Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 20  |  Issue : 4  |  Page : 281-293

Isolation, screening, and molecular identification of new lactic acid bacteria as potential probiotics


Department of Microbial Genetics, Biotechnology Research Institute, National Research Centre, Giza, Egypt

Date of Submission17-Jul-2021
Date of Decision14-Aug-2021
Date of Acceptance05-Sep-2021
Date of Web Publication24-Dec-2021

Correspondence Address:
Ph.D. Ahmed F El-Sayed
Department of Microbial Genetics, Biotechnology Research Institute, National Research Centre, Dokki, Giza, 12311
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/epj.epj_46_21

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  Abstract 


Background and objective Lactic acid bacteria (LAB) are the most useful microorganisms to society, LAB are used in the production of many fermented food products that stimulate digestion and immunity. Therefore, the objective study aimed to isolate, characterize, and identify new LAB members and determining their potential probiotic properties.
Materials and methods This study was performed through isolation, selection, and identification of LAB strains morphologically, biochemically, and genetically. Then, to determine their probiotics traits, which include acid resistance, bile-salt tolerance, β-galactosidase activity, heat sensitivity, salinity, cholesterol reduction, and antioxidant activity.
Results and discussion Eight LAB isolates were isolated and purified from milk and cheeses, out of eight isolates, K2 showed the highest resistance to acidic pH 2.0, where Log CFU/ml reduced from the initial count 8.79±0.17 at pH 7.0 to 4.57±0.10 at pH 2.0 with 51.99% survival rate. Also, the six bile-tolerant isolates (K1, K2, K3, K4, A11, and A5) were found tolerant to 1% bile-salt concentrations for 3 h with 92.50, 71.10, 64.35, 60.72, 71.11, and 72.21% rate of survival, respectively. Also, antibiotic susceptibility of A11 isolate was the most sensitive, but isolate A2 was the most resistant against antibiotics. Besides, heat sensitivity of selected LAB isolates (K1, K2, A11, and B1) were able to survive at temperatures 15, 37, and 45°C, but isolates (K3, K4, A5, and A2) did not grow at 45°C. Additionally, NaCl tolerance clearly revealed that both isolates (K2 and A11) were able to tolerate a wide range of NaCl (1–8% w/v), then the growth declined with the increase of salt (10%) concentration. The results showed that the cholesterol assimilation was reduced by 32.66 and 30.20% for A11 and K1 isolates, respectively. Also, K2 isolate showed the highest antioxidant activity (86.93%), and the best β-galactosidase producer (24.65 U/ml). Therefore, the 16S-rRNA sequencing was carried out to identify four promising LAB isolates that could be employed as a probiotic strain. Phylogenetic analysis showed that isolates belonged to three genera, including Lactobacillus, Lactococcus, and Enterococcus. Strains were Lactobacillus casei (A5) KU510332, Enterococcus faecium (A11) KU510333, Lactococcus lactis (K1) KU510334, and E. faecium (K2) KU510335.
Conclusion Our results presenting four new isolates as promising potential probiotics for further application in processing probiotic products.

Keywords: 16S-rDNA, antioxidant, bile salt, cholesterol, probiotic–lactic acid bacteria, β-galactosidase


How to cite this article:
El-Sayed AF, Abo-Sereih NA, El-Kawokgy TM. Isolation, screening, and molecular identification of new lactic acid bacteria as potential probiotics. Egypt Pharmaceut J 2021;20:281-93

How to cite this URL:
El-Sayed AF, Abo-Sereih NA, El-Kawokgy TM. Isolation, screening, and molecular identification of new lactic acid bacteria as potential probiotics. Egypt Pharmaceut J [serial online] 2021 [cited 2022 Aug 10];20:281-93. Available from: http://www.epj.eg.net/text.asp?2021/20/4/281/333681




  Introduction Top


Lactic acid bacteria (LAB) are Gram-positive, rods, aerotolerant, nonsporulating, and nonpathogenic organisms, mostly found in nature, its major industrial application in the era of foods and fermented products as Generally Regarded as Safe [1]. They are industrially used as starter cultures in the formation of dairy products, fermented vegetables, and meat products [2].

Probiotic was defined as ‘Vital microorganisms that gave critical effects on the human body by increasing its microbial balance’ [3]. Also, probiotic bacteria may secrete a large amount of metabolites that prevented the pathogen’s growth, and include organic acids and bacteriocins [4],[5]. Lactococcus also displays antifungal activity [6]. Lactobacilli living as commensal in the intestinal environment might possess probiotic activity; they may have antitumoral activity [7].

LAB have many important nutritional benefits, including increasing the nutritional value of food, preventing gastrointestinal infections, improving digestion of lactose, and controlling some types of cancer [8],[9]. Decreased cholesterol levels [10] help lactose metabolism and stimulate the immune system [11]. These health benefits can be attributed to numerous synergistic mechanisms and biological activities [12]. Also, these lactobacilli species have vital effects on health [13].

Screening of probiotics for their antioxidant potential by different methods like 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assays for evaluation of antioxidant activity was performed in vitro [14],[15]. β-galactosidase has long been accepted as an important ingredient in food-processing industries. In the dairy industry, β-galactosidase could be used to inhibit lactose crystallization, enhance sweetness, increase milk-product solubility, prepare low lactose-containing food products for low lactose-tolerant people, and recycling of the environmental pollutant, cheese whey [16].

Nowadays, along with the fast development of molecular biology and increasing knowledge on genomic structures of LAB, many molecular techniques, including 16S-rRNA gene-sequence analysis, have been developed [17],[18]. The large database of 16S-rRNA gene sequences enables us to identify LAB very easily [19]. The 16S-rRNA gene can be used for studying phylogenetic relationships between microorganisms and provides a more accurate and precise tool for microbial identification [20].

Here we present the isolation and identification of new four LAB strains on species level using the phenotypic, biotypic, and genotypic methods. Our isolates showed promising characteristics as probiotics by expressing acid and bile-salt tolerance, β-galactosidase activity, cholesterol reduction, and antioxidant activity.


  Materials and methods Top


Collection of samples

Isolation of LAB has been carried out from different sources: milk, cheese, and fermented milk.

Isolation of lactic acid bacteria

One gram of sample was immediately processed under aseptic conditions by suspending it in 9 ml of normal-saline NaCl (0.85%) and streaked on the de Mann Rogosa Sharpe (MRS) agar and incubated at 37°C for 48 h under CO2 5% and observed for the morphology and number of colonies [21].

Identification of selected isolates

Identification of the probiotic isolates was performed based on their morphological and biochemical characterizations, as described in Bergey’s Manual of Systematic Bacteriology [22]. Physiological properties were also studied [23].

Evaluation of the potential probiotic properties for lactic acid bacteria bacterial isolates

Tolerance to NaCl

MRS agar plates with different NaCl concentrations (2.0, 4.0, 6.0, 8.0, and 10%) were inoculated with tested isolates and incubated for 24 h at 37°C. The CFU was calculated for each concentration to denote NaCl tolerance [24].

Temperature sensitivity

The isolates were grown on MRS broth at varying temperatures, that is, 15, 25, 37, and 45°C for 48 h. Using the pour-plate method, LAB growth on MRS agar plates was used to denote temperature tolerance [24].

Acid tolerance

The isolates were grown for 24 h in MRS broth at 37°C. An aliquot of 0.1 ml from different cultures incubated for 24 h was inoculated into 10-ml MRS broth with a pH value adjusted to 2, 3, and 4, and incubated for 3 h at 37°C. pH tolerance of the strains was assayed on MRS agar plates by plate-count technique [25].

Bile-salt resistance

Overnight cultures from the tested isolates were inoculated into MRS broth with different concentrations of bile salt (Sigma Chemical Co., USA) (0.3, 0.5, and 1% w/v) for 3 h at 37°C. Then they were plated onto MRS agar plates and incubated for 24 h at 37°C. The MRS without bile salt was used as a control. The viable count was determined by plate count for each [25].

Bile-salt deconjugation

Bile-salt hydrolase (BSH) enzyme was screened. Briefly, 10-µl aliquots of cultures were spotted on MRS agar plates supplemented with 0.3, 0.5, and 1% (w/v) of taurodeoxycholic acid sodium salt (Sigma Chemical Co., USA). The hydrolysis zone surrounding colonies indicated the BSH activity of the tested bacteria [26].

Antibiotic susceptibility

The susceptibility to antibiotics was tested by the disk-diffusion method [27]. One percent of the test isolates inoculated in Mueller–Hinton agar (0.8% agar). Antibiotic disk was placed on it to allow the diffusion of antibiotics into the medium and then incubated at 37°C for 24 h. The inhibition zone around each antibiotic disk was measured to check the susceptibility of the isolate.

Antioxidant activity

The chemical assay that includes DPPH was used at a concentration of 0.2 mM in methyl alcohol. The sample was combined with an equivalent volume of freshly DPPH solution (0.2 mM). The mixture was vigorously shaken and left to react for 30 min in the dark. The scavenged DPPH was then monitored by determining the absorbance (Abs) at 515 nm against methanol blank using Agilent Technologies Cary100, UV-VI spectrophotometer [28].

In vitro cholesterol-reduction activity

Overnight isolates were inoculated (1%) into MRS broth supplemented with water-soluble cholesterol (100 mg/dl) and incubated for 24 h at 37°C. The cholesterol content was then measured in the supernatant by quantitative determination of cholesterol kit, ‘Spinreact Kits’ (Barcelona, Spain) according to the manufacturer’s instructions [29].

β-galactosidase activity

β-galactosidase activity in whole cells was determined according to the method of Miller and Brand [30].

Molecular identification of lactic acid bacteria using the 16 S ribosomal RNA gene

Extraction of genomic DNA: Genomic DNA was extracted from the overnight culture of the selected isolates using a Qiagen DNA purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

Amplification of 16s-rRNA by PCR and identification by sequence analysis: Genomic DNA of isolates was used as a template in PCR reactions using GoTaq Flexi DNA Polymerase (Promega Company, WI, USA) according to the manufacturer’s instructions and two primers, namely, 8 F (5-AGAGTTTGATCCTGGCTCAG-3) and 1495 R (5-CTACGGCTACCTTGTTACGA-3) [31]. A mixture of PCR conditions was as follows: 5× Go Taq Flexi buffer, 10 μl, Go Taq Flexi DNA polymerase, 0.5 μl, MgCl2, 25 mM, 2 μl, PCR nucleotide Mix, 10 mM, 1 μl of DNA, and 1.5 μl of double-distilled water mixed in a final volume of 50 μl. The program for PCR was as follows: 95°C for 5 min, 35 cycles of 95°C for 1 min, 50°C for 1 min, and 72°C for 2 min, and extension at 72°C for 10 min, +4oC∝. The PCR amplifications were performed by gene amp 9600 thermal cycler (Applied Biosystems, Foster City, CA, US).

Phylogenetic analysis of 16S-rRNA sequences: DNA sequencing of amplified fragments was carried out by the sequencing service of HVD, Germany. The length of 16s-rRNA gene was edited and trimmed with software BioEdit 7.2.5 and corrected sequences were compared with those stored in the GenBank database using BLAST. A phylogenetic tree based on 16S-rRNA genes was also constructed to determine the closest bacterial species by the neighbor-joining method using molecular evolutionary genetics analysis [32].

Statistical analysis

All experimental samples were tested in triplicate, and the resulting values are expressed as the mean±SD.


  Results Top


Isolation and identification of lactic acid bacteria isolates

From the samples, eight predominant colonies were isolated. As shown in [Table 1] and [Figure 1], the colonial and cell morphology of isolates of LAB were studied. The colonial morphology was either white or cream in color and their shape either circular or pinpoint in shape, and the margin of the colony was entire. All isolates showed a typical appearance of LAB on MRS medium (small, pinpointed colonies). Microscopic examination is the first criteria that provide information about genus level and purity of the isolates. The most important and widely used method is Gram staining. Among eight LAB isolates from cheddar cheese and goat milk, three strains were Gram-positive, rod-a shaped, and catalase-negative, while the remaining five strains were Gram-positive, cocci shaped, and catalase-negative according to their phenotype profile. By determining the cell morphology under the light microscope, all the selected isolates were Gram-positive, catalase-negative, and rod- or cocci-shaped bacteria. Cocci isolates were presumptively identified as Enterococcus or Lactococcus since they were coccus shaped and isolated from milk. Rod isolates were presumptively identified as the Lactobacilli group. Lactobacilli grow best in slightly acidic media with an initial pH of 6.0–4.0. Growth ceases commonly when pH 3.0–4.0 is reached. Isolates were first classified according to their growth at 45°C and 6.0% NaCl concentration; they were therefore identified as Enterococcus. The three isolates (K2, A11, and B1) were able to grow at 6.0% NaCl and 45°C and they were identified as Enterococcus spp. Whereas the two isolates (K1 and A2) could grow at 15 and 40°C, in 4% NaCl, and were able to ferment maltose, salicin, and/or saccharose. Therefore, K1 and A2 were identified as Lactococcus lactis subsp. lactis or L. lactis.
Table 1 Colonial and cell morphology of the selected isolates from dairy products

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Figure 1 Microscopic examination of some selected bacterial isolates. (a) Simple stain of isolate A5. (b) Simple stain of isolate K1. (c) Simple stain of isolate K4. (d) Simple stain of isolate K3. (e) Gram stain of isolate K2. (f) Gram stain of isolate A5. (g) Simple stain of isolate B1. (h) Simple stain of isolate A11.

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Data in [Table 2] revealed that all isolates were negative to oxidase, catalase, and nitrate reduction. Only the isolates (B1 and A5) showed the ability to utilize citrate by giving blue color but the other isolates indicating a negative reaction to citrate utilization. According to determination of their abilities to hydrolysis starch, all isolates were unable to hydrolyze starch, except isolates K3 and A2 that showed the ability to hydrolyze starch by giving a clear zone as positive results. All the isolates showed no ability to hydrolysis of casein, except the two isolates A2 and K1, which showed the ability to hydrolyze casein into simple amino acids by giving a clear zone. The isolates showed different types of sugar-utilization patterns, including glucose, fructose, galactose, lactose, sucrose, mannitol, sorbitol, and maltose. Their ability to ferment the sugar was confirmed by turning the media into yellow in the experimental tubes. [Table 2] explains that all isolates are able to ferment all monosaccharides such as glucose, fructose, galactose, and disaccharides (lactose). Furthermore, some of the chosen isolates (K3, A5, A2, and B1) can use sugar alcohol like mannitol and sorbitol, as well as disaccharides, while the other isolates cannot.
Table 2 Biochemical characteristics of the selected lactic acid bacteria isolates from dairy products

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Evaluation of the potential probiotic properties for lactic acid bacteria bacterial isolates

Tolerance to NaCl

Salt-tolerant probiotic strains were used in incorporation in probiotic products. Eight selected LAB strains in our study were tested for their growth under different salt concentrations. Data in [Table 3] revealed high growth of all isolates in the presence of 2% NaCl, but some isolates were able to grow in the presence of 4.0 and 6.0% NaCl with some differences as the growth was decreased when the concentration of NaCl was increased from 4.0 to 6.0%. The growth pattern at 4% NaCl concentration was also reported and concluded a wide variation in the growth of probiotic isolates. Only two strains showed weak growth at 8% NaCl, while no growth was detected at 10% NaCl concentration.
Table 3 Tolerance of lactic acid bacteria isolates to different concentrations of sodium chloride

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Acid tolerance

Acid tolerance is the second selection criterion for probiotics. Resistance to the acid condition was used as an essential property for probiotic strains. The ability of given LAB isolates to survive at low pH values is a mandatory criterion in the selection of probiotic. [Table 5] shows an acid-tolerance study for eight isolates after exposure for 3 h at pH 2.0, 3.0, and 4.0. Strain K2 was the most resistant to the acid environment among the isolates. The survival rate of selected isolates in acidic buffer (pH 2.0) was examined by the difference in viable cell counts after 0 min and 3 h of incubation, as shown in [Table 4]. All the isolates, except isolate k3, showed better tolerance to acidic pH of 2.0, as compared with normal conditions, isolates k1 and k2 showed a high survival rate at pH 4.0, while they showed 39.42 and 51.99% survival rate, respectively, in acidic pH 2.0, and other isolates showed intermediate levels of tolerance.
Table 4 Effect of acid on the survival of lactic acid bacteria isolates (Log CFU/ml)

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Table 5 Effect of bile salt on the survival of lactic acid bacteria isolates (Log CFU/ml)

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Bile resistance and bile-salt hydrolase activity

Bile tolerance is the third selection criterion for potential probiotic. In this study, isolates were inoculated in MRS media containing different concentrations of bile salts to check their ability to survive in it. MRS medium lacking bile salt was used as control, whereas 0.3, 0.5, and 1.0% bile-salt concentrations in MRS broth were used to check the tolerance of the isolates. As evident from [Table 5], the survival of selected isolates was also examined by the difference in viable cell counts following 0 min and 3 h of incubation in MRS containing 0.3, 0.5, and 1% bile salts and the results were subjected to statistical analysis (P<0.05). As given in [Table 5], isolate K1 showed the best survival rate (98.95% at 0.3% bile salt, 92.51% at 1% bile salt) in contrast to standard isolates (A2) (57.98% at 0.3% and 11.10% at 1% bile salt). Also, other isolates are intermediate between the two. Also, BSH was assayed with MRS agar containing 0.3, 0.5, and 1.0% bile-salt concentrations. According to [Table 6], isolate K1 was of the highest resistance to bile salt, high activity of BSH enzyme, and the highest clear zone at 1% concentration. While all the isolates showed no clear zone at 1% bile-salt concentration.
Table 6 Inhibitory activity of potentially probiotic bacterial isolates against bile salt hydrolase at different concentration (0.3, 0.4, 0.5, 1.0%) and different temperature (15–45°C)

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Temperature sensitivity

Temperature was considered a selection criterion for probiotic strain that grows at 37°C, which is an essential factor of probiotics to show their effectiveness. Also, growth at high temperatures was used as a technological feature in industries and packing of probiotic products. Therefore, selection of LAB isolates that grow under a broad range of temperature (37–45°C) was the target of our study. The results obtained in [Table 6] were positive for growth at the chosen temperature range (15–45°C). All the selected LAB isolates (K1, K2, A11, and B2) were able to survive at temperature ranges 15, 37, and 45°C. But other isolates (K3, K4, A5, and A2) do not grow at 45°C.

Antibiotic-susceptibility pattern of selected lactic acid bacteria isolates

Antibiotic therapy can significantly affect this microbial balance in the intestine by reducing the viability of indigenous microflora. The result presented in [Table 7] showed antibiotic susceptibility of probiotic culture ranging from sensitive (S), intermediate (I), and resistant (R) against different tested antibiotics. Isolate K1 was sensitive to rifampicin, tetracycline, chloramphenicol, neomycin, gentamicin, and streptomycin and resistant to kanamycin, vancomycin, ciprofloxacin, oxacillin, and cephradine and intermediate to ampicillin, amikacins, and norfloxacin. Isolate K2 was sensitive to vancomycin, oxacillin, kanamycin, and norfloxacin and resistant to ampicillin, amikacins, ciprofloxacin, and cephradine and intermediate to rifampicin, tetracycline, chloramphenicol, neomycin, streptomycin, and gentamicin. Both isolates (K3 and K4) were sensitive to tetracycline, chloramphenicol, and ciprofloxacin and resistant to norfloxacin, gentamicin, neomycin, and streptomycin. Both isolates (A11 and A5) were sensitive to tetracycline, chloramphenicol, and rifampicin and resistant to amikacins and streptomycin.
Table 7 Antibiotic susceptibility of the selected probiotic culture

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Antioxidant activity of selected lactic acid bacteria isolates

Accumulated evidence suggests that LAB strains have good potential for application in functional foods and health-related products. To augment these results, the present study researched the antioxidant activity of these strains. To elucidate the antioxidant activity of the eight strains, DPPH radical scavenging activities were determined. As shown in the results in [Figure 2], the isolate K2 was of the highest antioxidant activity (86.93%), while isolate K1 was of the lowest antioxidant activity (61.42%) as compared with all tested isolates.
Figure 2 Diagram of cholesterol assimilation and antioxidant activity of the selected isolates.

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Anticholesterol activity

Data presented in [Figure 2] indicated that all isolates can remove, and assimilated cholesterol ranged from 16 to 32%. Isolate A11 has the highest assimilation of cholesterol with 32.6%, in contrast, isolate K4 showed 16.5% of cholesterol assimilation. While isolates K1, K2, K3, A5, A2, and B1 have cholesterol-assimilation activity (30.2, 24.3, 27.6, 22.0, 18.7, and 24.9%), respectively, cholesterol assimilation was shown to be the highest for isolates A11 and K1 (up to 32%), whereas isolates K4 and A2 showed the least cholesterol removal (16.5 and 18.7%, respectively).

β-galactosidase activity of selected isolates

Data presented in [Figure 3] noted that all tested strains were grown in MRS medium containing 1% lactose as a medium used for colorimetric assay of β-galactosidase activity. When ONPG assay was used, isolate K2 was the highest enzyme producer of the isolates (24.65 U/ml) and considered to be the best β-galactosidase producer, while isolate K1 was of the lowest β-galactosidase activity about 6.68 U/ml, isolates K3, K4, A11, A5, A2, and B1 have β-galactosidase production (07.16, 19.30, 13.88, 19.92, 16.70, and 17.20 U/ml), respectively.
Figure 3 Diagram showing the β-galactosidase-enzyme activity (U/ml) of the selected isolates.

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Molecular identification of lactic acid bacteria using 16 S rRNA gene

Molecular identification of the selected isolates (K1, K2, A11, and A5) was carried out by amplification and sequencing their 16 S rRNA gene. The four PCR products were at an expected size 1500 bp ([Figure 4]). The amplified PCR products were purified, sequenced, and aligned using a blast with the published sequences of the 16 S rRNA gene of other strains deposited in NCBI databases. According to the blast results of the isolates with more than 99% similarities, they were identified as (K1) L. lactis, (A5) Lactobacillus casei, and (K2 and A11) Enterococcus faecium. Their partial sequences ([Figure 5],[Figure 6],[Figure 7],[Figure 8]) were submitted to the GenBank at NCBI database with accession numbers A5: KU510332.1, A11: KU510333.1, K1: KU510334, and K2: KU510335.1 ([Table 8]). Their phylogenetic trees are shown in [Figure 9].
Figure 4 About 1.5% agarose-gel electrophoresis of PCR products of 16 s rRNA gene for the isolates. Lane 1, DNA marker 1 Kb; lane 2, K1; lane 3, K2; lane 4, A11; lane 5, A5.

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Figure 5 The 16S-rDNA gene partial sequence of isolate (K1) Lactococcus lactis.

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Figure 6 The 16S-rDNA gene partial sequence of isolate (A5) Lactobacillus casei.

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Figure 7 The 16S-rDNA gene partial sequence of isolate (K2) Enterococcus faecium.

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Figure 8 The 16S-rDNA gene partial sequence of isolate (A11) Enterococcus faecium.

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Table 8 Identification percentage between selected potential probiotic bacterial isolates and related species

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Figure 9 Phylogenetic tree based on partial 16S-rDNA sequences, showing the relationship between the isolates A11, K1, K2, and A5 and other LAB. The tree was constructed using the MEGA 6.06 and neighbor-joining method. LAB, lactic acid bacteria.

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  Discussion Top


In the present work, dairy-product samples from milk, fermented milk, and cheese were selected to isolate LAB. The samples were gathered from different sources to get a wide diversity of bacterial strains. Eight isolates were selected randomly and purified. The selection is based on the change in the morphology of the colonies, such as color, texture, and margin. Phenotypic characters were used for the characterization of the isolates. For identification, biochemical characterization and molecular techniques were used. These characteristics are in agreement with LAB characteristics obtained by Axelsson [33] and Arasu et al. [34]. By comparing the results of the biochemical characterization of the bacterial isolates with Bergey’s manual of systematic bacteriology, the isolates were identified as Enterococcus spp., Lactococcus spp., and Lactobacillus spp. The survival under the environmental conditions of the gastrointestinal tract is an important characteristic feature of the isolate for potential use as a probiotic. Thus, the probiotic properties such as bile resistance and acid tolerance were assessed. Our isolates showed the ability to pass the basic criteria for probiotic features as they can tolerate to pH 2 and growth in 0.3% bile salts. Bile-salt tolerance varies significantly among LAB species even between strains themselves. LAB resistance to bile salt can be attributed to the activity of the enzyme BSH, which is responsible for the degradation of bile salts lowering its toxic effect [35].

In our work, data revealed that most of the strains could survive in the presence of different concentrations of bile salts ranging from 0.3 to 1.0% after 3 h of incubation in addition to their ability to hydrolyze the bile acids. The isolates proved their ability to grow at different pH values ranging from 2.0 to 7.0. The results obtained are in agreement with Soliman et al. [36] results with three isolates of Lactobacilli from Egyptian-fermented dairy products and showed that they can tolerate acid and bile salts. The ability of the LAB strains to tolerate salt concentration is commonly used as the selection for potential probiotic candidates. Our data indicated that all isolates could tolerate salt concentration (1–8%), while E. faecium isolate (K2) was tolerant to 8% and this result is in agreement with that reported by Adnan and Tan [37]. Also, Hoque et al. [24] observed the NaCl (2–10%) tolerance of Lactobacillus sp. isolated from yogurts.

The susceptibility of LAB isolate cultures was assessed against common antibiotics to determine the ability of the culture to maintain the microbial balance in the intestine during antibiotic treatment. The results obtained were in agreement with Zhou et al. [38] who showed that both Lactobacillus and Bifidobacterium strains were affected by β-lactam, Gram-positive spectrum, and broad-spectrum antibiotics. Some of the LAB species from Lactobacillus, Leuconostoc, and Streptococcus genera are sensitive to clinically relevant antibiotics such as ampicillin, tetracycline, penicillin, erythromycin, and chloramphenicol [39]. Also, our study was in agreement with Hashemi et al. [27] who found that probiotic strains were sensitive to different antibiotics, such as ampicillin, penicillin, chloramphenicol, erythromycin, and rifampicin, but they were resistant to vancomycin and streptomycin.

The antioxidant properties of the selected strains were determined by the DPPH radical scavenging activity method. Among the eight probiotic isolates, E. faecium isolate (K2) showed the highest antioxidant activity (86.9%), while L. lactis K1 isolate showed the least antioxidant activity (61.4%). These results are in harmony with Bing et al. [40], Wang et al. [10], and Shen et al. [41] who stated that CFS of Bifidobacterium animals gives potent antioxidant activities. Also, Wang et al. [10] showed that the filtrate from 35 LAB showed more potent antioxidant activities than bacterial suspensions.Cholesterol assimilation in the intestinal tract decreases the absorption of dietary cholesterol from the digestive tract, multiple mechanisms have been suggested for understanding the cholesterol-lowering activity of probiotic bacteria [42]. The data obtained showed that all the probiotic isolates tested could remove and assimilate cholesterol from media. These results are in harmony with Klaver and Van Der Meer [43], Lee et al. [44], and Tsai et al. [45].

β-galactosidase is widely used, especially in dairy technologies. As β-galactosidase plays an important role in the digestion of lactose, β-galactosidase-deficient individuals suffer from severe intestinal disorders, including bloating, flatulence, and abdominal pain. All the isolates in the study showed β-galactosidase activity, which allows them to be used in the dairy technology. E. faecium K2 isolate showed maximum β-galactosidase activity giving 24.65 U/ml. These results are in harmony with Lin et al. [46], and LAB probiotics are reported to accelerate lactose digestion lowering lactose-intolerance (LI) symptoms [47]. β-galactosidase possesses both hydrolyzation and transglycosylation activities, it can synthesize oligosaccharides simultaneously with lactose hydrolysis. It is stated that reliance on morphological, physiological, and biochemical characteristics for identification of LAB is often considered as misleading, since many morphological and nutritional requirements may be similar among different species [48]. Also, reliance on sugar-fermentation profile-based phenotypic characterization may not provide reliable identification of LAB; however, molecular-based methods such as 16S-rDNA were used to identify bacteria as an alternative to phenotypic methods [49]. In our study primarily, presumptive identification was done by biochemical assay and sugar-fermentation pattern according to Bergey’s Manual of Systematic Bacteriology. Further, the isolates were identified by 16S-rDNA sequencing, such as E. faecium, L. lactis, and L. casei, and these results are in harmony with Saeedi et al. [50] and Cibik et al. [51] as they showed that 16S-rDNA gene sequencing can be considered as a potent approach that enables both tracing phylogenetic relationships between bacteria and identification of bacterial strains isolated from various environmental sources and fermentation specimens.


  Conclusion Top


Milk products can act as a rich reservoir of LAB strains for their role in probiotic research and be used as probiotics. In this study, among several isolates, eight isolates of LAB were isolated, purified, and identified using morphological, biochemical, and physiological tests such as growth temperature, pH condition, salt concentration, and genotype-based method of 16S-rDNA. Then probiotic criteria, such as acid and bile-salt tolerance, bile-salt deconjugation, cholesterol removal, temperature sensitivity, antioxidant activity, and beta-galactosidase activity, were evaluated. Molecular-biology technology based on the 16S-rDNA-sequencing technique was followed to verify species’ nomenclature. We obtained four promising isolates as potential probiotics that showed probiotic characteristics and were identified as L. casei (A5), E. faecium (K2), E. faecium (A11), and L. lactis (K1) with accession numbers KU510332.1, KU510335.1, KU510333.1, and KU510334.1, respectively. These selected isolates have good probiotic traits and have many applications in the food industry.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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