Averages of the Lactobacilli and Bifidobacteria log numbers, in babies born by cesarean section and vaginally delivery, fed with pasteurized milk from human milk banks (HMB), formula (FM) and breast milk (BM).
The fact that living organisms play a key role on health, was put on a scientific basis at the beginning of the last century by Elie Metchinikoff, when working at the Pasteur Institute in Paris. The findings that Bulgarian peasants, who ingested large amounts of soured milks, also lived to a ripe old age led him to conclude about the beneficial effects of fermented milks.
One of the most convincing demonstrations of the role of the gut microbiota in resistance to disease was provided by Collins and Carter . These authors proved that germ-free guinea–pig was killed by 10 cells of
Probiotic was initially defined by Parker  as “Organisms and substances which contributes to intestinal microbial balance”. Fuller  redefined probiotics as “A live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance”. This definition clarifies the need for a probiotic to be viable.
The term prebiotic was subsequently adopted to define “non-digestible food ingredients that beneficially affect the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon that improve host health” Modification by prebiotics of the composition of the colonic microbiota leads to the predominance of a few of the potentially health-promoting bacteria, especially, but not exclusively, lactobacilli and bifidobacteria. Much of the work on prebiotics deals with the use of oligosaccharides, although the first demonstration of this type of effect was observed with a disaccharide, lactulose. Gibson and Roberfroid  also launched the concept of symbiotic by combining the rationale of pro- and prebiotics, is proposed to characterize some colonic foods with interesting nutritional properties that make these compounds candidates for classification as health-enhancing functional food ingredients.
The bacterial genera most often used as probiotics are lactobacilli and bifidobacteria. At present, probiotics are almost exclusively consumed as fermented dairy products such as yogurt or freeze-dried cultures, but in the future they may also be found in fermented vegetables and meats .
The microbial community inhabiting the gastrointestinal tract is characterized by its high population density, wide diversity, and complexity of interactions. Bacteria are predominant but a variety of protozoans, yeasts and bacteriophages are also found. Bacteria are not distributed randomly throughout the gastrointestinal tract but instead are found at population levels and species distributions that are characteristic of specific regions of the tract. The stomach and proximal small intestine contain relatively low numbers of microorganisms. Acid- tolerant lactobacilli and streptocococci predominate in the upper smal intestine. The distal small intestine (ileum) maintains a more diverse microbiota and higher bacterial numbers. The large intestine (colon) is characterized by large numbers of bacteria, low redox potential, and relatively high short-chain fatty acid concentrations. The prominent role played by anaerobic bacteria in this dynamic ecosystem is evident from the finding that more than 99% of the bacteria isolated from human fecal specimens are anaerobic or aerotolerant .
The intestinal tract is a dynamic ecosystem that is influenced by host, intrinsic, and environmental factors. Thus, our undestanding of gut microbial interactions and how the gastrointestinal activity is modulated, might help on establishing screening criteria to identify potentially probiotic bacteria suitable for human or animal use.
2. Microbial interactions in the gut
The nature of the microbial interaction can be predominantly by competition or mutualism . In the gut they can affect either the population level of a given strain or the metabolic activity of that strain. In addition, genetic transfers can occur between strains within the gut. The host and the diet cam modulate the expression of the microbial interactions. These interactions involve multiple mechanisms that are poorly understood. Such mechanisms are involved either in the size of subdominant microbial populations or in the metabolic activities of predominant populations. Diet and perhaps other environmental factors, such as stress, can modify their expression.
The gastrointestinal tract of neonates becomes colonized immediately after birth with environmental microorganisms, mainly from the mother by several processes including sucking, kissing, and caressing. The proximity of the birth canal and the anus, as well as parental expression of neonatal care, are effective methods of ensuring transmission of microbes from one generation to the next .The pattern and level of exposure during the neonatal period is likely to influence the microbial succession and colonization in the gastrointestinal tract. Infants from developing countries have an early colonization with enterobacteria whereas those born in countries with good obstetric and hygienic procedures, may result in a delayed development pattern or even the absence of certain groups of intestinal bacteria during succession .
After the birth process, neonates are continuously exposed to new microbes that enter the gastrointestinal tract with food. This begins with breast milk, which contains up to 106 microbes/mL in healthy mothers. The most frequently encountered bacterial groups include staphylococci, streptococci, corynebacteria, lactobacilli, micrococci, propionibacteria and bifidobacteria originated from the nipple and surrounding skin as well as the milk ducts in the breast [6, 9, 10].
A pronounced dominance of bifidobacteria was observed over the entire breast-feeding period, with a corresponding reduction in facultative bacteria [11, 12]. There is a strong evidence suggests that the early composition of the microbiota of neonates plays an important role for the postnatal development of the immune system [13, 14].
Both adults and neonates are regularly exposed to microorganisms via the diet, but are affected differently. The microorganisms entering newborns via milk are more likely to colonize than are those entering healthy adults [6, 15].
Bacterial species or strains that will be established in the infant bowel might be capable to utilize the substrates provided by the diet and the particular human host.
Protection against colonization of the intestinal tract by potentially pathogenic microorganisms, due to the gut microbiota, was called competitive exclusion , whose pioneering evidence had been obtained by Nurmi and Rantala , with birds. When these, soon after birth, were inoculated with cecal material of an adult bird, the frequency of Salmonella infections was significantly reduced.
Undoubtedly the main benefit attributed to probiotics is the competitive exclusion of pathogens that occurs by different mechanisms including: a) competition for receptors in the intestinal epithelium as occurs with lactobacilli that directly inhibits the binding of
Constituents of the normal microbiota and some pathogenic bacteria have the ability to colonize the mucosal surfaces  Some microorganisms seem to be able to securely attach to the intestinal epithelium , and is thought to be this an important prerequisite for probiotics in a long-term survival during competition against other microorganisms for specific niches and subsequent multiplication. However, no consensus among researchers exists about the fact that a probiotic should or should not adhere to mucosal surfaces, colonize and then exert a probiotic effect, being an alternative its regular consumption to maintain the levels needed to promote the effect, forming a transient microbiota .
Another desired effect of a probiotic includes altered metabolism of the intestinal microbiota as the reduction in the synthesis of toxins or carcinogenic substances or an increased production of short-chain fatty acids or other substances that improve the condition of the mucosa. Prebiotics may also be given to augment immune reaction, preferably those that have a protective effect without causing overt inflammation. The ability of lactic bacteria to inactivate mutagenic compounds, such as dyes and N-nitrosamines, has been attributed to cell wall components, such as peptidoglycan and polysaccharides .. The lactic acid bacteria also may mediate anticarcinogenic activities by reducing the activity of fecal bacterial enzymes such as nitroredutases, azoredutases and glucuronidase (EC 184.108.40.206) that convert procarcinogenic to carcinogenic compounds in the colon 
The ability to sense other bacteria may have important consequences for competitive and nutritional strategies controlling for example, entry into stationary phase, dispersal and the production of antimicrobial compounds. The ability to interfere with the signalling of bacteria will determine the fitness of the given organism to survive in the gut and may also have therapeutic potential. The study of cell-to-cell communication in gastrointestinal(GI) tract bacteria is not as advanced as it is for bacteria from other ecosystems. In Gram-negative bacteria the best-characterized systems involve
Probiotics may play an active role inflammatory bowel diseases by enhancing the intestinal barrier at the mucosal surface. Caballero-Franco et al.  investigated whether the clinically tested VSL#3 probiotic formula and/or its secreted components could augment the protective mucus layer in vivo and in vitro. For in vivo studies, Wistar rats were orally administered the probiotic mixture VSL#3 on a daily basis for seven days. After treatment, basal luminal mucin content increased by 60%. In contrast to the animal studies, cultured cells incubated with VSL#3 bacteria did not exhibit increased mucin secretion. However, the bacterial secreted products contained in the conditioned media stimulated a remarkable mucin secretion effect. Among the three bacterial groups (
The competitive exclusion of pathogens mediated by lactobacilli is usually performed by two mechanisms: (i) production of antimicrobial substances such as lactic acid and bacteriocins, and (ii) adhesion to the mucosa and coaggregation which can form a barrier which prevents colonization by pathogenic microorganisms .
Three mechanisms of aggregation have been reported so far. The first is related to the interaction between the components of the cell surface, as in the oral cavity with
Autoaggregation has been correlated with adhesion, which is known to be a prerequisite for colonization and infection of the gastrointestinal tract by many pathogens. Adherence to the epithelium is therefore a prerequisite for enterotoxigenic
Coaggregation is a process by which genetically distinct bacteria become attached to one another via specific molecules. Cumulative evidence suggests that such adhesion influences the development of complex multi-species biofilms. The coaggregation properties of probiotic strains with pathogens as well as their ability to displace pathogens are of importance for therapeutic manipulation of the aberrant intestinal microbiota. Aggregation abilities of a probiotic with the pathogen strains were strain-specific and dependent on time and incubation conditions 
Recently, the complement protein mannose-binding lectin (MBL) has been shown to play a role in the first line of defense against
In other cases, the adhesins are not lectins, such as in the case of
The second mechanism, described in lactobacilli, is dependent upon secretion of a protein of 32 kDa that promotes aggregation and a high frequency of conjugation  According to Collado, Meriluoto and Salminen  the ability to autoaggregate, together with cell-surface hydrophobicity and coaggregation abilities with pathogen strains can be used for preliminary screening in order to identify potentially probiotic bacteria suitable for human or animal use.Finally, in
3. Bioactive prebiotic components in milk
Many components of human milk are multifunctional, providing antimicrobial, antiinflammatory, antioxidant effect besides being growth factors .
Breast milk not only provides a range of substrates for bacterial growth, but it also appears to be a reservoir for some of the bacteria we inherit, including Lactobacillus sp. and
Although it is likely that antimicrobial components in human milk inhibit the growth of pathogenic bacteria, it is also likely that some substances stimulate the growth of beneficial bacteria,
The gut microbiota of breastfed infants is different from that of formula-fed infants. According to Penders , exclusively formula-fed infants were more often colonized with
Oliveira  studied the influence of diet and type of delivery in 68 neonates aged between seven and 21 days on both composition and evolution of the gut
Oliveira  also found that bifidobacteria numbers in infants born vaginally and fed with breast milk (BM) were higher than the others, while those who received pasteurized human milk from milk banks (HMB) showed a significant lower number of
The same negative pasteurization effect was observed by Oliveira  on the growth of lactobacilli (Table 1). Although breast-milk contains viable lactobacilli and bifidobacteria that might contribute to the initial establishment of the microbiota in the newborn, the negative effect of human milk pasteurization on the lactobacilli and bifidobacteria gut population, cannot be explained solely on the destruction of those bacteria by the pasteurization process. Milk formulas do not contain these bacteria, but favored the development of bifidobacteria and lactobacilli in the intestine reaching a number significantly higher, as compared to the gut microbiota of pasteurized human milk fed infants.
Indeed, the health-promoting effects of breast-milk have been linked partly to the presence of lactobacilli and bifidobacteria in breast-milk [10, 47], but clearly also to different milk bifidogenic components.
Both lactotobacilli and bifidobacteria benefit in environments with low redox potential and the presence of antioxidant compounds present in human milk. Anti-oxidants such as lactoferrin, α-tocopherol, β carotene, cysteine, ascorbic acid, uric acid, catalase and glutathione peroxidase are present in human milk . Most of these compounds are thermo-labile and might have been destroyed during milk pasteurization process. Whey protein is rich in
|HMB||2,4 a A||3,3 b A|
|FM||2,8 a B||5,7 a A|
|BM||3,8 a B||5,6 a A|
|HMB||5,6 a A||3,7 b A|
|FM||5,7 a A||6,5 ab A|
|BM||6,2 a A||7,4 a A|
3.1. Milk oligosaccharides
For many years, the oligosaccharides were considered for his role in the modulation of intestinal microbiota of infants. Currently, there is strong evidence that free oligosaccharides as well as glycoproteins are potent inhibitors of bacterial adhesion on the surface of the epithelium in the early stages of the infectious process. Therefore, the milk oligosaccharides have two important functions. The first as a source prebiotic stimulating the growth of probiotic bacteria and a second, operating in a non-specific defense mechanism inhibiting pathogens from adhering to the gastrointestinal mucosa. Although the exact pathophysiological mechanism of diarrhea is not yet fully elucidated, it seems that the ability of microorganisms to adhere to the mucosal surface is essential for spreading diarrheagenic bacteria in the duodenum .
Concentrations of total oligosaccharides in human milk (HMO) is 5,0-8,0 g per liter whereas just traces are found in cow’s milk. In cow’s milk, only small amounts of oligosaccharides are detectable, with sialyllactose being the major component .
Differences in the qualitative or quantitative aspects of term and preterm milk have not been observed, but compositional changes of oligosaccharides in term milk occurs during lactation with the largest amounts being found at early stages. The highest concentrations of HMOs can be found in colostrum (20 g/L), but even mature milk contains oligosaccharides in concentrations up to 13 g/L . Coppa  reported that lactose concentration (±SD) in human milk increased from 56 ± 6.06 g/L on day 4 to 68.9 ± 8.16 g/L on day 120. Oligosaccharide level decreased from 20.9 ± 4.81 g/L to 12.9 ± 3.30 gIL, respectively. Monosaccharides represented only 1.2% of total carbohydrates.
Although intact HMOs may be absorbed, ENGFER et al.  postulate that a majority of HOs reach the large intestine, where they serve as substrates for bacterial metabolism. Therefore, HMOs might be considered the soluble fiber fraction of human milk
Human milk compared with other milk species, is considered unique in terms of its complex oligosaccharides content. With few exceptions, HMOs have a core structure consisting of a lactose unit at the reducing end linked to
Within human milk oligosaccharides at least 10 containing GlcNAc are known as growth factors for a so-called bifidus biota in breastfed infants. Dietary modulation of the intestinal microflora is today one of the main topics of interest in the nutritional sciences. Fructo-oligosaccharides (FOS) and galacto-oligosaccharides (GOS) are prebiotics whose bifidogenic activity has been proven in adults. Moro and Arslanoglu  demonstrated that supplementation of infant formulas with a mixture of GOS and FOS modified the fecal flora of term and preterm infants, stimulating the growth of Bifidobacteria. In the trial with term infants, the bifidogenic effect of the prebiotic mixture was dose dependent and there was also a significant increase in the number of Lactobacilli in the supplemented group.
The similarities between epithelial cell surface carbohydrates and oligosaccharides in human milk strengthen the idea that specific interactions of those oligosaccharides with pathogenic microorganisms do occur preventing the attachment of microbes to epithelial cells. HMOs may act as soluble receptors for different pathogens, thus increasing the resistance of breast-fed infants. Some of the best-characterized adhesins of bacteria are those of
Rockova et al.  reported that two strains of
3.2. Milk proteins
Whey proteins constitute about 60-80% of the total protein content of human milk, but only 18% of bovine milk. Furthermore, the composition of whey proteins is different for each of the milks: beta-lactoglobulin, that is not found in human milk, predominates in bovine milk, while alfalactalbumin and lactoferrin predominate in human milk. The alfalactalbumin is necessary for the synthesis of lactose in the mammary gland, through the action of the lactose synthetase enzyme, their concentration in human milk ranges from 0.22 to 0.46 g/dl. The betalactoglobulin has been blamed for allergies to bovine milk .
Undenatured whey protein is rich in
Whey proteins present in human milk, such as secretory IgA, lactoferrin and lysozyme are very stable in acid medium, and reasonably resistant the action of proteolytic enzymes, it is believed, therefore, that over three quarters of these proteins appear intact in the feces of infants. Approximately 6-10% of lactoferrin is not digested by the intestinal tract, assuming that it can reach the colon and play prebiotic activities 
Lactoferrin, a glyco-protein, is a major protein in human milk (1.3-2.8 g/L) while it is present only in traces in cow´s milk. Lactoferrin inhibits the growth of bacteria and fungi due to its ability to bind iron, a function known as
In addition, lactoferrin also promotes the growth of beneficial bacteria such as
Lysozyme is an antimicrobial enzyme (EC 220.127.116.11) found in tears, saliva, human milk whey, mucus, neutrophil granules and egg- white. It hydrolyses b (1,4) linkage between N acetylglucosamine and N-acetylmuramic acid in bacterial cell wall. Gram positive bacteria are more susceptible to lysozyme than Gram negative. The enzyme synergistically interacts with other immunoprotective components like IgA, C3 complement components and lactoferin. Human milk contains up to 400 mg/mL of lysozyme, which is a concentration approx. 3000 times higher than in bovine milk.
Resistance to lysozyme and the ability to utilize human milk oligosaccharides (HMOs) were identified as the most important factors affecting the growth of bifidobacteria in human milk. Four out of 5 strains of human origin were resistant to lysozyme and utilized HMOs. In contrast,
According to Rockova et al.  the lysozyme-resistant
Lactoperoxidase makes up approximately 0.5% of the whey protein. In the presence of hydrogen peroxide (formed in small quantities by cells), catalyzes the oxidation of thiocyanate (part of saliva), forming hypothiocyanate, which can kill both gram-positive and gram-negative bacteria. Thus, lactoperoxidase in human milk may contribute to the defense against infection already in the mouth and upper gastrointestinal tract. Human milk contains active lactoperoxidase, but its physiologic significance is not yet known.
3.2.4. κ-Casein and glycomacropeptide
κ-Casein, a minor casein subunit in human milk, is a glycoprotein with charged sialic acid residues. The heavily glycosylated k-casein molecule has been shown to inhibit the adhesion of
Glycomacropeptide is resultant from the tryptic hydrolysis of human k-casein, containing sugars glucosamine and galactosamine. The molecular weight of intact human
Glycomacropeptide helps control appetite and inhibit the formation of dental plaque and dental cavities. It is a growth factor for bifidobacteria (bifidogenic factor 1) Levels of glycomacropeptide may range from 1% to 18% 
3.3. Milk fat
The main fatty acids present in human milk are restricted to those with 12-18 carbon atoms chains,namely lauric, myristic, palmitic, palmitoleic, stearic, oleic, linoleic and linolenic. Some of the long chain polyunsaturated acids such as arachidonic and others are derived from essential fatty acids linoleic and linolenic acids, totaling together with their precursors, about 15% of fat of human milk. This percentage is much higher than that found in bovine milk. Palmitic, oleic and linoleic add up together about 70% of total fatty acids of colostrum and 74% of that of mature milk 
Corcoran et al.  studied the effect of inclusion of various C18 fatty acids with 0–2 double bonds in either
Rosberg-Cody et al.  isolate different strains of the genus
4. Bioactive prebiotic components in honey
Most of the honey in the world is produced by bees from the nectar. Nectar is a sugar solution and water, may contain pure sucrose, a mixture of sucrose, glucose and fructose, or glucose and fructose only. The nectar is transported to the combs of the hive, where they will undergo physical and chemical changes responsible for their maturation (Crane, 1983). The chemical composition of honey, as well as aroma, color and medicinal properties, are directly related to the nectar source that originated with the bee species that produced it, with their geographic and climatic conditions. All these factors contribute to the wide variation found in honey .
Shin and Ustunol  defines honey as natural syrup containing mainly fructose (38.5%) and glucose (31.3%). Other sugars in honey include maltose (7.2%), sucrose (1,5%) and a variety of oligosaccharides (4.2%). In addition to the complex mixture of carbohydrates, are enzymes, minerals, pigments, waxes and pollen. More than one hundred eighty substances have been found in different honey types.
Honey is a complex product of easy digestion and assimilation, constituting a source of energy that contributes to the balance of biological processes in that it contains suitable proportions, enzymes, vitamins, fatty acids, amino acids, phenolic and aromatic substances . In addition contains oligosaccharides which stimulates the growth of probiotic bacteria in the gut [65, 66].
Leite et al. , found in various di-and trisaccharides in Brazilian honeys. Maltose showed up in higher levels in honeys surveyed followed by other five disaccharides, turanose, nigerose, melibiose, sucrose, isomaltose and four trisaccharides, maltotriose, panose, melezitose and raffinose..
Cellobiose, gentiobiose, isomaltose, kojibiose, laminaribiose, maltose, maltulose, melibiose, nigerose, palatinose, trehalose, trehalulose, turanose, and sucrose are the main disaccharides found in honey [66, 67]. However, it would be rather difficult to identify the predominant disaccharide or certain combinations in the previously studied honey types. For example, maltulose and turanose were found in many honey samples, however their concentrations varied to a wide extent. Thus, Sanz and others  found the highest amounts of maltulose and turanose (0.66 to 3.52 and 0.72 to 2.87 g/100 g of honey, respectively) in 10 samples of honey from different regions of Spain and commercially available nectar and honeydew honeys.
Carbohydrate degradation has been extensively studied in a variety of different
Pokusaeva et al.  describe the identification of two genes,
Proline is the main amino acid present in honey; it is added by the bee and its amount varies depending on the floral source..
Macedo et al.  studied the effect of the
It is well stablished the role of several oligosaccharides as prebiotic substances. The prebiotic effect of human milk, however, is not related to a single growth-promoting substance, but rather to a complex of interacting factors. In particular the prebiotic effect has been ascribed to several oligosaccharides, that is clearly proved. The role and the way milk fat and proteins such as lactoferrin, lysozyme stimulate the growth of probiotic bacteria is not yet clearly defined.