**2.5. Microscopy of honey**

As a bacterial control (without the addition honey), use well 10, and as a broth control (without the addition of honey and inoculum), use well 11. After adding the honey, inoculate 5 μL of a standardized suspension of the bacteria in question in each well, except the broth control

**Figure 8.** 96 well U bottom micro-plate with markings indicating the position of the lines (A–H) and columns (1–12).

plates, incubate in a bacteriological incubator at 35°C for 24 hour. After 24 hours of incubation the micro-plates are analysed to determine the MIC, which is defined as the lowest concentration of honey in which there is no visible growth after incubation. Finally, analyse the well

CFU/well. Identify the micro-

well, so that the end of test bacteria concentration is 5 × 10<sup>4</sup>

**Table 4.** Honey concentrations in percentage (%) for wells from 1 to 9.

**No of wells Honey (%)** 1 50.000 2 25.000 3 12.500 4 6.250 5 3.125 6 1.560 7 0.780 8 0.390 9 0.195

278 Honey Analysis

Microscopy of food is a technique used to identify foreign components in products, making it possible to check if they comply with standards. Several countries use government and health-related agencies to ensure food safety, by monitoring their supply chains. MERCOSUR GMC Resolution N° 15/1994 approved the Technical Regulations for the Identity and Quality of Honey based on resolutions N°. 18/1992 and N° 91/1993 of the Common Market Group [17]. Normative Instruction N°. 11 of October 20, 2000 approved the Technical Regulations for the Identity and Quality of Honey [18] and pursuant to Ordinance N°. 46 of 10 February, 1998 the Ministry of Health and the Ministry of Agriculture and Supply established the adoption of the Hazard Analysis and Critical Control Point (HACCP) system by the Food Industries for animal products [44]. This system is recommended by international bodies such as the World Trade Organization (WTO) and the World Health Organization [16], both forming part of the United Nations (UN), for Food and Agriculture.

In compliance with these standards, any problem identified in honey lots should be corrected immediately and possible causes should be identified. Once the failures are identified, the company must take corrective action to prevent new problems arising. These corrective actions must be validated through audits and microbiological tests that prove the definitive correction of the non-compliance.

According to the macroscopic and microscopic criteria established in Brazil [18] honey must be free from any foreign substance. In practice, dirt present in honey may come from two sources—the first occurs inside the beehive, and is more difficult to control as it is added to honey by bees which carry fragments of other insects, pollen and soil. Secondary sources are present from harvesting through the steps of obtaining, processing, and distribution of honey [45], and include wax fragments, propolis, larvae, wood fragments and among others. Use of Good Apicultural Practices reduces the risk of secondary contamination ensuring a quality product in accordance with standard rules [46]. Camargo [47] recommends the procedures of the Good Apicultural Practices should be applied during the processing of honey, including: use stainless steel trays for stacking wooden beehives, allow no contact between the wooden beehive and the ground; choose honeycombs free of bees, larvae, or pollen; open wooden beehives only in the reception of the honey house for prior cleaning (removing of adhered bees, wax and propolis); filter the honey with the aid of sieves with meshes of various diameters, pumps or filters; decant the honey for soil removal at lower densities.

**Method:** The analysis of dirt and foreign matter can be performed following the method of the Association of Official Analytical Chemistry AOAC [9] N°. 945.79, which uses filtration of the sample in the presence of nitric acid. The method is based on dissolving 100 g of the honey sample in 200 mL of distilled water which is heated and acidified with 5 mL of nitric acid (HNO<sup>3</sup> ) at a concentration of 6 M. Filter the sample in a Buchner funnel. Mark four quadrants on filter paper. Analyse using a stereoscopic microscope with a total multiplication of 100× and confirm the type of sediment between slide and cover slip under an optical microscope with a multiplication of 100–400×.

## **2.6. Clostridium botulinum**

The pathogenic microorganism of importance in honey is the *Clostridium botulinum* bacterium, which is capable of producing spores. Bacterial spores are latent and resistant to adverse environmental conditions and can thus endure processing and storage for long periods. Contamination of honey by *C. botulinum* spores occurs within the colony, making practical procedures for its prevention difficult.

In practice, the bees carry the spores of this bacterium in their legs and antennae, taken from the soil where they land constantly. These spores begin to grow in the colonies, and remain in the combs together with the honey. Contamination is also possible in the act of collecting the product if hygiene practices are poor, and further contamination can occur through contact with the ground. Once present in honey, it survives in the medium without competition from other microorganisms. The incidence of spores in honey may also be related to multiplication and sporulation in dead bees and their larval forms in the colonies [48].

Honey is the only food recognized as a risk factor for infant botulism. Although there have been many cases of occurrence of infant botulism from honey contaminated with *Clostridium botulinum*, literature on this topic remains scarce. Consequently, in Brazil the administration of honey to children is not recommended, especially in the breastfeeding phase. This practice is also adopted in the United States, the United Kingdom and Argentina, where spores were isolated [49].

This disease occurs in children under 12 months, and 95% of cases occur in the first 6 months of life, when honey is used as a sweetener for bottles and juices as well as to bathe pacifiers to soothe the child. A child's intestine possesses an immature flora. The intake of honey with spores leads to germination, multiplication and the production of *botulinum* neurotoxins in the intestinal lumen, causing many problems for the health of children [50]. The consumption of honey by adults or older children does not seem to provide any kind of risk in relation to botulism. Consequently, it is recommended by the World Health Organization and the US Centers for Diseases that honey should not be given to infants under 6 and 12 months, respectively [16, 31].

Honey added as an ingredient in commercial infant formulas for babies aged less than 1 year must be thermally processed to destroy *botulinum* spores. No reports exist about the use of honey as an ingredient in other foods which have caused botulism. The analysis of honey for *C. botulinum* is not recommended as a control measure [49].

The microbiological analysis of honey detects product contamination. The presence of microorganisms or their spores in honey can cause its deterioration and result in enzymatic changes, the production of mycotoxins and even consumer illness. Due to the therapeutic properties attributed to honey, antimicrobial evaluation is essential to contribute to the quality maintenance of this product, adding to its commercial value.
