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Cite this article: Alrubaye HH, Fakhry SS, Jebur ZA, Farhan F (2018) Biochemical and Molecular Characterization of Lactobacillus spp. Isolated from Dairy Products. JSM Microbiology 6(1): 1048.

*Corresponding authorHadeel H. Alrubaye, Food Contamination Research Center, Environment and Water Directorate, Ministry of Science and Technology, Aljadreha Street, Baghdad-Iraq, Tel +9647901291403; Email:

Submitted: 13 January 2018

Accepted: 16 April 2018

Published: 18 April 2018

Copyright© 2018 Alrubaye et al.

OPEN ACCESS

Keywords•Lactic Acid Bacteria; Primers; Antibiotic; PCR

Research Article

Biochemical and Molecular Characterization of Lactobacillus spp. Isolated from Dairy ProductsHadeel H. Alrubaye*, Saad S. Fakhry, Zahraa A. Jebur, and Farqed FarhanFood Contamination Research Center, Ministry of Science and Technology, Iraq

Abstract

Lactic acid bacteria (LAB) are significant to human health due to the production of some antimicrobial substances and ability to inhibit pathogenic bacteria. Furthermore, the bacteria are also used in the production of various food products. The aim of this study was isolation and characterization of Lactobacillus species from dairy products by using biochemical and molecular techniques

Twenty-eight (28) strains of lactic acid bacteria (LAB) were isolated from yogurt using MRS broth and agar cultured media. Strains isolated were characterized by biochemical properties. Other characterization like produce antimicrobial substances active against selected pathogens, isolates (NO.11,12,13,19,21 and 23) and antibiotic susceptibility of the isolates suggested those isolates could be used as probiotics for human use. For molecular characterization, Martin-Platero method was used for DNA extraction and purification and for16S rRNA amplification conventional was used. The 16S rRNA gene sequencing analysis identified and phylogenetic tree drawn by the Neighbor-Joining method (Mega 6).The study showed 16S rRNA gene sequence analysis is higher sensitivity to detect bacterial species than the conventional biochemical methods, then these sequence data have been submitted to the GenBank database (https://submit.ncbi.nlm.nih.gov) under accession numbers; MG890339, MG896122, MG847173, MG890341

ABBREVIATIONSPCR: Polymerase Chain Reaction; Spp : Species; CTX:

Cefataxime; C30: Chloramphenicol; AT: Aztreonam; CIP: Ciprofloxacin; E: Erythromycin; AMC: Amoxyclave; DO: Doxycycline Hydrochloride; TE: Tetracycline; AMP: Ampicillin; CN: Gentamycine; CL: Cephalxine; TCC: Ticracillin/Clavulanic Acid; MRS: De Man, Rogosa and Sharpe Agar; CFU/ml: Colony-Forming Unit/ Milliliter; DNA: Deoxyribonucleic Acid; rRNA: Ribosomal Ribonucleic Acid; EDTA: Ethylenediaminetetraacetic Acid; L (NO.) : Lactobacillus LAB Samples; L : Ladder; C: Control Negative; S: Lactobacillus Spp. Samples

INTRODUCTIONIn addition to the considerable technological and commercial

importance of their role in the manufacturing and preservation of many fermented food products, lactic acid bacteria (LAB) including Lactobacillus spp also play an important role in the protection of the intestinal and urogenital tracts [1,2]. Lactic acid Bacteria occur naturally as indigenous microflora in fermented milk products such as yogurt [3].

The genus Lactobacillus consists of a genetically and physiologically diverse groups of Gram-positive, rod-shaped, catalase negative and non-spore forming bacteria [4]. Many of Lactobacillus spp. are used in starter cultures for food and feed fermentations, and several species are frequently encountered in the human gastrointestinal tract [5]. Some strains of the LAB

are marketed as probiotics, which are claimed to have a positive effect on human and/or animal health [6,7].

Consumption of food containing live bacteria is the oldest and still most widely used way to increase the number of advantageous bacteria called “probiotics” in the intestinal tract [8].

Phenotypic characterization, traditionally the bacteria identified based on their morphological, biochemical and physiological properties. These characteristics can, however, vary within the same species based on growth conditions, and therefore can provide results in some cases not very reliable. Molecular methods can be effectively used for identification at the level of genus, species and even of strain. These methods based on the DNA sequence similarities of the microorganism to be identified with the DNA of a reference strain allow different levels of identification: strain, species, and genus. The main purpose of this work is the characterization of the Lactobacillus spp. found in the dairy product through the use of classical microbiological and molecular methods.

MATERIALS AND METHODS Collection of samples

A total of sixty (60) different Yoghurt samples were collected from local markets in the city of Baghdad in Iraq; based on their popularities among the consumers. The samples were stored

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aseptically at 4°C to prevent contamination immediately after collection.

Samples preparation and bacterial cultivationTen grams (10 g) of each yogurt sample were separately

diluted in 90 ml of sterile normal saline. Samples were enriched in MRS broth (PH6.2) for 24 h at 37°C under anaerobic conditions. Samples were then taken and streaked on to the MRS agar (PH6.2) plates and were incubated in an anaerobic jar at 37°C for 72 h. Suspected colonies were purified and streaked on MRS agar for further identification.

Identification of isolates Identification of the Lactobacillus spp. was performed

according to their morphological, cultural, physiological and biochemical characteristics. Macroscopic appearance of all the colonies was examined for cultural and morphological characteristics. Size, shape, color, and texture of the colonies were recorded. Isolates were characterized based on Gram’s stain reaction, cell morphology, the presence of capsule or endospore, motility, catalase reaction, oxidase reaction and by growth at 15°C and 45°C as described by Benson [9]. Tests of, nitrate reduction, sulfide, and indole production, and CO2

from glucose

and H2S production were performed according to [10]. Putative lactobacilli were identified to species level based on the sugar fermentation pattern of the API CHL50 System (bioMe’rieux).

Determination of antibiotic susceptibility of the isolates

Twelve commonly used antibiotics were used to determine the antibiotic susceptibility of the isolated Lactobacillus species using Kirby Bauer method. The antibiotic discs (Oxoid, England) were as follows: Cefataxime (CTX) 30 mcg, Chloramphenicol (C30) 30 mcg, Aztreonam (AT) 30 mcg, Ciprofloxacin (CIP) 5 mcg, Erythromycin (E) 15 mcg, Amoxyclave (AMC) 10mcg, Doxycycline hydrochloride (DO) 30 mcg, Tetracycline (TE) 30 mcg, Ampicillin (AMP) 10mcg, Gentamycin (CN) 10mcg, Cephalxine (CL) 30 mcg, Ticracillin/clavulanic Acid (TCC) 75/10 mcg.

Testing for antibacterial activity Antimicrobial action of all isolated Lactobacillus species

against selected indicator pathogenic bacteria was determined using the agar spot method. The isolated LAB species were grown in MRS broth at 37°C for 48-72 hrs and diluted to a density of 108 CFU/ml. Two microliters of each LAB species were spotted aseptically onto the MRS agar medium. The plates were then incubated anaerobically for 48-72 hrs. On the other hand, cultures of the pathogenic bacteria were prepared in nutrient broth and incubated at 37°C for 16-18 hrs. They were then transferred to the fresh nutrient broth and incubated at 37°C for 4 hrs. The pathogenic bacterial isolates were then adjusted to 108 CFU/ml. One ml of each of the pathogenic bacterial isolates was then inoculated into a flask with 100 ml molten nutrient agar at 50°C. Approximately 7 ml of the agar were poured as an overlay on the plate with the grown LAB, prepared as described above. The diameters of the clear zones (in the top agar layer) were measured to the nearest mm.

Isolation of DNAIn this study, four DNA extraction protocols were tested

(Martin-Platero et al. [11], Phenol–chloroform method [12]

Pospiech and Neumann modified method [13], and Modification of a salting- out procedure (MSOP) [14]). Martin–Platero method was the superior method used to extract DNA from bacteria with the modifications in the amount of lysozyme and incubation temperature. The pellet was suspended in 100 μl of TE buffer (10% w/v sucrose, 25 mM Tris–HCl pH 8.0, mM EDTA, 10 mg/ml, freshly made lysozyme and 40 μg/ml RNase A) and incubated for 30 min at 37°C. The protoplast cells were immediately lysed by adding 600 μl of lysis buffer (100 mM Tris–HCl pH 8.0, 100 mM EDTA, 10 mM NaCl and 1% w/v SDS) and incubated for 15 min at room temperature. The lysates were treated with 10 μl of proteinase K (10 mg/ml) and incubated for 15 min at 37°C. After incubation at 80°C for 5 min and cooling down to room temperature for 5‒10 min, 200 μl of sodium acetate (3 M, pH 5.2) were added, chilled on ice for 15 min and centrifuged at 6,000 rpm for 10 min. The supernatant was decanted and 600 μl of isopropanol was added to it to precipitate the DNA. Finally, genomic DNA was dissolved in distilled water and maintained at -20°C for further studies.

Amplification of 16S rRNA gene Amplification of 16S rRNA gene was performed using

GoTaq® Green Master Mix (Promega, USA) according to the manufacturer’s recommendations. The synthesized primers were used for amplification.

Temperature–time profile of PCR was the following: 95°C for 200 s, one cycle; 62°C for 50 s and 95°C for 20 s, 25 cycles; and 72°C for 120 s, one cycle. Analysis of PCR products was performed using electrophoresis in 1.5% gel, containing ethidium bromide. A marker contains DNA fragments of known size were used to know the expected product size. Then gel was visualized under ultraviolet (UV) light.

566 bp 16S rRNA gene sequences of aerobic gram-positive Lactobacillus spp.

The 566 bp sequences that should be amplified by the primers (Table 1). The universal primers for amplification of 566 bp-long 16S rRNA gene sequence were obtained from Prosekov et al., [15].The nucleotide sequences were used for the analysis of sequence similarity through Blast (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The percentage differences of the resultant partial 16S rRNA gene sequences among different species in the same group/genus of Lactobacillus spp. were determined by pairwise alignment using MEGA 6.

Phylogenetic treeThe phylogenetic relationship was convinced through

comparing all the 16S rRNA sequences of related species with those of isolates by pairwise alignment using MEGA 6 M [16,17].

RESULTS AND DISCUSSIONThe isolates were studied for their morphological

characterization. The colonies appeared small and large in their shape. The color of colonies ranged from off-white, shiny white to creamy white. Based on Gram staining and various biochemical tests, 28 isolates had been selected.

Antibiotic resistance

Antibiotic resistance of the isolated lactobacilli species is summarized in table (2). The species were sensitive towards

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most tested antibiotics. Notable observation is the resistance to Cefataxime (CTX), Chloramphenicol (C30) Tetracycline (TE), and Ampicillin (AMP), expressed by all isolates. Antibiotic resistance of microorganisms used as probiotic agents is an area of growing concern. Antibiotics are widely used in food-producing animals. It is believed that using these antibiotics contributes to the emergence of antibiotic-resistant bacteria present in the intestinal microflora. Then, these antibiotic-resistant bacteria can transfer resistance factors to other pathogenic bacteria through the exchange of genetic material [18]. One of the safety considerations in probiotics is verifying that a potential probiotic strain does not contain transferable resistance genes [19] (Figure 1, Table 2).

Testing for antibacterial activity The antagonistic effect of the isolated LAB species on some

common pathogenic bacteria was evaluated using the agar-spot method [20]. Results in the table (3) showed that all LAB species exhibit the antagonistic effect on both Gram-positive and Gram-negative bacteria.

Different reports showed that most Lactobacillus strains produce substances that inhibit pathogenic, non-pathogenic and spoilage organisms in fermenting foods and beverages. In general, the antimicrobial activity of lactobacilli may be due to lactic acid, acetic acid, formic acid, phenyllactic acid, caproic acid, organic acids, ethanol organic acids, hydrogen peroxide, bacteriocins or other inhibitory metabolites. Lactic acid and acetic acid are particularly important compounds, inhibiting a broad range of microorganisms [21] (Figure 2, Table 3).

Qualitative and quantitative assessment of DNAPurity and concentration of DNA solutions were measured

using Computerized NanoDrop-spectrophotometer. Results of DNA purity and concentration (ng/μl) were recorded and plotted automatically. The Nano Drop-spectrophotometer measures DNA purity and concentration according to the following equations:

DNA purity = Absorbance at 260 nm / Absorbance at 280 nm

DNA yield (µg) = DNA concentration (µg/µl) × total sample volume (ml).

DNA concentration is estimated by measuring the absorbance at the ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA. A ratio of ~1.8 is generally accepted as “pure” for DNA and the ratio 260/230; is used as a secondary measure of nucleic acid purity. The 260/230 values for “pure” nucleic acid are often higher than the respective 260/280 values. Expected 260/230 values are commonly in the range of 2.0-2.2. The quality of the isolated DNA was also evaluated by 0.8% Agarose gel electrophoresis. A 100 bp plus DNA ladder (SolGent, Korea) was used as a molecular weight standard to compare the intensity and approximate size of the isolated DNA. The purity of DNA extracted from Lactobacillus spp. indicated that the Martin–Platero method with several modifications was successful extraction method for Lactobacillus spp.

Polymerase chain reaction (PCR) of Lactobacillus spp Figure (3) shows that PCR of 9 amplicons for Lactobacillus

spp. with molecular size, 566 bp is associated with the calculated size of a target gene of Lactobacillus spp and shows the single band of PCR.

Figure 1 Antibiotic resistance: gentamycine (CN), Ticracillin/clavulanic Acid (TCC), Chloramphenicol (C30), Ampicillin (AMP), Tetracycline (TE), Amoxyclave (AMC), Ticracillin/clavulanic Acid (TCC).

Figure 2 Antagonistic effect of isolated LAB lactobacillus species with Shigella species (resistance of bacteria).

Figure 3 Gel electrophoresis of PCR for Lactobacillus spp.; L: Ladder; C: Control negative; 1-9: Lactobacillus Spp. samples.

Numerous studies have been done to evaluate microbial DNA extraction methods using various kinds of samples [22-24]. Efficient and reproducible methods for identification of Lactobacillus spp are needed, both for rapid identification and to facilitate the study of the complex microflora in a highly mixed environment [25].

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Figure 4 rRNA sequence chromatogram.

Figure 5 sequencing phylogenic tree, the similarity of Lactobacillus plantarum (L11 and L13) with Lactobacillus spp. M2(L12) and Lactobacillus fermentum (L19) was 93%.

Table 1: Amplification of 566 bp-long 16S rRNA gene sequence.

No. primer Sequence from 5' - 3'

1 16S Forward 5'- AGA GTT TGA TCC TGG CTC AGG A

2 16S Revers 5'- ACG CTT GCC ACC TAC GTA TTA C

Abbreviations: - : No Inhibition Zone; +: Complete Lysis

566 bp 16S rRNA gene sequences of aerobic Gram-positive Lactobacillus spp.

In the present study, 16S rRNA gene sequence analysis showed higher sensitivity to detect specific bacteria than the usual culture method. In addition, some bacteria strains that were not detected using the culture method. The culture method was not able to detect five to seven samples that the 16S rRNA gene sequencing analysis identified as positive. This is because

the samples did not obtain viable bacteria. On the other hand, 16S rRNA gene sequence analysis has been used to identify novel and emerging pathogens and to define complex microbial communities. Indeed, 16S rRNA gene sequence analysis provides a comprehensive assessment of microbial diversity compared with culturing alone and is an excellent complement to the culturing approaches (Figure 4).

The percentage similarities of the full and 566 bp 16S rRNA

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Table 2: Antibiotic resistance for Lactobacillus species.

No. of sample

Inhibition zoneCTX30mcg

C 30

30mcgAT30mcg

CIP5mcg

E15mcg

AMC30mcg

DO 30mcg

TE30mcg

AMP10mcg

CN10mcg

CL30mcg

TCC10mcg

1 2 1.7 X* X 1.4 1.1 X 2.3 1.3 X 1 2.62 2.2 2 X X 1.4 X X 2 0.8 X 0.9 33 3.4 2.5 X X 1.6 1.2 X 2.6 1.1 X 1.4 3.44 2 3.5 X 1 2.7 X X 2.5 0.7 X X X5 2.5 2.6 X 1.3 2.4 X 2.4 2.5 X X X X6 2.4 3.4 X 3.2 2.6 1.2 2.5 3 1.6 X X X7 2.2 3 X X 2.6 X 2.5 2.8 X X X X8 1.8 1.9 X X 1.3 0.9 X 1.9 0.9 X 1 2.59 2.2 3.2 X 3 2.4 0.9 2.5 2.1 1.2 X X X10 1.5 2.5 X 1.5 2.5 X 2 2 X X X X11 1.4 1.7 X X 1 X X 1.5 X X X 1.412 1.6 2.4 X X 2.5 X 2.5 2.5 X X X X13 3.2 2.4 X X 1.4 1.6 X 2.8 1.3 X X 314 1.8 1.3 X X 1.5 X X 1.8 X X 0.8 2.415 2.5 2.3 X X 1.2 1.3 X 2.5 1.2 X X 3.216 2 1.7 X X 1 0.9 X 1.9 0.9 X X 2.617 2 2 X X 2 X X 1.8 1 X X X18 2 2.5 X X 2.5 X 1.5 2 1.2 X X X19 2.2 2 X X 1.2 0.8 X 1.9 X X X 2.520 2.2 2 X X 1.4 X X 2 0.8 X 0.9 321 1.9 2.2 X X 1.1 0.8 X 1.9 0.9 X X 2.622 2.4 2.2 X X 1.4 1.2 X 2.7 1.2 X 0.7 3.323 2.2 3 X 0.7 2.2 X 2.5 2.5 1.2 X X X24 3 2 X X 1.2 1.6 X 2.8 1.4 X X 3.825 1.8 1.9 X X 1.3 0.9 X 1.9 0.9 X 1 2.526 3.4 2.5 X X 1.6 1.2 X 2.6 1.1 X 1.4 3.427 2.5 2.3 X X 1.2 1.3 X 2.5 1.2 X X 3.228 2 2.5 X X 2.5 X 1.5 2 1.2 X X XAbbreviations: Mcg: Microgram; CX: Cefataxime; C30: Chloramphenicol; AT: Aztreonam; CIP: Ciprofloxacin; E: Erythromycin; AMC: Amoxyclave; DO: Doxycycline Hydrochloride; TE: Tetracycline; AMP: Ampicillin; CN: Gentamycine; CL: Cephalxine(CL); TCC: Ticracillin/Clavulanic Acid; X: Negative Result

Table 3: Antagonistic effect of Lactobacillus spp. against pathogenic bacteria.

No. of sample proteus staphylococcus pseudomonas Shigella Salmonella

1 3.7 - + + +

2 cl 3.1 + + +

3 2.6 - + + +

4 3.2 - + + +

5 2.3 2.2 + + +

6 2.4 - + + +

7 2.8 2.5 + + +

8 2.4 - + 2.9 +

9 cl - + + +

10 2.8 3.2 + 3.4 +

11 2.5 - + 2.7 +

12 3 + + 2.5 +

13 2.7 + + 2.7 +

14 + + + 2.5 +

15 2.06 2.1 + 2.6 +

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16 2.7 0.6 + 2.6 +

17 2.8 2.8 + 0.6 +

18 2.83 2.3 + 2.7 +

19 2.5 2.6 + + +

20 2.2 2 + 1.5 +

21 2.4 2.5 + 2.7 +

22 4.4 2.8 + 1.7 +

23 1.7 + + + +

24 2.3 0.9 + 2.7 +

25 2.9 1.8 + 3.8 +

26 2.3 2.5 + 1.5 +

27 1.4 3.5 + 0.7 +

28 2.7 1.7 + 1.2 +

Abbreviations: - : No Inhibition Zone; +: Complete Lysis

Table 4: Lactobacillus isolates identified on the basis of percent similarity to 16S- rRNA region sequences in GenBank.Sample name source Molecule

typeQuery Length

Query cover Description Identities Accession

(Sequence ID)Program

L11 Yogurt nucleic acid 502 100%

Lactobacillus plantarum strain 30.2.12 16S ribosomal RNA gene, partial sequence

500/502(99%) KX649060.1 BLASTN 2.7.1+

L12 Powdered milk nucleic acid 507 100%

Lactobacillus spp. M2 16S ribosomal RNA gene, partial sequence

507/507(100%) KX131223.1 BLASTN 2.7.1+

L13 Yogurt nucleic acid 508 99%

Lactobacillus plantarum strain J_27.F_30609-4_8931Raw 16S ribosomal RNA gene, partial sequence

507/508(99%) MF980714.1 BLASTN 2.7.1+

L19 Yogurt nucleic acid 520 100%Lactobacillus fermentum strain BB102 16S ribosomal RNA gene, partial sequence

520/521(99%) KY940566.1 BLASTN 2.7.1+

Abbreviations: L: Lactobacillus LAB Samples

Table 5: Accession number in GenBank database.

Sample name Type of strain Accession number

L11 Lactobacillus plantarum MG890339

L12 Lactobacillus sp. M2 MG896122

L13 Lactobacillus plantarum MG847173

L19 Lactobacillus fermentum MG890341

gene sequences among the Lactobacillus spp. are shown in Supplementary (Table 4).

The 16S rDNA sequence of Lactobacilli isolates showed an average identity of 99% for on isolate with Lactobacillus plantarum (MF980714.1) and 100% for three isolates with Lactobacillus plantarum (KX649060.1), Lactobacillus spp. M2(KX131223.1) and Lactobacillus fermentum (KY 940566.1).

Phylogenetic treeA phylogenetic tree was drawn by the Neighbor-Joining

method (Mega 6). The 16S rRNA sequence of L11 showed an average identity of 99.% with Lactobacillus plantarum strain 30.2.12 (KX649060.1), L12 with an average identity of 100.% with Lactobacillus spp. M2 (KX131223.1), L13 with an

average identity of 99.% with Lactobacillus plantarum strain J_27.F_30609-4_8931Raw 16S and L19 with an average identity of 99.% Lactobacillus fermentum strain BB102 with through the BLAST2 analysis. A phylogenetic tree was constructed by using MEGA 6 software (Figure 5).

This average identity is 93% with 16S rRNA sequences of L11 and L13. The sequence was more similar to those of Lactobacillus plantarum strain 30.2.12 (KX649060.1) and Lactobacillus plantarum strain J_27.F_30609-4_8931Raw 16S with an average identity level of 99.5%.

Accession number

These sequence data have been submitted to the GenBank database (https://submit.ncbi.nlm.nih.gov) under accession

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numbers in the table (5).

CONCLUSIONThis work it was possible to increase knowledge related

to biodiversity and the dynamics of bacteria populations and interesting to test the effect of a co-culture of Lactobacillus spp. in the presence of another bacterial species, studying in more detail the interaction between microorganism inductor and producer would allow for to understand if the isolates from dairy products actually have the potential to produce antimicrobial substances which in fact are not generally active against Gram bacteria negative. Further studies would be needed to understand the nature and investigate the interactions between the microorganisms, as well as the identification based on 16S rDNA, combined with sequencing, were one effective and fast tool to identify the bacterial species present in dairy products.

ACKNOWLEDGMENTSThis work was carried out in the food contamination research

center, I would like to express my gratitude to Dr. Saad S. Fakhry for his interest and advice in the course of this work, and Mr. Farqed F. Abdulhameed for his skillful technical assistance.

CONFLICT OF INTERESTI certify that no funding has been received for the conduct of

this study and/or preparation of this manuscript.

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Alrubaye HH, Fakhry SS, Jebur ZA, Farhan F (2018) Biochemical and Molecular Characterization of Lactobacillus spp. Isolated from Dairy Products. JSM Micro-biology 6(1): 1048.

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