Isolation, Identification, and Characterization of Probiotic Properties of Lactic Acid Bacterial Strains Isolated from Rose Blossom of Rosa damascena Mill

Denkova, Zapryana; Zapryanova, Polina; Gaytanska, Yordanka; Goranov, Bogdan; Shopska, Vesela; Denkova-Kostova, Rositsa; Kostov, Georgi

doi:10.3390/pr13061644

Open AccessFeature PaperArticle

Isolation, Identification, and Characterization of Probiotic Properties of Lactic Acid Bacterial Strains Isolated from Rose Blossom of Rosa damascena Mill

by

Zapryana Denkova

¹,

Polina Zapryanova

²,

Yordanka Gaytanska

¹

,

Bogdan Goranov

²,

Vesela Shopska

²

,

Rositsa Denkova-Kostova

^3,* and

Georgi Kostov

²

¹

Department of Microbiology and Biotechnology, University of Food Technologies, 4002 Plovdiv, Bulgaria

²

Department of Wine and Beer Technology, University of Food Technologies, 4002 Plovdiv, Bulgaria

³

Department of Biochemistry and Nutrition, University of Food Technologies, 4002 Plovdiv, Bulgaria

^*

Author to whom correspondence should be addressed.

Processes 2025, 13(6), 1644; https://doi.org/10.3390/pr13061644

Submission received: 28 April 2025 / Revised: 21 May 2025 / Accepted: 21 May 2025 / Published: 23 May 2025

(This article belongs to the Special Issue Feature Papers in the "Food Process Engineering" Section)

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Abstract

:

This study on the isolation, identification, and characterization of the probiotic properties of lactic acid bacterial strains from the rose blossom of Rosa damascena Mill. (R. damascena) is crucial for discovering novel, plant-derived probiotics with potential health benefits and applications in food, medicine, and cosmetics. Nine lactic acid bacterial (LAB) strains were isolated from rose blossom of R. damascena, and they were identified to the species level by applying physiological and biochemical (API 50 CHL), and molecular genetic (16S rRNA gene sequencing) methods. The isolates were identified as belonging to the Lactobacillus helveticus, Lactobacillus acidophilus, and Lactiplantibacillus plantarum species. Some probiotic properties of the newly isolated and identified LAB strains were examined: their antibacterial activity against pathogens by the agar well diffusion method, and their antibiotic resistance profile by the agar paper disc diffusion method. The LAB strains studied demonstrated significant antibacterial activity against the Escherichia coli, Staphylococcus aureus, Salmonella Abony, Proteus vulgaris, Listeria monocytogenes, and Enterococcus faecalis pathogens and were resistant to most of the antibiotics used in clinical practice, which in turn suggested the possibility of their joint inclusion in therapy, in the composition of probiotic preparations. A batch fermentation process was conducted with Lactiplantibacillus plantarum 5/20, and the kinetic parameters of the batch fermentation process were determined in order to obtain a concentrate with a high viable cell count (10¹³CFU/cm³). The resultant concentrate was freeze-dried, and freeze-dried preparations with a high viable cell count (over 10¹² CFU/g) were obtained. Research on LAB strains isolated from R. damascena could reveal valuable LAB strains with significant probiotic properties. These strains will be suitable for various applications in the composition of starter cultures for functional beverages and foods, as well as probiotic preparations, showcasing the untapped potential of plant-associated microbiota.

Keywords:

lactic acid bacteria; Rosa damascena; isolation; identification; sequencing; antibacterial activity; pathogen; antibiotic; probiotic

1. Introduction

Lactic acid bacteria are recognized for their numerous probiotic properties, which can significantly enhance the health benefits of fermented foods. The isolation of LAB strains from diverse natural environments, such as rose blossom of R. damascena, opens up new perspectives offering insights into their probiotic potential and applications [1].

The antibacterial properties of LAB are particularly noteworthy [2]. The role of LAB in metabolic processes, particularly in carbohydrate fermentation, further emphasizes their probiotic benefits. LAB convert fermentable sugars into lactic acid and other metabolites, which can lower the gut pH, thus creating an inhospitable environment for pathogens and consequently inhibiting their growth, and also enhancing nutrient absorption due to improved digestibility and nutrient bioavailability for the host [3]. Apart from lactic acid, LAB also produce various metabolites, for example, bacteriocins, hydrogen peroxide, and other organic acids, which can also inhibit pathogenic microorganism growth [2,4,5]. The unique production of these antibacterial compounds allows LAB to outcompete harmful microorganisms for nutrients and colonization sites within the gut [4,6]. Thus, they both enhance food safety by preventing spoilage and contribute to overall health by maintaining a balanced gut microbiota.

The isolation of probiotic LAB strains from R. damascena may present novel opportunities for developing functional foods with enhanced protective effects against foodborne pathogens and spoilage microorganisms. The ability of these strains to produce lactic acid and other antibacterial agents while demonstrating resistance to adverse conditions suggests their potential utility in the development of novel probiotic-rich foods, such as fortified beverages, fermented vegetables, and even non-dairy alternatives [7]. Therefore, the exploration of LAB from such a unique source as rose blossom not only broadens the scope of probiotic research but also paves the way for innovative applications in functional food industries.

The significance of microbial interactions, particularly between LAB and other microorganisms, can amplify the health benefits provided by these probiotics. The use of LAB as starters in fermentation processes has been associated with improved sensory profiles and biological activity in food products [8].

Furthermore, laboratory screening of various LAB strains for specific probiotic properties can reflect a more focused approach to developing functional foods tailored to consumer health needs. For instance, selecting strains with pronounced antibacterial activity against common gut pathogens while maintaining gut epithelial adherence can produce more effective probiotics [9,10].

Research into the probiotic potential of natural and indigenous LAB strains is essential as they bear unique genetic makeups that may enhance their functional properties. Cultivating and utilizing strains specifically adapted to local environments could lead to the development of regionally significant functional foods that cater to the specific health needs of local populations [11]. This aligns with broader efforts toward sustainability and resilience in functional food production.

Last but not least, as consumer interest in functional foods continues to rise, the exploration of traditional sources such as R. damascena for LAB isolation could serve both health and economic purposes. Combining ancient ethnobotanical knowledge with modern microbiological techniques may yield new probiotic sources that not only benefit health but also honor cultural practices surrounding food production and fermentation. The exploration of LAB from such unique sources can indeed stimulate innovation in the field while preserving sustainable practices. Continued research is necessary to isolate and identify new LAB strains from plant sources and to explore their probiotic properties in order to identify specific applications in the development of both probiotic formulations and food bio-preservation strategies.

The objectives of the present study were the isolation and identification of LAB strains from the rose blossom of R. damascena and the examination of some of their probiotic properties, namely their antibacterial activity against pathogens and their antibiotic resistance profile.

2. Materials and Methods

2.1. Bacteria

2.1.1. Lactic Acid Bacteria

The research in the present work was performed with 11 LAB strains, designated as LAB 4/20, LAB 12/20, LAB 5/20, LAB 6/20, LAB 8/20, LAB 9/20, LAB 10/20, LAB 13/20, LAB 16/20, LAB 19/20, and LAB 22/20, isolated from rose blossom of R. damascena.

2.1.2. Pathogenic Test Bacteria

Gram-negative: Escherichia coli (E. coli) ATCC 25922, Proteus vulgaris (P. vulgaris) DSM 13387, Salmonella Abony (S. Abony) NTCC 6017; Gram-positive: Staphylococcus aureus (S. aureus) ATCC 25923, Enterococcus faecalis (E. faecalis) ATCC 29212, Listeria monocytogenes (L. monocytogenes) ATCC 19115.

2.2. Nutrient Media

The nutrient media used in the present study were MRS broth (Merck, Taufkirchen, Germany); MRS agar (Merck); 0.5% NaCl solution (Merck); LAPTg10 broth; LAPTg10 agar; LBG broth; and LBG agar [12]. The culture media were prepared according to a given composition by the Department of Microbiology and Biotechnology at the University of Food Technologies, Plovdiv and were supplied by Gentaur, Bulgaria.

2.3. Microbiological, Physiological, and Biochemical Methods

2.3.1. Isolation of Lactic Acid Bacteria

Lactic acid bacteria were isolated from the rose blossom of R. damascena in accordance with the method described in [13].

2.3.2. Determination of the Titratable Acidity

The Thörner method was used to determine the acid-forming ability of the bacteria [14].

2.3.3. Determination of the Number of Viable Bacteria

Appropriate tenfold dilutions of the sample for analysis in a saline solution were prepared, and 0.1 cm³ from the last three dilutions was spread plated on the corresponding solid nutrient medium. The inoculated Petri dishes were cultured for 48 to 72 h at the optimum growth temperature for the corresponding microorganism until countable single colonies appeared. The number of the single colonies was used to estimate the number of viable bacteria. The method was performed in accordance with the method described in [13].

2.3.4. Determination of the Biochemical Profile

The API 50 CHL system (BioMerieux SA, Marcy-l’Étoile, France) was used according to the manufacturer’s instructions. The results obtained were processed with the apiweb^® identification software.

2.3.5. Determination of the Antibiotic Susceptibility Profile

The antibiotic susceptibility profile was determined by the disc diffusion method [15] as described in [16]. A fresh 24 h culture of each strain in MRS broth was adjusted to a 0.5 McFarland standard and inoculated on MRS agar plates.

The paper discs were impregnated with antibiotics targeting the following:

Cell wall synthesis: penicillin, ampicillin, oxacillin, piperacillin, amoxicillin, bacitracin, and vancomycin;
Protein synthesis: tetracycline, doxycycline, tobramycin, amikacin, gentamicin, erythromycin, chloramphenicol, and lincomycin;
DNA synthesis and cell division: nalidixic acid, ciprofloxacin, novobiocin, and rifampin.

The Petri dishes were incubated at 37 ± 1 °C for 48 h. The diameters (in mm) of the formed inhibition zones around each of the antibiotic discs were recorded. The following designations were used when reporting the results: R—resistant (d_IZ < 8 mm), SR—intermediately sensitive (d_IZ 8–16 mm), S—sensitive (d_IZ > 16 mm).

2.3.6. Determination of the Antibacterial Activity Against Pathogens—Agar Well Diffusion Method

The agar well diffusion method was used in order to determine the antibacterial activity of the LAB strains studied against pathogenic bacteria [16]. Three samples were prepared for each LAB strain: culture liquid (CL), biomass in saline solution (BSS), cell-free supernatant (CFSN), and neutralized cell-free supernatant (NCFSN) (pH = 6.5), obtained from a 24 h culture of the respective LAB strains. The antibacterial activity was tested against the following test bacteria: E. coli ATCC 25922, L. monocytogenes ATCC 19115, S. aureus ATCC 25923, S. Abony NTCC 6017, E. faecalis ATCC 29212, and P. vulgaris DSM 13387.

Each of the test bacteria (suspension in saline solution at a concentration of 10⁷ CFU/cm³) was inoculated in Petri dishes with LBG agar medium, and after solidification of the agar, wells (d_well = 7 mm) were prepared. CL, BSS, and NCFSN in 0.1 cm³ quantities were pipetted into the wells and the Petri dishes were incubated for 1 h at 4 ± 2 °C for the pipetted samples to diffuse into the medium. Then, the Petri dishes with the test bacteria were incubated at 37 ± 1 °C for 24 h, and next, the diameters of the inhibition zones in mm were recorded.

2.4. Molecular Genetic Methods

Sequencing of the 16S rRNA Gene

The sequencing of the 16S rRNA gene was performed by the Macrogen Europe Laboratory, the Netherlands, by use of the Sanger method [17] and in accordance with the method described in [18].

2.5. Batch Cultivation

The cultivation of Lactiplantibacillus plantarum (Lp. plantarum) 5/20 took place in a laboratory bioreactor with a geometric volume of 2 dm³ and a working volume of 1.5 dm³. The control and monitoring of the main fermentation parameters were carried out by a Sartorius A2 control device, which included control loops for the stirring rate, temperature, pH, etc. The cultivation of the lactic acid bacteria was performed as described in [16]. The duration of the cultivation was 24 h, with samples of the culture fluid being periodically taken for analysis of the total number of viable cells of the respective strain (CFU/cm³) and the titratable acidity [19,20].

2.6. Modeling of the Process Kinetics and Identification of the Model Parameters

Since the main target product of the fermentation process is the biomass amount, the logistic curve in Equation (1), which contains a clear biological meaning, was used to model the growth kinetics. The duration of the induction period and the rate constant of adaptation were determined by Equation (2).

\frac{d X}{d τ} = μ_{m} X - β X^{2} = μ_{m} X - \frac{μ_{m}}{X_{f}} X^{2} = μ_{m} (1 - \frac{X}{X_{f}}) X

(1)

\ln \frac{M}{N_{0}} = μ_{m} τ + \ln {\frac{k_{0}}{k_{0} + μ_{m}} [1 + \frac{μ_{m}}{k_{0}} e^{[- (k_{0} + μ_{m}) τ]}]}

(2)

where µ_m is the maximum specific growth rate, h⁻¹; X_in and X_f are the initial and final concentrations of viable cells, CFU/cm³; β is the coefficient of intrapopulation competition cfu/cm³.h; M is the current biomass concentration, CFU/cm³; N₀ is the initial biomass concentration, CFU/cm³; τ_a is the induction period, h; and k₀ is the rate constant of the cell adaptation to the environment and the cultivation conditions, h⁻¹.

The logistic curve model (1) was solved numerically using the 4th-order Runge–Kutta method. For the identification of the model parameters, the Solver function in Excel was used. The model parameters were determined by minimizing the square of the difference between the experimental data and the data obtained from the corresponding model. The parametric identification of model (2) was performed in the Curve Expert Professional software product through nonlinear regression [21].

2.7. Freeze-Drying

The freeze-drying was conducted in accordance with the method described in [22,23].

2.8. Microbiological Studies of the Lyophilized Concentrate

Microbiological Status of the Native and Lyophilized Samples

According to BDS EN ISO 4833: 2004 [24].
Indicators:

Lactic acid bacteria, CFU/g [25];
Total number of mesophilic aerobic and facultative anaerobic bacteria: CFU/g [26];
E. coli in 0.1 g of the product [27];
Pathogenic microorganisms, including Salmonella sp. in 25.0 g of the product [28];
Coagulase-positive staphylococci in 1.0 g of the product [29];
Sulfite-reducing clostridia in 0.1 g of the product [30];
Microscopic mold spores, CFU/g [31];
Yeast, CFU/g [31].

2.9. Processing of the Results

The data from the triplicate experiments were processed using MS Office Excel 2013 software products, applying statistical functions to determine the standard deviation and the maximum error of the estimate at significance levels of α < 0.05.

3. Results and Discussion

3.1. Isolation, Identification, and Selection of LAB Strains from the Rose Blossom of R. damascena

3.1.1. Phenotypic Characteristics of the Newly Isolated LAB Isolates

The characterization of the newly isolated isolates began with an assessment of the purity of the culture through macroscopic and microscopic morphological control. The micro- and macromorphological characteristics of the 11 LAB isolates from rose blossom of R. damascena are presented in Table 1. When cultivated on an MRS agar medium, the isolates grew in the form of small, milky-white colonies with a star-shaped or biconvex lenticular shape, which were easily separated from the medium. The isolates were Gram-positive (Gr (+), non-spore-forming rods.

3.1.2. Physiological and Biochemical Characteristics of the Isolates

After characterization according to the main morphological criteria (Table 1), the isolates were subjected to determination of their biochemical profile with the API 50 CHL rapid identification system, and their ability to assimilate 49 carbon sources included in the system was determined (Table 2a,b). Based on the studies conducted with the API 50 CHL kits, the isolates were assigned to different LAB species with the corresponding percentage of confidence (Table 3).

The increasing use of molecular methods allows for more accurate, rapid, and reproducible differentiation between closely related species, which are difficult to distinguish based on phenotypic characteristics alone [32,33]. The similarity in the biochemical profiles of phylogenetically close species, as well as the influence of certain factors on the metabolic properties of lactic acid bacteria, necessitate additional characterization by applying molecular genetic methods [32,34].

3.1.3. Molecular Taxonomic Characterization—Genotypic Characteristics of the LAB Isolates Studied—Sequencing of the 16S rRNA Gene

For the complete species identification of the LAB isolates, a genetic method for genotyping was used: sequencing of the 16S rRNA gene. The results of the 16S rDNA sequence analysis referred LAB 4/20 to Lactobacillus helveticus (L. helveticus) with 99% similarity between the 16S rDNA sequence of LAB 4/20 and the partial 16S rDNA sequence of L. helveticus NBRC 15019 (Figure 1, Table 4), LAB 5/20 to Lp. plantarum with 99% similarity between the 16S rDNA sequence of LAB 5/20 and the partial 16S rDNA sequence of Lp. plantarum NBRC 15891 (Figure 2, Table 4), LAB 6/20 to L. helveticus with 99% similarity between the 16S rDNA sequence of LAB 6/20 and the partial 16S rDNA sequence of L. helveticus NBRC 15019 (Figure 3, Table 4), LAB 8/20 to L. helveticus with 99% similarity between the 16S rDNA sequence of LAB 8/20 and the partial 16S rDNA sequence of L. helveticus NBRC 15019 (Figure 4, Table 4), LAB 9/20 to L. helveticus with 99% similarity between the 16S rDNA sequence of LAB 9/20 and the partial 16S rDNA sequence of L. helveticus NBRC 15019 (Figure 5, Table 4), LAB 10/20 to Lactobacillus acidophilus (L. acidophilus) with 99% similarity between the 16S rDNA sequence of LAB 10/20 and the partial 16S rDNA sequence of L. acidophilus NBRC 13951 (Figure 6, Table 4), LAB 12/20 to L. helveticus with 99% similarity between the 16S rDNA sequence of LAB 12/20 and the partial 16S rDNA sequence of L. helveticus NBRC 15019 (Figure 7, Table 4), LAB 13/20 to Lp. plantarum with 99% similarity between the 16S rDNA sequence of LAB 13/20 and the partial 16S rDNA sequence of Lp. plantarum NBRC 15891 (Figure 8, Table 4), LAB 16/20 to Leuconostoc mesenteroides (L. mesenteroides) with 99% similarity between the 16S rDNA sequence of LAB 19/20 and the partial 16S rDNA sequence of L. mesenteroides ATCC 8293 (Figure 9, Table 4); LAB 19/20 to Lp. plantarum with 99% similarity between the 16S rDNA sequence of LAB 19/20 and the partial 16S rDNA sequence of Lp. plantarum NBRC 15891 (Figure 10, Table 4), and LAB 22/20 to L. mesenteroides with 99% similarity between the 16S rDNA sequence of LAB 22/20 and the partial 16S rDNA sequence of L. mesenteroides ATCC 8293 (Figure 11, Table 4).

3.2. Characterization of the Probiotic Potential of the Newly Isolated Lactic Acid Bacteria

3.2.1. Antibacterial Activity of the Isolated LAB Strains Against Pathogenic Bacteria

The antibacterial activity against pathogenic bacteria of the newly isolated LAB strains L. helveticus 4/20, Lp. plantarum 5/20, L. helveticus 6/20, L. helveticus 8/20, L. helveticus 9/20, L. acidophilus 10/20, L. helveticus 12/20, Lp. plantarum 13/20, and Lp. plantarum 19/20, isolated from rose blossom of R. damascena by the agar well diffusion method, was studied (Table 5a,b). All of the LAB strains examined had significant antibacterial activity against the E. coli ATCC 25922, S. aureus ATCC 25923, S. Abony NTCC 6017, P. vulgaris DSM 13387, L. monocytogenes ATCC 19115, and E. faecalis ATCC 29212 pathogenic bacteria (Table 5a,b). When neutralizing the organic acids released into the medium by the LAB strains, no antibacterial activity was found on the growth of pathogenic bacteria that cause foodborne infections and intoxications (Table 5a,b), indicating that the LAB strains studied did not produce any other substances besides organic acids that inhibit the growth of the test pathogenic bacteria.

L. helveticus 4/20, L. helveticus 6/20, L. helveticus 8/20 and L. helveticus 9/20 exhibited the highest antibacterial activity against L. monocytogenes and E. faecalis; L. helveticus 4/20 and L. helveticus 12/20 also manifested high antibacterial activity against S. aureus (Table 5a). Lp. plantarum 5/20 and Lp. plantarum 13/20 showed the highest antibacterial activity against L. monocytogenes, S. aureus, E. faecalis, and P. vulgaris. L. acidophilus 10/20 and Lp. plantarum 19/20 exhibited significantly lower antibacterial activity than all newly isolated LAB strains (Table 5b). Out of the five L. helveticus strains, L. helveticus 12/20 demonstrated the highest inhibitory activity against E. coli (Table 5a). Among the representatives of the Lp. plantarum species, Lp. plantarum 5/20 manifested the highest antibacterial activity against E. coli (Table 5b). Out of the five L. helveticus strains, L. helveticus 4/20 exhibited the highest inhibitory activity against S. aureus (Table 5a). Among the representatives of the Lp. plantarum species, Lp. plantarum 5/20 and Lp. plantarum 13/20 showed the highest antibacterial activity against E. coli (Table 5b). Out of the five L. helveticus strains, L. helveticus 4/20 and L. helveticus 12/20 demonstrated the highest inhibitory activity against S. Abony (Table 5a).

Among the representatives of the species Lp. plantarum, Lp. plantarum 5/20 exhibited the highest antibacterial activity against S. Abony (Table 5b). Out of the five Lactobacillus helveticus strains, Lactobacillus helveticus 4/20 and L. helveticus 12/20 demonstrated the highest inhibitory activity against P. vulgaris (Table 5a). Among the representatives of the Lp. plantarum species, Lp. plantarum 5/20 showed the highest antibacterial activity against P. vulgaris (Table 5b). Out of the five L. helveticus strains, L. helveticus 6/20, L. helveticus 9/20, and L. helveticus 12/20 manifested the highest inhibitory activity against L. monocytogenes (Table 5a). Among the representatives of the Lp. plantarum species, Lp. plantarum 5/20 exhibited the highest antibacterial activity against L. monocytogenes (Table 5b). Out of the five L. helveticus strains, L. helveticus 9/20 and L. helveticus 12/20 demonstrated the highest inhibitory activity against E. faecalis (Table 5a). Among the representatives of the Lp. plantarum species, Lp. plantarum 5/20 and Lp. plantarum 13/20 exhibited the highest antibacterial activity against E. faecalis (Table 5b). The most pronounced antibacterial activity against the test bacteria included in this study was demonstrated by L. helveticus 12/20 among the representatives of the L. helveticus species, and Lp. plantarum 5/20 among the representatives of the Lp. plantarum species (Table 5a,b).

When examining the antibacterial activity of each newly isolated LAB strain against each pathogen included in the experiment, inhibition zones were observed when the culture liquid (CL), the biomass in saline solution (BSS), and the cell-free supernatant (CFSN) were pipetted. For some strains, it was found that the CL and BSS had greater antibacterial activity than the CFSN, which means that LAB suppress pathogenic bacteria due to both competition for nutrients between them and inhibition by the lactic and other organic acids produced, as a result of which the pH decreases.

The present study highlights the significant antibacterial potential of LAB strains isolated from the rose blossom of R. damascena, as evaluated through the well diffusion method. All LAB strains tested exhibited pronounced inhibitory activity against a spectrum of foodborne pathogenic bacteria, including E. coli, S. aureus, S. Abony, P. vulgaris, L. monocytogenes, and E. faecalis. These findings reinforce the growing body of evidence supporting LAB as effective bio-preservatives and potential candidates for the development of functional foods and probiotics.

A key observation in this study was that the antibacterial activity of the LAB strains ceased upon neutralization of their organic acid content. This clearly suggests that the antibacterial effects observed are primarily attributable to organic acid production, mainly lactic acid, and not due to bacteriocins, hydrogen peroxide, or other antibacterial metabolites. This mode of inhibition, largely through acidification of the medium, aligns with previous findings reported for Lactobacillus and Lactiplantibacillus species, where the decrease in the environmental pH created unfavorable conditions for pathogen proliferation.

Among the strains tested, L. helveticus 12/20 and Lp. plantarum 5/20 consistently demonstrated the most potent antibacterial activity against nearly all tested pathogens. Notably, L. helveticus 12/20 exhibited strong inhibition against E. coli, S. aureus, S. Abony, P. vulgaris, L. monocytogenes, and E. faecalis, indicating its broad-spectrum efficacy. Likewise, Lp. plantarum 5/20 was particularly effective, showing significant inhibitory activity against S. aureus, L. monocytogenes, and P. vulgaris. These results underscore the strain-specific nature of antibacterial efficacy, even within the same species, further emphasizing the need for individual assessment of LAB candidates for food and health applications.

The comparative analysis of different LAB species and strains revealed a pattern: L. helveticus strains, L. helveticus 4/20, L. helveticus 6/20, L. helveticus 9/20, and L. helveticus 12/20, in particular, were more effective against Gram-positive bacteria such as L. monocytogenes and E. faecalis, while Lp. plantarum strains, especially Lp. plantarum 5/20 and Lp. plantarum 13/20, showed a broader spectrum, with potent activity against both Gram-positive and Gram-negative bacteria, including E. coli and S. Abony. This strain-level variability could be attributed to differences in organic acid profiles, acid tolerance, and metabolic capabilities.

The findings also emphasize the potential of underexplored natural sources, such as the rose blossom of R. damascena, as reservoirs of beneficial microorganisms. These results are particularly relevant for the food industry, where there is a growing demand for natural preservatives and health-promoting microbial strains. The ability of certain strains to effectively inhibit foodborne pathogens suggests their promising application as components in bio-preservatives, starter cultures, or next-generation probiotics.

The present study contributes valuable insights into the antibacterial potential of novel LAB strains, reaffirming the critical importance of strain-specific evaluation. Future research should aim to characterize the organic acid profiles of these strains, explore their performance in food matrix systems, and evaluate their safety and efficacy in vivo.

The present study demonstrates the significant antibacterial potential of lactic acid bacterial (LAB) strains newly isolated from rose blossom of Rosa damascena. Using the agar well diffusion method, all strains tested displayed inhibitory activity against a broad panel of foodborne pathogens, including E. coli, S. aureus, S. Abony, P. vulgaris, L. monocytogenes, and E. faecalis. These results align with previous findings that LAB exert strong antagonistic effects against pathogenic microorganisms through multiple mechanisms, primarily the production of organic acids such as lactic and acetic acid, which lower the environmental pH and inhibit pathogen growth [35].

A critical observation in our study is the complete loss of antibacterial activity following the neutralization of the LAB culture supernatants, indicating that the antibacterial effect is largely acid-mediated. This agrees with reports that the inhibitory capacity of LAB often results from pH-dependent mechanisms rather than the presence of proteinaceous bacteriocins or hydrogen peroxide under the conditions tested [36,37]. While many LAB are known to produce bacteriocins, their activity is often masked or diminished when the primary inhibition is due to acidic byproducts [38].

Among the strains studied, L. helveticus 12/20 and Lp. plantarum 5/20 demonstrated the most pronounced and consistent antibacterial effects, inhibiting nearly all target pathogens. This suggests their potential as promising candidates for applications in food preservation and probiotic development. The strain-specific nature of this activity supports findings by Dinev et al., 2018 [39], who emphasized that even within the same LAB species, antibacterial efficacy can vary widely depending on the strain’s metabolic output and environmental adaptability.

The comparatively broad-spectrum antibacterial activity observed in Lactiplantibacillus plantarum strains, particularly against Gram-negative bacteria such as E. coli and S. Abony, is consistent with earlier studies showing that certain Lp. plantarum isolates possess higher acid tolerance and metabolic flexibility, allowing them to survive and act across diverse ecological niches [40]. Conversely, L. helveticus strains, especially L. helveticus 4/20, L. helveticus 6/20, L. helveticus 9/20, and L. helveticus 12/20, were particularly effective against Gram-positive pathogens such as L. monocytogenes and E. faecalis, a trend similarly noted by Penderup Jensen et al., 2009 [41].

Moreover, the observed differences in the inhibitory activity between the cell-free supernatants, culture liquids, and biomass suspensions suggest that competitive interactions (e.g., nutrient depletion and co-aggregation) also play a role in pathogen inhibition, in addition to organic acid production. This multifactorial antibacterial strategy enhances the LAB utility in real-world applications where complex microbial ecosystems are present [42].

The isolation of such effective strains from R. damascena blossom, a relatively unexploited ecological niche, highlights the untapped potential of floral microbiota as sources of novel LAB with biotechnological applications. Previous studies have shown that flowers can host diverse and unique LAB communities, often with enhanced probiotic potential [1].

Taken together, the current findings support the inclusion of selected strains, particularly L. helveticus 12/20 and Lp. plantarum 5/20, in future studies aiming to develop functional foods or bio-preservative agents. Their ability to inhibit both Gram-positive and Gram-negative pathogens, coupled with their origin from a natural and culturally significant plant source, makes them strong candidates for commercial and clinical evaluation. Further investigations should include detailed metabolic profiling of the inhibitory compounds, whole-genome sequencing to identify probiotic and safety-related genes, and validation of efficacy in food systems and in vivo models.

3.2.2. Antibiotic Resistance of the Isolated LAB Strains

Understanding the antibiotic resistance profiles of lactic acid bacteria (LAB) with probiotic potential is crucial. This knowledge aids in the selection of strains suitable for combined antibiotic–probiotic therapies aimed at restoring normal gastrointestinal and urogenital microflora. However, LAB can harbor transferable resistance genes, posing a risk of spreading antibiotic resistance to pathogenic microorganisms. Therefore, assessing the antibiotic sensitivity of potential probiotic strains is essential with a view to ensuring both their efficacy and safety [43,44].

For this reason, 19 antibiotics with different mechanisms of action from the main groups used in medical practice were selected and the sensitivity of the newly isolated LAB strains was tested. The results obtained from the studies conducted using the agar diffusion method of Bauer et al., 1966 [15,45] for 24 h have been summarized in Table 6a,b.

The antibiotic susceptibility patterns observed among the L. helveticus and Lp. plantarum strains tested in this study revealed substantial variability, indicating the strain-specific nature of antibiotic resistance and susceptibility in this species. Given the increasing interest in LAB as probiotics and components of food starters, understanding their antibiotic resistance profiles is critical from both safety and therapeutic standpoints (Table 6a,b).

Regarding the L. helveticus strains, among the antibiotics targeting cell wall synthesis, a heterogeneous resistance pattern was observed. L. helveticus 4/20 and L. helveticus 9/20 exhibited resistance to multiple β-lactam antibiotics including penicillin, ampicillin, and oxacillin, while strains such as L. helveticus 8/20 demonstrated broad sensitivity within this group (Table 6a). Interestingly, vancomycin resistance was detected in L. helveticus 4/20, L. helveticus 9/20, and L. helveticus 12/20, which is noteworthy since LAB are often intrinsically resistant to glycopeptides due to the absence of the D-Ala-D-Lac target in their peptidoglycan precursors. The sensitivity of L. helveticus 6/20 to vancomycin may suggest a deviation from typical intrinsic resistance or experimental variability, warranting further genetic investigation. As regards protein synthesis inhibitors, all L. helveticus strains showed resistance to lincomycin and most of them to amikacin, in conformity with known intrinsic resistance mechanisms in LAB, including poor drug uptake or active efflux. However, variable responses to tetracyclines were observed: while L. helveticus 4/20 and L. helveticus 6/20 were resistant, L. helveticus 12/20 was sensitive.

Chloramphenicol sensitivity was broadly maintained across strains, aligning with its known efficacy against a wide range of Gram-positive bacteria. Erythromycin sensitivity in L. helveticus 6/20, L. helveticus 8/20, and L. helveticus 12/20 is particularly important, as macrolides are frequently used in clinical settings; resistance in L. helveticus 9/20 could reflect either acquired resistance or a variation in ribosomal binding sites. In the group of antibiotics that inhibit DNA synthesis or cell division, ciprofloxacin and nalidixic acid resistance was widespread, except in L. helveticus 8/20, which showed intermediate susceptibility. These findings may be due to mutations in the quinolone resistance-determining regions of DNA gyrase or topoisomerase IV, commonly implicated in fluoroquinolone resistance. Sensitivity to novobiocin and rifampin was more variable, L. helveticus 6/20 being uniquely sensitive to rifampin, suggesting differential mechanisms of resistance, potentially at the level of RNA polymerase structure or function. The strain-dependent differences in antibiotic resistance profiles observed underscore the importance of individual strain screening, especially when considering LAB strains for probiotic or food industry applications. Moreover, the presence of resistance to clinically relevant antibiotics such as vancomycin and ciprofloxacin raises potential concerns regarding horizontal gene transfer, particularly in environments where LAB co-exist with pathogenic bacteria. Further genomic analysis is warranted to determine whether the resistance observed is due to intrinsic mechanisms or the acquisition of resistance genes. This is especially important given the safety implications of using L. helveticus strains in food or pharmaceutical products (Table 6a).

Regarding the Lp. plantarum strains, among the cell wall synthesis inhibitors, resistance to β-lactam antibiotics was consistently observed across all Lp. plantarum strains. All three strains showed resistance to penicillin (Table 6b), ampicillin, oxacillin, and vancomycin—an expected finding considering the intrinsic resistance of many LAB to β-lactams and glycopeptides due to variations in their cell wall structure and low-affinity penicillin-binding proteins. However, strain-specific differences were noted in response to bacitracin, piperacillin, and amoxicillin. For instance, Lp plantarum 5/20 was sensitive only to amoxicillin, whereas Lp. plantarum 13/20 and Lp. plantarum 19/20 demonstrated intermediate susceptibility to amoxicillin and bacitracin, respectively. These subtle differences could reflect either adaptive phenotypic variation or distinct underlying resistance mechanisms, such as efflux pump activity or cell envelope permeability. In terms of protein synthesis inhibitors, tobramycin and amikacin resistance was universal across all L. plantarum strains, aligning with known intrinsic resistance in LAB to aminoglycosides due to their inability to penetrate the thick peptidoglycan layer in the absence of oxygen. Lincomycin resistance was also observed in all three Lp. plantarum strains, suggesting possible intrinsic mechanisms or conserved ribosomal protection elements. However, the response to other antibiotics such as tetracycline, doxycycline, erythromycin, and gentamicin varied among Lp. plantarum strains. Lp. plantarum 13/20 was sensitive to tetracycline and erythromycin, while Lp. plantarum 5/20 and Lp. plantarum 19/20 were resistant. Notably, chloramphenicol sensitivity was retained in all strains, a favorable observation given its historical effectiveness against Gram-positive bacteria and its use as a marker in probiotic screening. The intermediate responses to gentamicin and doxycycline across multiple strains suggest either partial resistance mechanisms or borderline susceptibility thresholds that warrant further MIC-based analysis. For DNA synthesis and cell division inhibitors, resistance to nalidixic acid was observed in all Lp. plantarum strains, which is consistent with previously reported resistance in LAB due to mutations in DNA gyrase or topoisomerase IV. Ciprofloxacin resistance was also prevalent but exhibited intermediate susceptibility in strains Lp. plantarum 13/20 and Lp. plantarum 19/20, suggesting potential variability in fluoroquinolone target affinity or efflux mechanisms. Rifampin sensitivity in Lp. plantarum 13/20 and intermediate susceptibility in others points toward differing rpoB gene sequences or promoter activity. Resistance to novobiocin in Lp. plantarum 13/20 and intermediate responses in the other two strains align with the strain-dependent nature of resistance against this DNA gyrase inhibitor. The data reveal that while Lp. plantarum strains share common resistance traits, particularly against aminoglycosides, β-lactams, and quinolones, there is clear inter-strain heterogeneity that could impact their use in functional foods or clinical applications. Once again, the occurrence of resistance to clinically important antibiotics such as erythromycin and ciprofloxacin in some strains is a potential concern, especially in the context of horizontal gene transfer within the gut microbiota. These findings emphasize the necessity of thorough safety assessments for each L. plantarum strain before inclusion in probiotic formulations, particularly when these strains may encounter antibiotic-selective environments in the host. Whole-genome sequencing, plasmid analysis, and conjugation assays would provide deeper insight into the genetic basis of resistance and the potential risk of horizontal gene transfer (Table 6b).

Resistance to β-lactam antibiotics, including penicillin, ampicillin, oxacillin, and amoxicillin, was evident in L. acidophilus 10/20, which aligns with previously reported intrinsic resistance mechanisms in lactobacilli (Table 6b). These mechanisms are thought to involve low-affinity penicillin-binding proteins (PBPs) and limited cell wall permeability to β-lactams. Interestingly, this strain showed sensitivity to piperacillin and intermediate sensitivity to bacitracin, suggesting partial or incomplete resistance mechanisms to certain β-lactams and polypeptide antibiotics.

This variability may reflect differences in the structural targets of these drugs or differential expression of resistance genes. In the category of protein synthesis inhibitors, L. acidophilus 10/20 demonstrated resistance to several commonly used antibiotics, including tetracycline, tobramycin, amikacin, lincomycin, and erythromycin. Resistance to aminoglycosides such as tobramycin and amikacin is well-documented in Lactobacillus species and is generally considered intrinsic due to their anaerobic or microaerophilic growth, which limits aminoglycoside uptake. Resistance to macrolides (erythromycin) and lincosamides (lincomycin), however, may be mediated by acquired genes such as erm(B) or lnu(A) and warrants further investigation to determine the risk of horizontal gene transfer. The strain retained sensitivity to chloramphenicol, a broad-spectrum antibiotic, and showed intermediate susceptibility to doxycycline and gentamicin, suggesting that some protein synthesis pathways remain vulnerable to inhibition. The intermediate susceptibility may indicate reduced expression of resistance mechanisms, low-level efflux activity, or partial permeability to these compounds. With respect to DNA synthesis and cell division inhibitors, L. acidophilus 10/20 was resistant to nalidixic acid and demonstrated intermediate sensitivity to ciprofloxacin, rifampin, and novobiocin. Resistance to quinolones such as nalidixic acid is common in lactobacilli and may arise from mutations in DNA gyrase or topoisomerase IV. The partial susceptibility to ciprofloxacin and rifampin may suggest incomplete resistance or adaptive tolerance, but further genomic analysis would be required to confirm whether these resistance traits are chromosomal or potentially mobile. The antibiotic profile of L. acidophilus 10/20 indicates a predominance of resistance to antibiotics across all three functional groups: cell wall synthesis, protein synthesis, and DNA replication inhibitors, with a few key exceptions. The retention of sensitivity to chloramphenicol and piperacillin, and intermediate sensitivity to several others, offers a promising basis for safe probiotic application, provided these traits are confirmed to be non-transferable (Table 6b).

3.3. Batch Cultivation of Lp. plantarum 5/20 and Kinetic Modeling in MRS Broth Medium

The suitability of Lp. plantarum 5/20 for the production of a probiotic biomass concentrate was evaluated through batch fermentation in MRS broth using a laboratory-scale bioreactor operated with mechanical stirring and controlled pH (maintained at 5.7). Throughout the cultivation, the growth kinetics were monitored (Figure 12).

The initial adaptation phase of the strain was minimal and difficult to delineate precisely. Therefore, based on equation (2), the induction time (τ₀), representing the period needed for metabolic adjustment, and the adaptation rate constant (k₀) were determined. The calculated values of τ₀ = 0.835 h and k₀ = 0.500 h⁻¹ suggested rapid metabolic transition of the strain to the new environmental conditions, consistent with previously observed behavior of similar strains under controlled fermentation conditions.

Between the 2nd and the 8th hours, the strain exhibited exponential growth, followed by a deceleration phase from the 8th to the 12th hour, entering a stationary phase at the 12th hour. At this point, a biomass density of approximately 7.1 × 10¹² CFU/cm³ was reached. Afterwards, only a slight increase was observed, culminating at approximately 2.4 × 10¹³ CFU/cm³ by the 24th hour.

Using the experimental data obtained, growth modeling was conducted to estimate key bioprocess parameters, including the maximum specific growth rate (µ_m) and the intrapopulation competition coefficient (β). The model outputs corresponded closely with the experimental data, demonstrating a high coefficient of determination (R²) and low prediction error (Table 7). The theoretical biomass peak of 13.63 log CFU/cm³ matched the observed maximum (13.36 log CFU/cm³), validating the model’s applicability for future process scaling and control.

Additionally, the model revealed a relatively high µ_m of 0.130 h⁻¹ and a low β value of 0.0095 cm³/(CFU·h), confirming the effective cultivation conditions and competitive potential of the strain in MRS broth. The pH trend presented in Figure 13 showed a rapid decrease from 6.19 to 5.7 by the fourth hour, after which pH was held constant. Meanwhile, the redox potential, initially stable (114–118 mV), gradually declined to −89 mV by the end of fermentation, reflecting increased metabolic activity and anaerobic environment formation.

3.4. Freeze-Drying

Freeze-drying is suitable for raw materials with a moisture content above 30% as many food products contain 70–90% free water. Like other preservation techniques, it requires high-quality materials. Pre-treatment of valuable bacterial strains or cultures is critical to ensure their survival during the process by creating protective conditions.

The post-drying vitality of lactic acid bacteria (LAB) depends on their ability to regain key biochemical activities, such as fermentation, proteolysis, aroma production, and antibacterial action, after rehydration. In their dry state, microorganisms enter anabiosis, where metabolic functions like nutrition, respiration, and reproduction nearly cease due to the absence of water and oxygen.

Survival rates of LAB during freeze-drying vary by species and are influenced by the growth phase at drying, the composition of the medium (some nutrients offer protection), and the cell concentration (higher viability is observed at densities above 10¹⁰ CFU/cm³). The heat transfer method also plays a role: conductive heating improves the survival of lactic streptococci, though L. bulgaricus remains particularly sensitive to the process [46,47,48].

Upon completion of the fermentation process, the biomass was immobilized in a hydrocolloid matrix. After freezing to a temperature of −40 °C, it was subjected to sublimation. The microbiological status of the resultant lyophilizate was determined (Table 8 and Figure 14).

The active cell titer in the dry product after freeze-drying was determined and the active cell concentration was found to be 2.3 × 10¹² CFU/g, with a survival rate of 92.52% (Figure 14 and Table 8). The high survival rate indicates that the strain was xerotolerant and was little affected by the freeze-drying conditions. The resistance of the strain to damaging factors during freeze-drying makes it valuable for production of different functional foods and beverages since not all lactic acid bacteria show high survival rates and resistance to production processes.

4. Conclusions

A total of eleven lactic acid bacteria (LAB) isolates were obtained from the rose blossoms of R. damascena and identified to the species level using a combination of cultural, microbiological, physiological–biochemical (API 50 CHL), and molecular genetic (16S rRNA gene sequencing) methods. The isolates included five strains of L. helveticus, three strains of Lp. plantarum, one strain of L. acidophilus, and two strains of L. mesenteroides. All isolates demonstrated notable antibacterial activity against common pathogenic microorganisms including E. coli, S. aureus, S. Abony, P. vulgaris, L. monocytogenes, and E. faecalis. Notably, Lp. plantarum 5/20, L. helveticus 6/20, and L. acidophilus 10/20 exhibited resistance to multiple antibiotics commonly used in clinical practice, indicating their potential application in combined probiotic–antibiotic therapies. Batch fermentation with Lp. plantarum 5/20 enabled the determination of key kinetic parameters for the production of a high-viability cell concentrate (~10¹³ CFU/cm³). A freeze-dried formulation was subsequently developed, retaining a high viable cell count (>10¹² CFU/g). These findings support the potential use of the newly isolated LAB strains in the development of probiotic formulations and starter cultures for functional foods and beverages, pending further evaluation of their probiotic properties.

Author Contributions

Conceptualization, Z.D. and R.D.-K.; methodology, R.D.-K. and B.G.; software, R.D.-K., G.K. and B.G.; validation, Z.D., R.D.-K. and G.K.; formal analysis, P.Z., Y.G., B.G. and V.S.; resources, Z.D.; data curation, R.D.-K. and G.K.; writing—original draft preparation, R.D.-K., V.S., P.Z., B.G. and Y.G.; writing—review and editing, R.D.-K., Z.D. and G.K.; supervision, Z.D.; funding acquisition, G.K. and P.Z. All authors have read and agreed to the published version of the manuscript.

Funding

The research in this article was funded by the project “Investigation of the synergistic action of plant extracts and lactic acid bacteria and ensuring sustainability in the production of fermented foods”, National Program “Young scientists and postdoctoral students—2”, Ministry of Education and Science, Bulgaria.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 4/20 and the partial nucleotide sequence of the 16S rDNA of L. helveticus NBRC 15019.

Figure 2. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 5/20 and the partial nucleotide sequence of the 16S rDNA of Lp. plantarum NBRC 15891.

Figure 3. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 6/20 and the partial nucleotide sequence of the 16S rDNA of L. helveticus NBRC 15019.

Figure 4. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 8/20 and the partial nucleotide sequence of the 16S rDNA of L. helveticus NBRC 15019.

Figure 5. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 9/20 and the partial nucleotide sequence of the 16S rDNA of L. helveticus NBRC 15019.

Figure 6. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 10/20 and the partial nucleotide sequence of the 16S rDNA of L. acidophilus NBRC 13951.

Figure 7. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 12/20 and the partial nucleotide sequence of the 16S rDNA of L. helveticus NBRC 15019.

Figure 8. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 13/20 and the partial nucleotide sequence of the 16S rDNA of Lp. plantarum NBRC 15891.

Figure 9. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 16/20 and the partial nucleotide sequence of the 16S rDNA of L. mesenteroides ATCC 8293.

Figure 10. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 19/20 and the partial nucleotide sequence of the 16S rDNA of Lp. plantarum NBRC 15891.

Figure 11. Comparison between the partial nucleotide sequence of the 16S rDNA of LAB 22/20 and the partial nucleotide sequence of the 16S rDNA of L. mesenteroides ATCC 8293.

Figure 12. Comparison of the experimental data with the logistic curve model for biomass accumulation during batch cultivation of Lp. plantarum 5/20 in a bioreactor with mechanical stirring at 37 °C.

Figure 13. Change in the oxidation–reduction potential and the pH in the batch cultivation of Lp. plantarum 5/20 in a bioreactor with mechanical stirring at 37 °C.

Figure 14. Survival of Lp. plantarum 5/20 cells during the freeze-drying process.

Table 1. Colonial characteristics and cell morphology of the LAB isolates.

Strain	Cellular Morphology		Colonial Characteristics
Strain	Description	Visualization	Description	Visualization
LAB 4/20	Long, thickened rods with rounded ends, arranged singly, in pairs and in short chains		Snowflake-shaped colonies, serrated ends, 2–3 mm in diameter
LAB 5/20	Fine, short rods with rounded ends, arranged in pairs and in short chains		Round colonies, with wavy ends, soft consistency, 2–3 mm in diameter
LAB 6/20	Long, thin rods with rounded ends, arranged singly and in long chains		Round colonies with wavy ends, soft consistency, 2–3 mm in diameter
LAB 8/20	Long, thin rods with rounded ends, arranged singly and in long chains		Round colonies with wavy ends, soft consistency, 2–3 mm in diameter
LAB 9/20	Short, thickened rods with rounded ends, arranged singly and in clusters		Round colonies with wavy ends, soft consistency, 2–3 mm in diameter
LAB 10/20	Long rods with rounded ends, arranged singly and in short chains		Snowflake-shaped colonies with wavy ends, soft consistency, 2–3 mm in diameter
LAB 12/20	Long thin rods with rounded ends, arranged singly and in short chains		Snowflake-shaped colonies with serrated ends, 2–3 mm in diameter
LAB 13/20	Short, thickened rods with rounded ends, arranged singly, in pairs, and in clusters		Round colonies with even ends, soft consistency, 2–3 mm in diameter
LAB 16/20	Cocci, arranged in pairs and in groups		Round colonies with even ends, soft consistency, 2–3 mm in diameter
LAB 19/20	Very short rods with rounded ends, arranged singly and in groups		Round colonies with even ends, soft consistency, 2–3 mm in diameter
LAB 22/20	Short rods with rounded, flat ends, arranged singly and in groups, soft consistency, 2–3 mm		Round colonies with even ends, soft consistency, 2–3 mm in diameter

Table 2. (a) Ability of the LAB 4/20, LAB 5/20, LAB 6/20, LAB 8/20, LAB 9/20, and LAB 10/20 isolates to assimilate 49 carbon sources included in the API 50 CHL identification system. (b) Ability of the LAB 12/20, LAB 13/20, LAB 16/20, LAB 19/20, and LAB 22/20 isolates to assimilate 49 carbon sources included in the API 50 CHL identification system.

(a)
#	Carbon source	LAB 4/20	LAB 5/20	LAB 6/20	LAB 8/20	LAB 9/20	LAB 10/20
1	Glycerol	-	-	-	-	-	-
2	Erythritol	-	-	-	-	-	-
3	D-arabinose	-	-	-	-	-	-
4	L-arabinose	-	+ (90–100%)	-	-	-	+ (90–100%)
5	Ribose	-	+ (90–100%)	+ (90–100%)	-	-	+ (90–100%)
6	D-xylose	-	-	-	-	-	+ (90–100%)
7	L-xylose	-	-	-	-	-	-
8	Adonitol	-	-	-	-	-	-
9	β-metil-D-xyloside	-	-	-	-	-	-
10	Galactose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
11	D-glucose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
12	D-fructose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
13	D-mannose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
14	L-sorbose	-	-	-	+ (90–100%)	-	-
15	Rhamnose	-	-	-	+ (90–100%)	-	-
16	Dulcitol	-	-	-	-	-	-
17	Inositol	-	-	-	-	-	-
18	Mannitol	-	+ (90–100%)	+ (90–100%)	+ (90–100%)	-	+ (90–100%)
19	Sorbitol	-	+ (90–100%)	+ (90–100%)	+ (90–100%)	-	+ (90–100%)
20	α-methyl-D-mannoside	-	+ (90–100%)	+ (90–100%)	-	-	+ (90–100%)
21	α-methyl-D-glucoside	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
22	N-acetyl-glucosamine	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
23	Amygdalin	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
24	Arbutin	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
25	Esculin	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
26	Salicin	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
27	Cellobiose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
28	Maltose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
29	Lactose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
30	Melibiose	-	+ (90–100%)	+ (90–100%)	+ (90–100%)	-	+ (90–100%)
31	Sucrose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
32	Trehalose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
33	Inulin	-	-	+ (90–100%)	-	-	-
34	Melezitose	-	+ (90–100%)	+ (90–100%)	+ (90–100%)	-	+ (90–100%)
35	D-raffinose	-	-	+ (90–100%)	-	-	+ (90–100%)
36	Starch	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	-
37	Glycogen	-	-	-	-	-	-
38	Xylitol	-	-	-	-	-	-
39	β-gentiobiose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
40	D-turanose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
41	D-lyxose	-	-	-	-	-	-
42	D-tagatose	-	-	-	-	-	-
43	D-fucose	-	-	-	-	-	-
44	L-fucose	-	-	-	-	-	-
45	D-arabitol	-	-	-	-	-	-
46	L-arabitol	-	-	-	-	-	-
47	Gluconate	-	+ (90–100%)	+ (90–100%)	-	-	+ (90–100%)
48	2-keto-gluconate	-	-	-	-	-	-
49	5-keto-gluconate	-	-	-	-	-	-
(b)
#	Carbon source	LAB 12/20	LAB 13/20	LAB 16/20	LAB 19/20	LAB 22/20
1	Glycerol	-	-	-	-	-
2	Erythritol	-	-	-	-	-
3	D-arabinose	-	-	-	-	-
4	L-arabinose	-	+ (90–100%)	+ (90–100%)	-	-
5	Ribose	-	+ (90–100%)	+ (90–100%)	-	-
6	D-xylose	-	-	+ (90–100%)	-	-
7	L-xylose	-	-	-	-	-
8	Adonitol	-	-	-	-	-
9	β-metil-D-xyloside	-	-	-	-	-
10	Galactose	+ (90–100%)	+ (90–100%)	+ (90–100%)	-	+ (90–100%)
11	D-glucose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
12	D-fructose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
13	D-mannose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
14	L-sorbose	-	-	-	-	-
15	Rhamnose	-	-	-	-	-
16	Dulcitol	-	-	-	-	-
17	Inositol	-	-	-	-	-
18	Mannitol	-	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
19	Sorbitol	-	+ (90–100%)	-	+ (90–100%)	+ (90–100%)
20	α-methyl-D-mannoside	-	+ (90–100%)	+ (90–100%)	-	-
21	α-methyl-D-glucoside	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
22	N-acetyl-glucosamine	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
23	Amygdalin	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
24	Arbutin	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
25	Esculin	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
26	Salicin	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
27	Cellobiose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
28	Maltose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
29	Lactose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
30	Melibiose	-	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
31	Sucrose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
32	Trehalose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
33	Inulin	-	-	+ (90–100%)	-	-
34	Melezitose	-	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
35	D-raffinose	-	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
36	Starch	+ (90–100%)	-	-	-	-
37	Glycogen	-	-	-	-	-
38	Xylitol	-	-	-	-	-
39	β-gentiobiose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	-
40	D-turanose	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)	+ (90–100%)
41	D-lyxose	-	-	-	-	-
42	D-tagatose	-	-	-	-	-
43	D-fucose	-	-	-	-	-
44	L-fucose	-	-	-	-	-
45	D-arabitol	-	-	-	-	-
46	L-arabitol	-	-	-	-	-
47	Gluconate	-	+ (90–100%)	-	+ (90–100%)	-
48	2-keto-gluconate	-	-	-	-	-
49	5-keto-gluconate	-	-	-	-	-

Table 3. Species identification of the LAB isolates after processing the API 50 CHL results with apiweb^®.

Isolate	Species Affiliation	Reliability, %
LAB 4/20	Lactobacillus crispatus	65.3
LAB 5/20	Lactiplantibacillus plantarum	99.9
LAB 6/20	Lactiplantibacillus plantarum	99.9
LAB 8/20	Lactiplantibacillus plantarum	99.9
LAB 9/20	Lactobacillus crispatus	65.3
LAB 10/20	Lactiplantibacillus plantarum	99.9
LAB 12/20	Lactobacillus crispatus	65.3
LAB 13/20	Lactiplantibacillus plantarum	99.9
LAB 16/20	Lactiplantibacillus plantarum	94.1
LAB 19/20	Lactiplantibacillus plantarum	99.9
LAB 22/20	Lactiplantibacillus plantarum	99.9

Table 4. Species identification of the LAB isolates, determined as a result of the 16S rRNA gene sequence analysis.

Isolate	Species Affiliation	Similarity, %
LAB4/20	L. helveticus	99%
LAB 5/20	Lp. plantarum	99%
LAB6/20	L. helveticus	99%
LAB8/20	L. helveticus	99%
LAB9/20	L. helveticus	99%
LAB10/20	L. acidophilus	99%
LAB12/20	L. helveticus	99%
LAB13/20	Lp. plantarum	99%
LAB16/20	L. mesenteroides	99%
LAB 19/20	Lp. plantarum	99%
LAB 22/20	L. mesenteroides	99%

Table 5. (a) Antibacterial activity of L. helveticus 4/20, L. helveticus 6/20, L. helveticus 8/20, L. helveticus 9/20, and L. helveticus 12/20 against pathogenic bacteria. d_well = 7 mm. The LAB concentration was 10¹¹ CFU/cm³. “-”—no inhibition of the growth of pathogenic bacteria by the LAB isolates was found. (b) Antibacterial activity of Lp. plantarum 5/20, Lp. plantarum 13/20, Lp. plantarum 19/20, and L. acidophilus 10/20 against pathogenic bacteria. d_well = 7 mm. The LAB concentration was 10¹¹ CFU/cm³. “-”—no inhibition of the growth of pathogenic bacteria by the LAB isolates was found.

(a)
d_IZ, [mm]		E. coli ATCC 25922, 6 × 10¹¹ CFU/cm³	S. Abony NTCC 6017, 2 × 10¹¹ CFU/cm³	P. vulgarisDSM 13387, 1.5 × 10¹¹ CFU/cm³	L. monocytogenes ATCC 19115, 4 × 10¹¹ CFU/cm³	S. aureus ATCC 25093, 1.7 × 10¹¹ CFU/cm³	E. faecalis ATCC 29212, 4.2 × 10¹¹ CFU/cm³
LAB strain		E. coli ATCC 25922, 6 × 10¹¹ CFU/cm³	S. Abony NTCC 6017, 2 × 10¹¹ CFU/cm³	P. vulgarisDSM 13387, 1.5 × 10¹¹ CFU/cm³	L. monocytogenes ATCC 19115, 4 × 10¹¹ CFU/cm³	S. aureus ATCC 25093, 1.7 × 10¹¹ CFU/cm³	E. faecalis ATCC 29212, 4.2 × 10¹¹ CFU/cm³
4/20	CL	14.67 ± 0.47	15.50 ± 0.41	17.00 ± 0.00	12.67 ± 0.47	17.17 ± 0.24	16.67 ± 0.47
	BSS	9.67 ± 0.47	12.33 ± 0.47	14.17 ± 0.24	11.17 ± 0.24	20.17 ± 0.24	13.33 ± 0.47
	CFSN	10.17 ± 0.24	12.00 ± 0.00	15.17 ± 0.24	10.50 ± 0.41	10.17 ± 0.24	11.33 ± 0.47
	NCFSN	-	-	-	-	-	-
6/20	CL	11.33 ± 0.47	14.33 ± 0.47	18.33 ± 0.47	12.17 ± 0.24	15.00 ± 0.00	16.50 ± 0.41
	BSS	8.00 ± 0.00	9.17 ± 0.24	14.00 ± 0.00	9.17 ± 0.24	16.17 ± 0.24	13.17 ± 0.24
	CFSN	9.50 ± 0.41	12.00 ± 0.00	15.67 ± 0.47	10.17 ± 0.24	13.67 ± 0.47	13.00 ± 0.00
	NCFSN	-	-	-	-	-	-
8/20	CL	10.17 ± 0.24	13.17 ± 0.24	16.50 ± 0.41	11.17 ± 0.24	13.50 ± 0.41	16.00 ± 0.00
	BSS	8.33 ± 0.47	8.17 ± 0.24	13.17 ± 0.24	8.00 ± 0.00	12.17 ± 0.24	12.17 ± 0.24
	CFSN	8.00 ± 0.00	10.00 ± 0.00	14.67 ± 0.47	10.50 ± 0.41	10.50 ± 0.41	13.17 ± 0.24
	NCFSN	-	-	-	-	-	-
9/20	CL	11.67 ± 0.47	12.50 ± 0.41	18.67 ± 0.47	11.50 ± 0.41	16.00 ± 0.00	18.67 ± 0.47
	BSS	8.17 ± 0.24	9.00 ± 0.00	12.17 ± 0.24	8.00 ± 0.00	10.17 ± 0.24	15.00 ± 0.00
	CFSN	9.50 ± 0.41	10.00 ± 0.00	15.17 ± 0.24	10.00 ± 0.00	10.67 ± 0.47	14.00 ± 0.00
	NCFSN	-	-	-	-	-	-
12/20	CL	14.00 ± 0.00	15.00 ± 0.00	18.00 ± 0.82	14.67 ± 0.47	16.17 ± 0.24	18.17 ± 0.24
	BSS	10.00 ± 0.00	10.00 ± 0.00	15.17 ± 0.24	9.17 ± 0.24	12.33 ± 0.47	13.17 ± 0.24
	CFSN	10.17 ± 0.24	13.50 ± 0.41	15.67 ± 0.47	12.00 ± 0.00	13.17 ± 0.24	13.00 ± 0.00
	NCFSN	-	-	-	-	-	8.00 ± 0.00
(b)
d_IZ, [mm]		E. coli ATCC 25922, 6 × 10¹¹ CFU/cm³	S. Abony NTCC 6017, 2 × 10¹¹ CFU/cm³	P. vulgarisDSM 13387, 1.5 × 10¹¹ CFU/cm³	L. monocytogenes ATCC 19115, 4 × 10¹¹ CFU/cm³	S. aureus ATCC 25093, 1.7 × 10¹¹ CFU/cm³	E. faecalis ATCC 29212, 4.2 × 10¹¹ CFU/cm³
LAB strain		E. coli ATCC 25922, 6 × 10¹¹ CFU/cm³	S. Abony NTCC 6017, 2 × 10¹¹ CFU/cm³	P. vulgarisDSM 13387, 1.5 × 10¹¹ CFU/cm³	L. monocytogenes ATCC 19115, 4 × 10¹¹ CFU/cm³	S. aureus ATCC 25093, 1.7 × 10¹¹ CFU/cm³	E. faecalis ATCC 29212, 4.2 × 10¹¹ CFU/cm³
5/20	CL	13.33 ± 0.47	17.00 ± 0.00	20.00 ± 0.00	13.67 ± 0.47	19.67 ± 0.47	18.50 ± 0.41
	BSS	9.17 ± 0.24	10.00 ± 0.00	16.17 ± 0.24	9.17 ± 0.24	17.33 ± 0.47	14.67 ± 0.47
	CFSN	11.33 ± 0.47	13.17 ± 0.24	17.67 ± 0.47	11.17 ± 0.24	14.67 ± 0.47	13.50 ± 0.41
	NCFSN	-	-	9.17 ± 0.24	8.17 ± 0.24	-	-
13/20	CL	10.17 ± 0.24	14.50 ± 0.41	17.17 ± 0.24	11.17 ± 0.24	19.50 ± 0.41	17.67 ± 0.47
	BSS	8.00 ± 0.00	10.17 ± 0.24	15.00 ± 0.00	9.00 ± 0.00	17.17 ± 0.24	14.17 ± 0.24
	CFSN	9.00 ± 0.00	13.17 ± 0.24	15.17 ± 0.24	10.50 ± 0.41	13.00 ± 0.00	13.00 ± 0.00
	NCFSN	-	-	8.00 ± 0.00	-	-	-
19/20	CL	9.67 ± 0.47	10.67 ± 0.47	11.00 ± 0.00	10.17 ± 0.24	10.17 ± 0.24	10.17 ± 0.24
	BSS	8.00 ± 0.00	8.17 ± 0.24	8.00 ± 0.00	8.00 ± 0.00	9.17 ± 0.24	8.67 ± 0.47
	CFSN	8.50 ± 0.41	9.00 ± 0.00	9.00 ± 0.00	8.67 ± 0.47	8.00 ± 0.00	9.17 ± 0.24
	NCFSN	-	-	-	-	-	-
10/20	CL	9.00 ± 0.00	10.17 ± 0.24	9.67 ± 0.47	10.17 ± 0.24	8.00 ± 0.00	8.17 ± 0.24
	BSS	8.17 ± 0.24	8.17 ± 0.24	8.17 ± 0.24	8.17 ± 0.24	8.17 ± 0.24	8.17 ± 0.24
	CFSN	9.00 ± 0.00	9.17 ± 0.24	9.17 ± 0.24	9.17 ± 0.24	8.00 ± 0.00	8.00 ± 0.00
	NCFSN	-	-	-	-	-	-

Table 6. (a) Antibiotic resistance of L. helveticus 4/20, L. helveticus 6/20, L. helveticus 8/20, L. helveticus 9/20, and L. helveticus 12/20. (b) Antibiotic resistance of Lp. plantarum 5/20, Lp. plantarum 13/20, Lp., plantarum 19/20, and L. acidophilus 10/20.

(a)
#	Mechanism of action	Antibiotic		Concentration	4/20	6/20	8/20	9/20	12/20
1	Inhibitor of cell wall synthesis	Penicillin	P	10 E/disc	R	S	S	R	R
2		Bacitracin	Cm	0.07 E/disc	R	S	S	SR	SR
3		Piperacillin	P	100 µg/disc	S	R	S	S	SR
4		Ampicillin	A	10 µg/disc	R	R	S	R	R
5		Oxacillin	O	1 µg/disc	R	S	R	R	R
6		Amoxicillin	Ax	25 µg/disc	S	S	S	R	SR
7		Vancomycin	V	30 µg/disc	R	S	S	R	R
8	Inhibitor of protein synthesis	Tetracycline	T	30 µg/disc	R	R	S	R	S
9		Doxycycline	D	30 µg/disc	R	SR	SR	SR	S
10		Gentamicin	G	10 µg/disc	SR	R	S	SR	SR
11		Tobramycin	Tb	10 µg/disc	R	S	S	R	R
12		Amikacin	Am	30 µg/disc	R	R	SR	R	SR
13		Lincomycin	L	15 µg/disc	R	R	SR	R	SR
14		Chloramphenicol	C	30 µg/disc	S	SR	S	S	S
15		Erythromycin	E	15 µg/disc	SR	S	S	R	S
16	Inhibitor of DNA synthesis and/or of cell division	Novobiocin	Nb	5 µg/disc	S	R	S	SR	R
17		Nalidixic acid	Nx	30 µg/disc	R	R	SR	R	R
18		Rifampin	R	5 µg/disc	SR	S	S	SR	S
19		Ciprofloxacin	Cp	5 µg/disc	R	R	SR	SR	SR
(b)
#	Mechanism of action	Antibiotic		Concentration	5/20	13/20	19/20	10/20
1	Inhibitor of cell wall synthesis	Penicillin	P	10 E/disc	R	R	R	R
2		Bacitracin	Cm	0.07 E/disc	R	SR	SR	SR
3		Piperacillin	P	100 µg/disc	R	SR	S	S
4		Ampicillin	A	10 µg/disc	R	R	R	R
5		Oxacillin	O	1 µg/disc	R	R	R	R
6		Amoxicillin	Ax	25 µg/disc	S	SR	R	R
7		Vancomycin	V	30 µg/disc	R	R	R	R
8	Inhibitor of protein synthesis	Tetracycline	T	30 µg/disc	SR	S	R	R
9		Doxycycline	D	30 µg/disc	S	S	SR	SR
10		Gentamicin	G	10 µg/disc	SR	SR	SR	SR
11		Tobramycin	Tb	10 µg/disc	R	R	R	R
12		Amikacin	Am	30 µg/disc	R	SR	R	R
13		Lincomycin	L	15 µg/disc	R	SR	R	R
14		Chloramphenicol	C	30 µg/disc	S	S	S	S
15		Erythromycin	E	15 µg/disc	R	S	R	R
16	Inhibitor of DNA synthesis and/or of cell division	Novobiocin	Nb	5 µg/disc	SR	R	SR	SR
17		Nalidixic acid	Nx	30 µg/disc	R	R	R	R
18		Rifampin	R	5 µg/disc	SR	S	SR	SR
19		Ciprofloxacin	Cp	5 µg/disc	R	SR	SR	SR

At d_IZ < 8 mm—R (resistant), at 8 < d_IZ < 16 mm—SR (intermediately sensitive), at d_IZ > 16 mm—S (sensitive).

Table 7. Kinetic parameters of the process of batch cultivation of Lp. plantarum 5/20 in a bioreactor with mechanical stirring at 37 °C.

Batch Cultivation of Lp. plantarum 5/20
µ, h⁻¹	β, cm³/(CFU.h)	X_kp, LogN	R²	Error
0.130	0.0095	13.63	0.9995	0.778

Table 8. Microbiological status of the lyophilized Lp. plantarum 5/20.

Type of Pathogenic Microorganisms Tested	According to BDS	Analysis Result
Total number of mesophilic aerobic and facultative anaerobic bacteria	No more than 800	180
E. coli in 0.l g of the product	None should be detected	Not detected
Sulfite-reducing clostridia in 0.l g of the product	None should be detected	Not detected
Salmonella sp. in 25.0 g of the product	None should be detected	Not detected
Coagulase-positive staphylococci in 1.0 g of the product	None should be detected	Not detected
Spores of microscopic molds, CFU/g	No more than 100	Not detected
Yeasts, CFU/g	No more than 100	Not detected

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Denkova, Z.; Zapryanova, P.; Gaytanska, Y.; Goranov, B.; Shopska, V.; Denkova-Kostova, R.; Kostov, G. Isolation, Identification, and Characterization of Probiotic Properties of Lactic Acid Bacterial Strains Isolated from Rose Blossom of Rosa damascena Mill. Processes 2025, 13, 1644. https://doi.org/10.3390/pr13061644

AMA Style

Denkova Z, Zapryanova P, Gaytanska Y, Goranov B, Shopska V, Denkova-Kostova R, Kostov G. Isolation, Identification, and Characterization of Probiotic Properties of Lactic Acid Bacterial Strains Isolated from Rose Blossom of Rosa damascena Mill. Processes. 2025; 13(6):1644. https://doi.org/10.3390/pr13061644

Chicago/Turabian Style

Denkova, Zapryana, Polina Zapryanova, Yordanka Gaytanska, Bogdan Goranov, Vesela Shopska, Rositsa Denkova-Kostova, and Georgi Kostov. 2025. "Isolation, Identification, and Characterization of Probiotic Properties of Lactic Acid Bacterial Strains Isolated from Rose Blossom of Rosa damascena Mill" Processes 13, no. 6: 1644. https://doi.org/10.3390/pr13061644

APA Style

Denkova, Z., Zapryanova, P., Gaytanska, Y., Goranov, B., Shopska, V., Denkova-Kostova, R., & Kostov, G. (2025). Isolation, Identification, and Characterization of Probiotic Properties of Lactic Acid Bacterial Strains Isolated from Rose Blossom of Rosa damascena Mill. Processes, 13(6), 1644. https://doi.org/10.3390/pr13061644

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