**6. Microbiological analysis of honey: fundamentals and objectives of the analysis and results for Brazilian samples**

determine the freshness of honey, as it is absent in fresh honey and its concentration increases during storage time [30]. Furthermore, high 5‐HMF content may indicate adulteration of honey by the addition of invert syrup [47]. Determination of 5‐HMF content may be performed employing a spectrophotometric method [43], or a chromatographic method by HPLC using calibration curves of 5‐HMF analytical standards to quantify this compound in honey [43, 53]. The Codex Alimentarius Committee on Sugars [8] stipulates 5‐HMF content at the maximum value of 40.00 mg/kg and, if honey is from a tropical region, accepts a maxi-

The HMF values found in Brazilian honey are higher than those found in nontropical countries, as Paulino et al. [42] that encountered 7.00–355.50 mg/kg in 13 samples from several cities of Ceará. Périco et al. [40] also found high HMF values (31.28 ± 0.2 to 581.4 ± 4.2 mg/kg) when analyzed 30 samples from Toledo, Paraná, and Soares et al. [41] found values ranging from 70.62 to 150.27 mg/kg. When honey from two distinct regions (São João do Cariri and Areia, both in Paraíba state) were analyzed by Rodrigues‐Evangelista et al. [44], the HMF

Diastases are enzymes present in honey, which are sensitive to heat and consequently, may be used to evaluate honey overheating [47]. Therefore, the measure of diastase activity is an indicative of honey's freshness and is useful to detect improper storage conditions [30]. Diastase activity may be also an indicative of honeybees fed artificially with glucose, as a diastase enzyme deficiency is observed in this case [54]. The determination of diastase activity is based on a spectrophotometric kinetic method, which measures the activity of diastasis enzymes present in honey, in order to monitor adulteration by the addition of sugar and evaluate storage time and conditions [4, 43]. For that, under specific conditions, the activity of diastase enzymes of honey is measured in a standard solution of starch. The Gothe unit is used to express diastase activity and is defined as the amount of enzyme which will convert 0.01 g of starch in 1 hour at 40°C [43]. The Codex Alimentarius Committee on Sugars [8] stipulates a minimum value of 8.00 Gothe; however, a minimum value of 3.00 Gothe is accepted for honey with low diastase activity if the 5‐HMF content is

The diastase activity was determined in 20 samples of honey from Ceará state by Sodré et al. [36] and found an average of 34.11 ± 8.41 (in Gothe scale). Sodré et al. [36] analyzed 36 honey samples from Bahia and found the value between 5.30 and 43.39. Marchini et al. [49] analyzed 205 honey samples from different localities of São Paulo state and found values ranging from 1.10 to 38.50, with an average of 8.14 for orange honey, 15.77 for eucalyptus honey, and 17.32 for wild honey. For different floral sources, the authors found values rang-

In complement to pollen microscopical analysis, physical‐chemical results for these Brazilian honey samples were conducted and which is presented below, where it is possible to demonstrate the identity and quality of some floral sources of Brazilian samples studied here.

mum value of 80.00 mg/kg.

**5.7. Diastase**

156 Honey Analysis

lower than 15 mg/kg.

ing from 7.80 to 19.00.

content was between 20.70 and 23.90 mg/kg.

Currently, safe food is a major global public health concern, since food may be contaminated by pathogenic microorganisms, which can cause severe diarrhea or debilitating infections. Furthermore, microorganisms can be responsible for the spoilage of food. Besides the contamination by microorganisms, food may be contaminated by chemical substances, such as toxins, environmental pollutants, and heavy metals [55].

Honey has low susceptibility to the proliferation of microorganisms due to its physicochemical characteristics, such as antimicrobial substances, low moisture content (low water activity), low pH, and oxidation reduction potential, among others [56]. Therefore, its antimicrobial properties discourage the growth or persistence of many microorganisms. Nevertheless, honey may be contaminated by primary and secondary sources of microbial contamination. Primary sources, including pollen, nectar, digestive tracts of bees, dust, air, and soil, are difficult to control. Secondary sources of contamination (after‐harvest) include cross‐contamination, equipment, food handlers, among others, and may be controlled by good manufacturing practices. Regarding the harvesting method, honey samples harvested using modern methods (colony established in man‐made bees' accommodation called hives) have lower yeast and bacterial counts than samples harvested using traditional methods (honey hunting, which use flame to destroy the insects and are used in honey bee colony established in wood logs), that is, modern methods are more hygienic and produce the better quality of honey. Furthermore, exposure of colony to fire also kills bees and hampers the process of cross‐pollination and may lead to consumption of the whole forest [57].

The honey samples should be subjected to quality control tests to evaluate their physicochemical and microbiological parameters. Thus, it is possible to assess whether the results are within specifications and detect if there was an adulteration of honey. In Brazil, the Ministry of Agriculture, Livestock, and Supply (MAPA) published the Technical Regulation of Identity and Quality of Honey (Brazil, 2000), which describes that the analysis of contaminants should follow the Technical Regulation of the Southern Common Market (MERCOSUL or MERCOSUR). Regarding microbiological criteria, the document "MERCOSUL/GMC/RES n° 15/94" has the following technical specifications for honey: total coliforms/g: absence; *Salmonella* spp. and *Shigella* spp./25 g: absence; enumeration of molds and yeasts: maximum of 100 CFU/g [58].

According to MAPA, microbiological methods recommended by the International Organization for Standardization (ISO) should be used [59]. The enumeration of coliforms is performed using the colony‐count technique (ISO 4832:2006) [60]. The total coliform group includes four genera: *Escherichia*, *Klebsiella*, *Citrobacter*, and *Enterobacter*. The presence of these bacteria in food indicates that there was fecal contamination. Therefore, they are commonly used as indicators of sanitary quality of honey. Some authors evaluated Brazilian honey samples from different regions of Brazil (states of Ceará, Bahia, Pernambuco, Piauí, Rio Grande do Norte, and the Rio Grande do Sul) and showed that there was absence of coliforms in these samples [9, 61–63], that is, the harvesting, the management, and the processing of the samples were made as recommended in order to obtain a good quality of honey.

The detection of *Salmonella* spp. should be performed using the International Standard ISO 6579:2002 [64]. The genus *Salmonella* includes several pathogenic serotypes, which can cause from gastroenteritis (fever, diarrhea, and abdominal cramps) to serious systemic infections (enteric fevers), like *Salmonella* typhi that causes typhoid fever. However, gastroenteritis is the most common form of salmonellosis and the major mode of transmission is by means of contaminated food. Some studies showed that there was an absence of *Salmonella* species in Brazilian honey samples from different regions of Brazil [9, 61, 63, 65].

The enumeration of yeasts and molds, in its turn, is performed using the colony‐count technique according to ISO 21527‐2:2008, which specifies a method for the enumeration of viable xerophilic molds and osmophilic yeasts in products that have a water activity less than or equal to 0.95 [66]. Luiz et al. [65] evaluated Brazilian honey samples produced in several cities of the state of Minas Gerais (Southeast region), and the yeast and mold counts varied from <10.0 to 3.3 x 10<sup>1</sup> CFU/g, that is, all samples were according to Brazilian law. In another study by Schlabitz et al. [63] with honey samples from state of Rio Grande do Sul (South region of Brazil), the majority of samples (10 samples) were within specifications, since the enumeration of yeasts and molds varied from <1.0 x 10<sup>1</sup> to 8.0 x 10<sup>1</sup> CFU/g. However, two samples had values above 100 CFU/g:1.3 x 10<sup>2</sup> and 6.1 x 10<sup>2</sup> CFU/g, respectively. Several honey samples produced in the state of Ceará (Northeast region of Brazil) were evaluated by Santos and Oliveira [61]. The authors showed that the majority of samples were within specifications, since yeast and mold counts varied from < 10.0 to 6.0 x 10<sup>1</sup> CFU/g. Only one sample had a count above 100 CFU/g, since it had 1.8 × 10<sup>2</sup> CFU/g.

Although not required by Brazilian law, the detection of *Clostridium* spp. (spore‐forming bacteria) also is important, since honey samples may be contaminated with spores of *Clostridium botulinum*, the etiological agent of botulism (potentially fatal disease). While the ingestion of these spores is considered harmless to healthy adults, the spores may germinate in the gut of infants under 6 months of age, multiply and produce botulinum toxins. This would not occur in children older than about 6 months and adults due to natural defenses that develop over time [55]. Ragazani et al. [68] evaluated honey samples from different regions of Brazil (states of São Paulo, Minas Gerais, Goiás, Ceará, Mato Grosso, and Santa Catarina) and isolated *C. botulinum* from 7% of the samples. In other studies, Schlabitz et al. [63] and Luiz et al. [65] showed that there was an absence of sulfite‐reducing clostridia in Brazilian honey samples from states of Rio Grande do Sul and Minas Gerais, respectively.
