**3. Beneficial microorganisms in honey for humans**

Human metabolism is dependent of symbiotic microorganisms, known as the indigenous microflora capable of favoring the production and absorption of essential nutrients to our body such as K and B12 vitamins, pentatonic acid, pyridoxine, and biotin, and acts by modulating the immune system [26]. This microbiota lives in the gut, due to high acidity of the stomach (pH 1.5); the most microorganisms are unable to grow, while in the gut we can found a lot of microorganisms with 500–600 different species [26]. There is no oxygen in gut; for this reason, the gut bacteria are aero-tolerant and facultative anaerobic. We can find bacteria of genus *Actinomyces*, *Bacteroides*, *Clostridium*, *Enterobacter*, *Enterococcus*, *Escherichia*, *Klebsiella*, *Lactobacillus*, *Proteus*, *Pseudomonas*, *Staphylococcus*, and *Streptococcus*; many of them are opportunistic pathogens when move to other parts of the body [26].

For honey production, honeybees ingest nectar and turn this with help of enzymes. Beyond the enzymes, they incorporate some symbiont microorganisms associated with gastrointestinal tract that can bring benefit to human health [27]. The natural human microbiota is stable; so it is necessary for daily intake of the new symbiont to be able to populate the human body and maintain its benefits [28]. These microorganisms are known as probiotics and, when they grow in human gut, can make nourishment benefits, like fermentation, and broke nutrients facilitating absorption of short-chain fatty acids, ions, amino acids, and vitamins; protective effect, preventing invasion of pathogenic microorganisms; and trophic effect in the gut epithelium and in the system [28].

Bacterium *Gluconobacter oxydans* was isolated from honey harvested directly from beehive. Also, *Pseudomonas* spp. and *Bacillus* spp. were found [29, 30]. However, *G*. *oxydans* is highlighted because they showed 100% of survival in pH of 5.0 and 50% of survival after 3 h of contact in pH of 2.0 and showed resistance in 2% of bile salts. This is atypical behavior for bacteria, because normally they have low resistance in acidic environments. For this way, a bacterium resistant to condition of the stomach is promising to arrive in the gut, where it will grow and will make benefit [29]. These bacteria can assimilate cholesterol reducing absorption of this component by the body, and it can be used as probiotics in food [29]. In addition to this, as honey is rich in fructose, some bacteria that live in there possess the ability to degrade fructose more easily; these bacteria are known as fructophilic lactic acid that prefer to metabolize fructose and not glucose as normally is observed. In the gut, these bacteria produce bacteriocins that act as a barrier to other microorganisms and contribute to the immune system. *Lactobacillus kunkeei*, fructophilic lactic acid bacteria, were found in the stomach of honeybees, as well as in their hives [27].

negative showed positive with molecular test [21, 22]. This microorganism enters the beehive through the contaminated water or even by contact of product with ground. This organism does not cause damage to honeybees, but it is responsible for the development of botulism in humans, especially in children or people with weakened immune systems and can lead to

Genus *Bacillus* comprises rod-shaped Gram-positive bacteria with the ability to form spores. There are 60 species of huge genetic diversity, and most of them are nonpathogenic; the pathogenicity associated with others is in opportunistic form. These pathogens belong to group *Bacillus cereus*, a subgroup *Bacillus subtilis*; however, *Bacillus licheniformis*, *Bacillus pumilus*, and *Bacillus majavensis* can cause poisoning by food too [24]. *Bacillus cereus* is an important pathogen in honey; it is an enterotoxin producer in pH 6.0–8.0 and tem-

Researchers isolated some bacteria in honey samples of different geographical and botanical origins. "They found *B*. *pumilus* (ML374), *B*. *licheniformis* (ML103A and ML104B), *B*. *amyloliquefaciens*, *B. subtilis*, *B*. *cereus*, *B. thuringiensis*, *B*. *licheniformis*, *B. megaterium*, and *B*. *pumilus* [13]." The bacteria of species *B*. *cereus* are enterotoxin producers; the others of *Bacillus* species are considered safe. Due to their ability of producing bacteriocins, they are promising in the study of new anti-

Human metabolism is dependent of symbiotic microorganisms, known as the indigenous microflora capable of favoring the production and absorption of essential nutrients to our body such as K and B12 vitamins, pentatonic acid, pyridoxine, and biotin, and acts by modulating the immune system [26]. This microbiota lives in the gut, due to high acidity of the stomach (pH 1.5); the most microorganisms are unable to grow, while in the gut we can found a lot of microorganisms with 500–600 different species [26]. There is no oxygen in gut; for this reason, the gut bacteria are aero-tolerant and facultative anaerobic. We can find bacteria of genus *Actinomyces*, *Bacteroides*, *Clostridium*, *Enterobacter*, *Enterococcus*, *Escherichia*, *Klebsiella*, *Lactobacillus*, *Proteus*, *Pseudomonas*, *Staphylococcus*, and *Streptococcus*; many of them are oppor-

For honey production, honeybees ingest nectar and turn this with help of enzymes. Beyond the enzymes, they incorporate some symbiont microorganisms associated with gastrointestinal tract that can bring benefit to human health [27]. The natural human microbiota is stable; so it is necessary for daily intake of the new symbiont to be able to populate the human body and maintain its benefits [28]. These microorganisms are known as probiotics and, when they grow in human gut, can make nourishment benefits, like fermentation, and broke nutrients facilitating absorption of short-chain fatty acids, ions, amino acids, and vitamins; protective effect, preventing invasion of pathogenic microorganisms; and trophic effect in the gut epi-

cells/mL to reach toxic

perature ranging from 6°C to 21°C, but it is necessary to ingest 107

**3. Beneficial microorganisms in honey for humans**

tunistic pathogens when move to other parts of the body [26].

thelium and in the system [28].

death [23].

236 Honey Analysis

effect [19].

microbial [25].

Besides these microorganisms is necessary consumption of substances that promote their development, known as prebiotics. These prebiotics are components, like oligosaccharides, that are not digested by humans, but they serve as a substrate for the growth and performance of probiotics [28, 31]. Currently, there is a great interest in combining probiotics with oligosaccharides acting like prebiotic. There are studies with probiotic *Lactobacillus* sp., which show that when they are grown in the presence of oligonucleotides, they show an increase in growth and antibacterial activity with production of bacteriocins [32].

The most-studied prebiotics are fructo-oligosaccharides, inulin, and oligofructose especially [33, 34]. However, there are others recognized as prebiotic, like galacto-oligosaccharides, trans-galactosylated oligosaccharide, isomalto-oligosaccharides, lactulose, pyrodextrin, and soy-oligosaccharides [28]. In honey we can find malto-oligosaccharides [35], specifically in Brazilian honey samples that were found in isomaltose, cellobiose, panose, maltotriose, melezitose, raffinose, maltose, turanose, and maltotriose, which are characterized as prebiotics [36].

In addition to probiotics, there are microorganisms associated with honey that can produce bacteriocins, which are substances able to reduce or eliminate competing microorganisms. These are peptides produced by bacteria producers of lactic acid, to reduce competition for nutrients, making inappropriate environment for development of other bacteria; for this reason, they are studied as an option for replacing antibiotics, and as usual these can cause harsh effects to humans also. Bacteriocins have high potency *in vivo* and *in vitro* and have low toxicity, and they can be produced in situ through consumption of probiotics or purified through bioengineering [37]. In 2013, a study was conducted with a new bacterium strain isolated from honey, able to produce bacteriocins fungicides called *Bacillus* BH072. These bacteriocins were tested and showed inhibitory character against *A*. *niger* CGMCC3.03928, *Fusarium oxysporum* CGMCC3.2830, *Pythium*, and *Botrytis cinerea* CGMCC3.4584 [25]. In another search, 13 lactic acid bacteria were isolated from honey and honeybees, and they were tested against bovine mastitis; they observed that the synergism between lactic acid bacteria and honey was able to inhibit growth of bacteria that cause mastitis, even those that were resistant to other antibiotics, and this is a promising preventive treatment to be studied [38].

Studies suggested that the antimicrobial character of honey is attributed to activity of these bacteria in honey; these are also present in the stomach of honeybees. *Lactobacillus* spp. were isolated from the stomach of honeybees and honey, they were then tested against *Escherichia coli* and *Salmonella enterica*, and they showed inhibitory effect. It is important to say that *Lactobacillus helsingborgensis* and *L*. *kunkeei* are the most candidate promisors like probiotic producers of bacteriocins [39]. Direct application of honey was also effective against *Serratia marcescens* and *Candida albicans* [40]. Beyond health benefits, discovery and application of microorganisms able to develop biotechnological products must be studied because they can improve lifestyle and human survival, becoming in this way beneficial microorganisms.

Besides the microbiota associated with honey, it is worth mentioning that this product alone is highly beneficial by features from its composition. This makes the honey effective activity like antimicrobial, antioxidant, anti-inflammatory, anticancer, antihyperlipidemic, cardioprotective properties, for ocular treatment, gastrointestinal tract disorders, neurological disorders and wound healing [1]. Honey has a series of phenolic acids like caffeic, ellagic, ferulic, and p-coumaric acids; flavonoids, such as apigenin, chrysin, galangin, hesperetin, kaempferol, pinocembrin, and quercetin; and antioxidants, such as tocopherols, ascorbic acid, superoxide dismutase, catalase, and reduced glutathione [41]. These compounds are known for their ability to reduce free radicals; this composition may vary depending on floral source that honeybees have visited for honey production [42]. Its antimicrobial activity makes it an important substance for the treatment of wounds as a result of carbon, lipids, amino acids, proteins, vitamins, and minerals active in healing. Components such as hydrogen peroxide, high osmolarity, acidity, non-peroxide factors, nitric oxide, and phenols are active in their healing effect. It also promotes growth of tissue in the human body, and it has anti-inflammatory activity [43]. However, it is important to note that honey directed to the treatment of wounds and inflammation should undergo irradiation treatment, so that microbiota will not interfere negatively on treatment [44].

Finally, it is important to note that consumption of foods able to bring health benefits, beyond nutrition, is a current practice that should be encouraged; honey is characterized as such, and it should be ingested daily.
