Comprehensive study of honey with protected denomination of origin and contribution to the enhancement of legal specifications

Molecules 2012, 17, 8561-8577; doi:10.3390/molecules17078561 molecules ISSN 1420-3049 www.mdpi.com/journal/molecules Article Comprehensive Study of Honey with Protected Denomination of Origin and Contribution to the Enhancement of Legal Specifications Antonio Iglesias 1, Xesus Feás 2, Sandra Rodrigues 3, Julio A. Seijas 2, M. Pilar Vázquez-Tato 2, Luís G. Dias 3 and Leticia M. Estevinho 3,* 1 Department of Anatomy and Animal Production, Faculty of Veterinary Science, University of Santiago de Compostela, Lugo, Galicia E-27002, Spain; E-Mail: [email protected] 2 Department of Organic Chemistry, Faculty of Veterinary Science, University of Santiago de Compostela, Lugo E-27002, Spain; E-Mails: [email protected]s (X.F.); [email protected] (J.A.S.); [email protected] (M.P.V.-T.) 3 CIMO-Mountain Research Centre, Agricultural College of Bragança, Polytechnic Institute of Bragança, Campus Santa Apolónia, Bragança E-5301-855, Portugal; E-Mails: srod[email protected] (S.R.); [email protected] (L.G.D.) * Author to whom correspondence should be addressed; E-Mail: [email protected]; Tel.: +351-2733-03342. Received: 29 May 2012; in revised form: 28 June 2012 / Accepted: 5 July 2012 / Published: 17 July 2012 Abstract: In this study the characterization of a total of 60 honey samples with Protected Denomination of Origin (PDO) collected over three harvests (2009–2011, inclusive), from the Northeast of Portugal was carried out based on the presence of pollen, physicochemical and microbiological characteristics. All samples were found to meet the European Legislation, but some didn’t meet the requirements of the PDO specifications. Concerning the floral origin of honey, our results showed the prevalence of rosemary (Lavandula pedunculata) pollen. The microbiological quality of all the analyzed samples was satisfactory, since fecal coliforms, sulfite-reducing clostridia and Salmonella were absent, and molds and yeasts were detected in low counts. Significant differences between the results were studied using one-way analysis of variance (ANOVA), followed by Tukey’s HSD test. The samples were submitted to discriminant function analysis, in order to determine which variables differentiate between two or more naturally occurring groups (Forward Stepwise Analysis). The variables selected were in this order: diastase activity, pH, reducing sugars, free acidity and HMF. The pollen spectrum has perfect discriminatory power. This is the first study in OPEN ACCESS Molecules 2012, 17 8562 which a honey with PDO was tested, in order to assess its compliance with the PDO book of specifications. Keywords: honey; microbiological; palynological; physicochemical; Protected Denomination of Origin 1. Introduction According to the European Union Legislation [1], the Codex Alimentarius [2] and the Portuguese Law Decree 214/2003 honey is the natural sweet substance produced by Apis mellifera bees from the nectar of plants, secretions of living parts of plants, or excretions of plant-sucking insects on the living parts of plants, which the bees collect, transform by combining with specific substances of their own, deposit, dehydrate, store and leave in the honeycomb to ripen and mature. More than 200 substances have been found in honey, the most abundant being carbohydrates (fructose, glucose, maltose, sucrose) [3]. However, minerals, proteins, vitamins, organic acids, flavonoids, phenolic acids, enzymes and other phytochemical substances are also important components of this natural product [4]. It is important to mention that honey’s composition depends on the floral origin, the climate, environmental and seasonal conditions, as well as on its handling and processing [5]. It has been used in ethno-medicine since the early humans, and in more recent times it is used in the treatment of burns, gastrointestinal disorders, asthma, infected and chronic wounds. It plays an important role in the human diet, and is also used in pharmaceutical and cosmetic industries [6]. As an easily assimilable food, honey makes a valuable nutritive product for children, athletes and convalescents [4,7]. In Portuguese agriculture, honey is a very important product. In fact, there are more than 26,000 beekeepers that produce about 11,000 tons per year of honey [8]. In agreement with the European Regulation [9] in Portugal there are nine Protected Denominations of Origin (PDO) for honey, which demonstrates that besides the investment on the formation of beekeepers, there is a growing interest on the assurance of the quality, with consequences not only in the economic dynamics of the rural areas in which the beekeepers operate, but also in honeys’ commercialization itself. Indeed, the honeys bearing the PDO logo have acquired a high added value, both within national and international level. In this context, it is mandatory to verify its compliance with the quality specifications of the European Union. Honey’s quality is related to its sensorial, physical, chemical and microbiological characteristics. The physicochemical properties depend on the nectar and the floral source, colour, flavours, moisture, amount of proteins and sugars [10,11]. These quality criteria are well specified by the EC Directive 2001/110 [1], being moisture content, electrical conductivity, ash content, reducing sugars, free acidity, diastase activity and hydroxymethylfurfural (HMF) content the major parameters of interest. However, EU legislation lacks specifications concerning microbial contamination and hygiene of the product. Indeed, many studies have been carried on the physicochemical parameters of honey all over the World [12–18], but few data about its microbiological safety is available, being the scarce studies essentially devoted to the detection of Clostridium botulinum, since honey is the only known dietary reservoir of spores of this Gram-positive bacteria [14,19]. In this context, and Molecules 2012, 17 8563 considering that honey has several sources of microbial contamination, among them, the pollen, the digestive tract of the Apis mellifera bees, dust, air, soil and the processing of the product, it is very important to assess the commercial quality and microbiological safety of honey (counts of moulds and yeasts, spores of Bacilus spp. and Clostridium spp.). Thus, the present study comprised several objectives. First of all, it aimed to characterize, in respect to floral origin, physicochemical parameters, microbiological safety and commercial quality, honey with PDO from the “Terra Quente” and from three different harvests: June of 2009, 2010 and 2011. The second goal was to test whether the product met the PDO products’ specifications. Last but not least, contribute to the introduction in the “book of specifications” of some parameters/analyses that are currently neglected: pH and microbiological indicators of safety and quality. 2. Results and Discussion 2.1. Pollen Analyses The analysis of the pollen profile of honey allows inferring its floral origin and confirms the identity of the honey source indicated by the beekeepers. The identified pollen and its frequency on the analyzed honeys from the different harvests are presented in Figure 1. Figure 1. Polinic spectrum of the “Terra Quente” honeys from the three harvests. The letters (a,b,c) represents which honeys are different by Tukey test with significance of α = 0.05. According to the “book of specifications”, in the spectrum of the “Terra Quente” honey pollen of the Papilionaceae family prevail (Cytisus multiflorus, Cytisus striatus, Retama sphaerocarpa, Genista florida), which is due to the wide spread of these botanic species in the Northeast of Portugal [20]. However, the special characteristics of “Terra Quente” honeys are associated with the presence of rosemary (Lavandula pedunculata). Our results showed that 55% of the samples harvested in 2009 had more than 35% of Lavandula pedunculata, being thereby denominated “rosemary honey”, in agreement with the book of specifications. In the same harvest, 45% of the samples had percentages of Lavandula sp. between 15% and 35%. The honey collected in 2010, 5% of the samples had less than Molecules 2012, 17 8564 15% of Lavandula sp., 30% had between 15% and 35% and 65% had more than 35% of this botanic specie. In the third harvest (2011), 25% of the samples had less than 15% of Lavandula sp., half of the samples contained between 15 and 35% and 25% showed more than 35% of this pollen. In the harvest of 2010, the pollen of Genista sp. was also very abundant, in contrast to the verified in the other years under study. 2.2. Colour Analysis Colour is considered as a quality parameter for honey. It depends on the floral origin and the minerals’ content [21]. Light-coloured honeys usually have low ash contents, while dark-colored honeys generally have higher [22]. The colour of the analyzed honey samples varied between light amber to amber. In the honey harvested in 2009 amber honey prevailed (71%), while in 2010 the most abundant was the amber type (41%). 58% of the honey harvested in 2011 was light amber (Figure 2). Figure 2. Colour of the samples in each harvest. 2.3. Physicochemical Analyses The results obtained from the physicochemical parameters are presented in the Table 1. The percentage of moisture ranged between 14.50 and 18.11, with mean values of 15.94 ± 0.70, 16.28 ± 0.46, 16.37 ± 0.70, in samples from 2009, 2010 and 2011, respectively. These results are bellow the limits of <20%, suggesting that the extraction period and the maturation of the product were adequate. In fact, honey moisture content depends on the environmental conditions and the manipulation from beekeepers at the harvest period, and can vary from year to year [23]. High moisture content in honey can increase its water activity and consequently make the preservation and storage more difficult. In addition, it may cause flavor loss and diminution of the “shelf-life”. Concerning this parameter, there were no significant differences, using the Tukey test (p < 0.05), that is one of several tests that can be used to determine which means amongst a set of means differ from the rest, between humidity values obtained for the honey samples of the three harvests. Molecules 2012, 17 8565 Table 1. Physicochemical results of “Terra Quente” honeys from the three harvests. Harvest (Year) Parameters Moisture (%) Electrical Conductivity (mS·cm−1) Ash (%) HMF (mg·kg−1) Diastase activity (Gothe Scale) pH Free acidity (meq·kg−1) Reducing sugars (%) Apparent sucrose (%) First (2009) 15.94 ± 0.70 a (14.50–17.21) 0.25 ± 0.07 b (0.13–0.36) 0.12 ± 0.06 b (0.02–0.21) 2.41 ± 1.45 b (1.31–7.02) 14.04 ± 3.51 b (10.06–25.00) 3.58 ± 0.14 a (3.32–3.81) 25.58 ± 6.12 a (13.94–33.63) 69.75 ± 4.19 a (63.71–76.89) 4.59 ± 1.51 a (2.60–7.53) Second (2010) 16.28 ± 0.46 a (15.40–17.02) 0.23 ± 0.06 b (0.15–0.36) 0.10 ± 0.05 b (0.03–0.21) 4.39 ± 2.64 a (2.63–13.84) 15.09 ± 1.29 ab (13.59–19.17) 3.25 ± 0.26 b (2.93–4.12) 25.16 ± 5.69 a (15.12–38.22) 66.96 ± 2.91 b (62.52–74.11) 4.73 ± 1.61 a (2.44–8.12) Third (2011) 16.37 ± 0.70 a (15.41–18.11) 0.29 ± 0.04 a (0.26–0.36) 0.15 ± 0.03 a (0.12–0.21) 3.61 ± 1.24 ab (1.71–7.28) 16.95 ± 2.26 a (11.99–19.10) 3.28 ± 0.25 b (2.97–3.64) 28.28 ± 3.93 a (21.52–36.80) 65.59 ± 2.08 b (60.21–68.57) 4.54 ± 1.28 a (2.34–7.36) p-value 0.082 0.006 <0.001 0.006 0.002 <0.001 0.143 <0.001 0.914 Results are given as the mean ± standard deviation (min–max values). The letters (a,b) represents which honeys are different by Tukey test with significance of α = 0.05. Molecules 2012, 17 8566 The electrical conductivity of the samples ranged between 0.13 and 0.36 mS·cm−1, suggesting that all samples are from nectar honey. This parameter of honey is closely related to the concentration of mineral salts, organic acids and proteins and shows great variability according to the floral origin. It is important for the differentiation of honeys, being therefore used in routine honey quality control [12]. The ash content is a complex function of the floral, geographical, soil and climatic characteristics and conditions [23]. The results found (0.02 to 0.21%) are much lower than the reported, by our team [15–16] and others worldwide [14], for other types of honey. This testifies the clearness of DPO honey samples and the lack of adulteration with molasses. The results obtained for electrical conductivity and ashes in the third harvest (2011) differed significantly from the results of the two previous extractions (p = 0.006 and p = 0.000, for 2010 and 2009). HMF content and diastase activity are widely recognized as indicators of honeys’ freshness [5]. The evaluation of the two parameters is very important as the changing behavior of diastase activity makes it an uncertain parameter to determine if honey has been submitted to heating [24]. Several factors influence the levels of HMF, such as temperature and time of heating, storage conditions, pH and floral source [25]. It is well known that honey heating results in the formation of HMF, which is produced during acid-catalysed dehydration of hexoses, such as fructose and glucose and Maillard reactions [23]. In fact, our results show that the analyzed honeys are fresh products and ensured that they weren’t submitted to heating or inadequate storing conditions, since the HMF content varied between 1.31 mg·kg−1 and 13.84 mg·kg−1. Concerning this parameter the highest value was obtained in the harvest of 2010 (4.39 ± 2.46 mg·kg−1). In the honey of 2011, the amount of HMF didn’t differ significantly from the obtained in the two previous harvests (p = 0.102). However, the values obtained for the first and second harvests were significantly different (p = 0.006). Diastase is an enzyme naturally occurring in honey. Its level depends upon geographic and floral origins of the product. The diastase activity found in the analyzed honeys (2009: 14.04 ± 3.51 mg·kg−1; 2010: 15.09 ± 1.29 mg·kg−1; 2011: 16.95 ± 2.26 mg·kg−1) was higher than 8 in the Gothe Scale (minimal value allowed by the regulations). The second harvest didn’t differ significantly from the others (p = 0.065), but there were significant differences between the one from 2009 and the one from 2011 (p = 0.002). Honey pH is affected by the conditions during extraction and storage, which also influences texture, stability and shelf-life. pH is indeed a useful index to predict the microorganisms’ growth, as the low pH of honey inhibits their presence [26]. It should be noted that honey’s pH is not directly related to the free acidity, due to the buffer action of the acids and minerals, which are naturally present. The results obtained for this parameter ranged from 2.93 and 4.12. These values are consistent with the published reported values of European monofloral honeys [27]. The free acidity didn’t differ significantly among the samples from the different harvests. In the honeys from the first harvest, the mean value was of 25.58 ± 6.12 meq·kg−1, in the second it was 25.16 ± 5.69 meq·kg−1 and in the third 28.28 ± 3.93 meq·kg−1. The presence of organic acids in equilibrium with their corresponding lactones, or internal esters, and some inorganic ions, such as phosphate, may influence the free acidity of honey. High acidity can be indicative of fermentation of sugars into organic acids. All the investigated samples met the requirements set by the regulation [1,2], which establish that the acidity shouldn’t exceed 50 meq·kg−1. Molecules 2012, 17 8567 Concerning the reducing sugars (fructose and glucose), the European Council Directive [1] imposes a limit of reducing sugars ≥60 g/100 g. The percentage of reducing sugars oscillated between 60.21% and 76.89%. The results obtained for this physicochemical parameter do not only meet the standards but also are identical to the observed by other authors in different types of honey [15,26,28]. Apparent sucrose (non-reducing sugar) varied between 2.34% and 8.12%. Significant differences among the samples from different harvests were not noted. This attests that the extraction of the product was adequate and that honey wasn’t adulterated [29]. As mentioned before, all the results were within the limits allowed by the European Council Directive [1] and the Codex Standard for Honey [2]. However, 16 samples (five from the first harvest; five from the second and six from the third) didn’t meet the requirements set by the PDO book of specifications, since in some of them the percentage of moisture was higher than 17% and in others the apparent sucrose exceeded 6%. In addition, some of those samples had less than 65% of reducing sugars. Our results suggest that some modifications in the PDO book of specifications should be introduced, in order to avoid devaluation of products that, despite being in agreement with the legislation, don’t exactly fit in the current specifications. This is particularly important as most of the “Terra Quente” PDO honey is exported. However, it is necessary to conduct further studies, since the number of samples tested does not allow generalization. 2.4. Microbiological Analyses Despite the stress conditions found in honey, many microorganisms can grow in this matrix. Microorganisms that survive in honey are those that withstand the concentrated sugar, acidity and other antimicrobial characters of honey. The presence of fungi in honey is linked to contact with the intestinal contents of bees, bee hive and grass. In any case this natural reservoir for microbes does not diminish the many important uses that honey is known for. The results obtained for the parameters that indicate the commercial quality (aerobic mesophiles, moulds and yeasts), sanitary quality (fecal coliforms) and safety (sulphite-reducing clostridia and Salmonella) are presented in Table 2. Table 2. Microbial analyses of “Terra Quente” honey samples. Harvest Aerobic mesophiles (cfu·g−1) Moulds and yeast (cfu·g−1) Fecal coliforms (MPN) Sulphite-reducing clostridia (in 0.01 g) Salmonella (in 25 g) First 38.20 ± 21.62 a 24.05 ± 11.81 a <1 Negative Negative Second 37.45 ± 19.99 a 30.95 ± 22.16 a <1 Negative Negative Third 37.45 ± 18.27 a 27.45 ± 18.33 a <1 Negative Negative p-value 0.681 0.482 n.a. n.a. n.a. Results are given as the mean ± standard deviation. The letter (a) represents that the honeys are not different by Tukey test with significance of α = 0.05. Aerobic mesophiles, moulds and yeasts in the analyzed honey ranged from 37.45 ± 18.27 to 38.20 ± 21.62 cfu·g−1 and from 24.05 ± 11.81 to 30.95 ± 22.16 cfu·g−1 for aerobic mesophiles and moulds and yeasts, respectively. No statistical differences were found, for all the analyzed parameters among the different harvests (aerobic mesophiles: p = 0.680; moulds and yeasts: p = 0.482). These results are generally superior than those reported previously [15] when analyzing commercial Molecules 2012, 17 8568 Portuguese honeys, what may be due to the pasteurization they go through. However, were lower than the reported by [30] in honeys from Argentina and by [31] in artisanal honeys from the Northeast of Portugal. From the microbiological point of view, these low values of aerobic mesophiles, molds and yeasts are most probably related to the environmental conditions, and are indicative of an appropriate management of apiaries. All the samples were negative in respect to fecal coliforms, sulphite-reducing clostridia and Salmonella. On the other hand, previous surveys about non-PDO honey reported moderate to high levels of contamination, by coliforms and sulphite-reducing clostridia [30,14]. It should be noticed that the European Union doesn’t have specifications/legislation concerning the microbiological parameters of honey, despite the negative effects that the presence of microorganisms, mainly C. botulinum can have in public health, especially when the honey is consumed by children, the elderly and immunodepressed patients. Thus, it is very important to conduct studies in order to establish microbiological criteria and analyses’ methodologies for honey. 2.5. Multivariate Analysis After applying the algorithm for selecting variables, the variable “pollen type” showed perfect discriminatory power in stepwise discriminant analysis performed (Table 3) (Wilks’ Lambda = 0.000). The first canonical discriminant function explained 74% of the total variance while the second accounted for 26%. The size of the coefficients indicates the discriminant power of the predictor variables. Thus it can be seen that in the function 1 the variables Echium sp., Cistus sp. and Rubus sp. discriminate best among the three groups. Function 2 was capable of discriminating Genista sp. and Prunus sp. The plot of the two canonical variables shows a complete separation among the third harvest with respect to the other two. Some samples of the first and second harvest are superposed (Figure 3). Table 3. Results of stepwise discriminant analysis (SDA) of palynological parameters in the three harvests of PDO honey. Wilks’ Lambda Partial Lambda F-remove (2,45) p-level Tolerance 1-Tolerance (R2) Echium sp. 0.000 0.847 4.054 0.240 0.561 0.439 Genista sp. 0.000 0.693 9.962 0.002 0.836 0.164 Populus sp. 0.000 0.714 9.008 0.005 0.158 0.842 Prunus sp. 0.000 0.905 2.360 0.106 0.837 0.163 Rubus sp. 0.000 0.962 0.898 0.415 0.476 0.524 F-remove = R to remove value. The physicochemical variables selected by stepwise discriminant analysis as the more discriminant were, in this order, diastase activity, pH, reducing sugars, free acidity and HMF. Wilks’ Lambda, indicates the contribution of each variable to the discrimination, as it can be seen that the five do not surpass 0.5 (Table 4). In this work the significance is high for HMF, free acidity and reducing sugars (p < 0.01). From this it can be concluded that the selected parameters have low discriminate power. The behaviour of the standardized canonical coefficients of the found discriminating function, as well as the analysis of the structure of the matrix, showed that the variables with greater weight, in sequence Molecules 2012, 17 8569 descendent, in the differences between the groups were: pH (0.53), reducing sugars (0.48), and diastase activity (−0.47), free acidity show lower values (0.03). Figure 3. Separation of harvest honeys using palynological analysis: plot of discriminant scores. ● Harvest 1 ■ Harvest 2 ▲ Harvest 3. Table 4. Results of stepwise discriminant analysis (SDA) of physicochemical parameters in the three harvests of PDO honey. Wilks’ Lambda Partial Lambda F-remove (2,53) p-level Tolerance 1-Tolerance (R2) HMF 0.485 0.633 15.349 <0.001 0.472 0.528 Diastase activity 0.332 0.924 2.188 0.122 0.795 0.205 pH 0.338 0.908 2.673 0.0783 0.924 0.076 Free acidity 0.383 0.802 6.562 0.003 0.861 0.139 Reducing sugars 0.373 0.823 5.695 0.006 0.466 0.534 F-remove = R to remove value. The general shape of the distribution of tree harvests scores on a scatter diagram whose axes are the first two canonical variables is shown in Figure 4, where the separation is correct for third harvest whereas some samples of first and second are overlapped. 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