1 Introduction

Functional and probiotic foods, such as fermented dairy products, may be thought to give extra health advantages or protection against diseases caused by ingesting contaminated food, in addition to being a source of nutrients. Food-borne illness prevention and treatment necessitate interdisciplinary methods that utilize beneficial living microorganisms (probiotics) to battle food-borne pathogens and the accompanying health hazards. Food-borne diseases pose several difficulties. Several studies have shown that probiotic LAB inhibits the growth of food-borne infections. Consuming a considerable number of probiotic living microorganisms with food has been shown to benefit consumers' health [1,2,3]. Because of customer desire for fewer chemical preservatives, the importance of employing helpful microbes to preserve food has grown. Furthermore, animals have been given preventative antibiotic doses for decades. The potential for people and cattle to promote cross-resistance and multiple antibiotic resistance in pathogenic microorganisms, as well as the harmful impact of encountering drug residues, are emerging concerns. One strategy for decreasing such difficulties is to use growth enhancers, such as probiotics, that have a good influence on host health and growth efficacy [4,5,6].

The term "lactic acid bacteria" (LAB) refers to a collection of gram-positive, acid-tolerant, nonsporulating, microaerophilic rods or cocci that have been classified based on their similar physiological and metabolic features. There are roughly 20 distinct genera within the phylum Firmicutes, with Lactobacillus, Lactococcus, Leuconostoc, Streptococcus, Enterococcus, Pediococcus, Aerococcus, Oenococcus, Tetragenococcus, Carnobacterium, Vagococcus, and Weissella being the main LAB. These microbes are found in nature in fermented foods such as cheese and yogurt. They improve the flavor and consistency of food while also inhibiting the growth of bacteria that cause degradation and sickness [7,8,9,10,11]. Recent research has demonstrated that some LAB isolates isolated from fermented foods exhibit desirable characteristics for probiotic cultures. Seven probiotic strains have been isolated from fermented foods including tiger nut milk, bovine milk [12] and natural whey starter used in cheese manufacture [13].

Egyptian traditionally fermented dairy products are consumed without heat treatment, making them good carriers for probiotic bacteria. Upper Egypt's probiotic sources include yogurt, Karish cheese (skim milk cheese), Rayeb milk (concentrated sour milk), and Kishk (wheat-based fermented milk). These things are used in large quantities [6, 14,15,16]. The probiotic strains isolated from these fermented foods and drinks show desirable functional properties for use as probiotics against food-borne illnesses, and they might be employed as a starting culture for the mass manufacture of the traditional product. Some LAB are probiotics, while others have the potential to be probiotics or are just fermentation cultures found in nature and used in the food industry. Both of these LAB groups have the potential to be advantageous [17].

The purpose of this study is to look at the in vitro probiotic properties of LAB isolated from three traditional Egyptian fermented dairy products (yogurt, Rayeb milk, buttermilk, and Kariesh cheese) in terms of their potential probiotic activity against different pathogens.

2 Materials and methods

2.1 Dairy samples

Forty fermented dairy samples were collected from numerous Fayoum governorate, Egypt, markets. Rayeb milk, yoghurt, Karish cheese, and buttermilk were included. Samples were put in clean containers and refrigerated until transport to the lab. When they entered the lab, they were separated.

2.2 Isolation of LAB

Each sample of dairy products was diluted in a saline solution with 0.85% sodium chloride before plating on MRS agar. The colonies were separated after incubation (37 °C/48 h). Randomly selected colonies (based on color, shape, elevation, and size) were striated on MRS agar-coated plates for future investigation. To retain culture, the initial colony was subcultured. Before being sealed, the isolates were inspected under a microscope. Gram-positive, catalase-negative cocci or bacilli are lactic acid bacteria. The bacteria were suspended in glycerol and kept at − 20 °C for further investigation.

2.3 Confirmation tests of isolates

Gram staining and morphology Using a light microscope with oil immersion lenses, fresh cultures of the isolated organisms were wet-mounted on microscopic transparencies. Cellular morphology involved cell shape and arrangement of the cells.

Catalase activity A single, uncontaminated colony was provided with a drop of 3% hydrogen peroxide solution to determine catalase activity.

Spore staining Gram-positive and catalase-negative isolates were examined for endospore formulation under a light microscope with oil immersion objectives after staining according to Prescott [15]. Therefore, isolates without endospores were studied.

Motility test The motility test was performed using the wet mount technique [18]. Non-motile microorganisms were chosen.

2.4 The probiotic properties LAB isolates

Antibacterial activity against pathogens According to Wolf and Wirth [19], The selected isolates were tested against food-borne pathogens Bacillus cereus, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella enteritidis, and Escherichia coli using the agar well diffusion assay. Inhibition zones were detected and measured.

Antibiotic susceptibility Antibiotic effectiveness was measured by LAB's zone of inhibition diameter (mm). [20]. In vitro diagnostics use Flummox, Ampiclox, Chlorophenicol, Penicillin, and Tarivid. These antibiotics were chosen since they have worked against several infections in the past. Most of them broadcast.

Screening for acidity and bile salt tolerance of viable cells The stress tolerance trials were modified from Parente et al. [21]. The OD600 readings of overnight cultures of thirteen distinct LAB isolates were reduced to approximately 0.6 and then normalised to 1. 1 N HCl was used to create MRS broth with pH values of 2.0, 3.0, and 4.0 (for acid stress) and 0.1, 0.3, and 0.5% bile salt (for bile salt stress). Bacteria were re-cultured in MRS broth at 37 °C for 48 h after treatment. Viability % calculation:

$$\% {\text{ viability}} = ({\text{OD sample}}/{\text{OD control}}) \times 100$$

2.5 Molecular identification (isolation of genomic and amplification of 16S rRNA)

LAB isolates were identified using morphological, phenotypic and biochemical characteristics. 16S rDNA sequencing was conducted to further confirm the identity of the isolates. All bacteria have the 16S rRNA gene, allowing relationship measurements so; 16S rRNA gene sequences identify strains by genus, species, and subspecies in all major bacterial phyla. Multiple well-known species with identical or similar 16S rRNA gene sequences are identified [22]. ABT DNA micro extraction reagent (Applied Biotechnology Co., Ltd., Egypt) extracted nucleic acid according to the manufacturer's instructions. Three isolates' 16S rRNA genes were amplified by PCR using universal primers (GAGTTTGATCCTGGCTCAG) and (GGTTACCTTGTTACGACT) to determine their species [23]. Solgent Co Ltd. (South Korea) gel-purified and sequenced PCR-amplified materials. Geneious (Biomatters) assembled the sequences. BLAST (GenBank) found shorter sequences. Phylogenetic analysis: MAFFT matched GenBank nucleotide sequences with identified sequences [24]. Neighbour joining method and Tamura-Nei were used to generate phylogenetic trees [25].

3 Results and discussion

3.1 Isolation and identification of LABs

A total of 13 isolates (from Kareish cheese, Rayeb milk, local yoghurt and buttermilk) were selected based on colony and cell morphology, staining, and the milk coagulation test. All of the selected isolates were Gram-positive, non-motile, non-spore producing, and catalase-negative, and they all produced distinctive tiny, whitish-creamy colonies on MRS agar. These strains' ability to develop in anaerobic environments suggests that they are linked to lactic acid bacteria, and hence they have been designated LAB-1 to LAB-13. Using a microscope, the cell morphology of all isolates was seen to be rod or coccus shaped (Table 1). LABs are bacteria with rod or coccus morphologies, negative catalase, homo or hetero fermentation, and grow in low acid environments [26, 27]. These are gram-positive bacteria that generate lactic acid as their primary fermentation product and are usually regarded as safe [28]. The milk coagulation test revealed that all of the isolates can coagulate milk at 37°C. Lactic acid bacteria can ferment milk to produce lactic acid and coagulate milk as result [27, 29].

Table 1 General characterization and morphology of LAB isolates
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4 Probiotic properties of LAB isolates

4.1 Antibacterial activity against pathogens

Antimicrobial activity against human pathogens is regarded as the most important characteristic of probiotic strains that promote gastrointestinal health. The antibacterial action of LAB may be attributable to the synthesis of numerous compounds during their growth and subsequent extracellular excretion. The LAB released several inhibitory compounds, including as organic acids, hydrogen peroxide, and bacteriocin, that are responsible for its antibacterial action [30, 31]. Hence, using the well diffusion technique, the antibacterial activity of the cell-free supernatant (CFS) of LAB isolates was evaluated. The results demonstrated that the CFS of all LAB isolates has an antibacterial action against selected pathogens, and the diameters of the inhibition zones differed as shown in Table 2. The diameter of inhibition zones demonstrated that crude extracts from each isolate exhibited significant (p < 0.001) antimicrobial action against each tested pathogen except LAB-10 which had no effect on B. cereus. Isolate LAB-3 had the greatest antagonistic activity against Staph. aureus, as measured by the inhibition zone's diameter of 23.33 mm. Similar results were reported in another studies [30, 32].

Table 2 Effect of interaction between LAB isolates and pathogenic strains on inhibition zone diameter
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Whereas, the main effect of several LAB isolates and tested pathogens was statistically examined (Table 3). LAB-6 had a significantly larger inhibition zone of 18.40 mm, followed by LAB-2 (18.07 mm). LAB-10 had the least antibacterial activity, resulting in a zone measuring 10.53 mm. Many investigations supported these findings by demonstrating that certain LAB strains may generate antimicrobial compounds that, through various methods, exert a potent inhibitory effect on numerous bacteria, including pathogens. Antimicrobial agents generated by LAB include organic acids, hydrogen peroxide, ethanol, diacetyl, acetaldehyde, acetoin, carbon dioxide, and bacteriocins. LAB-produced organic acid resulted in low pH numbers and hydrogen peroxide activity. Organic acids and other compounds demonstrate antibacterial action against several Gram-negative pathogens [3, 33,34,35,36,37]. In a recent investigation, four different strains of LAB isolated from colostrum were shown to have effective antibacterial properties against harmful microorganisms [38]. Moreover, The zone of inhibition produced by eight different lactobacilli isolates against various pathogens varied from 4.66 mm to 27.66 mm [39].

Table 3 Main effects of LAB isolates on inhibition zone diameter
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4.2 Antibiotic susceptibility

Due to the rising prevalence of multidrug-resistant bacterial isolates, antibiotic resistance is a global health problem. Given that LAB might operate as a reservoir of transferable antibiotic resistance genes, LAB strains used as food additives of dairy starters should be evaluated for its antibiotic susceptibility. So, the susceptibility of the thirteen isolates to five different antibiotics is displayed in Table 4. Tarivid and Flummox were both effective against every isolate. Whereas seven of thirteen isolates were Penicillin-resistant. Only LAB-3 exhibited strong chlorophenicol resistance. Moreover, LAB-6, LAB-4, and LAB-13 exhibited Ampiclox resistance. Other antimicrobials have a range of effects on isolates, from sensitivity to resistance. According to Table 5's main effect data, LAB-6 was the most resistant to all antibiotics tested, with the smallest inhibition zone diameter of 14.40 mm, followed by LAB-4 (14.80 mm). The most susceptible isolate was LAB-12, which exhibited the largest zone of inhibition (27.26 mm).

Table 4 Antibiotic susceptibility of selected LAB strains
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Table 5 Main effects of LAB isolates on inhibition zone diameter as affected by different antibiotics
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In recent study, Sixty-five LAB strains were used as probiotic additives or silage inoculants, were determined for susceptibility to eight clinically relevant antibiotics. Nineteen LAB strains showed phenotypic resistance to one antibiotic, while 15 bacteria were resistant to more than one antibiotic in the genera Lactobacillus and Pediococcus [40]. In another research by Nunziata et al., strains of Lactobacillus, Streptococcus, and Bifidobacterium that are now utilised for industrial dairy products are typically resistant to gentamycin, kanamycin, chloramphenicol, and tetracycline [41]. Also, eight isolates were examined for antibiotic sensitivity, including Lactobacillus plantarum, L. rhamnosus, L. acidophilus, L. salivarius, and L. paracasei, as well as Bifidobacterium longum, B. breve, and B. adolescentis. All of the lactobacilli strains were Vancomycin resistant, and all of them had intermediate Clindamycin resistance. Penicillin, Tetracycline, Ampicillin, Erythromycin, Streptomycin, Florfenicol, Chloramphenicol and Trimethoprim were all effective against all isolates [6].

Some Lactobacillus strains were evaluated for antibiotic susceptibility using disc diffusion [39]. These strains were sensitive to five different antibiotics, while L. plantarum GV69 was resistant to ampicillin and cefixime. One cell wall and protein-preventing antibiotic worked against the strains. Fonseca et al. [42] observed that L. brevis CCMA1284 was resistant to lincomycin, azithromycin, and penicillin, whereas two L. paracasei strains were mildly sensitive or susceptible. Also, some strains of Lactococcus lactis, Enterococcus spp., and Lactobacillus spp. were resistant to antibiotics, such as chloramphenicol, erythromycin, clindamycin, or tetracycline [43]. Numerous studies have documented a significant level of penicillin resistance in lactobacilli of fermented food origin. Resistance to penicillin and ampicillin varied by species. Strains of L. paracasei, L. casei, L. brevis, and L. plantarum have also been reported to be resistant to penicillin antibiotics at a high level [44,45,46,47].

4.3 Resistance to acid and bile salt

For a bacterial isolate to be considered a potential probiotic, it must be able to thrive under gastro-intestinal conditions and possess acid and bile tolerance, antimicrobial against pathogens, antibiotic susceptibility, and other characteristics. Acidity's effects on LAB isolates' viability are shown in Table 6. LAB-8 was the only isolate with a 100% viability at pH 4 and a 50% viability at pH 2, while being resistant to both of those conditions. Of the studied isolates, LAB-13 was the most acid-sensitive, dying out at pH 4, 3, and 2. This suggests that this isolate is particularly sensitive to the acidic environment of the intestines. Most isolates' viability (in percentage terms) reduced significantly (p 0.01) when pH fell.

Table 6 Effect of different pH and bile salt concentrations on viability (%) of LAB isolates
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In addition, every single isolate were tested for its tolerance to bile salt. The percentage of isolates that survived exposure to bile salts is shown in Table 6. The capacity of different cultures to thrive in MRS broth spiked with bile salts differed significantly (p > 0.01). It was discovered that LAB-8 was the strain least tolerant to bile, whereas LAB-5 was the most resistant. There was a statistically significant difference in the rate of growth between the sensitive and non-sensitive isolates (p > 0.05).

Probiotics must survive the stomach before accessing the small intestine so, tolerance to high bile salt levels and low pH are desired. In recent study, it was reported that 12 isolated LAB survived the stomach's acidic environment and high bile [48].

In two similar studies, some Lactobacillus spp. isolates' stomach pH and bile salt tolerance indicated their potential. Both studies found 0.3% bile salt resistance. However, Lactobacillus isolares were highly sensitive to stomach pH [49, 50]. Despite being lactic acid bacteria, fermented milks have high acidity at the end of production and during shelf life, which may explain their tolerance to low pH [51]. Mandal et al. found no significant difference in viable cell count for free and encapsulated L. casei cells at low pH and high bile salt concentration [52]. Moreover, L. brevis LB32 and L. pentosus LP05 could survive bile salt concentrations from 0.5 to 3%, however their coefficients of inhibition varied. Both isolates hydrolyzed bile well at pH 3, 4, and 5 [53].

According to a number of studies, acid and biliary resistance is an essential indicator in choosing probiotic strains. The most beneficial probiotic bacteria survive pH 3.0 for two hours according to Garriga et al. [54]. Consistent with this finding was the acid resistance of the selected isolates from our investigation. Some isolates were not resistant to pH levels below 2.0, possibly because probiotic bacteria are protected by food and other carrier matrix molecules in the stomach [55]. Moreover, 12 lactobacilli isolates including L. plantarum, L. sakei and L. bulgaricus have an excellent survival rate in bile salts ranging from 45 to 94% [56]. Since these microorganisms are naturally resistant to bile salts, their high resistance may also be explained by their pH tolerance.

5 Sequence analysis of 16S rRNA gene and phylogenetic analysis

Total genomic DNA was extracted from each of the three isolates LAB-2, LAB-3, and LAB-6 with the highest probiotic potential in order to identify their genotypic characteristics. This extraction was performed in order to identify the isolates via amplification of the bacterial-specific 16S rRNA gene sequencing which permits identification of bacteria into to the genus and species level as stated by Sadrani et al. [57]. The 16SrRNA gene sequences of selected isolates were compared with sequences in the Gene-Bank database. A phylogenetic tree based on 16SrRNA sequences (Fig. 1) revealed affiliation of isolate LAB 2 to the genus Lactobacillus, comprising 98.7% identity to L. rhamnosus OP268116 strain GCM20300, which is most closely related to L. rhamnosus. LAB 3 was revealed to genus Lactobacillus, comprising 98.9% identity to L. rhamnosus OP268117 strain 6481. While revealed affiliation of isolate LAB 6 to the genus Enterococcus, comprising 94.9% identity to Enterococcus durans OP268118 strain CAU5334; Comparative genomic research indicated minimal variations between this strain and Enterococcus durans.

Fig. 1
figure 1

Phylogenetic tree showing the relationship between selected isolates and its homologues strains in Genbank. The tree was constructed using MEGA6 sequence alignment programs

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Using phylogenetic estimation of 16S rDNA genes, other recent studies have identified at the strain level diverse lactic acid bacteria with potent probiotic properties isolated from fermented food products [2, 3]. Comparative 16S rRNA gene analysis conducted by Shokryazdan et al. [58] revealed that LAB belonged to L. fermentum, L. acidophilus, L. casei and L. buchneri. Using 16S rRNA gene analysis, Cho, Lee, and Hahm [59] have identified Lactobacillus strains with putative probiotic properties. In another recent study, two of the 60 isolates found in fermented foods showed the best probiotic results and were identified as Lactobacillus acidophilus CM1 and Lactobacillus delbrueckii OS1 where there are numerous Lactobacillus species in fermented foods, including yoghurt and kefir [60]. Seven lactobacilli isolates from human milk were identified by 16S rRNA gene analysis as Lacticaseibacillus paracasei and Lactobacillus gasseri. Overall, each strain demonstrated superior probiotic and technological properties for use in lactic fermentation [61]. Moreover, Coulibaly et al. [62] identified 12 LAB strains via 16S rDNA gene sequence homology analysis to be found from two genera, Pediococcus (P. acidilactici and P. pentosaceus) and Lactobacillus (L. plantarum) which selected considering to probiotics, functional, storage, and safety factors.

Our investigation led to the detection of three probiotic organisms, which were later described and genotypically confirmed to be members of the Lactobacillus and Enterococcus genera. Due to their unique properties, these indigenous probiotic strains may be beneficial to the food industries. This study demonstrates the importance of oral and dietary sources of new probiotics with desirable functional properties.

6 Conclusions

Due to their numerous health benefits, the study of LAB and their potential as probiotics has garnered significant attention in recent years. Lactobacillus species can be found in fermented foods. In the present research, probiotic properties of LAB isolated from fermented dairy products were evaluated. The thirteen selected isolates from the preliminary screening test met multiple requirements for use as probiotic microorganisms, including broad-spectrum antibacterial activity, susceptibility to antibiotics, and resistance to gastric pH and bile salts. The isolates LAB-2, LAB-3, and LAB-6 were identified to be L. rhamnosus OP268116, L. rhamnosus OP268117, and Ent. durans OP268118. Thus, these isolates can be considered potential probiotic strains with a beneficial role in maintaining good intestinal tract health in humans, and they could be used in novel strategies against the rising number of antibiotic-resistant pathogens in human infections, or even as food biopreservatives or a starter culture in the fermented dairy products and cheese industry. To complete the safety evaluation and probiotic properties of these newly characterized LAB strains, however, in vivo and clinical trials, additional in vitro studies, and genomic characterization are necessary.