**1.1. Sampling plan for the microbiological analysis of honey lots**

**1. Introduction**

260 Honey Analysis

Standardization [12, 13].

CFU/g, and can even reach 10<sup>3</sup>

instruction no

–10<sup>4</sup>

and Quality of Honey [18] and normative instruction no

Most of the analysis techniques described in this chapter were recommended by the Agência Nacional de Vigilância Sanitária (the National Health Surveillance Agency) (ANVISA) [1], including the Official Analytical Methods for the Microbiological Analysis and Control of Products from Animal and Water Sources [2], those of the American Public Health Association; described in the fourth edition of the Compendium of Methods for the Microbiological Examination of Foods [3–5], the International Commission on Microbiological Specifications for Foods [6, 7]; the Food and Drug Administration (FDA), recommended by the Ministry of Agriculture for the analysis of foods of animal origin, in accordance with Normative Instruction number 62, dated August 26th, 2003 [2]; the Food Safety and Inspection Service of the US Department of Agriculture [8], the Association of the Official Methods of Analysis of AOAC International [9], the Bacteriological Analytical Manual (FDA) [10], the Microbiology Laboratory Guidebook [11] and the latest editions of the International Organization for

Among the various parameters that indicate the quality and safety of honey, the most important are those that define its microbiological characteristics. Honey, as with any other raw material of vegetable or animal origin, naturally presents microbial contaminants of commercial importance formed by microorganisms adapted to the characteristics of the honey, such as high-sugar content, low acidity and the presence of natural antimicrobial substances. Because of these characteristics, the microbial load in honey is generally low, below 10<sup>2</sup>

reducing the shelf-life of the product. It presents floral indicators of the possible presence of pathogenic microorganisms, and so can be harmful to the health of the consumer. Protecting food products from any kind of contamination or adulteration which can cause harm to public health or economic disorder is a global concern [8] and specific methods of analysis are required to evaluate this type of raw material. Moreover, the risks represented by the poor handling conditions used by workers responsible for the harvest, extraction and preparation of this product require effective interventions and procedures to minimize these risks [14].

Aiming to control the quality of honey, the World Trade Organization recommends the adoption of standards, guidelines and norms developed by Codex Alimentarius—the revised codex standard for honey 2001 [15]. This is an international public agency created by the Food and Agricultural Organization (FAO) and the World Health Organization (WHO) [16], both of which form part of the United Nations Organization (UNO). MERCOSUL 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], in which honey can contain a maximum of 100 colony forming units of fungus per gram (CFU/g). Normative

the Technical Regulations for the Identity and Quality of bee apitoxin, beeswax, royal jelly, lyophilized royal jelly, bee pollen, propolis and propolis extract [19], as previously microbiological standards had not been established for these apiculture products. To ensure the

11 approved, on 20 October 2000, the Technical Regulations for the Identity

CFU/g. Consequently, it can cause undesirable changes by

3 dated January 19th 2001 approved

Sampling plans allow the evaluation of the microbiological conditions of honey lots. These were proposed by the International Commission on Microbiological Specifications for Foods [6] and their application supports the acceptance or rejection of a honey lot as a whole, describing the hygienic sanitary conditions under which this food was obtained, processed, stored, distributed for consumption, as well as its shelf life and the risk posed to consumer health.

For the microbiological analysis of a honey lot, it is necessary to define some important concepts, such as: lot, *n, c, m* and *M*. A 'lot' is the total units of honey pots produced, handled or stored under the same conditions, within a certain period; 'n' is the number of units taken randomly from a lot to be analysed individually. For honey, '*n*' is equal to five sample units and constitutes a representative sample of the lot; '*m*' is the set of microbiological standards established for a microorganism in a given food; '*c*' is the maximum acceptable number of units in which microbial counts in the lot are above the minimum threshold (*m*) and below the maximum tolerated limit (*M*); ' *M*' is the tolerable limit, above standard, which can be reached by (*c*) sample units, but cannot be exceeded by either [6].

Brazilian legislation on the microbiological requirements of food includes Ordinance n<sup>o</sup> 101 of 1993 of the Ministry of Agriculture, Livestock and Supply and RDC-12 Resolution 2001 of the National Health Surveillance Agency of the Ministry of Health [1]. In the case of honey (molasses and similar) a value of *n* = 5 is adopted, while values of *c, m* and vary according to the microorganism considered: coliforms at 45°C/g (*n* = 5, *c* = 2, *m* = 10 and *M* = 10<sup>2</sup> ) and *Salmonella sp*/25 g (*n*=5 *c* = 0; *m* = absent) under this legislation are more flexible than the levels established by Mercosul [17] in which honey must meet the following microbiological characteristics: Coliforms at 35°C/g (*n* = 5, *c* = 0, *m* = 0); *Salmonella* spp - *Shigella* spp/25 g (*n* = 10, *c* = 0, *m* = 0); Fungi and Yeast CFU/g (*n* = 5, *c* = 2 *m*=10, *M* = 100). Therefore, a maximum of 100 colony forming units of fungus per gram of honey (CFU/g) is acceptable.

For the analysis of *Salmonella sp*/25 g, a two-class plan is applied, as this trial investigates the presence or absence of this microorganism. In this case, '*c*' is equal to zero, absence is acceptable and the presence of any sample unit is unacceptable. In these tests a single sample analysis is performed. For analysis of coliform 45°C, a three-class plan is applied, which classifies lots into three categories: acceptable, intermediate and unacceptable. In this case, the standard is not absence but values within a range between *m* and *M*. In the two-class plan, *M* separates acceptable from unacceptable lots. In a three-class plan this value separates an acceptable lot from an intermediate lot [10, 11].
