**1. Introduction**

Manuka honey is a dark monofloral honey derived from the New Zealand manuka tree (*Leptospermum scoparium*, *L. scoparium*); known for its distinctive taste and aroma, it plays a significant role in modern medical applications, especially as an antibacterial, antiviral, and antibiotic agent [1,2]. Manuka honey showed, for instance, bactericidal potency against *Pseudomonas aeruginosa* and *Staphylococcus aureus* (strains responsible for the inflammation of the mucous lining of the paranasal sinuses) and antiviral ability against Varicella zoster (the virus that causes chickenpox and shingles) [3,4]. In turn, a study of the combination of five novel antibiotics and manuka honey revealed the improved activity of these antibiotics against wound pathogens [5]. In addition, the honey from New Zealand promotes the growth of certain types of probiotics such as *Lactobacillus reuteri* and *Lactobacillus rhamnosus* [6]. In addition, manuka honey exhibits strong antioxidant and anti-inflammatory properties due to the presence of various bioactive compounds such as phenols, flavonoids, and enzymes, which makes it effective in the treatment of chronic ulcer and topical clinical inflammation [7–9]. Furthermore, numerous data highlight the anticancer effect of manuka honey [10,11]. A cytotoxic influence on human lung, breast, colon, and metastatic cancer cell lines, as well as on murine melanoma, colorectal carcinoma, and human hepatocarcinoma was observed [12–14].

Although the importance of manuka honey for medicinal purposes is well documented in the literature, information regarding its physicochemical characteristics remains limited. The European Union has established regulations governing the physicochemical parameters of honey in order to ensure its quality, authenticity, and safety [15]. These properties

**Citation:** S ˛ek, A.; Por˛ebska, A.; Szcz ˛esna, T. Quality of Commercially Available Manuka Honey Expressed by Pollen Composition, Diastase Activity, and Hydroxymethylfurfural Content. *Foods* **2023**, *12*, 2930. https://doi.org/10.3390/ foods12152930

Academic Editors: Liming Wu and Qiangqiang Li

Received: 30 June 2023 Revised: 26 July 2023 Accepted: 30 July 2023 Published: 2 August 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

includes moisture content, apparent reducing sugars content, apparent sucrose content, water-insoluble solids content, ash content, acidity, diastase activity, hydroxymethylfurfural (HMF) content, and electrical conductivity [15]. Specifying the above-mentioned characteristics of honey is also intended to protect consumers against deceptive practices of sellers of this rarely cheap product. Two physicochemical parameters of honey, which deserve particular attention as they are crucial for honey quality control, are not clearly defined for manuka honey: the diastase activity (diastase number, DN) and the HMF content. Diastase (α-amylase) is one of the main enzymes found in honey; its activity level is considered an indicator of honey freshness and its proper processing [16]. Similarly, the content of HMF, a furanic compound formed when sugar-containing products are acidified or heated, informs about the quality of honey. Since a high level of HMF can be a result of adulteration with sugar additives, the HMF amount is also useful for assessing the authenticity of honey samples [17].

Most of the studies on the DN and HMF content in manuka honey carried out so far have not linked these experiments with melissopalynological analysis [17–24]. Only a few papers discuss this issue using these three methods [25–27]. However, in these studies, the results are presented for individual samples of manuka honeys without using the relevant statistics. It was found that the manuka honey with 75.8% of *L. scoparium* pollen grains contains a DN and HMF content equal to 19.03 (Schade units) and 16.42 mg·kg<sup>−</sup>1, respectively [25]. In turn, the honey samples with an *L. scoparium* pollen content of about 31 and 51% are characterized by a diastase activity of about 7.2 and 9.1 Schade, respectively, and by an HMF amount of 27.9 and 33.5 mg·kg−1, respectively [26]. Another study indicates that the manuka honey DN and HMF content was equal to 8.0 ± 1.0 Schade and 18.1 ± 3.2 mg·kg<sup>−</sup>1, respectively, but declared that the pollen analysis was performed; however, the percentage composition of pollen grains in the tested honey was not given [27]. Importantly, since the manuka pollen was considered over-representative and the manuka tree shares similar pollens with the kanuka bush, Moar stated that to be called manuka honey, honey must contain at least 70% of manuka pollen grains [28]. Thus, it is likely that some of the honeys mentioned should not be called manuka honey.

The problem of manuka honey falsification has been recognized earlier; in response to this issue, the New Zealand Ministry for Primary Industries reported the combination of five attributes required to distinguish manuka honey from other honey types, as well as to identify monofloral and multifloral manuka honeys [29,30]. Four of the attributes refer to the four specific compounds found in manuka honey by liquid chromatography–tandem mass spectrometry, i.e., 2 -methoxyacetophenone, 2-methoxybenzoic acid, 3-phenyllactic acid, and 4-hydroxyphenyllactic acid; the other relates to the DNA markers of manuka pollen determined by the multiplex qPCR assay [29,30]. Other studies have described the potential of compact atmospheric solids analysis probe mass spectrometry, liquid chromatography–high resolution mass spectrometry, fluorescence spectroscopy, and 1H NMR spectroscopy for the evaluation of manuka honey authenticity [31–34]. Another unique feature of manuka honey is its high methylglyoxal (MGO) content [35]. This compound can be formed in foods through five pathways. The first pathway is the Maillard reaction, i.e., a reaction between amino acids and reducing sugars that occurs during food storage at room temperature and during thermal processing [36]. The other pathways include the autoxidation of hexoses, the oxidation of unsaturated fatty acids in lipids, the dehydration of dihydroxyacetone (DHA), and the microbial metabolism of dihydroxyacetone phosphate [36]. In manuka honey, MGO is naturally formed as a result of the dehydration of dihydroxyacetone, a compound present in the nectar of manuka tree flowers [36]. The chemical transformation of DHA to MGO occurs in moderate heat, and the period of storage, which means that the MGO in manuka honey has already been produced in the beehive, as well as being produced in storage drums after humans collect the honey [37]. It is worth mentioning that manuka honey is frequently stored for several years without any temperature regulation [37]. In some cases, this is to intentionally increase the amount of MGO formed, especially since the MGO is thought to be responsible for

the antibacterial activity of manuka honey [37,38]. It is easy to determine that advanced and often sophisticated equipment is needed to apply above described techniques used for verifying the authenticity of manuka honey; therefore, pollen analysis appears to be the most suitable first-choice method for manuka honey recognition. Thus, in this paper, using the example of the Polish market, we demonstrate the variation in the percentage of *L. scoparium* pollen in honeys labeled and sold as manuka honeys. Then, we present the DN value and HMF content based on relevant statistics. The pollen analysis results can be regarded as an alert both for consumers of manuka honey and for sellers purchasing the product from unproven suppliers, and above all for scientists who conduct research on manuka honeys without confirmation of their authenticity. In turn, the DN and HMF content results point to the questionable quality of the imported manuka honey.
