Characterization of nuvita biosearch center (NBC) isolated lactic acid bacteria strains from human origin and determination of growth kinetic profiles of selected cultures under bioreactor

Abstract

Backgorund

In recent years, there has been an increasing interest in the field of research into the isolation and characterization of probiotics in the prevention of diseases and the need to maintain the continuity of healthy microbiota. Therefore, the aim of this study is to isolate and identify bacteria found in maternal colostrum, breast milk, adult and infant feces, analyze possible probiotic potential, and reveal the developmental kinetics of selected strains.

Results

We isolated 40 bacterial species from 4 different sources and identified 19 bacteria in the form of bacilli through molecular biology and carried out studies with 11 of them. Five of the selected strains showed the better results considering bile salt resistance and ability to survive at different pH and antimicrobial effect. When the adhesion capacity in cell culture is examined, the better 2 strains are; Lactobacillus pontis ZZ6780 and Lactobacillus reuteri NBC2680 were selected and the growth kinetics of these strains were demonstrated at the 3 L bioreactor scale. Finally, the growth kinetics of selected strains were determined and the maximum specific growth rate of selected Lactobacillus pontis ZZ6780 and Lactobacillus reuteri NBC2680 was calculated as 0.412 h^− 1 and 0.481 h^− 1, respectively. In addition, the dry cell matter amounts were found to be and 4.45 g/L and 5.23 g/L, respectively.

Conclusion

This study established the groundwork for the selection of safety probiotics for the development and application of LAB. It is thought that the two strains obtained as a result of this study can be considered as potential probiotic strains in the food, pharmaceutical and cosmetic industries.

Introductıon

The human microbiota consists of a population of beneficial and harmful bacteria. Probiotics are live microorganisms that are beneficial for human health (Anadón et al. 2021). Probiotic microorganisms most frequently colonize in the gastrointestinal (GI) tract (Nithya et al. 2023). Probiotic bacteria, which maintain the balance in the microbiota, are located in epithelial cells, supporting the development of these cells and regulating the functions of these cells (Toupal and Cosansu 2023).

Lactobacillus spp. is the most common lactic acid bacteria that is gram positive, facultative anaerobic or microaerophilic, rod-shaped and nonsporing (Makaravo et al. 2006). Lactobacilli bacteria have been proven to have many positive effects on health. These effects include protection against intestinal and infections (Karami et al. 2017), regulation of the indigenous microbial population, reduction of lactose intolerance, immunotherapeutic effect (Harahap et al. 2023), increasing the nutritional value of foods, lowering the serum cholesterol level (Nami et al. 2019) and strengthening the immune system. The effectiveness of probiotics is proven through tests such as sensitivity to antibiotics, resistance to bile salts and low pH, thus ensuring consumer safety. Probiotic bacteria are considered safe food (GRAS) due to their bioactive molecules (Srinivash et al. 2023).

Lactic acid bacteria isolated from breast milk have been attractive for their probiotic potential, human origin, and the ability to reside in the intestine (Fernández et al. 2013). Breast milk has been shaped by millions of years of evolution that has resulted in a perfect multifunctional fluid. Actually, beyond the supply of nutrients and vitamins, breast milk provides bioactive factors, including immunoglobulins, cytokines, antimicrobial proteins, hormones, and oligosaccharides, which work in concert to fortify mucosal immunity, shape the gut microbiota, stimulate body growth and it is considered the best source of nutrition for infants (Hennet and Borsig 2016; Verhasselt et al. 2008). Breast milk has long been thought to be aseptic, and the presence of bacteria was assumed to be a result of contamination. Breast milk obtained from healthy women contains approximately 10³-10⁴ colony forming units / mililitre (cfu/ml) bacterial flora and is a source of microbiota for infants (Murphy et al. 2017). Breast milk is an important factor in the initiation, development, and composition of the neonatal gut microbiota (Gaudana et al. 2010). Commonly isolated facultative anaerob bacteria from breast milk belong to the genera Staphylococcus spp., Streptococcus spp., Enterococcus spp., Lactobacillus spp. and Bifidobacterium spp. (Martin et al. 2012; Reis et al. 2016).

Probiotic bacteria must adhere to the mucus layer and epithelial cells covering the intestinal lumen in order to prevent them from sliding out of the small intestine with peristaltic movements (Fernández et al. 2003). Adhesion to the intestinal epithelium is a crucial step for a probiotic bacteria to benefit the host. The ability of probiotic bacteria to adhere to intestinal mucosa is very significant in establishing temporary colonization of probiotics by maintaining the balance of the intestinal microflora. The high adhesion capacity of probiotic bacteria increases their residence time in the intestine. According to literature, Lactobacillus species probiotic strains adhere to the intestinal epithelium and function as a microbial barrier against pathogenic bacteria (Coconnier et al. 1997; Hudault et al. 1997; Servin 2004). Due to the difficulties that may be encountered during in vivo bacterial adhesion studies in human, an in vitro model system for bacterial adhesion to human intestinal cells has been developed (Ouwehand et al. 2002). Caco-2, HT29 are frequently used as epithelial colon cells and HT29-MTX cells are usually used as mucus-secreting cells in vitro studies (Archer et al. 2018; Gagnon et al. 2013).

Recently, understanding of the importance of probiotics has increased interest in large-scale fermentation studies of lactic acid bacteria (Chang and Liew 2013). In this regard, microbial growth kinetics studies are crucial opportunity for advances in biotechnology. Growth kinetic are very useful in the design and control of biotechnological processes because they provide a high degree of information about the growth behavior of microorganisms (Khalfallah et al. 2023). The growth of lactic acid bacteria has a complex structure and many components such as carbon source, nitrogen source, vitamins, inorganic salts and various nutrients are required in the growth medium. Thus, the production of viable cells, biomass and metabolites of lactic acid strains are notably affected by the cultivation conditions and medium components (Brinques et al. 2010). In order to be produced on an industrial scale and used effectively for subsequent processes; It is very important to reveal the specific growth rate (µmax), doubling time (g), number of generations and productivity values (Ucok and Sert 2020).

The aim of present study is to characterize the probiotic properties of lactic acid bacteria isolated from breast milk, maternal colostrum, adult and infant feces, and to reveal the growth characteristics of selected cultures for industrial production.

Materials and methods

Sample collection

In order to provide isolation from microbial matrices, four different sources were studied: maternal colostrum, breast milk, adult and infant feces. Maternal colostrum was collected 10 days after birth, breast milk and infant feces were collected within 6 months after birth, adult feces from a 28 year old female individual. The study protocol was approved by the Ümraniye Training and Research Hospital Clinical Research Ethics Committee (B.10.1.TKH.4.34.H.GP.0.01/110). All samples was collected as independent each other. Milk samples were collected manually in sterile tubes after disinfecting the teats and the surrounding area, but the first few drops were discarded. In addition, feces samples were collected with sterile swabs. The samples were kept at 4 °C until delivery to the Microbiology Laboratory of the Nuvita Biosearch Center (NBC) (Alyors Inc.) and immediately processed.

Isolation of LAB

For the isolation and identification of lactic acid bacteria, 1 gram of sample was taken from the isolation sources and transferred to 9 ml of 0.9% NaCl solutions and homogenized with a stomacher for 2 min. Six-fold dilutions of the samples were made with NaCl solution and 0.1 ml of sample for each dilution was planted on MRS-Cys agar plates. Agar plates were incubated at 37 °C under anaerobic conditions for 48 h (Dunne et al. 2003). An anaerobic jar with an anaerobic gas generating kit (Mitsubishi AneeoroPack, USA) was used to maintain the anaerobic condition. Colonies with different morphologies were purified and tested for catalase reaction, gram staining and microscopic examination. Then, 40 isolates were selected for further studies and stored in 30% glycerol stocks at − 80 °C.

Preliminary characterization of LAB isolates

The isolated bacteria were characterized by gram staining, catalase test, bacillus shape and non-spore-forming morphological and biochemical characteristics. Bacterial colonies that had gram-positive and catalase-negative characteristics and were rod-shaped on microscopic examination were selected for genotypic identification (Adugna and Andualem 2023).

Catalase test

For the catalase test, 3% hydrogen peroxide was used to select catalase-negative isolates. In the catalase test, bacteria taken from solid agar with the help of a loop tip were mixed with hydrogen peroxide (H₂O₂) on the slide, and it was observed that gas was released in those with catalase positive (+) and there was no gas in those with catalase negative (-) (Khalil et al. 2018).

Genotypic identification of lactic acid bacteria isolates

Genomic DNA isolation

Stock bacterial isolate kept at -80 °C were activated in deMan, Rogosa, and Sharpe (MRS) broth (Merck, Darmstadt, Germany) with cysteine (add 0.05% cysteine-hydrochloride). DNA isolation was performed with the Thermo Scientific GeneJet Genomic DNA Purification Kit (Thermo Fisher Scientific, US). Determination of the amount of DNA obtained after isolation was made using a NanoPhotometer (Implen, Germany). The obtained DNA isolate was stored at -20 °C until the next step.

Identification of isolates by RAPD PCR gene sequence

For the discrimination total of 19 LAB strains were grouped by random amplification of polymorphic DNA (RAPD) with primer M13 (5c-GAGGGTGGCGGTTCT-3c), as reported by Rossetti and Giraffa (2005). For this purpose, sterile water, primer (M13), master mix and DNA template were added to 100 µl PCR tubes, with a total final volume of 50 µl, and all components were mixed thoroughly with a micropipette. DNA fragments were amplified in a thermal cycler (Bio-Rad Laboratories, Hercules, CA) under the following conditions: Initial denaturation occurred at 94 °C for 3 min, followed by 30 cycles of denaturation at 95 °C for 30 s, annealing for 30 s, extension at 72 °C for 1 min and a 10 min final extension step at 72 °C. The products resulting from the PCR reaction were loaded onto a 1% agarose gel stained with ethidium bromide and DNA separation was performed at 90 V for 1 h. The gel obtained as a result of electrophoresis was subjected to imaging on a gel imager (Bio-Rad Laboratories, Hercules, CA) (Dertli et al. 2016).

Identification of isolates by 16 S PCR gene gequence

16 S rRNA analysis was applied to 11 bacteria distinguished by RAPD-PCR analysis. The PCR mixture prepared for 16 S rRNA analysis; A PCR mixture targeting 1,500 bp was prepared with 1-µl DNA of the relevant bacterium, 25 µl Q5 High-Fidelity 2X Master Mix (New England Biolabs), 20 mmol AMP_F (5’GAGAGTTTGATYCTGGCTCAG − 3’) and AMP_R (3’AAGGAGGTGATCCARCCGCA − 5’). This mixture was completed with 50 µl of sterile pure water. Program used for 16 S rRNA PCR analysis: 2 min at 98 °C, 30 s at 98 °C, 20 s at 57 °C. and in the last stage, 5 min at 72 °C. This process was carried out for 25 cycles. The products resulting from the PCR reaction were loaded onto a 1% agarose gel and DNA separation was performed at 90 V for 40 min. The gel obtained as a result of electrophoresis was subjected to imaging in the Bio-Rad Gel Doc EZ Imager. Reaction products were prepared as described above, and PCR products were sequenced and identified by Medsantek (Istanbul, Türkiye). Sequences obtained based on the 99-100% similarity criterion were identified using the BLAST algorithm. Sequences representing the best matches were retrieved and aligned using the clustal method (Kumar et al. 2018; Serrano-Niño et al. 2016; Tamura et al. 2021). The 16 S rRNA sequences of lactic acid bacteria strain NBC2680 and ZZ6780 of human origin identified in this study have been recorded in NCBI GenBank under the number OR999593-PP059538, respectively.

Probiotic potential characterization of bacterial isolates

The criteria that can be base on the selection microorganisms to be used as potential probiotic strains; selection of strain source, biosafety, resistance to low pH and bile salt in in vitro conditions, adhesion to and colonization of intestinal epithelium, antifungal activity, antimicrobial activity, stimulation of immune response, suitability for industrial production processes and survival in shelf life.

Tolerance to low pH

To determine the tolerance to low pH, the potential probiotic bacterial strains were grown in appropriate MRS or MRS-Cys medium with 1% inoculation for 24 h. Each bacterial suspension was adjusted to a concentration of 10⁶ cfu/ml. The bacteria were incubated in MRS or MRS-Cys broth adjusted to pH 2.0 and pH 3.0 with 1 M (Molar) HCl, and control broth for 6 hours. After 0, 3, and 6 hours of incubation, serial dilutions were prepared for bacterial viability and planted on MRS or MRS-Cys agar medium. The analyzes were performed in duplicate.

Resistance of bile salts

To determine the resistance of bile salts, the potential bacteria were grown in appropriate MRS or MRS-Cys medium with 1% inoculation for 24 h. Each bacterial suspension was adjusted to a concentration of 10⁶ cfu/ml. The bacteria were incubated in MRS or MRS-Cys medium supplemented with bile salt (Bovine bile, Sigma-Aldrich) at concentrations of 0.3% and 2% (v/v), and control broth for 6 hours. After 0, 3, and 6 hours of incubation, serial dilutions were prepared for bacterial viability and planted on MRS or MRS-Cys agar medium. The experiment was studied in duplicate.

Haemolytic activity

The bacterial isolates were inoculated on agar containing 5% sheep blood and the petri dishes were incubated at 37 °C for 24–48 h. At the end of incubation, zone formation in the petri dishes was observed. Staphylococcus aureus strain was used as a positive control. The formation of a green appearance around the colony indicates alpha hemolytic activity, the formation of a clear zone indicates beta hemolytic activity, and the absence of a zone indicates gamma hemolytic activity. This test was performed in duplicate (Maragkoudakis et al. 2009).

Gelatinase activity

The gelatinase activity of Lactobacillus strains was tested according to the method of Daliri et al. (2022) using MRS media containing 3% (w/v) gelatin (NEOGEN, USA). Petri plates containing the medium were spot inoculated with the strains and incubated at 37ºC for 48 h. The plates were flooded with saturated ammonium sulfate solution; The presence of a clear zone around the colonies was considered to be a positive indication of gelatinase production. Duplicates were preperad for each isolate studied. S. aureus strain was used as positive control.

DNase activity

The selected LAB isolates were cultured in duplicate on DNase agar medium (Sigma-Aldrich, EUA) to check production of DNase enzyme and the plates were incubated at 37ºC for 48 h. The plates were flooded with 1 M HCl; The presence of clear pink zone around the colonies was considered to be a positive indication of DNase production (Fonseca et al. 2021).

Antibiotic susceptibility

The susceptibility of potential probiotic strains to Vancomycin (VA, 30 µg) and Ofloxacin (OFX, 5 µg), Clindamycin (C, 2 µg), Erythromycin (E, 15 µg), Trimethoprim-sulphamethoxazole (TMP-SMX, 1.25 µg/23.75 µg), Levofloxacin (LEV, 5 µg), Streptomycin (S, 10 µg), Gentamycin (CN, 10 µg) was determined with antibiotic disks. The bacterial strains were inoculated at 1% into MRS or MRS-Cys broth and incubated at 37 °C for 24 h. The bacteria were planted on agar petri dishes using the spread plate technique. The antibiotic discs were placed in the middle of the petri dish and were incubated for 24–48 h at 37 °C. The resulting zone diameters were measured and the results were evaluated according to Clinical and Laboratory Standards Institute (CLSI) parameters. The analysis was duplicated.

Antimicrobial activitiy

Antimicrobial activity of probiotic isolates was determined using the agar well diffusion method. The probiotic isolates were incubated in appropriate broth for 24 h at 37 °C. Escherichia coli, Staphylococcus aureus, Salmonella enterica, Listeria monocytogenese pathogenic bacteria were inoculated in Brain Heart Infusion broth (Merck, Darmstadt, Germany). These selected pathogenic bacteria are common microorganisms that harm human health. The density of probiotic and pathogenic bacteria was adjusted according to the McFarland (0.5%) standard. 100 µl of each pathogen to be tested was spread on the BHI agar surface. A corkbor was used to puncture the agar surface. The probiotic bacterial strains in the broth medium were centrifuged at 10,000 rpm for 10 min and the cell-free supernatant (CFS) was collected and its pH was neutralized. 100 µl of CFS was added to the wells on the agar and the petri dishes were incubated at 37 °C for 16 h. The clean zone diameters formed around the wells by probiotic bacterial isolates against pathogens were measured in millimeters (mm). The experiment was duplicated (Tsega et al. 2023).

Cell culture

Culturing of caco-2 and HT29-MTX cells

The mucus-secreting cell line HT29-MTX was purchased from European Collection of Authenticated Cell Cultures (ECACC) and the human colonic cell line Caco-2 was obtained from The American Type Culture Collection (ATCC). The Caco-2 cells were routinely grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) with 4.5 g/L glucose (PAN-Biotech, Germany), supplemented with 10% (v/v) fetal bovine serum (FBS) inactivated for one hour at 56 °C (PAN-Biotech, Germany), with 1% (v/v) non-essential amino acids (PAN-Biotech, Germany), and 1% (v/v) penicillin-streptomycin (PAN-Biotech, Germany). The culture medium was changed 1 to 2 times per week. The HT29-MTX cells were grown in Dulbecco’s modified Eagle’s minimal essential medium (DMEM) with 4.5 g/L glucose (PAN-Biotech, Germany), supplemented with 10% (v/v) fetal bovine serum (FBS) inactivated for one hour at 56 °C (PAN-Biotech, Germany), with 1% (v/v) L-Glutamine 2 mM (PAN-Biotech, Germany), 1% (v/v) non-essential amino acids (PAN-Biotech, Germany), and 1% (v/v) penicillin-streptomycin (PAN-Biotech, Germany). The culture medium was changed 2 to 3 times per week. All experiments were carried out at 37 °C and cells were maintained in a 5% CO₂:95% air atmosphere. Fully differentiated cells were obtained 21 days and used in the adhesion assays. At least 24 h before the tests, Caco-2 and HT29-MTX cells inthe DMEM medium without serum and antibiotic (DMEM w/o) were seeded in 12-well culture plates at a concentration of 2 × 10⁵ and 3 × 10⁵ cells per well, respectively.

Bacterial adhesion assay

The adhesion ability of selected probiotic microorganisms to Caco-2 and HT29-MTX cells was appraised by plate counting method. The bacterial strains were activated in MRS broth under appropriate conditions 24 h before the assays. The Lactobacillus spp. cells were recovered by centrifugation at 8000 g for 5 min, washed twice with sterile 0.85% Sodium chloride (NaCI) solution and suspended at a concentration of 1 × 10⁸ (cfu) /ml in DMEM w/o medium. Initial viable bacteria were counted by plating on MRS agar. The bacterial suspension was added to each well of the cell lines and incubated in a 5% CO₂:95% air atmosphere at 37 °C for 3 h. After incubation, the Caco-2 and HT29-MTX monolayers were gently washed five times with PBS (pH 7.4) to remove unattached bacteria. The cells with adherent bacteria were treated with 0.05% trypsin-EDTA (PAN-Biotech, Germany) for 3 min at 37 °C followed by addition of culture medium containing FBS to stop the trypsin reaction. The supernatants containing adherent bacterias were collected and determined by plating serial dilutions on MRS agar. The enumeration was done after 48 h incubation at 37 °C in a suitable atmosphere. The results of the adhesion assays were expressed as the ratio of adherent bacteria to the total number of bacteria added to each well. The experiments were performed in duplicate.

Growth kinetics of selected lactobacilli cultures

Strain and inoculum preparation

Following the characterization of lactobacilli for their technological properties and cell culture, NBC2680 and ZZ6780 were further selected for determination of their growth kinetics properties. NBC2680 and ZZ6780 strains were stored in 15% glycerol at -80 °C. Frozen cells were precultured in MRS broth for 37 °C and kept stirring at 200 rpm for 16 h before inoculation in the bioreactor.

Fermentation process and calculation of kinetic parameters

NBC broth medium was used as culture medium for Lactobacillus speices in the glass bioreactor (Minifors 2, Infors HT, Switzerland). The composition of NBC medium is 96.06 g/L glucose, 40.76 g/L yeast extract, 19.43 g/L inorganic salts, and 11.01 ml/L Tween 80 (Kavak et al. 2022). The inorganic salt component is as follows: sodium acetate 45%; dipotassium hydrogen phosphate 20%; di-ammonium hydrogen citrate 20%; magnesium sulfate 10% and manganese sulfate 5%. NBC medium was used as a single medium. Batch cultivations were carried out with 1.5 L total volume at 37 ⁰C and during 18 h, in two parallels. pH was adjusted to pH 5.8 by the automatic addition of 3 N NaOH. Stirrer speed was 150 rpm and after sterilization, oxygen was removed by sparging N₂ for 30 min. The inoculum ratio was 1:20 (v/v) of the initial volume.

To determine the kinetic parameters, the specific growth rate µ value is calculated mathematically with the following expression below:

$${{dX} \over {dt}}\; = \;\mu \cdot X\;Xt\; = \;X0 \cdot {e^{\mu \cdot t}}\;\mu \; = \;{{(\ln X1 - \ln X0)} \over t}$$

where X (cells per mL) is the microbial population at time t (hours); X₀ (cells per mL) is the initial microbial population; µ is the specific growth rate, defined as the tangent in the inflection point (per hour). The value of td (is also referred to as the doubling time) which indicates the generation time is expressed as follows:

$\:X1\:=\:2X0$$\:{\upmu\:}\:=\:ln\:2\:/\:td\:\:$$\:\:\:td\:=\:0.693\:/\:{\upmu\:}\:\:\:\:$ Eq. (2)

where n, which is the number of generations, was obtained by dividing the period of logarithmic growing time to the doubling time is expressed as follows:

$$\:n\:=\:t\:/\:td\:n\:=\:\varDelta\:t\:/\:td\:\:\:\:\:\:\:\:\:\:\:\:\:\:\text{E}\text{q}.\:\left(3\right)$$

Productivity (φ) is obtained as the biomass production per unit time per unit culture volume is expressed as follows:

$\:\phi\:\:biomass\:=\frac{\left(\:Wbm1\:\--\:Wbm0\:\right)}{\left(t1\:\--\:t0\right)}$ Eq. (4)

where W, is the weight of biomass calculated from absorbance values at 600 nm in the Optical Density (OD) graph. OD600 measurements were performed on a UV-VIS spectrophotometer (Shimadzu UV-1900, Japan).

Dry cell weight is the weight of biomass calculated from the wet matter value in 1 g of sample in the moisture analyzer (Ohaus MB120, Switzerland).

$\eqalign{& Dry\;cell\;weight\;\left( {{g \over L}} \right) = \; \cr & Wet\;weight\left( {{g \over L}} \right) \cdot Dryweight(\% ) \cr}$ Eq. (5)

Data analysis

The data were analyzed statistically using the SPSS Statistics software package (version 29.0, IBM Corp., Armonk, NY, USA). The quantitative data were analyzed using One-way analysis of variance (ANOVA) with the Duncan multiple range tests (p < 0.05). The total bacteria cell counts (cfu/ml) was converted to a logarithmic value before statistical analysis.

Results

Preliminary characterization of lab isolates

A total of 40 bacteria were isolated from four different sources of human origin, and 32 bacterial colonies were identified as gram positive and catalase negative from these isolates. It was determined that 19 of the 32 isolates obtained were in the form of bacilli and 13 had cocci morphology.

Genotypic identification of lactic acid bacteria isolates

As a result of RAPD PCR, 7 of 19 bacteria obtained from four different sources of human origin were determined to be similar. Phylogenetic analysis of the 11 strains obtained based on the 16 S ribosomal RNA (rRNA) gene sequence was performed and bacterial names were identified from the National Center for Biotechnology Information (NCBI) BLAST system. Molecular identification of bacteria isolated from human sources using 16 S rRNA gene sequencing is shown in Table 1.

According to the genotypic identification and 16 S rRNA sequencing results of bacteria obtained from human origin sources; 1 Lactobacillus rhamnosus, 2 Lacticaseibacillus rhamnosus, 1 Limosilactobacillus fermentum, 1 Lactobacillus mucosae, 1 Lentilactobacillus parabuchneri, 1 Lactobacillus reuteri, 1 Lacticaseibacillus paracasei, 1 Lactiplantibacillus plantarum, 1 Lactobacillus oris, 1 Lactobacillus pontis strains were identified.

The 16 S rRNA genes of different lactic acid bacteria isolated from human sources were aligned using the MEGA11 program, and the phylogenetic relationship of these strains was determined by creating different subgroups.

Multiple sequence alignment analysis showed that the 16 S rRNA sequences of Lacticaseibacillus rhamnosus and Lactobacillus rhamnosus strains isolated from three different sources are close, and similarly, the sequences of Lactobacillus mucosae and Lentilactobacillus parabuchneri strains isolated from two different sources showed closeness. Although Limosilactobacillus fermentum and Lactobacillus mucosae strains were isolated from two different sources, their sequences were related. The similarities of Lactobacillus oris strains isolated from infant feces and Lactobacillus pontis strains isolated from adult feces are shown on the phylogenetic tree based on 16 S rRNA gene sequence analysis (Fig. 1).

Table 1 Molecular identification of isolates using 16 S rRNA gene sequencing

Fig. 1

Phylogenetic tree based on 16 S rNA gene sequence analysis of lactobacilli isolates. The neighbour-joining method was used to generate the tree. Bootstrap values based on 1000 replications are indicated at the nodes of the tree. The scale bar indicated 0.05 substitutions per nucleotide position. Accession numbers for sequences obtained from the NCBI database

Probiotic potential characterization of bacterial isolates

Tolerance to low pH

Probiotic strains must survive under gastrointestinal stress factors to maintain their biological activity within the host (Mbye et al. 2020). The viability analysis results of 11 selected lactobacilli isolates under pH 2.0 and pH 3.0 acidic conditions are shown in Fig. 2. The viability of bacteria treated under pH 2.0 acidic conditions decreased compared to the control group. At the 6th hour of incubation at pH 2.0, 5 probiotic bacteria strains showed viability. It was determined that NUV81, one of the probiotic bacteria whose viability was examined under pH 3.0 acidic conditions, decreased compared to the control group at the 3rd hour and did not show viability at the 6th hour. Among the probiotic bacteria whose viability was examined in pH 3.0 acidic environment, an increase in the viability of NUV160, NUV185, SA74 and ZZ6780 strains was observed, while a decrease was observed in other strains.

Fig. 2

Viability of the strains expressed as log cfu/ml after 0, 3 and 6 h incubation at control (A), pH2 (B) and pH 3 (C)

Resistance of bile salts

It has been reported that high bile tolerance benefits probiotic strain colonization in the host gastrointestinal tract (Luo et al. 2012). In this regard, it is important to evaluate the ability of potential probiotics to survive in the presence of bile salt. In the present work, two different bile concentrations were evaluated (0.3-2% w/v). The results determined by the plate count method are given in Table 2. When the results were examined, it was seen that the strains maintained their viability in the medium containing bile salt. In general, an increase in the viability of all strains except NUV81 was observed at both concentrations at the 6th hour of incubation. It was determined that NBC2680, NUV135, SA74 and ZZ6780 strains showed the highest resistance to bile salt at both concentrations.

Table 2 Selected Lactobacillus strains viability after 0, 3 and 6 h incubation in 0.3% or 2% bile salts

Hemolytic activity

Hemolytic activity is an important criterion in evaluating potential probiotics in terms of safety. The reason is that hemolytic activity indicates the potential virulence of pathogenic bacteria (Gebre et al. 2023). As a result of the analysis, potential probiotic strains did not form zones and showed gamma hemolytic activity.

Gelatinase and DNase activity

For gelatinase and DNase activity assays, it was observed that no zones were formed around the colonies at the end of the 48 h incubation period when compared to the positive control strain S. auerus. In this studies, none of the strains were found to be positive for gelatinase and DNase activity, validating their relative safety as probiotic candidates.

Antibiotic susceptibility

One of the most important safety considerations for a potential probiotic strains that it does not contain transferable antibiotic resistance genes to the pathogenic microorganisms which may bring about danger. It was observed that all strains were resistant to vancomycin, oflaxin and TMP-SMX, and only the NUV160 strain was determined to have intermediate sensitivity to the TMP-SMX antibiotic. All strains except NUV180 strain were found to be sensitive to clindamycin. While all strains showed sensitivity to erythromycin, strain NBC2680 was observed to have the intermadiate sensitive gene. In general, there is a group of antibiotics to which all strains are sensitive and resistant. The results are show in Table 3.

Table 3 Antibiotics susceptibility test results of the selected Lactobacillus strains for various antibiotics

Antimicrobial activity

Cell-free supernatant (CFS) is a liquid containing metabolites such as organic acids, fatty acids, bacteriocin released into the medium used during microbial growth. LAB demonstrate antimicrobial activity with these metabolites they produce (Mani-López et al. 2022; Tenea et al. 2018). The ability to inhibit pathogenic microorganisms growth is one of the mechanisms by which probiotic bacteria perform to protect the host. The analysis results of the antimicrobial effects of potential probiotic isolates against the pathogens E. coli, S. aureus, S. enterica and L. monocytogenes are given in Table 4.

Table 4 Antimicrobial activity profile of strains against various pathogens

The NUV7 strain showed inhibitory effects on all four different pathogens studied. In general, probiotic strains showed the most inhibition against the S. enterica pathogen. It was determined that the pathogen with the lowest inhibition zone diameter was E. coli. NUV135 strain formed the highest zone diameters against the S. aureus pathogen, NUV160 and NUV180 strains against the S. enterica pathogen, and NUV7 and NUV17 strains against the L. monocytogenes pathogen.

Cell culture

The adhesion ability to Caco-2 and HT29-MTX cells was evaluated for the 11 selected LAB and the results are presented in Table 5. The seven isolates showed a very low percentage of adhesion ability. However ZZ6780, NBC2680 and NUV81 showed higher percentages (12.04%, 10.67% and 10.63% of adhesion, respectively) of adhesion to Caco-2 cells compared to the positive control L. rhamnosus GG (10.04%). Similar results were obtained for the adhesion to HT29-MTX cells. While the percentage of adhesion to HT29-MTX cells of the control strain L. rhamnosus GG was 9.24%, the adhesion percentages of ZZ6780, NBC2680 and NUV81 strains were determined 10.03%, 9.70% and 9.61%, SA74 and NUV180 were characterized by its moderate adhesion capacity to Caco-2 and HT29-MTX cells compared to LGG control strain (8.51% and 8.74%, 7.22% and 5.48% of adhesion, respectively). It was observed that the adhesion percentage of five strains showing low capacity adhesion to HT29-MTX cells was higher than the adhesion percentage to Caco-2 cells.

Table 5 Percentage values of Caco-2 and HT-29 MTX cell lines adhesion

Growth kinetics of selected culture

NBC2680 and ZZ6780 were further selected for determination of their growth kinetics properties. Both strains were tested for their growth kinetics under laboratory-scale on 3 L bioreactor fermentation conditions and µmax (specific growth ratemax), t_d (doubling time), n (number of generations), φ and φmax (productivity) were determined. The growth kinetic parameters and biomass value are show in Table 6.

Table 6 Growth kinetic parameters and biomass value

The growth versus time, OD600, and dry weight amount per 1 g/L curves of NBC2680 and ZZ6780 are given in Fig. 3. Mainly, L. reuteri and L. pontis growth was characterized by a delay phase of about 3–4 h, a steep exponential phase for 12 h, followed by a fixed phase for 2 h. The maximum increase in the count of bacteria was found as 3.1 log₁₀ cfu/ml when NBC2680 had reached the stationary phase. Also, the maximum increase in the count of bacteria was found as 2.25 log₁₀ cfu/ml when the ZZ6780 had reached the stationary phase.

Fig. 3

OD values and viable cell count level and dry weight of NBC2680 (A, B) and ZZ6780 (C, D)

Discussion

The identification of lactic acid bacteria with probiotic properties based on physiological and biochemical properties, their identification on the basis of species and genus using international specific primers has been widely published in the literature. Since the 16 S rRNA gene is highly conserved among different bacteria and archaea, phylogenetic studies have been handled in this context. Genomic DNA was isolated by the method described by Dertli et al. (2016). The amplified PCR product was purified and sequenced to identify the isolated bacteria.

Isolation and identification of strains human origin revealed the presence of L. mucosae, L. reuteri, L. paracasei, L. plantarum, L. oris, L. rhamnosus, L. parabuchneri, L. pontis, L. fermentum strain. When the studies in isolation from breast milk were examined, it was determined that the L. rhamnosus strain was generally included in each study (Damaceno et al. 2023; Kang et al. 2020; Li et al. 2017).

L. reuteri, L. paracasei and L. plantarum bacteria obtained by isolation of infant feces Zhang et al. 2018; Ahrné et al. 2005; while it was similar to the study results in the articles by Khalil et al. 2007; it was determined that there was no article stating that L. oris and L. mucosae bacteria were found in infant feces. As a result of the isolation studies, it was concluded that L. paracasei bacteria was commonly found in infant feces. The presence of L. fermentum and L. rhamnosus bacteria in the maternal colostrum in our study was similar to the study of Liu et al. (2020), and both studies determined the presence of the L. rhamnosus strain.

L. parabuchneri bacteria isolated from adult feces was similar to our study because it was also found in the results of previous studies (Dal Bello et al. 2003). When the isolation results were examined, it was concluded that L. parabuchneri bacteria was not found very frequently in adult feces. The results of the L. rhamnosus strain isolated from adult feces was supported by the studies of Kang et al. (2017). While L. pontis bacteria was not isolated from adult feces, it was determined to be present in the isolation study from vaginal flora (Shazadi et al. 2022; Mehta et al. 2020).

It was determined that the lactic acid bacterium common in isolations from adult feces, breast milk and maternal colostrum was L. rhamnosus. While L. rhamnosus obtained from breast milk and maternal colostrum was found on the same branching with its similarities, L. rhamnosus isolated from adult feces was located on different branching. The similarity of L. oris and L. paracasei bacteria isolated from infant feces was revealed as a result of phylogenetic branching. It was determined that L. mucosae and L. parabuchneri bacteria isolated from different sources were located in the same branch.

The characterization as probiotics is determined by their ability to survive in the low pH of the stomach and the high concentration of bile salt in the gastrointestinal tract (Kobierecka et al. 2017). The pH in human stomach ranges from 1 during fasting, to 4.5 after a meal (Wang et al. 2009). Since Lactobacillus strains are known to survive at pH 4.6, which is the common final acidity of many fermented dairy products, lower pH values (2.0 and 3.0) were examined. The pH value of 3.0 has been most investigated to use probiotic strains (Shokryazdan et al. 2014), because strain viability is reduced at pH 2.0 (Bhatt et al. 2012). In our study, the highest viability at the end of the 6th hour at pH 2 was observed in NBC2680, while at pH 3 it was also observed in ZZ6780. In the studies of De Angelis et al. in 2006, it was reported that the L. mucosae strain isolated from pig feces showed resistance at pH 2.0 and pH 3.0 in 180 min; similar results were seen in our study. (De Angelis et al. 2006). Bile released in the small intestine damages bacteria by destroying the bacterial cell membrane. Probiotics has a bile salt hydrolase enzyme (BSH), which hydrolyzes bile salts and reduces their solubility (Jannah et al. 2014; Hwanhlem et al. 2010). Gondaliya and Ramani analyzed the bile salt resistance of L. fermentum and L. oris strains isolated from breast milk at 0.5% and 1.50% concentration, and as a result of the analysis, it was observed that bacterial viability decreased with increasing concentration (Gondaliya and Ramani 2021). It was reported by Jose et al. that the tolerance of Lactobacillus spp. isolated from dairy food product and animal rumen to 0.3% bile salt was higher than the tolerance to 2.0% bile salt (Jose et al. 2015). Overall, Lactobacillus strains are tolerant to bile salts (Liu et al. 2020; Mohammedi et al. 2018; Khalkhali and Mojgani 2017).

The present study assessed safety of the isolated strains. Gelatinase and DNase are the enzymes mostly produced by pathogenic microorganisms (Zhao et al. 2011). It was determined that the strains did not have any potential virulence properties such as hemolytic, gelatinase and DNase activities. Previous studies of hemolytic activity have reported that lactobacilli group were non-hemolytic (Halder et al. 2017; Kaktcham et al. 2012). It was observed that Lactobacillaceae family isolated from milk by Maragkoudakis and colleagues showed γ hemolytic activity (Maragkoudakis et al. 2006). It has been reported in previous studies that most Lactobacillus strains do not show gelatinase and DNase activity (Abouloifa et al. 2020; Fonseca et al. 2021; Huligere et al. 2023).

Antibiotic susceptibility studies showed that was determined 7 strains of the selected probiotic bacteria were resistant to Levoflaxin and 4 strains had intermediate sensitivity. It was determined that 8 strains were resistant to the antibiotic streptomycin, 2 strains had intermediate sensitivity and 1 strain was sensitive. It was observed that strain NBC2680 had intermediate sensitivity to the antibiotic gentamicin, while all other strains had resistance genes. Previous stuides, different resistance rates to ciprofloxacin (60–70%), gentamicin (0–100%), and streptomycin (70–80%) have been reported among Lactobacillus spp., including L. helveticus, L. casei, and L. plantarum (Hummel et al. 2007; Guo et al. 2017; Li et al. 2019). This indicates the existence of considerable differences in antibiotic resistance both on an intergenus and interspecies level as well as a species dependency in Lactobacillus spp. similar to our study results, Florez et al. reported that the L. plantarum strain showed high sensitivity to the antibiotic Clindamycin (Florez et al. 2006). Chang et al. observed that the L. reuteri strain was sensitive to erythromycin but resistant to gentamicin (Chang et al. 2023).

CFS, which contains metabolites such as hydrogen peroxide, short-chain fatty acids and low molecular weight proteins produced by lactic acid bacteria, shows anti-microbial properties by inhibiting the growth of pathogens (Tanaka et al. 1999; Torshizi et al. 2008). It was observed by Wang et al. that the L. plantarum strain showed high antimicrobial activity against S. aureus, E. coli and S. enterica pathogens (Wang et al. 2011). These results are similar to the results in our study. According to the antimicrobial results of Ambalam et al., L. rhamnosus strain showed high activity against L. monocyctogenes and our analysis results were supported (Ambalam et al. 2009). In the study conducted by Jomehzade et al., with lactic acid bacteria isolated from infant feces in southwest Iran, it was determined that L. plantarum, L. fermentum, L. rhamnosus and L. paracasei strains were effective against E. coli and S. enterica pathogens (Jomehzade et al. 2020). The activity of the lactic acid bacteria tested in this study against pathogens has been supported by previous studies (Jovanovic et al. 2015; Mollova et al. 2024).

Probiotic bacterial strains must be able to survive passage through the gastrointestinal tract and colonize the small intestine and colon for a long period to provide host health benefits (Suskovic et al., 2001). We analyzed the ability of 11 selected probiotic strains to adhere to the intestinal epithelial cells Caco-2 and the mucus-secreting cells HT29-MTX. In general, it was observed differences in the adhesion capacities of probiotic isolates depending on the type of probiotic bacteria strain and the isolated source. It was determined that NBC2680 strain, which showed the highest tolerance to 0.3% bile salt, and ZZ6780 strain, which showed high resistance in pH 3 environment, showed high adhesion ability to both cell lines. Moreover, It has been observed that bacteria isolated from adult and infant feces show higher adhesion ability compared to the other sources. It has also been associated in previous studies that probiotic bacteria that are resistant to low acid environment and bile salt have a higher capacity to adhere to the intestinal epithelium and mucosa (Bao et al. 2010; Guo et al. 2015; Jin et al. 1998; Ross et al. 2005). L. plantarum probiotic bacteria strains, which is known to have high adhesion ability (Duary et al. 2011), showed average adhesion capacity compared to the L. rhamnosus GG control strain in our adhesion tests. When the findings of the L. rhamnosus species obtained from three different sources were evaluated, the probiotic isolated from adult feces showed higher adhesion capacity than the other sources. L. rhamnosus strains have a high capacity to adhere to the intestinal mucosa (Chae et al. 2022). In our results, it was observed that L. rhamnosus strains had increased adhesion efficiency in mucus-secreting cells compared to intestinal epithelial cells. Similar to our findings, it was found previously that the L. paracasei strain isolated from feces showed lower adhesion capacity than the L. rhamnosus strain (Verdenelli et al. 2009). According to the result of our adhesion assays, it was found that L. pontis, L. reuteri and L. mucosae species showed high adhesion ability compared to the selected other species. The result is similar to that observed previously (Jensen et al. 2014; Pearce et al. 2018; Repally et al. 2018; Roos and Jonsson 2002). However, there was no study about the adhesion to epithelial cells and intestinal mucosa of L. pontis and L. oris strains.

Determination of the growth parameters in a reasonable accepted medium is a crucial step for the commercial application of a probiotic strain. Considering the previous studies, many factors such as pH, air supply rate, fermentation temperature, fermentation operation system (batch, fed-batch or continuous) affected lactic acid bacteria growth although the same synthetic medium (MRS broth) was used as the fermentation medium (Kayacan et al. 2023; Mechmeche et al. 2017; Zheng et al. 2018). Additionally, determining growth kinetics is an important issue for biotechnological processes. In the present study, µmax value was calculated to be 0.481 h^− 1 for L. reuteri NBC280 with a doubling time (t_d) of 1.21 h^− 1. Additionally, number of generations (n) was calculated to be 6.4 for strain NBC2680 productivity (φ) and maximum specific productivity (φ max) values were 0.572 g L^− 1 and 1.02 g L^− 1, respectively. In terms of growth kinetics of ZZ6780 µmax value was calculated 0.412 h^− 1 whereas the doubling time (t_d) 1.12 h^− 1. The number of generations (n) was calculated to be, 5.5 and productivity (φ) and maximum specific productivity (φ max) value for ZZ6780 were 0.498 g L^− 1 and 0.764 g L^− 1, respectively. These findings revealed that maximum specific growth rate of both strains was higher in comparison to the growth kinetics of other LAB strains (Gao et al. 2009; Rezvani et al. 2017; Ucok and Sert 2020). According to the fermantation results, the wet weight of dry biomass NBC2680 and ZZ6780 was determined as 29.80% and 27.91%, repectively. Based on this, biomass production was 5.23 g/L and 4.45 g/L on dry cell matter basis, repectively. In previous studies; Hwang et al. (2011) reported that during the batch fermentation of LP02 isolated from infant feces, 2.2 g/L dry cell was produced. Śliżewska and Chlebicz-Wójcik (2020), developed L.reuteri LOCK 1092 strain on MRS and SSM medium and found the specific growth rate 0.14 and 0.31 h^− 1, respectively. Burge et al. the was determined maximal specific growth of the three L. reuteri strains cultivated in MRS medium supplemented with 20 g/L of glucose. Maximum specific growth (µmax ^− 1) rates were equal to 0.82, 0.80, and 0.50 h^− 1 respectively for the DSM 20,016, DSM 17,938 and ATCC 53,608 strains (Burge et al. 2015). Choi et al. (2021), find out that the maximum biomass was 4.3 g/L amount of L. plantarum 200,655. Fonteles et al. (2011), DSM 20,016 strain of determined the cell viability was 8.3 log cfu m/L and biomass concentration 3.96 g/L at the end of the fermentation. In different study, Lactobacillus casei biomass was produced at a maximum dry cell of 0.45 g/L in batch, semi-batch and continuous cultures (Aguirre-Ezkauriatza et al. 2010). Furthermore, L. plantarum strain Pi06 cultured in MRS-Cys (0.05% L cysteine) medium was reported produced 4.60 g/L biomass (Hwang et al. 2012). Determination of the growth kinetics of NBC2680 and ZZ6780 in the study revealed their suitability for industrial scale growth applications. Because the growth kinetic data both dry cell matter, maximum specific growth rate and cell viability amount obtained as a result of the study are promising when compared to previous studies.

Conclusion

To summarize ın the present study, 40 bacteria were isolated from 4 different sources of human origin: breast milk, maternal colostrum, adult feces and infant feces. 19 of these isolates were determined to be lactobacilli as a result of analyzes and microscopic analyzing, and 7 of them were determined to be similar as a result of RAPD PCR. Phylogenetic analysis based on the 16 S rRNA gene sequence of 11 strains obtained as a result of eliminating similar bacterial strains was performed. According to the findings obtained after isolation and identification, human feces; regardless of adult or infant, is reach in terms of containing various lactobacilli than human milk. Lactobacillus pontis, Lentilactobacillus parabuchneri, Lacticaseibacillus rhamnosus from adult feces; Lactobacillus oris, Lactiplantibacillus plantarum, Lacticaseibacillus paracasei, Limosilactobacillus reuteri, Lactobacillus mucosae from infant feces; Lacticaseibacillus rhamnosus, Limosilactobacillus fermentum bacteria were isolated from maternal colostrum and Lactobacillus rhamnosus bacteria were isolated from breast milk. These results show the importance of the isolation source in obtaining different types and numbers of potential probiotic bacterial strains.

After the identification step, characterization studies were carried out to determine the potential probiotic properties of 11 strains. As a result of the investigation of the potential probiotic properties of 11 different Lactobacillus strains obtained from 4 different sources, the most prominent strains were SA74, NUV180, NUV81, NBC2680 and ZZ6780. NBC2680 and ZZ6780 strains were selected through cell culture studies and their fermentation was studied. Finally, determination of the growth kinetics of NBC2680 and ZZ6780 demonstrated their suitability for industrial-scale growth applications. As a result of the study, NBC2680 and ZZ6780 strains, which are considered to be commercialized, were registered in NCBI Genbank. It is thought that the results of this study will play an important role in introducing potential probiotic strains to the industry.