Lactic acid bacteria (LAB) are heterogenous group of nonsporulating, aerotolerant, gram-positive, microorganisms arranged in rods or cocci1. Pediococcus, Streptococcus, Leuconostoc, Lactococcus, Enterococcus and Lactobacillus, are the main identified LAB groups based on morphology and carbohydrates fermentation2. However, these biochemical based identification methods are not conclusive as LAB groups have common nutritional and growth requirements. Gene sequencing using 16S rDNA had been the most reliable identification method, despite it sometimes fail to differentiate LAB species3.
Some LAB are very good examples of probiotics bacteria, which are live microorganisms that are beneficial to health. However, other LAB may have some probiotics potentials or only can be used as fermentation cultures. Probiotic properties are; capability to tolerate acid and bile salt, gastric fluids tolerance; adherence to intestinal mucosa; bacteriostasis on pathogenic bacteria and inhibition by antibiotics4. LAB are usually found in many food sources such as dairy products, fermented milk, fish from fresh water and fermented meat or vegetables5.
Ingestion of probiotic microorganisms with food is one of the approaches to combat the problem of food-borne diseases6. Therefore, this study studied the probiotic properties of LAB strains isolated from commercial Rayab milk, as they may have a beneficial role against food-borne pathogens. Moreover, to compare API identification method for some LAB isolates versus 16S rDNA sequencing molecular method.
MATERIALS AND METHODS
Study area: This study was carried during 2 month period from beginning of September to end of October, 2019, samples were collected from local market of Alexandria city, Egypt.
Samples and media: In this investigation, 20 fermented Rayab milk samples (1 L) produced by 4 different manufactures were collected from local market in Alexandria, Egypt. Lactobacillus MRS Agar and MRS broth media (HIMEDIA, India) were applied to selectively isolate LAB.
Isolation of LAB: Serial dilutions were prepared from each sample (up to105). Pour plate method was done using MRS and M17 agar media, at 37°C for 24-48 h in anaerobic CO2 incubator (Sysmedical, China Anaerobic Culture Incubator). Bacterial isolates were subcultured twice on fresh MRS plates for purity check7.
Identification of bacterial isolates
Preliminary phenotypic characterization of isolates: Gram stain reaction, microscopic cell morphology, cellular arrangement and catalase activity were identified for all isolates8,9.
Confirmation by API systems: API systems was used to identify isolates that are gram-positive and catalase negative to species level; API 20 STREP (Bio Mérieux, France) was done for gram positive cocci (Lactococcus) On other hand, identification of gram positive rods (Lactobacilli) was accomplished by using API 50 CHL micro-identification systems (Bio Mérieux, France).
Molecular confirmatory identification of (LAB) 16S ribosomal DNA: Both biochemical and molecular identifying of 10 randomly selected LAB isolates were done, to compare results of API systems versus 16S rDNA sequencing.
Extraction of genomic DNA: One and half ml overnight LAB culture grown in MRS broth was centrifuged at 12000 rpm. Supernatant was discarded, pellet was used to purify total genomic DNA by Gene JET genomic DNA purification kit (Thermo Fisher, USA). Purified DNA stored at -20°C till use.
16S ribosomal DNA (rRNA) molecular marker identification: LAB isolates were identified through 16S rDNA gene sequenced protocol. DreamTaq Green PCR Master Mix (2X) (K1081, Thermo Fisher, USA) was used for specific gene amplification according to manufacturer protocol through CreaCon (Holland, Inc) Universal 16S rDNA primers were employed to amplify specific amplicons as shown in Table 1. Polymerase chain reaction (PCR) system cycler was used, PCR initiated with 5 min of initial denaturation at 94°C. then, repeated at a total of 30 times (at 94 for 30 sec, at 55 for 30 sec, at 72 for 1 min) and finally at 72 for 7 min10. Using DNA ladder (peqGOLD 1 kb DNA-Ladder, Peq (LAB), VWR), 1% agarose gel was used for amplicons visualization via gel documentation system (Geldoc-it, UVP, England).
Sequencing and identification of PCR products: Specific DNA bands were eluted from agarose gel. Products of PCR were purified with EZNA ®Gel Extraction Kit, (D2500-01, Omega BIO-TEK, USA). Specific 16S rDNA amplicon were sequenced using the ABI PRISM® 3100 Genetic Analyzer (Micron-Corp. Korea). To detect LAB genera, sequence homology analysis was performed for all sequences through submission into www.ncbi.nlm.nih.gov and search in GeneBank database via BLAST (Basic Local Alignment Search Tool).
Construction of phylogenetic tree: 16S rDNA sequences of (LAB) were retrieved from GeneBank database, Clusteral W software analysis was applied to construct Phylogenetic tree based on multiple sequence comparison alignments using Pairwise Distance method.
Tolerance to acidity and bile salts
Acid tolerance: Pour plate method was used to determine bacterial count variation. LAB isolates were inoculated onto 5 mol HCL supplemented MRS and M17 media at pH 2.0 and 3.0 and normal MRS and M17 agar media (pH 6.5)11.
Bile salt tolerance: Bacterial broth cultures were inoculated into MRS broth with and without 0.3% (w/v) bile, in water bath at 37°C. Growth was observed by measuring the absorbance at 600 nm. Delay of growth due to inhibition by the bile salts was calculated as the difference (d) in the length of time between the two samples (with and without bile)4.
Antibiotic susceptibility: Agar disk diffusion assay was applied to evaluate antibiotic susceptibility of identified LAB, using ten antibiotics discs (ceftriaxone/30 μg, amoxicillin/25 μg, tobramycin/10 μg, penicillin G/10 units, cefoxitin/30 μg, neomycin/30 μg, gentamicin/10 μg, vancomycin/256 nitrofurantoin/300 and nalidixic acid/30 μg (bio TRADING, Netherland)12.
Hemolytic activity: Hemolysis was evaluated via culturing LAB isolates on Columbia blood agar (Oxoid, Thermo Fisher Scientific, USA), supplemented with 5% (v/v) human blood, for 24 and 72 h periods at 37°C in anaerobic jars9.
Evaluation of antibacterial activity of LAB isolates: Agar well diffusion assay was applied using pour plated MHA plates inoculated with four food borne pathogenic bacteria indicators; Salmonella typhimurium strain (ATTC 13311), Staphylococcus aureus strain (NCINB 50080), Listeria monocytogenes strain (ATTC19111) and E. coli O157 strain (ATTC 700728) After solidification, sterile cork borer was applied to made agar wells which filled with 100 μL of filtered LAB supernatant (centrifugation at 10000×g for 15 min). After Plates incubation at 37°C for 24 h, inhibition zones diameter (mm) were measured13.
Statistical analysis: Data were collected, tabulated and analyzed using statistical software SPSS version 24 (IBM Corp., Chicago, Illinois, USA).
From total 20 fermented Rayab milk products, 95 lactic acid bacteria were isolated. Colonies were circular in shape with varied color ranged from white to creamy. All isolates were catalase negative, 69 of them were Gram positive bacilli and the remaining 26 isolates were Gram positive cocci.
Based on interpretation of the API database, 45 LAB isolates were satisfactorily identified into species level, API 50 CHL identified 22 lactobacilli and another 23 lactococcus were identified using API 20 STREP as shown in Table 2.
The 45 isolates grouped into four main genera; as illustrated in Fig. 1.
When the phenotypic method using API systems compared to the 16S rDNA sequencing results, evidence of non-concomitant result is illustrated in Table 4. 16S rDNA identified higher numbers of Enterococcus spp. On the other hand, Lactobacillus genus was mostly identified by API 50CHL. API system correctly identified 6 isolates to genus level (60%), however it showed divergent result in species level identification, where only one isolate (10%) was correctly identified to species level.
Tolerance to acidity and bile salt: In this investigation, 45 LAB reflected varied acid tolerance patterns. Twenty eight (62.2%) out of 45 isolated lactic acid bacteria showed distinguish resistance for surviving on low pH conditions.
At pH 3.0, 19 strains showed survival rates >90%. On the other hand, 21 strains reflected survival rates >90% at pH 2.0. Furthermore, only ten strains showed good tolerance at both pH 2.0 and 3.0. Two LAB isolates (6, 25) considered the highest acid tolerant at pH 3.0 and 2.0 with a survival rate of 96 and 94% respectively. Comparing with control LAB isolate, viable counts were decreased at both pH 2.0 and 3.0 after 2 h.
Regarding bile salt tolerance, varied inhibition values were recorded for 15 (53.5%) out of 28 acid tolerant LAB isolates according to Gilliland standards14. Only one LAB isolate considered resistant strain, 4 strains reflected tolerant pattern. Furthermore, ten strains classified as bile weakly tolerant. Two LAB isolates (13 and 21) showed bile-sensitive response (Table 5).
Antibiotic susceptibility and hemolytic activity of LAB: Different susceptibility response remarked tested 45 LAB isolates against 10 applied antibiotics. Highest inhibition influences against LAB were observed for gentamicin, tobramycin and neomycin with largest inhibition zones ranges) respectively.
Reduction of inhibition effects against 45 LAB isolates seen with penicillin, nitrofurantoin and vancomycin respectively. Interestingly, lowest inhibition influences were recorded for amoxicillin, ceftriaxone, cefoxitin and nalidixic acid.
Thirty three out of 45 LAB isolates (73.3%) were classified as nonhemolytic (ɣ-hemolysis), while 26.7% reflected α-hemolytic activity as shown in Table 6.
Antibacterial activity of isolated LAB: Variable spectra of inhibition zones of LAB strains against the tested pathogenic bacteria. Thirty-eight (84.4%) out of 45 tested LAB strains showed antibacterial activity against all 4 tested bacteria. Varied antibacterial activity was recorded for LAB isolates against Salmonella typhimurium (ATTC 13311), with highest inhibition zones 20.0-30.0 mm for 19 LAB isolates.
Against E. coli O157 (ATTC 700728) 13 LAB isolates showed highest inhibition zones ranging from 10.1-15.0 mm. Regarding Staphylococcus aureus (NCINB 50080), 26 LAB isolates expressed highest inhibition zones ranging from 10.1-15.0 mm. Twenty LAB isolates showed highest inhibition zones from 20.0-30.0 mm of against Listeria monocytogenes (ATTC19111) (Table 6).
The inhibitory effect of LAB with probiotic activity against pathogens causing food disease has recommended their use as an alternative to chemical drugs15. In the present study two isolates out of 45 LAB tested for acid tolerance at both pH 2.0 and 3.0 for 2 h had a very high survival rate of 94 and 96% respectively. Survival rates >90% to pH 2.0, 3.0 were found in 21, 19 LAB isolates respectively. The percentages of tested strains with survival rates >90% to pH 2.0, 3.0 were 46.6 and 42.2%, respectively. Tulumoglu et al.16 observed closer percentage of 45%. Also Zhang et al.4 who reported that 21, 17 strains had survival rates >90% to pH 2.0, 3.0 for 2 h respectively and lower percentages of tested strains with survival rates >90% to pH 2.0, 3.0 which were 30 and 25%, respectively. Similarly, Rajoka et al.17 showed a survival rate above 80% at pH 2 for 3 h. Lower survival rates 55, 49, 65 and 57%, to pH 2.0 for 2 h demonstrated by Mourad and Nour-Eddine18.
Bile salt tolerability is the criteria by which probiotic bacteria can survive within the intestinal conditions. An important probiotic criterion in order to one out of 28 acid tolerant LAB demonstrated resistant pattern for bile salt (d<15 min). Similarly, Zhang et al.4 considered only 2 out of 21 isolates to be bile resistant. Higher percentage of resistant isolates was recorded by Jacobsen et al.19 where all the three studied lactic acid bacteria isolated from Ghanaian fermented maize were bile resistant.
The other bile tolerant patterns found in this study were; tolerant pattern (4 isolates) and bile weakly tolerant (10 isolates). Hyronimus et al.20 reported higher percentage of tolerant LAB, where all studied strains isolated from cow excrement were bile tolerant. However more relevant results found by Zhang et al.4 where 6 out of 21 LAB were tolerant strains and 13 out of 21 isolates were weekly tolerant.
Antibiotic resistance can be horizontally transferred from LAB, when used as probiotics, to pathogenic bacteria in the intestine21. In the present study, gentamicin, tobramycin and neomycin showed the highest inhibition effects on LAB. Penicillin, nitrofurantoin and vancomycin inhibited 85, 75 and 71% of tested 45 LAB isolates respectively. De Almeida Júnior et al.22 and Dasen et al.23 reported similar high sensitivity to vancomycin 84 and 100%, respectively. These results disagree with the concept of natural resistance of Lactobacillus against vancomycin24.
Probiotics LAB possess a bacteriostasis role by inhibiting pathogenic bacteria and changing the distribution of bacterial community within intestine16,25.Thirty-eight (84.4%) out of 45 tested LAB strains showed antibacterial activity against all four tested food pathogens. Tadesse et al.26 and Bassyouni et al.27 verified stronger antibacterial effects of LAB where all isolates showed antibacterial effects against Salmonella typhimurium and E. coli.
Considering LAB isolates identification, several molecular and phenotypic methods are available: Species-specific PCR reaction,16S rDNA sequencing, Biolog and API 50CHL. In this study, 10 selected LAB isolates out of 45 identified by API phenotypic method system were subjected to identification using molecular method of 16S rDNA sequencing.
API results differed from the molecular reference method for the majority of the tested isolates: 9 (90%) out of 10 isolates showed non concomitant results when compared to 16S rDNA sequencing. Similar result was verified by Moraes et al.28 where 86.2% of isolates showed high divergent results between these two methods. However other studies should compare different identification approaches of LAB using more numbers of LAB isolates. Some LAB isolates from Rayab milk in Egypt had potential probiotic activity, so it can be used to promote health and to combat food borne diseases. LAB identification to species level using conventional biochemical identification method including API systems may be not reliable enough, therefore better to use molecular identification and sequencing to identify LAB species. Limitation of the study was that it needs to compare more numbers of isolates using both biochemical and molecular identification.
Lactic acid bacteria can be used as probiotic supplements to treat infectious diarrheal diseases. Rayab milk consumed in lower Egypt is a source of plenty of lactic acid bacteria isolates that had probiotic activity. Use of biochemical method in identification of lactic acid bacteria gave inconsistent results with molecular gold standard method.
This study discovered that LAB isolated from Rayab milk can be beneficial for use as probiotics to promote health. This study will help the researchers to uncover the critical areas of probiotic medicine that many researchers were not able to explore. Thus, a new theory to combat food borne diseases may be arrived at.