**1. Introduction**

Bee pollen is collected from plant flowers and agglutinated into pellets by honeybees via oral secretion. As an indispensable nutrient source for honeybee development, bee pollen is rich in carbohydrates, proteins, amino acids, polyphenols, lipids, minerals, and vitamins [1]. Its chemical composition varies considerably according to its botanical origins [1,2]. The large amounts of bioactive constituents endow bee pollen with health beneficial properties, such as antioxidant, anti-inflammatory, anti-allergen, anti-aging, and anti-cancer effects [3–5]. Owing to its nutritional and therapeutic properties, bee pollen has gained increasing attention worldwide and is commercially consumed as a natural dietary supplement for human health promotion [6,7].

Camellia bee pollen (CBP) is among the most important bee pollen products that are extensively consumed in China. It is gathered by honeybees from the flowers of tea plants (*Camellia sinensis* L.), the leaves of which can be made into tea, a popular beverage worldwide with various health benefits. It has been reported that the chemical constituents of tea flowers are similar to those of tea leaves [8]. A share of some common bioactive constituents and functional properties between CBP and tea can thus be expected. Indeed, CBP has a special fragrance, similar to the aroma of tea. Moreover, it has been demonstrated that CBP possesses higher anti-inflammatory, antioxidant, and anti-tyrosinase activities relative to other types of bee pollen [9–11]. In recent years, the identification of bioactive

**Citation:** Qi, D.; Lu, M.; Li, J.; Ma, C. Metabolomics Reveals Distinctive Metabolic Profiles and Marker Compounds of Camellia (*Camellia sinensis* L.) Bee Pollen. *Foods* **2023**, *12*, 2661. https://doi.org/10.3390/ foods12142661

Academic Editor: Olga Escuredo

Received: 13 June 2023 Revised: 5 July 2023 Accepted: 6 July 2023 Published: 11 July 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/).

constituents responsible for the observed functional properties has been attracting growing interest. Among them, caffeine, kaempferol, levulinic acid, and 5-hydroxymethyl furfural are reported to contribute partly to the anti-tyrosinase activities of CBP [12–14]. However, the metabolic basis for its functional properties is still far from being fully understood, thereby impeding the use of CBP in the cosmetics, food, and pharmaceutical industries.

The aforementioned superior functional properties promote increasing demand for CBP, which leads to fraudulent practices in the market [15]. To identify bee pollen of different botanical origins, sensory testing (e.g., color, aroma, and taste characteristics) and microscopic examination (e.g., size, form, and color of pollen grains) are widely used [16]. However, such subjective judgments based on sensory evaluation are easily biased by personal preference. Moreover, even with a microscope, it is still difficult to distinguish between different types of bee pollen with similar morphological and structural attributes [17,18]. The situation is even worse for CBP, which shows substantial morphological variation between tea cultivars [19]. The lack of accurate identification methods represents a loophole for the current adulteration chaos of bee pollen. A more sensitive method is, thus, urgently needed to ensure accurate identification for the long-term development of the bee product industry.

Non-targeted metabolomics based on high-resolution mass spectrometry provides a convenient method for the simultaneous analysis of hundreds or thousands of small molecules in food products, including various bee products [20–23]. This approach, combined with targeted metabolomics, plays a key role in screening and quantifying marker compounds for food authenticity [24–26]. For bee pollen authenticity, however, such research is currently limited [27].

Our study aimed to uncover distinctive metabolic components of CBP and to explore efficient metabolite indicators to identify authentic CBP. To this end, non-targeted metabolic profiling of CBP and other types of bee pollen was performed. We proposed epicatechin gallate (ECG) and L-theanine as marker compounds of CBP, and measured their content based on ultra-high performance liquid chromatography-quadruple-Exactive Orbitrap mass spectrometry (UHPLC-Q-Exactive Orbitrap-MS).
