Article pubs.acs.org/JAFC
A Possible Freshness Marker for Royal Jelly: Formation of 5‑Hydroxymethyl-2-furaldehyde as a Function of Storage Temperature and Time Marco Ciulu,† Ignazio Floris,‡ Valeria M. Nurchi,§ Angelo Panzanelli,† Maria I. Pilo,† Nadia Spano,† and Gavino Sanna*,† †
Università degli Studi di Sassari, Dipartimento di Chimica e Farmacia, via Vienna 2, 07100 Sassari, Italy Università degli Studi di Sassari, Dipartimento di Agraria, via De Nicola 9, 07100 Sassari, Italy § Università di Cagliari, Dipartimento di Scienze Chimiche e Geologiche, Cittadella Universitaria, 09042 Monserrato-Cagliari, Italy ‡
ABSTRACT: In this article we present a study of the variability of the concentration of 5-hydroxymethyl-2-furaldehyde (HMF) in natural royal jelly (RJ) as a function of its storage temperature (−18, 4, and 25 °C) and time (up to 9 months after harvesting). For this work HMF is evaluated using an RP-HPLC method we previously assessed. While all RJ samples stored at 4 and −18 °C always showed levels of HMF under the limit of detection (0.13 mg kg−1), samples kept at 25 °C showed an exponential increase in the concentration of HMF as a function of the storage time. This behavior and a number of desirable features of the analytical method used (ease of use in routine laboratories, availability of a complete validation protocol specifically developed for RJ, based on consolidated chemical knowledge) allow us to hypothesize the use of HMF as a possible, reliable freshness marker for RJ. KEYWORDS: royal jelly, freshness marker, HMF, 5-hydroxymethyl-2-furaldehyde, RP-HPLC, Maillard reaction
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store RJ under refrigerated conditions,5,7 though for longer periods of time the only effective way to preserve the product’s quality is to store it at −20 °C.8 Although international standards for RJ freshness have not yet been established, studies have proposed a number of markers that in principle are able to reveal its exposure to inadequate storage conditions.4 The proposed markers include physical−chemical parameters,5,9−11 classes of compounds such as sugars,9 amino acids,12,13 proteins,5,8,9,14 and water-soluble vitamins,5,6 molecules such as furosine7 and adenosine-5′-triphosphate,15 and enzymes such as glucose oxidase.10 On the other hand, another study relevant to this topic was conducted by Antinelli and co-workers16 and demonstrated that 10-hydroxy-2-decenoic acid, an unsaturated fatty acid specific to RJ, is not suitable as a freshness marker for this matrix. Nevertheless, all of these proposed markers suffer from important drawbacks: e.g., the analytical method is not completely validated, it requires uncommon instrumentation, it is unable to clarify all the phases along the entire shelf life of RJ, or the marker is also present in fresh RJ and thus requires a tedious and preliminary study aimed at evaluating its natural variability. Recently, our research groupwhich has been active for years devising, assessing, and validating analytical methods to measure the concentration of trace species in apiary products17−25has developed and validated an HPLC method for the determination of HMF (5-hydroxymethyl-2-furaldehyde) in RJ.26 HMF is the freshness parameter for honey adopted by both Codex Alimentarius (Alinorm 01/25 2000)27
INTRODUCTION Royal jelly (RJ) constitutes one of the most important products of beekeeping. It is secreted from the hypopharyngeal and mandibular glands of the worker honeybees to feed the queen during the entire course of her life, and it is considered to be the principal vehicle for caste differentiation inside the beehive.1 Many biologically active substances have been identified in the chemical composition of RJ, and several examples of functional properties have been reported in the literature.2,3 From a chemical point of view, RJ is a very complex matrix. The most abundant compounds (or chemical classes) that compose it are water (50−70%), proteins (9−18%), mono- and disaccharides (7−23%), and lipids (3−8%).2−4 Among the minor species we find mineral salts, free amino acids, and vitamins, which are present at levels of 0.8−3%, 0.6−1.5%, and several hundreds of milligrams per kilogram, respectively.2−4 Fresh RJ also contains enzymes such as glucose oxidase, invertases, and phosphatases.3 In addition, RJ contains a large number of molecules with strong bioactive properties. Among these, the most renowned are the unsaturated fatty acid 10-hydroxyl-2-decenoic acid, which has been shown to possess antibacterial, antiulcer, antidiabetic, anticancer, and antihyperlipoidemic characteristics,3 and royalactin, a 57 kDa bioactive peptide capable of inducing queen differentiation in honeybees.5,6 Indeed, RJ is known to exhibit a number of biological and pharmacological properties; these have been reported in a recent and detailed review by Bogdanov.3 The freshness of RJ is one of the most important factors determining its quality, since RJ contains a large variety of labile bioactive molecules. Thus, improper or prolonged storage of RJ can cause changes to its physical and chemical features, determining a loss of its functional properties and, therefore, its commercial value. For this reason, it is highly recommended to © 2015 American Chemical Society
Received: Revised: Accepted: Published: 4190
October 31, 2014 April 8, 2015 April 10, 2015 April 10, 2015 DOI: 10.1021/acs.jafc.5b00873 J. Agric. Food Chem. 2015, 63, 4190−4195
Article
Journal of Agricultural and Food Chemistry
these findings, we have repeated the evaluation of the LOD and LOQ with a better choice of the HMF standard solutions. Hence, four 4% TCA aqueous solutions containing respectively 4, 6, 8, and 10 μg L−1 of HMF have been analyzed in triplicate, now giving LOD and LOQ values of 0.13 and 0.39 mg kg−1, respectively. Figure 1 gives a comparison of selected portions of chromatograms (retention time between 5.5 and 6.5 min) of
and the European Union (EU Directive 110/2001).28 Preliminary analyses on a number of fresh RJ samples showed that HMF concentrations were over the limit of quantification (LOQ) only in samples stored at room temperature. Hence, the principal aim of this study is to monitor the evolution of the concentration of HMF in fresh RJ stored for months at different temperatures in order to ascertain the potential suitability of HMF as a freshness marker for this foodstuff.
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MATERIALS AND METHODS
Chemical and Reagents. The chromatographic solvents used were a 0.05 mol dm−3 H2SO4 aqueous solution and methanol (HPLC grade, Riedel de Haen, Milan, Italy). Before their use, all solvents were filtered through a 0.45 μm membrane from Millipore (Bedford, MA, USA) to remove any particulates. 5-Hydroxymethyl-2-furaldehyde was purchased from Lancaster (Milan, Italy). Trichloroacetic acid (TCA) was obtained from Sigma-Aldrich (Milan, Italy). All aqueous solutions were prepared using type I ultrapure water (New Human Power II Scholar UV, Human Corporation, Seoul, Korea). Instrumentation. The HPLC equipment used for the experiments consisted of a Series 200 binary pump, a sampling valve, a 20 μL sample loop, a Series 200 LC column oven, and a Series 200 UV−vis variable wavelength detector, all from PerkinElmer, Milan, Italy. Separation was performed using a 250 mm × 4.6 mm Ascentis C18 column, 5 μm particle size (Supelco, Bellefonte, PA, USA), thermostated at 20 °C and fitted with a guard cartridge packed with the same stationary phase. Data were processed using Turbochrom Workstation Software (PerkinElmer, Milan, Italy). Experimental Protocol. A freshly harvested RJ sample was obtained from “Organizzazione Produttori Apicoltori Sardi Terrantiga” (San Sperate, Sardinia, Italy). Once harvested, the RJ was homogenized, divided into 12 dark glass bottles (1−12), and kept at −18 °C until the beginning of the experimental protocol. The initial HMF content of RJ was determined within 5 days from delivery. To study the evolution of the level of HMF in RJ stored under different conditions, the 12 bottles were divided into three groups of four samples. The groups were stored at −18, 4, and 25 ± 2 °C (room temperature), respectively. Each sample was analyzed in triplicate after 30, 90, 180, and 270 days from delivery. Samples stored at −18 and 4 °C were equilibrated at room temperature before analysis. Prior to each analysis, all of the samples were homogenized for 5 min. Determination of HMF Content in Royal Jelly. The HMF concentration was determined using the same RP-HPLC procedure previously developed and validated for RJ by our research group.26 Briefly, the analytical steps involved first a pretreatment of the sample with an aqueous solution of trichloroacetic acid followed by a gradient elution RP-HPLC analysis and analyte detection at 280 nm.
Figure 1. Overlapped chromatographic portions (retention time between 5.0 and 6.5 min) of a blank solution (line 1), sample 9 after 1 month of storage at 25 °C (line 2), and the same sample 9 spiked with 50 μL of a freshly prepared 4 mg/L HMF aqueous solution (line 3).
the blank solution (line 1), of the RJ sample 9 after 1 month of storage at 25 °C (line 2), and of the same RJ sample spiked with 50 μL of a freshly prepared 4 mg/L HMF aqueous solution (line 3). Monitoring of HMF in RJ on Variation of the Storage Temperature and Storage Time. In our previous study on the assessment and validation of the analytical method to measure the HMF concentration in RJ,26 we observed proportionality between the amount of HMF measured and the age of RJ samples. In particular, whereas HMF was not quantifiable in any fresh samples, its concentration was higher than 9 mg kg−1 in the oldest RJ sample stored at room temperature. In this study, in order to verify the fitness of HMF as a potential marker of freshness of RJ, we carefully monitored the evolution of this analyte in RJ samples stored at different temperatures for the 9 months following their harvest. The RJ samples were always kept in dark glass bottles to prevent any degradation effect promoted by light. The initial concentration of HMF was under the LOD of the method for all samples, as shown in Table 1. Samples stored at 4 and −18 °C showed only negligible levels of HMF for the entire duration of the study. On the other hand, in samples stored at room temperature we observed an exponential increase of the HMF concentration as a function of storage time. The dependence of the HMF concentration in RJ samples kept at room temperature and the storage time are shown in Figure 2.
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RESULTS Review of the LOD and LOQ Values According to the Experimental Results of the Study. The calculation of the LOD in the method recently proposed by us was accomplished using the ULA1 approach.29 This choice was supported both by the preliminary experience gained in the validation of HPLC methods devoted to measure HMF concentration in honey samples of different origin19,21 and by the knowledge that the ULA1 method is optimal for reducing any decision error approach.25 It is crucial, however, that the concentration of the most diluted standard solution used to evaluate LOD turns out to be close enough to the real LOD value. In this connection, the improved knowledge of the method acquired during this study has allowed us to observe that both the LOD and LOQ values previously measured were overestimated (i.e., the effective concentration of analyte associated with both qualitative and quantitative information was lower than that evaluated during the relevant validation step26). On the basis of
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DISCUSSION The choice of a proper freshness marker for functional foods is a very complex problem that requires the careful and simultaneous consideration of a wide number of parameters, related to both the candidate marker and to the analytical method used to determine it. First, the optimal freshness marker should not be traceable in the fresh food product,7,30 so that the mere presence of the marker is indicative of spoilage. If, on the other hand, the marker is present in the fresh matrix, 4191
DOI: 10.1021/acs.jafc.5b00873 J. Agric. Food Chem. 2015, 63, 4190−4195
Article
Journal of Agricultural and Food Chemistry Table 1. Concentration of HMF in RJ at Varying Storage Temperatures and Times av HMF content (mg kg−1), n = 3 temp −18 °C 4 °C room temp
a
sample a
1−4 5−8a 9 10 11 12 av
0 days b
