Original Article

Isolation and characterization of the lactobacillus strain from honey and its probiotic properties


Background and Objectives: The lactobacilli are abundant in honey, helping protect against pathogens and providing antimicrobial properties. This study aimed to isolate lactobacillus species from different honey regions and evaluate their potential probiotic properties.
Materials and Methods: Eighty-eight samples were collected from different regions, including the northern, central, and southern areas, and obtained through retail stores. All samples were independently examined for the presence of Lactobacillus using both culture and real-time PCR methods. Probiotic tests were performed on the isolated Lactobacillus strains, including hemolytic activity, bile, acid, and pepsin resistance. Additionally, the antibiotic resistance of the obtained strains was investigated using seven different antibiotics.
Results: Thirteen Lactobacillus isolates were obtained from 7 (8.0%) honey samples. Of these, eight isolates were identified as L. plantarum (61.54%), four isolates as L. rhamnosus (30.77%), and one isolate as L. acidophilus (7.69%). All strains were devoid of hemolytic activity, and three isolates (23.07%) were found to be resistant to acid, while 2 (15.38%) showed resistance to bile and pepsin. All isolates were resistant to vancomycin (100%). Additionally, only one strain exhibited resistance to all tested antibiotics. Furthermore, the present study demonstrates a significant association (p-value<0.05) between the presence of Lactobacillus in various regions of Iran.
Conclusion: Various factors, such as climatic conditions and geographical location, can influence honey's composition and microbial diversity. Identifying and isolating potential probiotic species in honey could significantly expand their use in the food and pharmaceutical industries, offering numerous health benefits and potential therapeutic applications.

1. Vit P, Vargas O, Lopez T, Valle F M. Meliponini biodiversity and medicinal uses of pot-honey from El Oro province in Ecuador. Emir J Food Agric 2015; 27: 502-506.
2. Oryan A, Alemzadeh E, Moshiri A. Potential role of propolis in wound healing: Biological properties and therapeutic activities. Biomed Pharmacother 2018; 98: 469-483.
3. Almasaudi S. The antibacterial activities of honey. Saudi J Biol Sci 2021; 28: 2188-2196.
4. Carnwath R, Graham E, Reynolds K, Pollock P. The antimicrobial activity of honey against common equine wound bacterial isolates. Vet J 2014; 199: 110-114.
5. Olaitan P B, Adeleke O E, Iyabo O. Honey: a reservoir for microorganisms and an inhibitory agent for microbes. Afr Health Sci 2007; 7: 159-165.
6. Pećanac M, Janjić Z, Komarčević A, Pajić M, Dobanovački D, Mišković-Skeledžija S. Burns treatment in ancient times. Med Pregl 2013; 66: 263-267.
7. Olofsson T C, Vásquez A. Detection and identification of a novel lactic acid bacterial flora within the honey stomach of the honeybee Apis mellifera. Curr Microbiol 2008; 57: 356-363.
8. Neveling D P, Endo A, Dicks L M. Fructophilic Lactobacillus kunkeei and Lactobacillus brevis isolated from fresh flowers, bees and bee-hives. Curr Microbiol 2012; 65: 507-515.
9. Endo A, Futagawa-Endo Y, Dicks L M. Isolation and characterization of fructophilic lactic acid bacteria from fructose-rich niches. Syst Appl Microbiol 2009; 32: 593-600.
10. Vásquez A, Olofsson TC, Sammataro D. A scientific note on the lactic acid bacterial flora in honeybees in the USA — A comparison with bees from Sweden. Apidologie 2009; 40: 26-28.
11. Olofsson T C, Alsterfjord M, Nilson B, Butler È, Vásquez A. Lactobacillus apinorum sp. nov., Lactobacillus mellifer sp. nov., Lactobacillus mellis sp. nov., Lactobacillus melliventris sp. nov., Lactobacillus kimbladii sp. nov., Lactobacillus helsingborgensis sp. nov. and Lactobacillus kullabergensis sp. nov., isolated from the honey stomach of the honeybee Apis mellifera. Int J Syst Evol Microbiol 2014; 64: 3109-3119.
12. Vásquez A, Forsgren E, Fries I, Paxton R J, Flaberg E, Szekely L, et al. Symbionts as major modulators of insect health: lactic acid bacteria and honeybees. PLoS One 2012; 7(3): e33188.
13. Forsgren E, Olofsson T C, Vásquez A, Fries I. Novel lactic acid bacteria inhibiting Paenibacillus larvae in honey bee larvae. Apidologie 2010; 41: 99-108.
14. Syed Yaacob S N, Huyop F, Kamarulzaman Raja Ibrahim R, Wahab RA. Identification of Lactobacillus spp. and Fructobacillus spp. isolated from fresh Heterotrigona itama honey and their antagonistic activities against clinical pathogenic bacteria. J Apic Res 2018; 57: 395-405.
15. Cotter P D, Ross R P, Hill C. Bacteriocins—a viable alternative to antibiotics? Nat Rev Microbiol 2013; 11: 95-105.
16. Yu J, Wang H, Zha M, Qing Y, Bai N, Ren Y, et al. Molecular identification and quantification of lactic acid bacteria in traditional fermented dairy foods of Russia. J Dairy Sci 2015; 98: 5143-5154.
17. Xu Y, Xie M, Xue J, Xiang L, Li Y, Xiao J, et al. EGCG ameliorates neuronal and behavioral defects by remodeling gut microbiota and TotM expression in Drosophila models of Parkinson’s disease. FASEB J 2020; 34: 5931-5950.
18. Markazi A, Luoma A, Shanmugasundaram R, Mohnl M, Murugesan G R, Selvaraj R. Effects of drinking water synbiotic supplementation in laying hens challenged with Salmonella. Poult Sci 2018; 97: 3510-3518.
19. Jomehzadeh N, Javaherizadeh H, Amin M, Rashno M, Teimoori A. Quantification of intestinal Lactobacillus species in children with functional constipation by quantitative real-time PCR. Clin Exp Gastroenterol 2020; 13: 141-150.
20. Kim E, Yang S-M, Cho E-J, Kim H-Y. Novel real-time PCR assay for Lactobacillus casei group species using comparative genomics. Food Microbiol 2020; 90: 103485.
21. Huang C-H and Huang L. Rapid species-and subspecies-specific level classification and identification of Lactobacillus casei group members using MALDI Biotyper combined with ClinProTools. J Dairy Sci 2018; 101: 979-991.
22. Kim E, Yang S-M, Kim D, and Kim H-Y. Real-time PCR method for qualitative and quantitative detection of Lactobacillus sakei group species targeting novel markers based on bioinformatics analysis. Int J Food Microbiol 2021; 355: 109335.
23. Muthaiyan A, Ricke SC. Current perspectives on detection of microbial contamination in bioethanol fermentors. Bioresour Technol 2010; 101: 5033-5042.
24. Ramiah K, Van Reenen C, Dicks L. Expression of the mucus adhesion gene mub, surface layer protein slp and adhesion-like factor EF-TU of Lactobacillus acidophilus ATCC 4356 under digestive stress conditions, as monitored with real-time PCR. Probiotics Antimicrob Proteins 2009; 1: 91.
25. Lashani E, Davoodabadi A, Dallal MMS. Some probiotic properties of Lactobacillus species isolated from honey and their antimicrobial activity against foodborne pathogens. Vet Res Forum 2020; 11: 121-126.
26. Humphries R, Bobenchik AM, Hindler JA, Schuetz AN. Overview of changes to the clinical and laboratory standards institute performance standards for antimicrobial susceptibility testing, M100, 31st Edition. J Clin Microbiol 2021; 59(12): e00213-21.
27. Anderson KE, Sheehan TH, Mott BM, Maes P, Snyder L, Schwan MR, et al. Microbial ecology of the hive and pollination landscape: bacterial associates from floral nectar, the alimentary tract and stored food of honey bees (Apis mellifera). PLoS One 2013; 8(12): e83125.
28. Ambika Manirajan B, Ratering S, Rusch V, Schwiertz A, Geissler‐Plaum R, Cardinale M, et al. Bacterial microbiota associated with flower pollen is influenced by pollination type, and shows a high degree of diversity and species‐specificity. Environ Microbiol 2016; 18: 5161-5174.
29. Mustar S, Ibrahim N. A sweeter pill to swallow: A review of honey bees and honey as a source of probiotic and prebiotic products. Foods 2022; 11: 2102.
30. Seraglio S K T, Schulz M, Brugnerotto P, Silva B, Gonzaga L V, Fett R, et al. Quality, composition and health-protective properties of citrus honey: A review. Food Res Int 2021; 143: 110268.
31. Kwon H-S, Yang E-H, Yeon S-W, Kang B-H, Kim T-Y. Rapid identification of probiotic Lactobacillus species by multiplex PCR using species-specific primers based on the region extending from 16S rRNA through 23S rRNA. FEMS Microbiol Lett 2004; 239: 267-275.
32. Aween MM, Hassan Z, Muhialdin BJ, Noor HM, Eljamel YA. Evaluation on antibacterial activity of Lactobacillus acidophilus strains isolated from honey. Am J Appl Sci 2012; 9: 807-817.
33. Razmgah N, Mojgani N, Torshizi M. Probiotic potential and virulence traits of Bacillus and Lactobacillus species isolated from local honey sample in Iran. IOSR J Pharm Biol Sci 2016; 11: 87-95.
34. Feizabadi F, Sharifan A, Tajabadi N. Isolation and identification of lactic acid bacteria from stored Apis mellifera honey. J Apic Res 2021; 60: 421-426.
35. Tajabadi N, Mardan M, Saari N, Mustafa S, Bahreini R, Manap MYA. Identification of Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus fermentum from honey stomach of honeybee. Braz J Microbiol 2014; 44: 717-722.
36. Khan K A, Ansari M J, Al-Ghamdi A, Nuru A, Harakeh S, and Iqbal J. Investigation of gut microbial communities associated with indigenous honey bee (Apis mellifera jemenitica) from two different eco-regions of Saudi Arabia. Saudi J Biol Sci 2017; 24: 1061-1068.
37. López AC, Alippi AM. Phenotypic and genotypic diversity of Bacillus cereus isolates recovered from honey. Int J Food Microbiol 2007; 117: 175-184.
38. Saccà M, Lodesani M. Isolation of bacterial microbiota associated to honey bees and evaluation of potential biocontrol agents of Varroa destructor. Benef Microbes 2020; 11: 641-654.
39. Cianciosi D, Forbes-Hernández TY, Afrin S, Gasparrini M, Reboredo-Rodriguez P, Manna PP, et al. Phenolic compounds in honey and their associated health benefits: A review. Molecules 2018; 23: 2322.
40. Escuredo O, Míguez M, Fernández-González M, Seijo MC. Nutritional value and antioxidant activity of honeys produced in a European Atlantic area. Food Chem 2013; 138: 851-856.
41. Alvarez-Suarez J M, Tulipani S, Romandini S, Bertoli E, Battino M. Contribution of honey in nutrition and human health: a review. Med J Nutrition Metab 2010; 3: 15-23.
42. C HC, T R K. Probiotic potency of Lactobacillus plantarum KX519413 and KX519414 isolated from honey bee gut. FEMS Microbiol Lett 2018; 365: 10.1093/femsle/fnx285.
43. Stojančević M, Bojić G, Al Salami H, Mikov M. The influence of intestinal tract and probiotics on the fate of orally administered drugs. Curr Issues Mol Biol 2014; 16: 55-68.
44. Anisimova EA, Yarullina DR. Antibiotic resistance of Lactobacillus strains. Curr Microbiol 2019; 76: 1407-1416.
45. Campedelli I, Mathur H, Salvetti E, Clarke S, Rea M C, Torriani S, et al. Genus-wide assessment of antibiotic resistance in Lactobacillus spp. Appl Environ Microbiol 2019; 85(1): e01738-18.
IssueVol 15 No 3 (2023) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijm.v15i3.12905
Honey; Probiotics; Lactobacillus; Real-time polymerase chain reaction; Antibiotics

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Goli Mehdi Abadi ME, Hosseini-Safa A, Habibi S, Dehghan M, Forouzani-moghaddam MJ, Oshaghi M. Isolation and characterization of the lactobacillus strain from honey and its probiotic properties. Iran J Microbiol. 2023;15(3):439-447.