An innovative molecular approach towards the cost-effective entomological authentication of honey

Abstract

Honey authentication and traceability are crucial not only for economic purposes but also for ensuring safety. However, the widespread adoption of cutting-edge technologies in practical applications has been hampered by complex, time-consuming sample pre-treatment processes, the need for skilled personnel, and substantial associated expenses. This study aimed to develop a simple and cost-effective molecular technique to verify the entomological source of honey. By utilizing newly designed primers, we successfully amplified the mitochondrial 16S ribosomal RNA gene of honey bees from honey, confirming the high quality of the extracted DNA. Employing RFLP analysis with AseI endonuclease, species-specific restriction patterns were generated for honey derived from six closely related honey bees of the Apis genus. Remarkably, this method was proven equally effective in identifying heat-treated and aged honey by presenting the same RFLP profiles as raw honey. As far as we know, this is the initial research of the simultaneous differentiation of honey from closely related honey bee species using the restriction endonuclease AseI and mitochondrial 16S rRNA gene fragments. As a result, it holds tremendous potential as a standardized guideline for regulatory agencies to ascertain the insect origins of honey and achieve comprehensive traceability.

Fig. 1. Agarose gel electrophoresis of PCR products of honey bee species (or honey) obtained with 16S rRNA-F/16S rRNA-R primers.

a PCR products from honey bee species. Lane M: 100 bp marker. AM1, AM2: A. mellifera; AC1, AC2: A. cerana; AL1, AL2: A. laboriosa; AD1, AD2: A. dorsata; AF1, AF2: A. florea; AA1 and AA2: A. andreniformis; b PCR products from honey. Lane M: 100 bp marker. AMH1, AMH2: A. mellifera honey; ACH1, ACH2: A. cerana honey; ALH1, ALH2: A. laboriosa honey; ADH1, ADH2: A. dorsata honey; AFH1, AFH2: A. florea honey; AAH1 and AAH2: A. andreniformis honey; NC negative control.

Fig. 2. A phylogenetic tree was constructed based on the alignment of 470~479 bp mitochondrial 16S rRNA gene sequences obtained from honey bees and honeys.

The construction was performed using the neighbor-joining method with the Kimura 2-parameter model. The sequences with NCBI accession numbers represent published honey bee sequences retrieved from GenBank. The numbers displayed above the branches indicate the percentage bootstrap values generated from 1000 replicates.

Fig. 3. Aligned DNA sequences of the mitochondrial 16S rRNA gene obtained from six honey bee species during the present study.

The restriction site for AseI is highlighted.

Fig. 4. RFLP patterns of PCR products amplified from honey bee (or honey) using AseI.

a PCR-RFLP profile of honey bee. Lane M: 50 bp marker. AM1, AM2: A. mellifera; AC1, AC2: A. cerana; AL1, AL2: A. laboriosa; AD1, AD2: A. dorsata; AF1, AF2: A. florea; AA1 and AA2: A. andreniformis. b PCR-RFLP profile of honey. Lane M: 50 bp marker. AMH1, AMH2: A. mellifera honey; ACH1, ACH2: A. cerana honey; ALH1, ALH2: A. laboriosa honey; ADH1, ADH2: A. dorsata honey; AFH1, AFH2: A. florea honey; AAH1 and AAH2: A. andreniformis honey; NC: negative control.

Fig. 5. Analysis of PCR products (or PCR-RFLP profile) obtained from A. mellifera honey under different processing conditions.

a PCR products of A. mellifera honey. Lane M: 100 bp marker. 1: raw; 2: room temperature for 1 year; 3. room temperature for 5 years; 4: −20 °C for 6 years; 5: −20 °C for 8 years; 6: crystalline honey; 7: 60 °C for 1 h; 8: 70 °C for 1 h; 9: 80 °C for 1 h; 10: 90 °C for 1 h; 11: 100 °C for 1 h; 12: commercial honey. b PCR-RFLP profile of A. mellifera honey. Lane M: 50 bp marker. 1: raw; 2: room temperature for 1 year; 3. room temperature for 5 years; 4: −20 °C for 6 years; 5: −20 °C for 8 years; 6: crystalline honey; 7: 60 °C for 1 h; 8: 70 °C for 1 h; 9: 80 °C for 1 h; 10: 90 °C for 1 h; 11: 100 °C for 1 h; 12: commercial honey.