 ± 0.01	8.19 ± 0.03
Lactococcus lactis FBM_1331	2.0	8.27 ± 0.01	2.01 ± 0.08	N.D.	N.D.	9.09 ± 0.07	9.02 ± 0.13	9.01 ± 0.02	7.04 ± 0.11
	3.0		5.56 ± 0.09	3.72 ± 0.04	2.80 ± 0.07
	7.0		8.27 ± 0.05	8.29 ± 0.01	8.28 ± 0.02
Lp. plantarum FBM_1422	2.0	8.62 ± 0.01	4.47 ± 0.01	2.00 ± 0.06	N.D.	9.66 ± 0.03	9.52 ± 0.11	9.40 ± 0.22	6.65 ± 0.01
	3.0		6.91 ± 0.06	6.47 ± 0.01	6.20 ± 0.16
	7.0		8.62 ± 0.01	8.63 ± 0.02	8.61 ± 0.05
Lc. rhamnosus FBM_1423	2.0	8.63 ± 0.03	4.15 ± 0.2	N.D.	N.D.	9.45 ± 0.05	9.38 ± 0.05	9.28 ± 0.08	7.64 ± 0.02
	3.0		7.02 ± 0.1	3.93 ± 0.11	3.14 ± 0.02
	7.0		8.62 ± 0.01	8.64 ± 0.02	8.62 ± 0.01
Lc. rhamnosus FBM_1433	2.0	8.15 ± 0.01	2.60 ± 0.04	N.D.	N.D.	9.21 ± 0.08	9.15 ± 0.09	8.69 ± 0.14	6.90 ± 0.15
	3.0		4.75 ± 0.07	3.36 ± 0.04	3.06 ± 0.07
	7.0		8.15 ± 0.04	8.16 ± 0.05	8.15 ± 0.01
Lc. rhamnosus FBM_1434	2.0	8.33 ± 0.09	3.11 ± 0.01	N.D.	N.D.	9.42 ± 0.04	9.37 ± 0.01	8.90 ± 0.01	7.49 ± 0.08
	3.0		6.70 ± 0.06	5.54 ± 0.01	3.14 ± 0.04
	7.0		8.32 ± 0.01	8.34 ± 0.05	8.33 ± 0.01

N.D., not detected.

).

Incubation at pH 2.0 for longer than 1 h resulted in a significant (p < 0.05) decrease in cell viability (≤4.55 Log CFU/mL). Likewise, incubation at pH 3.0 caused a significant (p < 0.05) reduction in cell counts after 2 h, apart from Lp. plantarum FBM_1422 strain, which survived at satisfactory levels (>6 Log CFU/mL). According to the work of Liong et al. [66], maintenance of cell levels at >6 Log CFU/mL after incubation at pH 3.0 for 2 h is an important feature; however, no official survival limit has been set by the European Authorities (EFSA). The resistance of LAB to low pH can be attributed to a mechanism based on F₀F₁-ATPase activity, as it has a catalytic part (F₁) and a membrane part (F₀), serving as a channel for the transport of protons. Its action lies in the fact that in conditions, where the pH of the environment is low, it increases the internal pH, balancing the difference between cytoplasmic and external pH [67,68].

3.4.2. NaCl Tolerance

As survival and growth of functional cultures in the presence of a high NaCl concentration is one of the desired characteristics for dairy products, the survival of the wild-type strains during incubation in a nutrient medium enriched with different NaCl concentrations was evaluated. The results are presented in Table 4.

The exposure of the strains to a NaCl concentration ≤ 4% had no significant (p > 0.05) effect on cell viability, except from Lactococcus lactis subsp. lactis FBM_1321, Lc. rhamnosus FBM_1433, and Lc. rhamnosus FBM_1434, where significantly lower (p < 0.05) cell loads were recorded (8.66 ± 0.08, 8.69 ± 0.14, and 8.90 ± 0.01 Log CFU/mL, respectively) compared to control values (0% NaCl). Increasing the concentration of NaCl in the medium led to a gradual decrease in the cell levels of all strains (p < 0.05). Thus, incubation at an 8% NaCl concentration resulted in cell loads ranging from 5.66 to 8.28 Log CFU/mL. In this case, the highest (p < 0.05) cell loads were observed in Lc. paracasei FBM_1327 (8.28 ± 0.11 Log CFU/mL), while the lowest (p < 0.05) cell loads were found in Lactococcus lactis subsp. lactis FBM_1321 strain (5.66 ± 0.07 Log CFU/mL). Similar results were reported in other studies evaluating the survival of strains at different NaCl concentrations [39,69,70]. For example, for LAB strains, satisfactory levels of cell viability were recorded during incubation in the presence of NaCl concentration ≤ 6.5%, but weak growth was observed at 10% NaCl concentration [70].

3.5. Cell Immobilization and Preparation of Functional Dairy Products

Of all strains tested, Lc. paracasei FBM_1327 was selected for the manufacture of dairy products. This selection was based on the combination of safety profile, functional, and technological properties, as shown above. The addition of an adjunct culture in dairy products can interfere with the starter culture, due to the competition for nutrients, production of inhibitory substances, or pH changes [71,72], resulting in complex microbial associations. Given the strain-specific characteristics, assessment of several factors for novel strains to be added to foods is highly recommended.

3.5.1. Preparation of Freeze-Dried Immobilized Cells of Lc. paracasei FBM_1327 on Oat Flakes

Immobilization of Lc. paracasei FBM_1327 on oat flakes and subsequent freeze-drying led to the production of freeze-dried immobilized cells with a concentration of 8.88 Log CFU/g. Cell immobilization on oat flakes was confirmed by electron micrographs (Figure 3). The bacterial cells were clearly attached to the food matrix, as reported elsewhere [11,14,42].

3.5.2. pH, TA, Sugar, and Organic Acid Content of Functional Yoghurt and Ayran Products

The pH and the TA which are considered important physicochemical parameters were determined at different storage intervals, and the results are summarized in

Table 5. Effects of Lc. paracasei FBM_1327 on pH and TA (%) in functional yoghurt and ayran products during storage at 4 °C.

Parameter	Storage Time (Days)	YC	YC_O	Y_WF	Y_WIO	Y_FDF	Y_FDIO
pH	1	4.32 ± 0.03	4.38 ± 0.02	4.48 ± 0.04 b	4.49 ± 0.01 b,c	4.57 ± 0.01 b	4.60 ± 0.01 b,c,d
	10	4.31 ± 0.02	4.35 ± 0.01	4.29 ± 0.01 a	4.33 ± 0.02 a	4.30 ± 0.02 a	4.36 ± 0.01 a
	20	4.30 ± 0.02	4.27 ± 0.02	4.41 ± 0.02 a,c	4.46 ± 0.02 a,b,c	4.44 ± 0.03 b,c	4.28 ± 0.05 a,d,e,f
	30	4.31 ± 0.02	4.34 ± 0.01	4.30 ± 0.01 a	4.30 ± 0.06 b,c,d	4.46 ± 0.01 a,e	4.41 ± 0.04 a
TA (%)	1	0.68 ± 0.06	0.59 ± 0.06	0.59 ± 0.06	0.69 ± 0.01	0.68 ± 0.06	0.66 ± 0.06
	10	0.73 ± 0.01	0.83 ± 0.03	0.86 ± 0.06 a	0.82 ± 0.01	0.72 ± 0.01	0.75 ± 0.08
	20	0.90 ± 0.13	0.86 ± 0.06 a	1.09 ± 0.01 a	0.99 ± 0.13 a,d	0.78 ± 0.08	0.77 ± 0.07 d
	30	0.50 ± 0.06	0.77 ± 0.06 b	1.10 ± 0.08 a,b,c	0.77 ± 0.06 b	0.95 ± 0.06 a,b,d	0.87 ± 0.08 b
		AC	AC_O	A_WF	A_WIO	A_FDF	A_FDIO
pH	1	4.19 ± 0.05	4.21 ± 0.03	4.14 ± 0.02 b	4.14 ± 0.02 b	4.04 ± 0.03 b,c,d,e	4.14 ± 0.02 b,c,f
	10	4.13 ± 0.01	4.15 ± 0.03	3.96 ± 0.01 a,b,c	3.96 ± 0.01 a,b,c	3.92 ± 0.03 a,b,c	3.96 ± 0.01 a,b,c
	20	4.11 ± 0.01	4.16 ± 0.01	3.89 ± 0.04 a,b,c	3.89 ± 0.04 a,b,c	3.86 ± 0.01 a,b,c,e	3.89 ± 0.04 a,b,c
	30	4.11 ± 0.01	4.12 ± 0.01	3.82 ± 0.04 a,b,c	3.82 ± 0.04 a,b,c	3.76 ± 0.04 a,b,c,d,e	3.82 ± 0.04 a,b,c,f
TA (%)	1	0.59 ± 0.07	0.88 ± 0.04	1.20 ± 0.07 b,c	0.96 ± 0.03 b,d	0.97 ± 0.07 b,c,d	1.62 ± 0.03 b,c,d,e,f
	10	1.08 ± 0.03	1.21 ± 0.01	1.47 ± 0.04 a,b,c	1.17 ± 0.09 a,d	1.47 ± 0.04 a,b,c,e	1.69 ± 0.04 b,c,d,e,f
	20	1.20 ± 0.07	1.26 ± 0.01	1.45 ± 0.04 a,b,c	1.28 ± 0.03 a,d	1.66 ± 0.06 a,b,c,d,e	1.69 ± 0.01 b,c,d,e
	30	1.15 ± 0.01 a	1.17 ± 0.09 a	1.49 ± 0.01 a,b,c	1.29 ± 0.04 a,d	1.64 ± 0.00 a	1.90 ± 0.04 a,b,c,d,e

The values are expressed as means ± standard deviation. Different superscript letters show significantly differences: ^a p < 0.05 vs. day 1; ^b p < 0.05 vs. C; ^c p < 0.05 vs. C_O; ^d p < 0.05 vs. _WF; ^e p < 0.05 vs. _WIO; ^f p < 0.05 vs. _FDF. YC, control yoghurt; YC_O, control yoghurt + oat flakes; Y_FDF, yoghurt with freeze-dried free Lc. paracasei FBM_1327 cells; Y_FDIO, yoghurt with freeze-dried immobilized Lc. paracasei FBM_1327 cells on oat flakes; Y_WF, yoghurt with wet free Lc. paracasei FBM_1327 cells; Y_WIO, yoghurt with wet immobilized Lc. paracasei FBM_1327 cells on oat flakes; AC, control ayran; AC_O, control ayran + oat flakes; A_FDF, ayran with freeze-dried free Lc. paracasei FBM_1327 cells; A_FDIO, ayran with freeze-dried immobilized Lc. paracasei FBM_1327 cells on oat flakes; A_WF, ayran with wet free Lc. paracasei FBM_1327 cells; A_WIO, ayran with wet immobilized Lc. paracasei FBM_1327 cells on oat flakes.

. In yoghurts, the pH values on the 1st day of storage ranged from 4.32 to 4.60 in all samples. In products with Lc. paracasei FBM_1327, the pH was significantly reduced at day 10 (p < 0.05 compared to on the 1st day), but an increase was observed at day 20. Regarding TA, it ranged from 0.59 to 1.10%, in line with the suggestions of Codex Alimentarius (CXS 243-2003), according to which a minimum value of 0.6% is recommended. In products containing Lc. paracasei FBM_1327, TA values were significantly increased (p < 0.05) on day 10, except for that of the Y_FDIO sample, where no difference was observed (p > 0.05). In contrast, a significant (p < 0.05) decrease was observed in the case of the Y_FDF sample. Similar results have been reported in yogurts [73] and when Lc. paracasei was added to yogurt products [74,75].

Similarly, in ayran products, the pH ranged between 4.04 and 4.21 on the 1st day of storage at 4 °C, in line with a previous study [26]. By the end of the storage period (day 30), a significant reduction (p < 0.05) in pH values was observed in all samples containing the Lc. paracasei FBM_1327 strain, while in the control samples (AC and AC_O), pH values remained similar (p > 0.05) to those recorded on day 1. The TA ranged between 0.59 and 1.90%, as also reported in similar studies [76,77,78,79]. Of note, in all samples, the TA significantly increased during product storage at 4 °C, with the highest values recorded on day 30 [80]. Moreover, higher acidity values were generally observed in ayran products containing freeze-dried Lc. paracasei FBM_1327 cells, either free or immobilized on oat flakes (A_FDF and A_FDIO samples), compared to the control samples and products containing wet Lc. paracasei FBM_1327 cultures (AC, AC_O, A_WF, and A_WIO). Increased TA consisted of a convenient factor for the growth of LAB [81]. The decrease in the pH value and the increase in TA in products that contained Lc. paracasei FBM_1327 cells could imply that the strain was metabolically active during storage in the case of ayran products, resulting in organic acid production (mainly lactic acid) [81].

In yoghurt products, lactose was the predominant sugar followed by galactose and glucose. Lactose and galactose ranged in similar values in all samples, while glucose was significantly lower in both control samples (YC and YC_O) (p < 0.05) (

Table 6. Effects of Lc. paracasei FBM_1327 on residual sugars and organic acids in functional yoghurt and ayran products. The concentration is expressed as mg/g.

		Sugars		Acids
Samples	Glucose	Galactose	Lactose	Lactic acid	Citric acid
YC	1.75 ± 0.01	7.22 ± 0.19	21.08 ± 0.58	5.24 ± 0.04	1.43 ± 0.03
YC_O	1.80 ± 0.14	6.61 ± 0.23	19.66 ± 0.43	5.04 ± 0.32	1.52 ± 0.04
Y_WF	1.15 ± 0.03 a,b	6.77 ± 0.18	19.92 ± 0.51	4.88 ± 0.15	1.77 ± 0.05 a,b
Y_WIO	1.16 ± 0.01 a,b	6.44 ± 0.36	18.49 ± 0.87	4.71 ± 0.25	1.72 ± 0.09 a
Y_FDF	1.31 ± 0.08 a,b	7.20 ± 0.04	19.00 ± 0.49	5.04 ± 0.02	1.79 ± 0.07 a,b
Y_FDIO	1.10 ± 0.13 a,b	6.45 ± 0.07	18.99 ± 0.90	4.65 ± 0.33	1.74 ± 0.07 a
AC	0.58 ± 0.01	3.03 ± 0.14	9.07 ± 0.30	3.58 ± 0.08	1.10 ± 0.02
AC_O	0.61 ± 0.01	2.90 ± 0.05	8.80 ± 0.04	3.60 ± 0.03	1.01 ± 0.01
A_WF	0.51 ± 0.02 b	2.25 ± 0.11 a,b	10.95 ± 0.05 a,b	3.40 ± 0.07	1.05 ± 0.03
A_WIO	0.56 ± 0.01	2.34 ± 0.06 a,b	10.84 ± 0.19 a,b	3.44 ± 0.05	1.05 ± 0.03
A_FDF	0.52 ± 0.03	2.18 ± 0.12 a,b	10.62 ± 0.59 a,b	4.07 ± 0.23 a,b,c,d	1.05 ± 0.04
A_FDIO	0.62 ± 0.04 c,e	2.13 ± 0.13 a,b	11.17 ± 0.80 a,b	4.12 ± 0.10 a,b,c,d	1.04 ± 0.02

The values are expressed as means ± standard deviation. Different superscript letters show statistically significant differences: ^a p < 0.05 vs. C; ^b p < 0.05 vs. C_O; ^c p < 0.05 vs. _WF; ^d p < 0.05 vs. WIO; ^e p < 0.05 vs. LF. YC, control yoghurt; YC_O, control yoghurt + oat flakes; Y_FDF, yoghurt with freeze-dried free Lc. paracasei FBM_1327 cells; Y_FDIO, yoghurt with freeze-dried immobilized Lc. paracasei FBM_1327 cells on oat flakes; Y_WF, yoghurt with wet free Lc. paracasei FBM_1327 cells; Y_WIO, yoghurt with wet immobilized Lc. paracasei FBM_1327 cells on oat flakes; AC, control ayran; AC_O, control ayran + oat flakes; A_FDF, ayran with freeze-dried free Lc. paracasei FBM_1327 cells; A_FDIO, ayran with freeze-dried immobilized Lc. paracasei FBM_1327 cells on oat flakes; A_WF, ayran with wet free Lc. paracasei FBM_1327 cells; A_WIO, ayran with wet immobilized Lc. paracasei FBM_1327 cells on oat flakes.

). Lactic and citric acids were also identified. Lactic acid concentration ranged in similar levels in all samples (p > 0.05), while citric acid in yoghurt products enriched with the adjunct culture was significantly higher (p > 0.05) compared to the control samples.

In ayran products, galactose concentration was significantly lower (p < 0.05), while lactose concentration was significantly higher (p < 0.05) in all products containing Lc. paracasei FBM_1327 cells, compared to the control samples (AC and AC_O). Citric acid was not affected by the addition of the functional culture; however, in ayran products with the freeze-dried Lc. paracasei FBM_1327 culture (free or immobilized), higher lactic acid was noted, compared to the control samples and samples with the wet cultures (p < 0.05). The higher lactic acid concentration in these samples is consistent with the increased cell counts (expressed as Log CFU/g) of the Lc. paracasei FBM_1327 strain, implying an active metabolism at the day of production. Similar results were reported in the literature [82] for such products.

3.5.3. Minor Volatile Analysis in Functional Yoghurt and Ayran Products

To determine the quality characteristics of the functional dairy products, minor volatile compounds were determined by HS-SPME GC/MS analysis (Supplementary Figure S2). The analysis revealed the presence of acetaldehyde, acetone, 2,3-butanedione, 2-butanone, ethyl acetate, 2,3-pentanedione, 2-pentanone, 2-methyl-3-pentanone, 3-hydroxy-2-butanone, 3-methyl-1-butanol, hexanal, 2,4-dimethyl-1-heptane, 1-ethyl-3-methyl-benzene ή 1,3,5-trimethyl-benzene, 1-hexanol, hexanoic acid, 2-ethyl-1-hexanol, octanoic acid, and 1,3-bis (1,1-dimethylethyl)-benzene in yoghurt products and acetaldehyde, acetone, 2,3-butanedione,2-butanone, 2-pentanone, 2-methyl-3-pentanone, 3-hydroxy-2-butanone, hexanal, 2,4-dimethyl-1-heptane, 1-ethyl-3-methyl-benzene ή 1,3,5-trimethyl-benzene, 1-hexanol, 2-heptanone, hexanoic acid, 2-ethyl-1-hexanol, octanoic acid, and 1,3-bis (1,1-dimethylethyl)-benzene in ayran products. The concentrations of the identified compounds ranged from 0.01 to 0.70 mg/kg for yoghurt and from 0.01 to 0.20 mg/kg for ayran products, while no significant differences were observed between the samples (p > 0.05). The main volatiles identified in yoghurts were acetaldehyde [83,84], whereas in ayran, acetaldehyde and diacetyl [85]. A similar concentration of diacetyl, along with a higher concentration of acetaldehyde, as found in our study, was reported by Bonczar et al. [86], comparing plain and probiotic yoghurts. Flavor compounds in yoghurts in similar concentrations expressed as μg/L [84], in line with our study, have been also reported [87]. According to a review, more than 100 volatile compounds have been detected in yogurt and acetaldehyde, diacetyl, acetoin, acetone, and 2- butanone are responsible for the characteristic and typical attributes of the product [88]. S. thermophilus was found to produce 53 volatile compounds during milk fermentation [89], whereas other studies reported that L. delbrueckii subsp. bulgaricus is mainly responsible for producing the majority of volatile compounds present in yoghurt and ayran products. S. thermophilus is more responsible for the lipolytic activity [83,86].

Several studies have focused on the unique flavor characteristics that probiotic supplementation can impart to fermented milks [85,89,90]. In contrast, the results of the present study highlighted that the addition of the functional ingredient had no effect on the volatiles produced in the final products.

3.5.4. Survival of Lc. paracasei FBM_1327 in Functional Yoghurt and Ayran Products

While there is no universal guideline, to meet both regulatory and quality standards (as specified by Codex Alimentarius and US FDA standards), the concentration of S. thermophilus and L. delbrueckii subsp. bulgaricus in yoghurt should remain above 10⁶ to 10⁷ CFU/g throughout its shelf life [91]. The viability of both starter and adjunct cultures is crucial [92,93], given that high cell loads of the functional cultures (>7 Log CFU/g) have to be achieved upon consumption [94].

In yoghurt products, the initial levels of Lc. paracasei FBM_1327 strain (day 1) ranged from 7.69 to 8.13 Log CFU/g (8.13 ± 0.01, 8.13 ± 0.01, 7.69 ± 0.01, and 7.80 ± 0.01 in Y_WF, Y_WIO, Y_FDF, and Y_FDIO samples, respectively) (Figure 4). Importantly, cell counts of Lc. paracasei FBM_1327 throughout storage remained stable (p > 0.05) in all samples. The levels of the strain in the yoghurt products produced with wet cells (Y_WF and Y_WIO) were significantly higher (p < 0.05) at all timepoints compared to those in the products produced with the freeze-dried cultures (Y_FDF and Y_FDIO), probably due to stress during the freeze-drying process [95]. In all cases, cell loads of the adjunct culture remained above the suggested levels (7 Log CFU/g) [94], achieving a daily dose of >9 Log CFU, which is necessary for providing the health-promoting effects, considering that typically 200 g of yoghurt are contained per cup [95].

Cell levels of L. delbrueckii subsp. bulgaricus on the day of production ranged from 7.92 to 8.22 Log CFU/g (Figure 4). Cell loads in the samples produced with wet cultures (Y_WF and Y_WIO) on the 20th and 30th days of storage were significantly higher (p < 0.05) than the levels of the products with the freeze-dried cultures (Y_FDF and Y_FDIO samples). In all products enriched with Lc. paracasei FBM_1327, cell levels of L. delbrueckii subsp. bulgaricus on the 20th and 30th days of storage were significantly higher (p > 0.05) than those of the control samples. Ιn the control samples (YC and YC_O samples), the levels of L. delbrueckii subsp. bulgaricus were significantly (p < 0.05) lower after 20 and 30 days of storage compared to day 1. Of note, in the control samples with oat flakes and after 30 days of storage, cell levels of L. delbrueckii subsp. bulgaricus were higher (p < 0.05) compared to the yoghurts with no oat flakes. All these results highlighted the prebiotic effects of beta-glucans present in oats, as also discussed elsewhere [96].

On day 1, the levels of S. thermophilus ranged from 8.66 to 9.12 Log CFU/g, in all products. In the case of Y_WIO, Y_FDF, and Y_FDIO samples, the levels remained stable during storage (p > 0.05). The levels of S. thermophilus cells in yogurt products with wet or freeze-dried immobilized Lc. paracasei FBM_1327 cells on oat flakes (Y_WIO and Y_FDIO samples) were significantly lower (p < 0.05) compared to the control samples. Of note, no microbial contamination was observed during storage in any product.

Ayran is an alternative to yoghurt supplemented with salt (NaCl) and water. Hence, ayran could serve as an alternative functional beverage for delivering functional cultures. Higher amino acid composition [97] or better sensory results during storage [98] have been reported in ayran containing probiotics compared to yoghurt. Due to the refreshing attributes of this drink [99], the development of novel products using immobilized Lc. paracasei FBM_1327 cells on oat flakes was also investigated.

The levels of Lc. paracasei FBM_1327 strain after production (day 1) ranged from 8.53 to 8.91 Log CFU/g (8.77 ± 0.01, 8.53 ± 0.12, 8.72 ± 0.04, and 8.91 ± 0.11 in A_WF, A_WIO, A_FDF, and A_FDIO samples, respectively) (Figure 5). The highest initial value (p < 0.05) was recorded in the ayran product containing freeze-dried immobilized cells in oat flakes (A_FDIO sample). After 30 days of storage, higher values (p < 0.05) were recorded in the samples containing the freeze-dried culture (A_FDF and A_FDIO samples) compared to those with the wet cells of the adjunct strain (A_WF and A_WIO samples). Additionally, in ayran products supplemented with wet or freeze-dried immobilized cultures oon oat flakes (A_WIO and A_FDIO samples), the levels of the Lc. paracasei FBM_1327 strain remained consistently high (8.56–9.00 Log CFU/g) after 30 days, compared to the samples with wet or freeze-dried free cells (A_WF and A_FDF samples) (8.38–8.68 Log CFU/g), underlying the positive contribution of immobilization on oat flakes to cell viability. Similar efforts have been reported in the literature [20,100], highlighting the favorable environment of such products towards functional cultures’ survival [93].

After 30 days of storage at 4 °C, no significant reduction in the cell counts of S. thermophilus and L. delbrueckii subsp. bulgaricus was observed in the samples containing the Lc. paracasei FBM_1327 strain (p > 0.05) compared to values on day 1. However, in the control samples, cell levels of S. thermophilus and L. delbrueckii subsp. bulgaricus significantly (p < 0.05) decreased after 30 days of storage and were slightly lower than those reported in another study [100].

Lc. paracasei is a mesophilic bacterium with an optimal growth temperature of 30–40 °C and the ability to grow at 45 °C and in a pH range of 4.5–6.5 [101], which makes the fermentation conditions of the yogurt favorable for its growth and survival. Additionally, the contribution of Lc. paracasei FBM_1327 to the viability of L. delbrueckii subsp. bulgaricus is noteworthy, as higher survival rates were recorded throughout storage, compared to the control samples. However, in the samples containing freeze-dried functional cultures, lower cell levels were recorded compared to those containing wet cells. Similar results have been reported in previous investigations concerning the use of Lc. paracasei strains in yogurt production [74,75].

Notably, after 30 days of storage, no contamination by Enterobacteriaceae, coliforms, E. coli, or fungi was observed in the functional ayran samples produced (A_WF, A_WIO, A_FDF, and A_FDIO samples). On the other hand, in the control samples (AC and AC_O), contamination by Staphylococcus (1.41–1.60 Log CFU/g) after 30 days of storage was noticed. It is important to mention that for fermented dairy products, the limits for E. coli, enterobacteria, and Staphylococcus are <10², <10⁴, and 10³ CFU/mL, respectively, according to European Union Regulation 2073/2005.

The principal component analyses (PCAs) presented in Figure 6 illustrated the variability in pH, TA, and microbial populations, as well as the distribution of samples across groups during storage. Control samples (with or without oat flakes) were clustered on the left part of the plot, whereas products containing Lc. paracasei FBM_1327 formed a distinct cluster on the right.

3.5.5. Effects of Lc. paracasei FBM_1327 on Sensory Properties of Functional Yoghurt and Ayran Products

The sensory properties of the new products prepared with Lc. paracasei FBM_1327 were evaluated and compared to similar commercial products. The results are summarized in Tables S1 and S2 (Supplementary Data). The appearance of all yoghurt products was characterized as smoother, though higher syneresis was observed compared to the commercial product. The aroma of YC, YC_O, Y_FDF, and Y_FDIO samples was described as milk-like, in contrast to the neutral aroma of the commercial product. Regarding the texture, products containing Lc. paracasei FBM_1327 exhibited less cohesion and stickiness, with higher syneresis compared to the commercial product. When tasted, lower cohesion and syneresis were noted in products YC, YC_O, Y_FDF, and Y_FDIO, whereas the commercial yoghurt and the YC_O sample were characterized as sticky. Notably, the presence of solid parts was witnessed in products that contained the oat flakes (YC_O and Y_FDIO samples). The yoghurt containing immobilized cells on oats was perceived as sweet, while all other products (including the commercial product) were described as sour. Although the commercial yoghurt received the higher overall evaluation score, no significant (p > 0.05) differences were identified, ascertaining the acceptability of the new products.

Sensory evaluation of ayran beverages containing freeze-dried Lc. paracasei FBM_1327 cells on oat flakes revealed a milky aroma with a cereal-like scent and a sweet and sour taste (Table S2). Additionally, the texture of these beverages was characterized as creamier than the control samples and the commercial product. Overall, the new products were well received by the panel, achieving scores comparable to those of the commercial product (p > 0.05).

4. Conclusions

Raw goat and ewe’s milk samples were used for the isolation of seven lactic acid bacteria strains. The newly isolated strains were tested for cholesterol-lowering ability, antagonistic activity against foodborne pathogens, survival in acidic and osmotic environments, and adhesion to differentiated Caco-2 cells. Lc. paracasei FBM_1327 strain exhibited the highest cholesterol assimilation percentage (up to 28.78%), inhibited the growth of foodborne pathogens (by 5.32–6.76 Log CFU/mL), and demonstrated high survival rates (up to 8.28 Log CFU/mL) in the presence of 8% NaCl. The presumptive beneficial strain Lc. paracasei FBM_1327 was then immobilized on oat flakes, and the immobilized cells were incorporated into yogurt and ayran products. Cell loads of Lc. paracasei FBM_1327 remained stable and exceeded 7 Log CFU/g during storage, whereas its addition did not affect the physicochemical and sensory attributes of the products, highlighting its suitability as an adjunct ingredient.

Our findings suggested that immobilized Lc. paracasei FBM_1327 on oat flakes may serve as a potent functional ingredient for developing widely consumed dairy products. The incorporation of such a functional ingredient in yoghurt and ayran products could be easily adopted by the industry, as the novel products produced displayed similar characteristics to commercial products. However, future in vivo studies and clinical trials are necessary to confirm the functional effects observed in vitro.