Microbiological Quality of Apis mellifera L. Honey Samples from Western Paraná, Southern Brazil

Because honey is a saturated solution of sugar obtained from nectar which undergoes dehydration of up to 15 to 18% humidity, it is considered a naturally safe food against microorganisms. The high concentration of carbohydrates, as an antimicrobial factor, limits the amount and activity of water to microorganisms, causing a low reduction potential and high viscosity that limits the entry of oxygen into honey. These factors contribute to the stability of honey, which prevents microorganisms from developing and multiplying, resulting in senescence until their mortality (Molen et al., 1997; Wen et al., 2017). Therefore, only a limited number of microorganisms are to expected survive in honey. Yeasts, fungi, and such spore-forming bacteria as Clostridium botulinum are found in honey, but their high presence is indicative of immature honey harvest as it presents conditions for them to survive and proliferate (Snowdon & Cliver, 1996). The primary source of these microorganisms are pollen grains, bee-collected nectar and such factors related to the digestive tract of bees and natural environment as dust, air and soil (Kačániová et al., 2009; Anderson et al., 2013; Corby-Harris, Maes, & Anderson, 2014; Wen et al., 2017). The hygiene of the manipulators of honey, honey house, equipment, and physical structure can also be a source of microorganisms (Fernández et al., 2017). The quality control of honey is determined through international regulations for the physical-chemical parameters of moisture, sugars, hydroxymethylfurfural, diastase activity, acidity, electrical conductivity and color (Codex, 2001).

However, many countries have their own regulations, which generate some divergences in these parameters (Silva et al., 2016; Pascual-Maté et al., 2018). Although the microbiological analysis of honey is not necessary as routine, it improves the product's food safety and helps to diagnose possible hygienic-sanitary flaws in colony management and product handling. Thus, importance of Brazil's honey production and increased exports, its commercial honey is governed by the laws of the Southern Common Market (MERCOSUR), which accepts a maximum of 100 CFU/g for fungi and yeast and does not allow the presence of Salmonella and Shigella or of total coliform bacteria in honey.

The western region of Paraná in the year 2016 produced 705 tons, which represents 15.6% of state production (IBGE, 2018). Because beekeeping is important in this region, the beekeepers working together with Beekeeping Cooperative of the West Coast of Paraná, in partnership with the University State of the West of Paraná, ITAIPU-Binational, have been awarded the Indication of Origin (IP) seal, a type of Geographical Indication (GI), with the National Institute of Intellectual Property (INPI, 2017). These institutions and others that have joined the group have been working to obtain, another GI, the origin denomination quality label for honey produced in this region through research that proves its quality and relate the characteristics of the product to its phytogeographic origin (Camargo et al., 2014; Jacobus et al., 2019). In this study, we characterize the microbiological situation of the honey samples to assess whether beekeepers are applying and following food safety rules. Then, Non-metric Multidimensional Scaling analysis was applied to establish multifactorial relationships and observe the behavior of sample clusters from the region.

Microbiological analyses showed that all samples presented values within the range determined by the RDC-12 technical regulation of 2001. The microbial counts in the samples are shown in Tab. 1. In general, the mean incidence of aerobic mesophilic bacteria, yeasts, and fungi was lower than that reported by other studies (Tab. 1). The total count of aerobic mesophilic had a mean value of 2.52 log CFU/g (Tab. 1), ranging from 0 to 4.4 log CFU/g among samples from different locations (Tab. 2).

The total yeast and mold counts, present mean values of 0.46 log CFU/g and 2.95 log CFU/g suggest adequate management by the great majority of beekeepers (Tab. 1). However, the Entre Rios do Oeste and Itaipulândia samples had a mean between 7.1 and 8.9 log CFU/g for molds. Regarding the sanitary quality of the samples, was a reduced incidence of Clostridium spp. 1.46 log CFU/g, total coliforms 0.78 log CFU/g, and 0.50 log CFU/g fecal coliforms (Tab. 1). Despite the reduced microbial load in the evaluated samples, the incidence of aerobic mesophilic was observed in 70% of the samples, Clostridium spp. in 77%, total coliforms in 60%, fecal coliforms in 62%, total yeasts in 34% and total molds in 83% (Tab. 1).

Table 3. Shows the genera of isolated fungi. The genus that presented the highest incidence was Cladosporium spp., 77% of the total of fungi, representing. This may be due to the cosmopolitan characteristics. The other fungi Phoma spp. had an incidence of 49%, Aspergillus spp. 31%, Fusarium spp. 22%, and Penicillium spp. 13% (Tab. 3).

Multivariate analysis

NMDS analysis visualized the formation of a general structure of the bacterial groups in the honey samples and indicated a similarity between them (Fig. 2). A e stress value of 0.154 indicated that the distance between the points in the ordering chart is representative of the degree of similarity between the samples and the bacterial communities. Samples from the upper left quadrant had higher values for molds, yeasts, Fusarium ssp., Penicillium spp., Cladosporium spp., Aspergillus spp., Phoma spp. and fecal coliforms. The right quadrant showed higher values for Clostridium spp. and aerobic mesophilic (PCA), and, the lower part between the two quadrants presented the highest values for total coliforms.

Fig. 2

However, NMDS was not sufficient to distinguish whether the separation of the cluster was due to some microbial communities showing a degree of overlap. Thus, the application of the method of the width of the silhouette determines the optimal point for the formation of two groups (Fig. 3). Occurring a cophenetic correlation coefficient (CCC) of 0.76 between the original similarity matrix and the resulting matrix of similarity. Estevinho et al. (2016) believes this CPCC value is reasonable as a representative factor of the hierarchy.

Fig. 3

After the ordination of the NMDS graph and the application of the silhouette-width method, the formation of two groups was determined. Together with the clustering analyses (UPGMA), using the Euclidean distance, the difference of the groups between the samples was determined more accurately.

The new NMDS projection (Fig. 4) demonstrated that despite the large projection area, there was a similarity between the microbiological parameters. Although the profiles were visually separable, the samples were separated into two groups. The largest group presented a group of sixty-four samples covering all the microbiological parameters except for total coliforms, which were grouped in the group within the circle, since these three samples presented the highest values of total coliforms with the other parameters.

Fig. 4

Honey is a saturated solution of sugar, which limits the quantity and activity of water for the microorganism, whose development and multiplication is prevented its, leading to its senescence until its mortality (WEN et al., 2017). Other antimicrobial factors of honey are low protein content, acidity, and reduced oxidation-reduction potential together with the high viscosity that limits the entry of oxygen into the honey (SILVA et al., 2016). This research showed that the honey produced by beekeepers was very similar with some exceptions, which are being corrected with technical monitoring. The observed values of the microbial population of aerobic mesophiles from this study are below those found in the literature. The samples of honey from Benin ranged from 2.25 to 2.46 log CFU/g (Azonwade et al., 2018) and those of organic honey from Portugal around 1.3×10² ± 75.5×10¹ CFU/g (Estevinho et al., 2012). These values demonstrate that the incidence of these microorganisms occurs even in regions with high sanitary control.

The microorganisms found in this study for aerobic mesophiles, molds, and yeasts are probably related to the primary contamination by the intestinal contents of the bees, the environment of the colony and the plants foraged by them (Anderson et al., 2013; Wen et al., 2017). These same authors affirmed that a microbial flow occurs inside the colony, because they observed the same microorganisms in the flowers, the bee bread, the environment of the hive and the intestine of the bees.

This study indicated the incidence of Clostridium spp., the most important micro-organism for human health. The contamination may have occurred due to foraging, air, dust or energy supply provided by beekeepers during winter (Snowdon & Cliver, 1996). Jay, Loessner, & Golden (2005) reported Bacillus and Clostridium may be important bacterial contaminants of sugar cane (Saccharum officinalis) and beet (Beta vulgaris), which are used in the winter feeding of colonies in this region.

The incidence of Clostridium spp. was also observed by Pucciarelli et al. (2014), in 70% of the samples when evaluating the growth of this microorganism in the honey of Tetragonisca angustula, although they were not able to isolate Clostridium botulinum in the same samples through biochemical tests. This suggests that in the present study the incidence of Clostridium spp. does not indicate the presence of C. botulinum. Although the antibacterial conditions honey with low pH (3.4 to 5.5) and low water activity (0.5 to 0.6) acts as an antimicrobial factor inhibiting the survival of bacteria and is frequently associated with infant botulism (Aureli, Franciosa, & Fenicia et al., 2002). Due to such characteristics, the World Health Organization (WHO, 2002) and National Health Surveillance Agency (Anvisa, 2011) do not recommend honey for infants and children under 12 months of age, because they have limited intestinal microflora and low immunity.

The low yeast value demonstrated limited growth of these microorganisms, indicating the good hygienic-sanitary quality of the samples because yeasts are transferred to honey through primary and secondary contamination (Snowdon & Cliver, 1996). Furthermore, physicochemical analyses indicated low moisture content (<20%) of the collected honey, which makes bacterial growth difficult because the above 21% moisture is known to favor the proliferation of these microorganisms which survive in acidic conditions, cause fermentation and the formation of carbon dioxide and alcohol and reduce the quality of honey (Snowdon & Cliver 1996; White, 2010). Sereia et al. (2010) evaluated the hygienic-sanitary quality of organic and non-organic honey samples from the Paraná River islands and observed that organic honey presented lower quality than conventional honey to hygienic-sanitary quality and that the humidity of the samples was the main factor for the reduced product quality.

The incidence of these microorganisms indicated secondary contamination for the sanitary quality (total coliforms and fecal coliforms) and safety (Clostridium spp.) of the samples (Snowdon & Cliver, 1996). The values of coliforms indicated direct or indirect contamination of fecal origin since the natural habitat of coliforms in the enteric tract of humans and animals and the absence of these microorganisms indicate adequate hygiene during collection, extraction, and handling of honey (Sanz et al., 1995).

Total coliforms and fecal coliforms in the literature (Pucciarelli et al., 2014) were present in a greatest variety in honey samples in Argentina, Italy (Sinacori et al., 2014) and Mexico (Vázquez-Quiñones et al., 2018) and absent in the samples evaluated from Spain (Sanz et al., 1995), Argentina (Iurlina & Fritz, 2005), Portugal Estevinho et al. (2012) and Mexico Fernández et al. (2017). Therefore, this is first study on honey in the process of designation of origin in Brazil and provides information for future work to reduce these microorganisms in honey. Increased microbiological values in the locations furthest from the cooperative (Santa Helena municipalities) such as Terra Roxa, Marechal Cândido Rondon, Entre Rios do Oeste, and Francisco Alves may be due to less technical assistance and training of beekeepers. These skills are important as good production practices improve the quality of the honey produced. Evaluation microorganisms at different points in honey processing (honeycombs, honeycomb uncapping, honey extractor, honey sump, and honey drum), demonstrated that in eight honey extraction units, when good manufacturing standards were not followed observed contamination increased per coliforms at all points of evaluation (Fernández et al., 2017). Sereia et al. (2011) concluded the same in the evaluation of organic honey production systems and reported a reduction of molds and yeasts on samples of honey processed adequately to those whose beekeepers did not follow good hygiene practices, by Administrative Rule No. 367 of September 4, 1997 (Brasil, 1997).

The genera of molds identified in this study corroborate the data of the literature, since the genus Penicillium, Cladosporium, Aspergillus, and Alternaria, are considered common microorganisms in honey samples (Nasser, 2004; Kacaniová et al., 2009; Wen et al., 2017). Sinacori et al. (2014) and Knazovická et al. (2015) verified that the microflora of multiflora honey had higher rates of biodiversity and species richness compared to monofloral ones. The highest percentage of Cladosporium spp. observed may be due to this genus having a cosmopolitan distribution with 772 names (Ducan et al., 2004), and its spores can be found in the air, soil, water, and plants (Ogórek et al., 2012). Kačániová et al. (2009) reported a high incidence (25,000 CFU/g) of the genus Cladosporium and the genus Penicillium (12.66%), in honey, Cladosporium (13.27%) and Penicillium (72.24%) in the environment of the colony. With an increase in molds and yeasts in the enteric tract of the bees between the season of the summer and the winter, suggesting a microbial flow within the colony (Kačániová et al., 2009).

Wen et al. (2017) evaluated the microbial load in the first fifteen days of honey maturation and observed that while the ripening process and reduction of the moisture content of honey occurred there was a reduction in the quantity and richness of microorganism species. Besides, these authors pointed out that, after five days of maturity, there was a reduction of the genera Cladosporium, Alternaria, Aspergillus, Penicillium, Phoma while yeast after that period remained in a small quantity. This suggested that samples collected ahead of time, with high moisture content, become more favorable to the multiplication of these microorganisms which leading honey deterioration.

Thus, it was concluded that the majority of the samples were within the parameters accepted by the legislation. The largest microbial communities were of mesophilic aerobes and molds. The analyses showed that honey samples are similar to one another for most microbial communities. These results indicate that honey produced in the western region of Paraná are very similar. The results reported in this study show a sampling of the situation of the bee chain in the region, and with this information it is possible to set up an activity plan to improve technology transfer to beekeepers.