Isolation and biological evaluation of lactic acid bacteria from bee pollen and bee bread

Document Type : Research Paper

Authors

1 Department of Animal Sciences, Faculty of Agriculture, Bu Ali Sina University, Hamedan, Iran.

2 Department of Honeybee, Animal Science Research Institute of Iran, Agricultural Research Education and Extension Organization (AREEO), Karaj, Iran.

Abstract

The present research aimed to isolate lactic acid-producing bacteria from pollen or bee bread. For this purpose, bacterial strains were isolated from various pollen and bee bread samples using an MRS culture medium. The characteristics of the isolates were assessed through Gram staining, catalase activity tests, fermentation of specific sugars, growth curve analysis, pH reduction in the culture medium, and gas production. The results from the growth curve analysis of the isolated bacteria demonstrated significant differences in their growth rates under identical culture conditions, indicating varying capacities for pollen fermentation. Specifically, bacteria MB1, MB2, MB4, and MP1 exhibited robust growth during the initial 48 hours. In contrast, bacteria MB3 and MP2 remained in the lag phase throughout this period; however, MP2 and MB4 subsequently experienced a marked increase in growth rate, with the highest optical density (OD). The maximum OD recorded for bacteria MB1, MB3, and MB4 were 1.15, 0.602, and 0.939 respectively after 115 hours, while bacterium MP2 achieved its plateau optical density of 1.314 after 65 hours. In the next phase of the study, sterilized and ground pollen was inoculated with the most effective bacterial strains identified in the initial isolation phase. Following incubation of the pollen-containing culture medium, bacterial activity in fermenting pollen was evaluated based on lactic acid production. All isolated bacteria were confirmed to be Gram-positive and catalase-negative, demonstrating the ability to ferment certain simple sugars. The isolated strains exhibited varying efficiencies in both growth rates and lactic acid production, suggesting differences in their capacity to utilize the nutrients found in pollen. However, no significant differences were observed among the bacterial strains concerning gas production.

Keywords

Extended Abstract

Introduction

Pollen is the primary source of protein for honeybees, and its quality and digestibility are crucial factors for their health. There is a direct correlation between pollen supply and the successful rearing of broods. The morphology of pollen grains features a natural protective layer against external factors, which limits access to its nutrients. Consequently, bees within the hive convert it into bee bread through a solid-state fermentation process. Lactic acid bacteria, a group of bacteria that typically found in the normal flora of healthy organisms, play a significant role in this convertion. The pollen brought to the colony by honeybees is converted into a valuable substance known as bee bread, a process that occurs through the addition of honey, digestive enzymes, and lactic acid fermentation during the storage of pollen in honeycomb cells. This fermentation process protects pollen from losing its properties and enhances the compounds resulting from enzymatic changes; however, the precise mechanism remains unclear.

Materials and methods

A mixture of 10 grams of bee bread and 10 grams of dried pollen was prepared in 250 cc of sterilized distilled water containing 0.1% (w/v) peptone using a sterilized mixer at 300 rpm for 2 minutes. Two cc of the resulting stock solution was injected into MRS broth culture medium. Ten samples from the pollen stock solution and ten from the bee bread stock solution were inoculated into Hungate tubes. Based on gas production after 72 hours, one sample from each culture (pollen and bee bread) was selected for further testing and inoculated into solid culture media. After another 72 hours, the bacteria with the most significant growth (in terms of colony size on plates) were transferred to liquid culture for subsequent subculturing, which was repeated five times for bacterial purification. Ultimately, 20 Hungate tubes containing bacteria derived from pollen and bee bread were selected for further tests, including Gram staining, sugar fermentation tests, catalase tests, growth curve analysis, fermentation capacity of pollen by bacteria, and lactic acid measurement.

 

Results and Discussion

The results indicated that all 12 purified bacteria were catalase-negative and Gram-positive. Sugar fermentation tests showed that all isolates had a high capability to ferment the specified sugars. Notably, all bacteria exhibited greater growth in fructose. Growth curve analysis revealed significant differences in growth rates among isolates in identical culture conditions, suggesting varying abilities to ferment pollen. Different isolates demonstrated diverse levels of lactic acid production, indicating functional diversity among the isolated bacterial populations. The honeybee gut serves as a substantial reservoir for lactic acid-producing bacteria. Isolating these bacteria from bee bread and pollen that are associated with the honeybee gut, can provide valuable insights into their potential roles in fermentation processes and their relationship with honeybee health and nutrition. The metabolic activity of lactic acid bacteria is often linked to their growth rates; fast-growing strains can metabolize nutrients more efficiently and produce beneficial compounds such as organic acids that support host health while inhibiting pathogen growth. The production of lactic acid by these bacteria has been studied in various sections of the honeybee gut, particularly in digestive areas. The amount of lactic acid produced is crucial as it can indicate aspects such as fermentation activity (the efficiency of carbohydrate fermentation by various bacteria in the honeybee gut), pH regulation (preventing pathogen growth at low pH), nutritional benefits (using lactic acid as an energy source), and immune system modulation (supporting immunity through lactic acid and other metabolites). Furthermore, the ability of lactic acid bacteria to produce lactic acid is an important factor in their selection as probiotics. Studies have shown that lactic acid bacteria isolated from honeybees can produce lactic acid, with some strains exhibiting high production capacity. Some isolates obtained in our study also demonstrated good lactic acid production potential, indicating their high fermentative capacity. These differences in lactic acid production may reflect functional diversity among isolated bacteria. all isolated lactic acid bacteria were capable of producing lactic acid and biomass; however, production levels varied among strains.

 

Conclusion:

Lactic acid bacteria isolated from the digestive tract or honeybee products such as pollen and bee bread possess the ability to ferment raw pollen and may offer good probiotic potential for use in honeybee and human nutrition. Further studies aimed at improving the isolation of these bacteria and optimizing fermentation techniques are necessary to maximize the potential of these beneficial microbes.

Author Contributions

Mojtaba Sharifinia: Methodology, Software, Formal analysis, Investigation, Writing - Original Draft.

 Daryoush Alipour: Conceptualization, Validation, Supervision, Writing - Review & Editing, Project administration.

Hasan Aliarabi: Formal Analysis, Writing - original draft.

Naser Tajabadi: Formal Analysis, Data curation, Writing - original draft.

All authors contributed equally to the conceptualization of the article and writing of the original and subsequent drafts.

Data Availability Statement

The data supporting reported results are collected and used from published articles, to them we have referred in the manuscript.

Acknowledgements

The authors would like to thank all participants of the present study.

Conflict of interest

The author declares no conflict of interest.

 

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Iranian Journal of animal Science
Volume 56, Issue 3
September 2025
Pages 585-596

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History

  • Receive Date: 30 November 2024
  • Revise Date: 13 January 2025
  • Accept Date: 20 January 2025

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