Asian Journal of Dairy and Food Research, volume 41 issue 4 (december 2022) : 468-473

Identification and Antibacterial Activity of Lactobacillus Isolated from Different Raw Honey in Mostaganem Province, Algeria

D.S. Radja1,*, M. Homrani1, A.A. Dahou1, A. Haddad1, N. Rechidi1, A. Homrani1
1Laboratory of Sciences and Technics of Animal Production, Abdelhamid Ibn Badis University, Mostaganem, Algeria.
Cite article:- Radja D.S., Homrani M., Dahou A.A., Haddad A., Rechidi N., Homrani A. (2022). Identification and Antibacterial Activity of Lactobacillus Isolated from Different Raw Honey in Mostaganem Province, Algeria . Asian Journal of Dairy and Food Research. 41(4): 468-473. doi: 10.18805/ajdfr.DRF-264.
Background: The increase of multidrug-resistant pathogens causes increased concerns about the efficacy of conventional treatment, which requires the search for new alternatives to antibiotics. The present study aimed to evaluate the antibacterial activity of lactobacilli isolated from eight (n=8) typical Apis mellifera honey produced in Mostaganem region against four pathogenic bacteria responsible of bovine mastitis.

Methods: Lacbacilli strains, isolated from different media were firstly presumptively identified by catalase test, Gram staining and cells morphology, than confirmed by partial 16S rRNA sequence analysis. Antibacterial activity of isolates was determined by the overlay agar method. Antibiotic susceptibility of mastitis pathogens was tested by disk diffusion technique.

Result: Two species of lactobacilli were isolated and identified as Lactobacillus plantarum and Lactobacillus pentosus. Antibiotic sensitivity test demonstrated that mastitis pathogens showed the resistance to antibiotics tested. All the isolates showed antimicrobial activity with inhibition zone diameters ranged from 5±0.02 to 21±2.12 mm. The results revealed that honey can be considered as a source of Lactobacillus with antibacterial activities which can be used as alternative treatment of bovine mastitis.
Lactic acid bacteria (LAB) are one of the most microorganisms group studied by researchers. They are fluently used in food and pharmaceutic industries. These bacteria are widely present in nature and can be isolated from different sources such as fermented foods or gastrointestinal tract of humain and animals (Lashani et al., 2020). Lactobacillus is the largest genus within the group of lactic acid bacteria with more than 237 species (Zare et al., 2018). Lactobacilli are characterized by, Gram-positive rods, anaerobic but aerotolerant, non-sporulating and catalase negative. They are generally recognized as healthy, of 3  GRAS 3 (Generally Recognized As Safe) status and play an important role in the fermentation and preservation of foods. In addition, due to their capacity of bioactive compounds production, such as organic acid (lactic acid, acetate acid and formic acid), hydrogen peroxide (H2O2), ethanol, enzymes, benzoate, antimicrobial peptides (AMPs), free fatty acid and volatile compounds some Lactobacillus species can be developed as natural drugs and novel antimicrobial agent (Niode et al., 2019). On the other, some strains of lactobacilli have beneficial effects on human health and are used as a “probiotic”.
Honey is a natural food produced by honey-bees (Apis mellifera) from blossoms nectars or honeydew. Due to their nutritional, energetic and therapeutic proprieties this product is very popular by consumers (Fabazadi et al., 2020).  Recently, some researchers have been interested to lactic acid bacteria from honey and mainly Lactobacillus genus. Different species of lactobacilli were detected in honeys such as Lb kunkeei, Lb plantarum, Lb pentosus, Lb paracasei, Lb casei, Lb brevis, Lb rhamnosus, Lb fermentum (Hasali et al., 2015; Mounia et al., 2018; Lashani et al., 2020). The community of LAB found in honey is related to digestive tract of honeybees, nectar, pollen, propolis and flowers (Silva et al., 2017).
Currently, the conventional treatment of infections is based on antibiotic therapy. However, the increase of multidrug-resistant pathogens, due to the overuse of antibiotics in human medicine and its intensive use in the animal industry, causes increased concerns about the efficacy of this treatment, which requires the search for new alternatives to antibiotics (Abdul Hafeez et al., 2019; Geeta et al., 2021; Aazami et al., 2016).
In the present study we identified the species of Lactobacillus detected in honey samples from Mostaganem region in Northwest Algeria by the repetitive element palindromic-polymerase chain reaction (REP-PCR) and we also, evaluated their antimicrobial activity against some pathogenic bacteria responsible of bovine mastitis.
During the honey harvesting season starting, between May and August 2019, a total of eight (n=8) raw honey samples were collected, under aseptic conditions, from different localities of Mostaganem region situated in north west of Algeria. All honey samples, were directly obtained from beekeepers and then were stored in sterile bottles at -20°C. Floral origins of these samples were provided by the beekeepers’ in relation to the locations where the beehives were situated (Table 1).

Table 1: Locality, floral origin and harvesting season of studied honeys.

Isolation of Lactobacillus
Approximately ten grams (10 g) of fresh honey samples were aseptically weighed into a sterile stomacher bag and mixed with 90 mL of sterile 0.1% (w/v) peptone water for 2 min using a stomacher homogenizer. Then, one milliliter (1 ml) of the resulting solution was added to 9 ml of MRS broth and incubated at 30°C for 48 h, followed by serial dilutions with sterile peptone water (0.1% w/v). A volume of 0.1 ml was spread on several specific modified media, namely MRS agar (de Man, Rogosa, Sharpe), MRS agar with (0.8% CaCO3), MRS agar with (0.1% L-cysteine and 2% fructose) and Rogosa agar. These specific seeded isolation media were incubated at 37°C, under anaerobic conditions for 72 h (Feizabadi et al., 2020; Homrani et al., 2019).
Biochemical screening of Lactobacillus
Pure colonies grown on microbiological agar medium were tested biochemically for catalase activity by adding a drop of 3% hydrogen peroxide (H2O2). Only catalase-negative colonies (not releasing gas bubbles in the presence of H2O2) were retained for Gram staining and cell morphology tests by light microscopy. All catalase-negative, Gram-positive and non spore-forming bacteria were selected and stored in MRS broth supplemented with 15% glycerol and stored at -20°C (Mahmoud Elzeini et al., 2020).
Molecular identification
Bacterial identification by the repetitive element palindromic-polymerase chain reaction (REP-PCR) was done as follows:
DNA extraction
The total bacterial DNA of selected isolates and reference strains used was checked and extracted using the method developed by BIO-RAD laboratories and adopted by (Cholet, 2006; Gevers et al., 2001; Koenraad et al., 2008; Dahou et al., 2021).
From a well individual colony, a 1 ml suspension is prepared in 1 ml of sterile distilled water, then centrifuging for 10 min at 3000 rpm. The bacterial pellet is stored at -20°C for 1 hour, the pellet obtained is washed with 1 ml of TES buffer with its resuspensed in 300 µl of STET buffer (1 litter= 80 g Sucrose, 50 ml Triton, 18.6 g EDTA, 6.05 g Tris). An addition of 40µl of an SDS solution (sodium dodecyl sulphate solution) at 20% of a TE buffer composed of 10 Mm Tris-HCL and 1 mM EDTA, pH=8.0, the extraction of the lysate was carried out with an equal distributed between phenol, chloroform and iso-amyl alcohol, then phase separation is carried by centrifugation for 20 min at 3000 rpm. After mixing of the aqueous phase with 70 µl of 5 M NaCl and 1 ml of isopropanol, by the following the DNA precipitate is collected by centrifugation at 3000 rpm for 20 min. Concentration and purity of DNA extract was determined spectrophotometrically by weigh 260 nm.
DNA amplification by REP-PCR
The amplification of the DNA fragments obtained from the extraction was performed on a cycler from BIORAD (BIORAD, USA) by the use of specific primers wich will amplify the DNA fragment encoding the 16 S region.
Agarose gel electrophoresis
Visualization of the REP-PCR amplicons was performed by electrophoresis on 1.2% agarose electrophoresis was achieved using a fluorescent dye ethidium bromide (BET).
Comparison of DNA sequences obtained
 The acquisitions obtained by the REP-PCR are compared with reference species Lactobacillus plantarum subsp. plantarum ATCC 14917 and Lactobacillus pentosus ATCC 8041 and with those from the Gene Bank data base using the program blast ( from NCBI.
Pathogenic bacterial strains
Four pathogenic strains (n=4) responsible for bovine subclinical mastitis which are Staphylococcus xylosus, Staphylococcus simulans, Klebsiella pneumonea and Enterobacter spp, provided by the Laboratory of Sciences and Technics of Animal Production, University of Mostaganem were used as indicator strains for assessment of antimicrobial activity. These strains were identified by standard biochemical test (Bio-Merieux, France), API Staph and API 20E.
Antibiotic susceptibility test of bacterial strains
Antibiotic susceptibility, for the four pathogen strains, was performed using the disc diffusion method, according to the national committee of clinical laboratory standards (NCCLS) (CLSI. 2018). Broth cultures, of each pathogen strains were diluted in saline solution to about 107 CFU/ml (0.5 Units of McFarland turbidity standard). Subsequently, 100 μL of these growing cultures was spread on Mueller-Hinton (MH) agar. The antibiotic discs were then, placed on the surface of plates. After incubation at 37°C for 24 h, the diameters of the zone of inhibition around the discs were measured in millimeter using a ruler. The tested antibiotics and their concentrations were as follows: Amoxicillin + Acid clavulanic 20+10 µg, Penicillin 6 µg, Oxacillin 1 µg, Bacitracine 130 µg, Ampicillin 10 µg, Streptomycin 300 µg, Trimetoprim + Sulfametoxazol 1.25+23.75 µg.
Antibacterial activity of Lactobacillus isolated from honey
The overlay method, described by Fleming et al., (1975) was used to evaluate the antibacterial potential of Lactobacillus isolates on selected pathogenic bacteria. Fresh culture of Lactobacillus realized in MRS broth was inoculated in spot on MRS agar plates and grown at 30°C for 18 h under anaerobic conditions.  The plates were then, overlaid with 8 mL of MRS soft agar (0.7% agar) seeded by 100 µl of indicator strain cultivated overnight. After 24 h of aerobic incubation at 30°C the diameter of inhibition zone was measured in millimeters (mm).
Isolation and screening of Lactobacillus
After pre-enrichment in MRS broth followed by plating in four microbiological media a total of fifty seven (n=57) from one hundred and forty four (n=144) isolates were identified as presumed of Lactobacillus based on their catalase negative, gram positive and bacilli form (Table 2). The results found proved the presence of lactobacilli in raw honey produced in Mostaganem region. Majority of lactobacilli were isolated from MRS medium followed by MRS supplemented by CaCO3 and MRS supplemented by fructose and cysteine. The origin of lactobacilli detected in honey was honey stomacher of honey bees and the variation of lactobacilli number depending on honey bee health and nectar source (Olofsson et al., 2016).

Table 2: Isolation of Lactobacillus from Mostaganem honeys.

Molecular identification
A total of eleven (n=11) isolates were selected and performed by partial analysis of 16S rRNA sequence using the NCBI blast program ( to the research for 16S rRNA sequence similarities. Results confirmed that all isolates belonging to the Lactobacillus genus, three isolates were identified as Lactobacillus pentosus (lb38, lb174, lb176) and eight as Lactobacillus plantarum (lb75, lb108, lb133, lb87, lb21, lb234, lb109, lb175) with 100% genetic sequence similarities. The results from this study are in agreement with previous studies of (Homrani et al., 2019) who also, detected the species of Lactobacillus plantarum and Lactobacillus pentosus in Algerian honeys. Feizabadi et al., (2020) proved that the predominance of Lactobacillus species is mainly by Lactobacillus plantarum, followed by Lactobacillus pentosus in Apis mellifera honeys produced in Iran. Other species of Lactobacillus were isolated from honeys such as Lb acidophilus, Lb brevis, Lb fermentum, Lb paracasei, Lb kunkeei (Aween et al., 2012; Hasali et al., 2015; Bulgasem et al., 2016; Olofsson et al., 2016; Lashani et al., 2020).
Antibiotic susceptibility test
Sensitivity of bacteria isolated from bovine subclinical mastitis to antibiotics are presented in Table 3. We noticed that Klebsiella pneumonia strain showed a resistance to all antibiotics tested. Enterobacter spp strain was susceptible only to ampiciline. Staphylococcus simulans and Staphylococcus xylosus showed resistance to penicillin, streptomycin and oxacillin. The same results was also reported by Singh et al., (2016) who observed that the bacteria isolated from bovine mastitis resists to cefotaxime, ampicillin-sulbactam, cefixime and ceftriaxone. Bolte et al., (2020) reported that some mastitis pathogens on German dairy farms have developed resistance to frequently used antibiotics.

Table 3: Antibiotic susceptibility of bacterial isolates from bovine sub clinical mastitis.

Antibacterial activity of Lactobacillus isolated from honey
The eleven (n=11) Lactobacillus produce different zones of inhibition by the double agar overlay method and the results are presented in Table 4. The diameter of the inhibition zones ranged from 5 to 21.5 mm (Klebsiella pneumonae), from 8 to 19.5 mm (Enterobacter spp), from 6 to 14.5 mm (Staphylococcus simulans) and from 5 to 12.5 mm (Staphylococcus xylosus).

Table 4: Antibacterial activity of lactobacilli against bacterial isolates of bovine mastitis.

The highest zone of inhibition was observed by lb176 (21.5±2.12 mm) against Klebsiella pneumonae and lb133 (19±1.41 mm) against Enterobacter spp while the minimum antibacterial effect was observed in lb87 against Staphylococcus xylosus (5±0.02 mm) and Staphylococcus simulans (6±0.01 mm). Piccart et al., (2016) reported that 13 lactic acid bacteria (Lactobacillus and Bifidobacterium) isolated from honey and honey bees havehigh and promising in vitro antibacterial activity against bovine mastitis pathogens. Olofsson et al., (2016) tested lactic acid bacteria symbionts in honey bees against severe pathogens such as methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa and vancomycin-resistant Enterococcus (VRE). LAB from honey bees digestive tract produce broad-spectrum antibacterial compounds such as Serratia marcescens, Eschericia coli, methicillin-resistant Staphylococcus aureus, Klebsiella aerogenes, Salmonella typhi, Pseudomonas spp, Klebsiella spp, Proteus spp and Bacillus subtilis (Niode et al., 2019). Geetha et al., (2015) demonstrated the efficacy of purified antimicrobial peptides of LAB isolated from partially decayed of food against pathogenic bacterial strains of bovine mastitis.
The antibacterial effect of Lacobacillus plantarum and Lactobacillus pentosus isolated from Algerian honeys against gram negative bacteria is showed by Homrani et al., (2019). Lashani et al., (2018) showed that Iranian honey can be a source for different Lactobacillus such as Lb plantarum and Lb paracasei and their inhibitory effects against pathogen bacteria as Staphylaococcus aureus. Ugras et al., (2017) found that Lb Kunkeei isolated from the stomach of bee inhibits Escherichia coli ATCC 35218, Listeria monocytogenes ATCC 7644, Yersinia pseudotuberculosis ATCC 911, Pseudomonas aeruginosa ATCC 27853.
This preliminary experimental study revealed the antibacterial potential of two different Lactobacillus species, isolated from honeys produced in Mostaganem region (Lactobacillus plantarum and Lactobacillus pentosus) against the agents of bovine subclinical mastitis. The conventional treatment by antibiotic therapy has showed insufficiencies against the pathogenic agents of the bovine mastitis and in this frame, the lactobacilli strains isolated could be promising alternative treatments of the bovine mastitis. In this perspective, it will be necessary to confirm the antibacterial effect of the combination of honey and lactobacilli by the development of experimental curative tests in vivo.
We would like to thank the Directorate General of Scientific Research and Technological Development “DGRSDT” for its support in the development of our scientific research results.

  1. Aazami, N., Kalantar, E., Poormazaheri, H., Setayesh, V., Pou, N., Salehi, J.G. (2016). Selection and characterization of potential probiotic Lactobacilli spp isolated from chicken feces may be used as a potent antibacterial agent. Asian Journal of Dairy and Food Research. 35(1): 50-57.

  2. Abdul-Hafeez, M.M. (2019). Testimony for veterinary apitherapy. International Journal of Complemenary and Alternative Medicine. 12(1): 15-22. DOI: 10.15406/ijcam.2019.12.00442.

  3. Aween, M.M., Hassan, Z., Muhialdin, B.J., Eljamel, Y.A., Al-Mabrok, A.S., Lani, M.N. (2012). Antibacterial activity of Lactobacillus acidophilus strains isolated from honey marketed in Malaysia against selected multiple antibiotic resistant (MAR) gram-positive bacteria. Journal Food Science 77(7): M364-M371. 

  4. Bolte, J., Zhang, Y., Wente, N., Krömker, V. (2020). In vitro susceptibility  of mastitis pathogens isolated from clinical mastitis cases on northern German dairy farms. Veterinary Sciences. 7(1): 10-16.

  5. Bulgasem, B.Y., Lani, M.N., Hassan, Z., Wan, Y.W.M., Fnaish, S.G. (2016). Antifungal activity of lactic acid bacteria starains isolated from natural honey against pathogenic candida species. Mycobiology. 44(4): 302-309.

  6. Cholet, O. (2006). Etude de l’écosystème fromager par une approche biochimique et moléculaire. INRA de Paris-Grignon, Thèse  de doctorat en sciences des aliments, 193 p.

  7. Dahou, A.A., Bekada, A., Homrani, A. (2021). Identification of a Lactococcus lactis isolated from a fresh local cheese of the western Algerian steppe « J’ben of Naama ». Asian Journal of Dairy and Food Research. 40(1): 40-44.

  8. Feizabadi, F., Sharifan, A., Tajabadi, N. (2020). Isolation and identification of lactic acid bacteria from stored Apis mellifera honey. Journal of Apicultural Research. 60(3): 421-426.

  9. Fleming,  H.P., Etchells, J.L., Costilow, R.N. (1975). Microbial inhibition by an isolate of Pediococcus from cucumber brines. Journal of Applied Microbiology. 30(6): 1040-1042.

  10. Geetha, R., Sathian, C.T., Prasad, V., Gleeja, V.L. (2015). Efficacy of purified antimicrobial peptides from lactic acid bacteria against bovine mastitis pathogens. Asian Journal of Dairy and Food Research. 34(4): 259-264.

  11. Geeta, Ajit, S.Y., Suchismita, P., Ravi, R., Asok, K.M., Gopi, M., Naveen, K.N., Ranjana, P. (2021). Probiotic attributes of Lactobacillus fermentum nkn51 isolated fromyak cottage cheese and the impact of its feeding on growth, immunity, caecal microbiology and jejunal histology in the starter phase of broiler birds. Indian Journal of Animal Research. (55): 451-456.

  12. Gevers, D., Huys, G., Swings, J. (2001). Applicability of rep-PCR fingerprinting for identification of Lactobacillus species. J.F.E.M.S.M.L., 205(1): 31-6. doi: 10.1111/j.1574-6968.2001. tb10921.

  13. Hasali, N.H.M., Zamri, A.I., Lani, M.N., Mubarak, A., Suhaili, Z. (2015). Identification of lactic acid bacteria from meliponine honey and their antimicrobial activity against pathogenic bacteria. A.E.J.S.A. 9: 1-7.

  14. Homrani, M., Dalache, F., Bouzouina, M., Nemiche, S., Homrani, A. (2019). Antibacterial activities of Algerian raw honeys and isolated Lactobacillus against gram-negative bacteria. Advances Bioresearch. 10(1): 31-39.

  15. Koenraad, V.H., Peter, V., Geert, H. (2008). Molecular identification and typing of lactic acid bacteria associated with the production of two artisanal raw milk cheeses. Dairy Science andTechnology. 88: 445-455.

  16. Lashani, E., Davoodabadi, A., Soltandallal, M.M. (2018). Antimicrobial  effects of Lactobacillus plantarum and Lactobacillus paracasei isolated from honey against Staphylococcus aureus. Journal of Babol University of Medical Science. 20(3): 44-49.

  17. Lashani, E., Davoodabadi, A., Soltan, D.M.M. (2020). Some probiotic properties of Lactobacillus species isolated from honey and their antimicrobial activity against foodborne pathogens. Veterinary Research Forum. 11(2): 121-126.

  18. Mahmoud Elzeini, H., Abdel-atti, A.A., Fawzy, N.N., Essam, E.Y., Abdel, M.HA. (2020). Isolation and identification of lactic acidbacteria from the intestinal tracts of honey bees, Apis mellifera L. in Egypt. Journal of Apicultural Research. 60(3): 1-9.

  19. Mounia, H., Dalache, F., Bouzouina, M., Nemmiche, S., Homrani, A. (2018). Antibacterial activity of Lactobacilli detected in Algerian raw honeys against gram-negative bacteria. SAJEB. 3(8). DOI: p83-90.

  20. Niode, N.J., Salakin, C.L., Rumokoy, L.J.M., Tallei, T.E. (2019). Lactic acid bacteria from honey bees digestive tract and their potential as probiotics. Advances in Biological Sciences Research. 8(1): 236-241.

  21. Olofsson, T.C., Butler, È., Markowicz, P., Lindholm, C., Larsson, L., Vásquez, A. (2016). Lactic acid bacterial symbionts in honeybees - An unknown key to honey’s antimicrobial and therapeutic activities. International Wound Journal. 13(1): 668-679.

  22. Piccart, K., Vásquez, A., Piepers, S., De, V.S., Olofsson, T.C. (2016). Short communication: Lactic acid bacteria from the honey bee inhibit the in vitro growth of mastitis pathogens. Journal of Dairy Science. 99(4): 2940-2944.

  23. Silva, M.S., Rabadzhiev, Y., Eller, M.R., Iliev, I., Ivanova, I., Santana, W.C. (2017). Microorganisms in Honey. In: Intech, editor. Honey Analysis. Toledo VA. p.233-258.

  24. Singh, V.K., Kumar, A., Yadav, S.K. (2016). Antimicrobial susceptibility  profiling of milk samples from bovine clinical mastitis. International Journal of Medical Microbiology and Tropical Diseases. 2(2): 52-55.

  25. Ugras, S. (2017). Isolation, identification and characterization of probiotic properties of bacterium from the honey stomachs of Yigilca honeybees in Turkey. Turkich Journal of Entomology. 41: 253-261.

  26. Zare, M.E., Lashani, E., Davoodabadi, A. (2018). Antimicrobial properties of lactic acid bacteria isolated from traditional yagurt and milk against Shigella strains. German Medical Science Hygiene and Infection Control. 13: Doc:10.3205/ dgkh000307,URN :urn :nbn :de :0183-dgkh0003078.

Editorial Board

View all (0)