Proteomics is becoming increasingly relevant in honey authenticity testing. For several years, we have therefore been in close scientific exchange with a research group in Prague, Czech Republic, which developed the foundations of this proteomics-based approach. At the same time, we began early on to establish and evaluate the method in our own laboratory. The procedure has since been validated and accredited at FoodQS. As interest in the use of proteomics for honey analysis has grown considerably and we are receiving an increasing number of questions on the subject, we would like to provide a concise overview of the scientific background, potential applications and limitations of the method.
What does proteomics mean in honey analysis?
Proteomics refers to the targeted analysis of proteins. Since almost all enzymes are proteins, the method can be used, among other applications, to detect specific naturally occurring or foreign enzymes in honey.
For the analysis, proteins present in the honey are first extracted and enzymatically digested into smaller fragments known as peptides. Characteristic peptides are then determined using LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry).
Proteins of different biological origins differ in their amino acid sequences and, consequently, in the peptides generated from them. Proteomics can therefore be used not only to identify a particular protein but, where suitable markers are available, also to determine the organism from which it originates.
Why are foreign enzymes in honey of interest?
Authentic honey naturally contains a variety of proteins and enzymes, some of which originate from the honey bee (Apis mellifera). During the processing of nectar or honeydew, bees add enzymes from their glandular secretions. These enzymes are involved in various processes during honey maturation and consequently become natural constituents of honey.
Enzymes are also produced on an industrial scale for a wide range of technical applications. Fungi and bacteria such as Aspergillus and Bacillus species are commonly used in fermentation processes to produce specific enzymes. Such industrially produced enzymes are not part of the natural protein profile of honey.
Foreign enzymes can enter a honey sample in different ways. They may be added directly to increase enzymatic properties such as diastase activity. Enzymes such as amylases and glucoamylases are also used in the industrial production of certain sugar syrups. One of their functions is to break down starch into smaller sugar molecules. Residues of these enzymes may remain in the finished syrup and can subsequently enter honey if that syrup is added. Under certain circumstances, their detection may therefore provide an indication of possible syrup adulteration.
What can proteomics detect?
At present, our laboratory can differentiate between a range of foreign enzymes, including:
- α-amylase (Aspergillus niger)
- α-amylase (Aspergillus oryzae)
- α-amylase (Bacillus licheniformis)
- α-amylase (Bacillus amyloliquefaciens)
- β-amylase (Hordeum vulgare)
- glucoamylase (Aspergillus niger)
- invertase (Saccharomyces cerevisiae)
Furthermore, proteins that naturally originate from the honey bee can be investigated:
- α-glucosidase (Apis mellifera)
- MRJP 1 (Apis mellifera)
In addition to their presence, the amount of these proteins may provide important information. If proteins that are typically present in honey are found only at unusually low levels, this could indicate dilution with syrup. Such findings must always be assessed in the context of natural variability, including possible differences between honey varieties, and together with other analytical results.
How does proteomics differ from conventional enzyme activity-based tests?
Conventional foreign-enzyme tests, which have been established for many years, primarily determine whether a particular enzymatic activity is present. Proteomics takes a different approach by detecting characteristic components of the protein itself.
This provides two major advantages.
Firstly, proteomics offers a high degree of specificity. It can distinguish between α-amylases of different biological origins, such as an enzyme originating from the honey bee and an industrially produced α-amylase from a fungus.
Secondly, under suitable conditions, proteomics can also detect denatured enzymes that are no longer active. This may be particularly relevant after heat treatment. Once an enzyme has lost its activity due to heat exposure, an activity-based assay can no longer detect it through its enzymatic function. Proteomics, by contrast, may still identify characteristic peptides of the protein.
What are the limitations of proteomics?
In particular, highly refined syrups may have been purified to such an extent that proteins and enzymes have largely been removed and no suitable protein markers remain detectable. The absence of foreign proteins therefore does not, by itself, prove that no syrup has been added to a honey.
Conversely, a positive foreign protein finding must always be interpreted in the context of the individual sample. One possible source is residues from bee feeding, as feeding syrups or fondants may also contain enzymes that are foreign to honey. Small traces of such residues can occur even when good beekeeping practices are followed and should not automatically be interpreted as evidence of adulteration. The assessment therefore depends not only on whether a marker is detected, but also on the level at which it is present and on the overall analytical context of the sample. A similar principle is applied in other methods used for the detection of exogenous sugars, where low-level traces potentially originating from bee feeding are distinguished from findings indicative of actual adulteration or inappropriate beekeeping practices.
The assessment of the amounts of naturally occurring bee-derived proteins also requires a sufficiently broad reference database. Typical levels may vary between authentic honeys and different honey varieties, and this natural variation needs to be better understood before unusually low levels can be interpreted reliably.
How should proteomics be viewed in the context of honey authenticity testing?
Proteomics is not a universal stand-alone test that provides a simple answer to the question of whether a honey is “authentic or adulterated”. Rather, it represents another useful piece of the puzzle in honey authenticity testing and can complement established analytical methods by providing additional, highly specific information.
For those interested, proteomics analysis is available in our laboratory under Code 840. If you have any questions about the method or whether it may be suitable for a particular analytical question, please feel free to contact us.
Image: Maniturm / Pixabay
