**3. Honey as probiotic source**

The great majority of the dry weight of honey (95–98%) consists of carbohydrates, mainly glucose and fructose, but also sucrose, maltose and other oligosaccharides. A minor portion (2–5%) is made up of various secondary metabolites, such as polyphenols and flavonoids, minerals, proteins, amino acids, enzymes, organic acids, minerals, vitamins, fatty acids, pol‐ len and other solid particles from the process of obtaining honey [1, 2]. It also contains traces

Prebiotics are substances that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, the probiotic bacteria. Honey is often used as a sweetener but its use in medical preparations date from ancient cultures [4, 5]. Such functional properties includes antibacterial, antioxidant, antitumor, anti‐inflammatory, antibrowning and antiviral [6, 7]. More recently, it was also found to be

Antioxidant activity is defined as the capability of a compound to protect an organism from oxidant attack. Two widely used methods to verify this capability are the diphenylpicrylhy‐ drazyl (DPPH) and the 2,2′‐azinobis (3‐ ethylbenzthiazoline‐6‐sulfonic acid) (ABTS) assays. Both of them share the same mechanism of the reduction of the stable free radical but not measure the effect of an antioxidant on cell survival [9]. The biological yeast‐based method can also measure the ability of a compound to induce cellular resistance to the damaging

This chapter describes the main bioactive components of honey, with emphasis on phenolic

Honeybees exist before human inhabits the Earth. It is formed due to action of honey‐ bee's enzymes (diastase, invertase and glucose oxidase) on nectar or secretions of flowers. Honey is composed of various sugars, mainly glucose and fructose, but also sucrose, maltose and other oligosaccharides. In addition, honey contains proteins, amino acids, enzymes, organic acids, minerals and pollen. Besides, it can also contains traces of fungi, algae, yeasts and other solid particles from the process of obtaining honey [12] and lactic

Overall, honey contains acids, such as gluconic, succinic, malic, acetic, citric and butyric acid. Gluconic acid is found in greater amounts and is produced by action of glucose oxi‐ dase enzyme on the glucose to produce gluconic acid and hydrogen peroxide. Eighteen free amino acids occur in honey. Proline is the most abundant. Honey has small amount of vitamins that are negligible in the nutritional point of view. Therein includes ascorbic acid, niacin, pantothenic acid, riboflavin and thiamine. The minerals found in honey are potassium, sodium, calcium, magnesium, chlorine, iron, copper, manganese, phosphorus, sulfur and silica. Its content level of minerals is very variable and depends on the nectar source. Besides honey contains small amount of vegetable substances that contribute to the

of fungi, algae, yeasts and lactic acid bacteria (LAB) [3].

prebiotic and even a source of probiotic microorganisms [8, 9].

compounds, antioxidant activity and assay methods.

effects of oxidants [10, 11].

288 Honey Analysis

**2. Honeybee composition**

acid bacteria (LAB) [3].

aroma and taste.

Probiotic was originally defined by Parker [13] as "organisms and substances which con‐ tribute to intestinal balance." Later, Fuller [14] redefined as "viable microbial supplement which beneficially affects the host by improving the intestinal microbial balance, having specific effect in preventing pathological condition." Fuller's definition showed the need for the viability of probiotics in the food matrices and after passing the gastrointestinal tract. Probiotic definition has been expanded, not restricting to the health effects on the indigenous microbiota. According to Schaafsma [15], "oral Probiotics are microorganisms which upon ingestion in certain numbers, exert health effects beyond the inherent basic food nutrition."

The honey relationship with probiotic microorganisms is already in the generation of hon‐ eybees, when honeybees to be fed with honey over the 21 days of generation are stimulated immunologically due to probiotics contained in honey [16].

For a long time, researchers believed that the source of lactic acid bacteria in the honey was pollen and secretions of flowers that arrived to honey transported by honeybees. However, later studies proved that the lactic acid bacteria are present in the stomach of the honeybees; therefore, it is a source of lactic acid bacteria. The colonization mechanism is not fully clari‐ fied yet [8].

In the honey production process, the enzyme glucose oxidase is responsible for the trans‐ formation of the glucose in galacturonic acid. This causes the natural acidification of honey and therefore its preservation. Then, the majority of pathogenic and spoilage microorganisms are inhibited [12]. Due to honey acidity, yeasts and lactic acid bacteria are the predominant microorganisms. Among the lactic acid bacteria, there are probiotic microorganism, espe‐ cially those belonging to *Lactobacillus* and *Bifidobacterium* genus.

Within the most isolated species of *Lactobacilli* genus are those belonging to the species *L. apis, L. insects, L. alvei, L. plantarum, L. pentosus, L. parabuchneri, L. kunkeei, L. kefiri* [17], and *Lactobacillus acidophilus*. Among *Bifidobacterium* genus, novel species were identified, *B. asteroids* and *B. coryneform* [8]. LAB symbionts within honeybees are responsible for many of the antibacterial and therapeutic properties of honey [3].

Olofsson et al. [3] reported that 13 lactic acid bacteria symbionts from the honey stomach of honeybees (*Apis mellifera*) were also found in large concentrations in fresh honey as well as having a wide spectrum of antimicrobial activity against various honeybee pathogens and bacteria and yeasts from flowers. According to these authors, many of the unknown healing and antimicrobial properties of honey are linked with these LAB symbionts. Every single member of the LAB microbiota of honeybees produces different bioactive metabo‐ lites. Organic acids were produced by all tested strains but in different amounts. Lactic, formic and acetic acids were produced as well as a wide variety of other interesting metab‐ olites such as benzene and 2‐heptanone and also putative lactic acid bacteria proteins in different honey types, suggesting their importance in honey production and antimicrobial activity.
