Effects of Probiotics, Prebiotics, and Synbiotics on Human Health

The human gastrointestinal tract is colonised by a complex ecosystem of microorganisms. Intestinal bacteria are not only commensal, but they also undergo a synbiotic co-evolution along with their host. Beneficial intestinal bacteria have numerous and important functions, e.g., they produce various nutrients for their host, prevent infections caused by intestinal pathogens, and modulate a normal immunological response. Therefore, modification of the intestinal microbiota in order to achieve, restore, and maintain favourable balance in the ecosystem, and the activity of microorganisms present in the gastrointestinal tract is necessary for the improved health condition of the host. The introduction of probiotics, prebiotics, or synbiotics into human diet is favourable for the intestinal microbiota. They may be consumed in the form of raw vegetables and fruit, fermented pickles, or dairy products. Another source may be pharmaceutical formulas and functional food. This paper provides a review of available information and summarises the current knowledge on the effects of probiotics, prebiotics, and synbiotics on human health. The mechanism of beneficial action of those substances is discussed, and verified study results proving their efficacy in human nutrition are presented.


Introduction
Nowadays, besides the basic role of nutrition consisting in the supply of necessary nutrients for growth and development of the organism, some additional aspects are becoming increasingly important, including the maintenance of health and counteracting diseases. In the world of highly processed food, particular attention is drawn to the composition and safety of consumed products. The quality of food is very important because of, i.e., the problem of food poisoning, obesity, allergy, cardiovascular diseases, and cancer-the plague of the 21st century. Scientific reports point to the health benefits of using probiotics and prebiotics in human nutrition. The word "probiotic" comes from Greek, and it means "for life". Most probably, it was Ferdinand Vergin who invented the term "probiotic" in 1954, in his article entitled "Anti-und Probiotika" comparing the harmful effects of antibiotics and other antibacterial agents on the intestinal microbiota with the beneficial effects ("probiotika") of some useful bacteria [1]. Some time after that, in 1965, Lilly and Stillwell described probiotics as microorganisms stimulating the growth of other microorganisms [2]. The definition of probiotics has been modified and changed many times. To emphasise their microbial origin, Fuller (1989) stated that probiotics must be viable microorganisms and must exert a beneficial effect on their host [3]. On the other hand, Guarner and Schaafsma (1998) indicated the necessary use of an appropriate dose of probiotic organisms required to achieve the expected effect [4]. The current definition, formulated in 2002 by FAO (Food and Agriculture Organization of the United Nations) and WHO (World Health Organization) working group experts, states that probiotics are "live strains Absence of genes responsible for antibiotic resistance localised in non-stable elements.

Functionality
• Competitiveness with respect to the microbiota inhabiting the intestinal ecosystem. • Ability to survive and maintain the metabolic activity, and to grow in the target site.

•
Resistance to bile salts and enzymes.

•
Resistance to low pH in the stomach.

•
Resistance to bacteriocins and acids produced by the endogenic intestinal microbiota.

•
Adherence and ability to colonise some particular sites within the host organism, and an appropriate survival rate in the gastrointestinal system.

Technological usability
• Easy production of high biomass amounts and high productivity of cultures. • Viability and stability of the desired properties of probiotic bacteria during the fixing process (freezing, freeze-drying), preparation, and distribution of probiotic products.

Probiotic Microorganisms
Probiotic products may contain one or more selected microbial strains. Human probiotic microorganisms belong mostly to the following geni: Lactobacillus, Bifidobacterium, and Lactococus, Streptococcus, Enterococcus. Moreover, strains of Gram-positive bacteria belonging to the genus Bacillus and some yeast strains belonging to the genus Saccharomyces are commonly used in probiotic products [21].
Probiotics are subject to regulations contained in the general food law, according to which they should be safe for human and animal health. In the USA, microorganisms used for consumption purposes should have the GRAS (Generally Regarded As Safe) status, regulated by the FDA (Food and Drug Administration). In Europe, EFSA introduced the term of QPS (Qualified Presumption of Safety). The QPS concept involves some additional criteria of the safety assessment of bacterial supplements, including the history of safe usage and absence of the risk of acquired resistance to antibiotics [22,23]. Table 2 presents probiotic microorganisms contained in pharmaceutical products and as food additives. Table 2. Probiotic microorganisms used in human nutrition [24][25][26].

Type Lactobacillus
Type Bifidobacterium

Mechanism of Action of Probiotics
A significant progress has been observed lately in the field of studies on probiotics, mostly in terms of the selection and characteristics of individual probiotic cultures, their possible use, and their effect on health.
Probiotics have numerous advantageous functions in human organisms. Their main advantage is the effect on the development of the microbiota inhabiting the organism in the way ensuring proper balance between pathogens and the bacteria that are necessary for a normal function of the organism [27,28]. Live microorganisms meeting the applicable criteria are used in the production of functional food and in the preservation of food products. Their positive effect is used for the restoration of natural microbiota after antibiotic therapy [29,30]. Another function is counteracting the activity of pathogenic intestinal microbiota, introduced from contaminated food and environment. Therefore, probiotics may effectively inhibit the development of pathogenic bacteria, such as Clostridium perfringens [31], Campylobacter jejuni [32], Salmonella Enteritidis [33], Escherichia coli [34], various species of Shigella [35], Staphylococcus [36], and Yersinia [37], thus preventing food poisoning. A positive effect of probiotics on digestion processes, treatment of food allergies [38,39], candidoses [40], and dental caries [41] has been confirmed. Probiotic microorganisms such as Lactobacillus plantarum [42], Lactobacillus reuteri [43], Bifidobacterium adolescentis, and Bifidobacterium pseudocatenulatum [44] are natural producers of B group vitamins (B1, B2, B3, B6, B8, B9, B12). They also increase the efficiency of the immunological system, enhance the absorption of vitamins and mineral compounds, and stimulate the generation of organic acids and amino acids [18,[45][46][47]. Probiotic microorganisms may also be able to produce enzymes, such as esterase, lipase, and co-enzymes A, Q, NAD, and NADP. Some products of probiotics' metabolism may also show antibiotic (acidophiline, bacitracin, lactacin), anti-cancerogenic, and immunosuppressive properties [45,[48][49][50].
Molecular and genetic studies allowed the determination of the basics of the beneficial effect of probiotics, involving four mechanisms: (1) Antagonism through the production of antimicrobial substances [51]; (2) Competition with pathogens for adhesion to the epithelium and for nutrients [52]; (3) Immunomodulation of the host [53]; (4) Inhibition of bacterial toxin production [54].
The first two mechanisms are directly associated with their effect on other microorganisms. Those mechanisms are important in prophylaxis and treatment of infections, and in the maintenance of balance of the host's intestinal microbiota. The ability of probiotic strains to co-aggregate, as one of their mechanisms of action, may lead to the formation of a protective barrier preventing pathogenic bacteria from the colonisation of the epithelium [27]. Probiotic bacteria may be able to adhere to epithelial cells, thus blocking pathogens. That mechanism exerts an important effect on the host's health condition. Moreover, the adhesion of probiotic microorganisms to epithelial cells may trigger a signalling cascade, leading to immunological modulation. Alternatively, the release of some soluble components may cause a direct or indirect (through epithelial cells) activation of immunological cells. This effect plays an important role in the prevention and treatment of contagious diseases, as well as in chronic inflammation of the alimentary tract or of a part thereof [28]. There are also suggestions of a possible role of probiotics in the elimination of cancer cells [55].
Results of in vitro studies indicate the role of low-molecular-weight substances produced by probiotic microorganisms (e.g., hydroperoxide and short-chain fatty acids) in inhibiting the replication of pathogens [28]. For example, Lactobacillus genus bacteria may be able to produce bacteriocins, including low-molecular-weight substances (LMWB-antibacterial peptides), as well as high-molecular-weight ones (class III bacteriocins), and some antibiotics. Probiotic bacteria (e.g., Lactobacillus and Bifidobacterium) may produce the so-called de-conjugated bile acids (derivatives of bile acids), demonstrating stronger antibacterial effect than the bile salts produced by their host [28,56]. Further studies are necessary to explain the mechanism of acquiring resistance to their own metabolites by Lactobacillus genus bacteria. The nutrient essential for nearly all bacteria, except for lactic acid bacteria, is iron. It turns out that Lactobacillus bacteria do not need iron in their natural environment, which may be their crucial advantage over other microorganisms [57]. Lactobacillus delbrueckii affects the function of other microbes by binding iron hydroxide to its cellular surface, thus making it unavailable to other microbes [58].
The immunomodulatory effect of the intestinal microbiota, including probiotic bacteria, is based on three, seemingly contradictory phenomena [53,59]: (1) Induction and maintenance of the state of immunological tolerance to environmental antigens (nutritional and inhalatory); (2) Induction and control of immunological reactions against pathogens of bacterial and viral origin; (3) Inhibition of auto-aggressive and allergic reactions.
Probiotic-induced immunological stimulation is also manifested by the increased production of immunoglobulins, enhanced activity of macrophages and lymphocytes, and stimulation of γ-interferon production. Probiotics may influence the congenital and acquired immunological system through metabolites, components of the cellular wall, and DNA, recognised by specialised cells of the host (e.g., those equipped with receptors) [28]. The principal host cells that are important in the context of the immune response are intestinal epithelial cells and intestinal immune cells. Components of the cellular wall of lactic acid bacteria stimulate the activity of macrophages. Those, in turn, are able to destroy microbes rapidly by the increased production of free oxygen radicals and lysosomal enzymes. Probiotic bacteria are also able to stimulate the production of cytokines by immunocompetent cells of the gastrointestinal tract [60]. On the other hand, the immunological activity of yeast is associated with the presence of glucans in their cellular wall. Those compounds stimulate the response of the reticuloendothelial system [61].
The last of the abovementioned probiotic effects-inhibition of the production of bacterial toxins-is based on actions leading to toxin inactivation and help with the removal of toxins from Nutrients 2017, 9, 1021 6 of 30 the body. Help in detoxification from the body can take place by adsorption (some strains can bind toxins to their cell wall and reduce the intestinal absorption of toxins), but can also result from the metabolism of mycotoxins (e.g., aflatoxin) by microorganisms [62][63][64]. However, not all probiotics exhibit detoxifying properties, as it is a strain-related characteristic. Studies should therefore be conducted to select strains with such characteristics. The effectiveness of some probiotics in combating diarrhoea is probably associated with their ability to protect the host from toxins. The reduction of metabolic reactions leading to the production of toxins is also associated with the stimulation of pathways leading to the production of native enzymes, vitamins, and antimicrobial substances [28].
Gut microbiota play a significant role in host metabolic processes (e.g., the regulation of cholesterol absorption, blood pressure (BP), and glucose metabolism), and recent metagenomic surveys have revealed that they are involved in host immune modulation and that they influence host development and physiology (organ development) [65][66][67]. Nutritional programming to manipulate the composition of the intestinal microbiota through the administration of probiotics continues to receive much attention for the prevention or attenuation of the symptoms of metabolic-related diseases. Currently, studies are exploring the potential for expanded uses of probiotics for improving health conditions in metabolic disorders that increase the risk of developing cardiovascular diseases such as hypertension. Further investigations are required to evaluate the targeted and effective use of the wide variety of probiotic strains in various metabolic disorders to improve the overall health status of the host [65].
In order to confirm the beneficial role of probiotics in improving cardiovascular health and in the reduction of BP, more extensive studies are needed to understand the mechanisms underlying probiotic action. Most probably, all of the abovementioned mechanisms of probiotic action have an effect on the protection against infections, cancer, and the stabilization of balance of the host's intestinal microbiota. However, it seems unlikely that each of the probiotic microorganisms has properties of all four aspects simultaneously and constitutes a universal remedy to multiple diseases. An important role in the action of probiotics is played by species-and strain-specific traits, such as: cellular structure, cell surface, size, metabolic properties, and substances secreted by microorganisms. The use of a combination of probiotics demonstrating various mechanisms of action may provide enhanced protection offered by a bio-therapeutic product [68]. Figure 1 summarises the mechanisms and effects of action of probiotics.

Probiotics for Humans
In the face of widespread diseases and ageing societies, the use of knowledge on microbiocenosis of the gastrointestinal tract and on the beneficial effect of probiotic bacteria is becoming increasingly important. The consumption of pre-processed food (fast food), often containing excessive amounts of fat and insufficient amounts of vegetables, is another factor of harmful modification of human intestinal microbiota. There is currently no doubt about the fact that the system of intestinal

Probiotics for Humans
In the face of widespread diseases and ageing societies, the use of knowledge on microbiocenosis of the gastrointestinal tract and on the beneficial effect of probiotic bacteria is becoming increasingly important. The consumption of pre-processed food (fast food), often containing excessive amounts of fat and insufficient amounts of vegetables, is another factor of harmful modification of human intestinal microbiota. There is currently no doubt about the fact that the system of intestinal microorganisms and its desirable modification with probiotic formulas and products may protect people against enteral problems, and influence the overall improvement of health.
Probiotics may be helpful in the treatment of inflammatory enteral conditions, including ulcerative colitis, Crohn's disease, and non-specific ileitis. The aetiology of those diseases is not completely understood, but it is evident that they are associated with chronic and recurrent infections or inflammations of the intestine. Clinical studies have demonstrated that probiotics lead to the remission of ulcerative colitis, but no positive effect on Crohn's disease has been observed [69,70]. Numerous studies assessed the use of probiotics in the treatment of lactose intolerance [71,72], irritable bowel syndrome, and the prevention of colorectal cancer [73] and peptic ulcers [74].
Considering their role in the inhibition of some bacterial enzymes, probiotics may reduce the risk of colorectal carcinoma in animals. However, the same effect in humans has not been confirmed in clinical trials [75]. On the other hand, a positive effect on the urogenital system (prevention and treatment of Urinary Tract Infections (UTIs) and bacterial vaginitis) constitutes an excellent example of the benefits associated with the use of probiotics [76][77][78]. There were attempts to apply probiotics to pregnant women and neonates in order to prevent allergic diseases such as atopic dermatitis. However, the scope of action is controversial in this kind of case [79]. There is evidence that the consumption of probiotics-containing dairy products results in the reduction of blood cholesterol, which may be helpful in the prevention of obesity, diabetes, cardiovascular diseases, and cerebral stroke [80]. The reduction of cholesterol level achieved due to probiotics is less pronounced compared to the effect of pharmaceutical agents, but leads to a significant minimisation of side effects [80]. Other studies confirmed the effect of the probiotic formula VSL#3 and of the Oxalobacter formigenes bacterial strain on the elimination of oxalates with urine, which may potentially reduce the risk of urolithiasis [81]. Studies on animals demonstrated that orally administered Lactobacillus acidophilus induces expression of µ-opioid and cannabinoid receptors in intestinal cells and mediate analgesic functions in the intestine, and that the observed effect is comparable to the effect of morphine [82]. However, the effect has not been demonstrated in humans.
There are many reports on the application of probiotics in the treatment of diarrhoea. The application of Saccharomyces boulardii yeast to patients with acute, watery diarrhoea resulted in the cure and reduced frequency of that type of complaints in two subsequent months [83]. The efficacy of probiotic strains in the therapy of nosocomial, non-nosocomial, and viral diarrhoeas has also been documented. It turns out that probiotics may increase the amount of IgA antibodies, which leads to the arrest of a viral infection [84].
Antibiotic-associated diarrhoea (AAD) is a common complication of most antibiotics and Clostridium difficile disease (CDD), which also is incited by antibiotics, and is a leading cause of nosocomial outbreaks of diarrhoea and colitis. The use of probiotics for these two related diseases remains controversial. A variety of different types of probiotics show promise as effective therapies for these two diseases. Using meta-analyses, three types of probiotics (Saccharomyces boulardii, Lactobacillus rhamnosus GG, and probiotic mixtures) significantly reduced the development of antibiotic-associated diarrhoea. Only S. boulardii was effective for CDD [85].
Studies performed in a foster home in Helsinki (Finland) demonstrated that the regular use of Lactobacillus rhamnosus GG in the form of a probiotic resulted in a reduced number of respiratory tract infections [86]. Other studies demonstrated that the application of a diet depleted of fermented foods caused a reduction of congenital immunological response, as well as a significant reduction of stool Lactobacillus count and of the stool amount of short-chain fatty acids. Moreover, the reduction of phagocytic activity of leukocytes was observed after two weeks of the diet, which could have a negative impact on the organism's ability to protect against infections [87]. The effect of a fermented product containing Lactobacillus gasseri CECT5714 and Lactobacillus coryniformis CECT5711 strains on blood and stool parameters was studied in a randomised, double-blind trial on 30 healthy volunteers. No negative effects were observed in the group of subjects receiving the probiotic strains. Some positive effects were observed, including: the production of short-chain fatty acids, humidity, frequency and volume of stools, and subjective improvement of intestinal function [88]. Studies by Alvaro et al. (2007) demonstrated a significant reduction of Enterobacteriaceae count and increased galactosidase activity in the alimentary tract of yoghurt consumers, compared to those who did not eat yoghurt [89]. Table 3 lists the results of studies focusing on the effect of probiotics on human health. There are examples of clinical trials during which the probiotics group received the probiotic prophylactically or in addition to the standard therapy.  Significantly reduction eczema in high-risk for a minimum of 2 years provided that the probiotic was administered to the infant within 3 months of birth. [123] 50 children with AD B. animalis subsp lactis 8 weeks Significant reduction in the severity of AD with an improved ration of IFN-γ and IL-10.

Prebiotics
Different prebiotics will stimulate the growth of different indigenous gut bacteria. Prebiotics have enormous potential for modifying the gut microbiota, but these modifications occur at the level of individual strains and species and are not easily predicted a priori. Furthermore, the gut environment, especially pH, plays a key role in determining the outcome of interspecies competition. Both for reasons of efficacy and of safety, the development of prebiotics intended to benefit human health has to take account of the highly individual species profiles that may result [129].
Fruit, vegetables, cereals, and other edible plants are sources of carbohydrates constituting potential prebiotics. The following may be mentioned as such potential souces: tomatoes, artichokes, bananas, asparagus, berries, garlic, onions, chicory, green vegetables, legumes, as well as oats, linseed, barley, and wheat [130]. Some artificially produced prebiotics are, among others: lactulose, galactooligosaccharides, fructooligosaccharides, maltooligosaccharides, cyclodextrins, and lactosaccharose. Lactulose constitutes a significant part of produced oligosaccharides (as much as 40%). Fructans, such as inulin and oligofructose, are believed to be the most used and effective in relation to many species of probiotics [131].

Prebiotic Selection Criteria
According to Wang (2009), there are five basic criteria for the classification of food components such as prebiotics (Figure 2) [132]. The first criterion assumes that prebiotics are not digested (or just partially digested) in the upper segments of the alimentary tract. As a consequence, they reach the colon, where they are selectively fermented by potentially beneficial bacteria (a requirement of the second criterion) [133]. The fermentation may lead to the increased production or a change in the relative abundance of different short-chain fatty acids (SCFAs), increased stool mass, a moderate reduction of colonic pH, reduction of nitrous end products and faecal enzymes, and an improvement of the immunological system [134], which is beneficial for the host (the requirement of the third criterion). Selective stimulation of growth and/or activity of the intestinal bacteria potentially associated with health protection and wellbeing is considered another criterion [8]. The last criterion of the classification assumes that a prebiotic must be able to withstand food processing conditions and remained unchanged, non-degraded, or chemically unaltered and available for bacterial metabolism in the intestine [132]. Huebner et al. (2008) tested several commercially available prebiotics using various processing conditions. They found no significant changes of the prebiotic activity of the tested substances in various processing conditions [135]. Meanwhile, Ze et al. (2012) showed that it was possible to alter the ability of gut bacteria by utilising starch in vitro [136]. The structure of prebiotics should be appropriately documented, and components used as pharmaceutical formulas, food, or feed additives should be relatively easy to obtain at an industrial scale [137]. colon, where they are selectively fermented by potentially beneficial bacteria (a requirement of the second criterion) [133]. The fermentation may lead to the increased production or a change in the relative abundance of different short-chain fatty acids (SCFAs), increased stool mass, a moderate reduction of colonic pH, reduction of nitrous end products and faecal enzymes, and an improvement of the immunological system [134], which is beneficial for the host (the requirement of the third criterion). Selective stimulation of growth and/or activity of the intestinal bacteria potentially associated with health protection and wellbeing is considered another criterion [8]. The last criterion of the classification assumes that a prebiotic must be able to withstand food processing conditions and remained unchanged, non-degraded, or chemically unaltered and available for bacterial metabolism in the intestine [132]. Huebner et al. (2008) tested several commercially available prebiotics using various processing conditions. They found no significant changes of the prebiotic activity of the tested substances in various processing conditions [135]. Meanwhile, Ze et al. (2012) showed that it was possible to alter the ability of gut bacteria by utilising starch in vitro [136]. The structure of prebiotics should be appropriately documented, and components used as pharmaceutical formulas, food, or feed additives should be relatively easy to obtain at an industrial scale [137].
Prebiotics may be used as an alternative to probiotics or as an additional support for them. Longterm stability during the shelf-life of food, drinks, and feed, resistance to processing, and physical and chemical properties that exhibit a positive effect on the flavour and consistence of products may promote prebiotics as a competition to probiotics. Additionally, resistance to acids, proteases, and bile salts present in the gastrointestinal tract may be considered as other favourable properties of prebiotics. Prebiotic substances selectively stimulate microorganisms present in the host's intestinal ecosystem, thus eliminating the need for competition with bacteria. Stimulation of the intestinal microbiota by prebiotics determines their fermentation activity, simultaneously influencing the SCFA level, which confers a health benefit on the host [139,140]. Moreover, prebiotics cause a reduction of intestinal pH and maintain the osmotic retention of water in the bowel [134]. However, it should be considered that an overdose of prebiotics may lead to flatulence and diarrhoea-these effects are absent in the case of excessive consumption of probiotics. Prebiotics may be consumed on a longterm basis and for prophylactic purposes. Moreover, when used at correct doses, they do not stimulate any adverse effects, such as diarrhoea, susceptibility to UV light, or hepatic injuries caused by antibiotics. Prebiotic substances are not allergenic and do not proliferate the abundance of antibiotic-resistance genes. Of course, the effect of the elimination of selected pathogens achieved by the use of prebiotics may be inferior to antibiotics, but the properties mentioned above make them a natural substitute for antibiotics [134].

Prebiotic Substances
The majority of identified prebiotics are carbohydrates of various molecular structures, naturally occurring in human and animal diets. The physiological properties of potential prebiotics determine their beneficial effect on the host's health. Prebiotics may be classified according to those properties as [134]:  not digested (or only partially digested);

Prebiotic selection criteria
Resistance to digestion in the upper sections of the alimentary tract.

Fermentation by intestinal microbiota.
Beneficial effect on host's health.

Selective stimulation of growth of probiotics.
Stability in various food/feed processing conditions.
Prebiotics may be used as an alternative to probiotics or as an additional support for them. Long-term stability during the shelf-life of food, drinks, and feed, resistance to processing, and physical and chemical properties that exhibit a positive effect on the flavour and consistence of products may promote prebiotics as a competition to probiotics. Additionally, resistance to acids, proteases, and bile salts present in the gastrointestinal tract may be considered as other favourable properties of prebiotics. Prebiotic substances selectively stimulate microorganisms present in the host's intestinal ecosystem, thus eliminating the need for competition with bacteria. Stimulation of the intestinal microbiota by prebiotics determines their fermentation activity, simultaneously influencing the SCFA level, which confers a health benefit on the host [139,140]. Moreover, prebiotics cause a reduction of intestinal pH and maintain the osmotic retention of water in the bowel [134]. However, it should be considered that an overdose of prebiotics may lead to flatulence and diarrhoea-these effects are absent in the case of excessive consumption of probiotics. Prebiotics may be consumed on a long-term basis and for prophylactic purposes. Moreover, when used at correct doses, they do not stimulate any adverse effects, such as diarrhoea, susceptibility to UV light, or hepatic injuries caused by antibiotics. Prebiotic substances are not allergenic and do not proliferate the abundance of antibiotic-resistance genes. Of course, the effect of the elimination of selected pathogens achieved by the use of prebiotics may be inferior to antibiotics, but the properties mentioned above make them a natural substitute for antibiotics [134].

Prebiotic Substances
The majority of identified prebiotics are carbohydrates of various molecular structures, naturally occurring in human and animal diets. The physiological properties of potential prebiotics determine their beneficial effect on the host's health. Prebiotics may be classified according to those properties as [134]: • not digested (or only partially digested); • not absorbed in the small intestine; • poorly fermented by bacteria in the oral cavity; • well fermented by seemingly beneficial intestinal bacteria; • poorly fermented by potential pathogens in the bowel.
Carbohydrates, such as dietary fibre, are potential prebiotics. Prebiotic and dietary fibre are terms used alternatively for food components that are not digested in the gastrointestinal tract. A significant difference between those two terms is that prebiotics are fermented by strictly defined groups of microorganisms, and dietary fibre is used by the majority of colonic microorganisms [141]. Therefore, considering one of the basic classification criteria, it turns out that using those terms alternatively is not always correct. Prebiotics may be a dietary fibre, but dietary fibre is not always a prebiotic [138]. The following non-starch polysaccharides are considered to be dietary fibre: cellulose, hemicellulose, pectins, gums, substances obtained from marine algae, as well as lactulose, soy oligosaccharides, inulins, fructooligosaccharides, galactooligosaccharides, xylooligosaccharides, and isomaltooligosaccharides. Based on the number of monomers bound together, prebiotics may be classified as: disaccharides, oligosaccharides (3-10 monomers), and polysaccharides. The most promising and fulfilling criteria for the classification of prebiotic substances, as evidenced by in vitro and in vivo studies, are oligosaccharides, including [142,143]: fructooligosaccharides (FOS), galactooligosaccharides (GOS), isomaltooligosaccharides (IMO), xylooligosaccharides (XOS), transgalactooligosaccharides (TOS), and soybean oligosaccharides (SBOS).
Also, polysaccharides such as inulin, reflux starch, cellulose, hemicellulose, or pectin may potentially be prebiotics. Examples of prebiotics that are most commonly used in human nutrition are presented in Table 4. The use of glucooligosaccharides, glicooligosaccharides, lactitol, izomaltooligosaccharides, stachyose, raffinose, and saccharose as prebiotics requires further studies [144]. Table 4. Examples of prebiotics and synbiotics used in human nutrition [134,145,146].

Mechanism of Action of Prebiotics
Prebiotics are present in natural products, but they may also be added to food. The purpose of these additions is to improve their nutritional and health value. Some examples are: inulin, fructooligosaccharides, lactulose, and derivatives of galactose and β-glucans. Those substances may serve as a medium for probiotics. They stimulate their growth, and contain no microorganisms. Figure 2 presents the principal mechanisms of prebiotic action and some of their effects on the host's health. Prebiotics are not digested by host enzymes and reach the colon in a practically unaltered form, where they are fermented by saccharolytic bacteria (e.g., Bifidobacterium genus). The consumption of prebiotics largely affects the composition of the intestinal microbiota and its metabolic activity [147]. This is due to the modulation of lipid metabolism, enhanced absorbability of calcium, effect on the immunological system, and modification of the bowel function [147]. It is highly probable that providing an energy source that only specific species in the microbiota can utilize has a greater impact on microbiota composition and metabolism than these other factors. The molecular structure of prebiotics determines their physiological effects and the types of microorganisms that are able to use them as a source of carbon and energy in the bowel [134]. It was demonstrated that, despite the variety of carbohydrates that exhibit the prebiotic activity, the effect of their administration is an increased count of beneficial bacteria, mostly of the Bifidobacterium genus [148,149].
The mechanism of a beneficial effect of prebiotics on immunological functions remains unclear. Several possible models have been proposed [150]: (1) Prebiotics are able to regulate the action of hepatic lipogenic enzymes by influencing the increased production of short-chain fatty acids (SCFAs), such as propionic acid. The main aim of prebiotics is to stimulate the growth and activity of beneficial bacteria in the gastrointestinal tract, which confers a health benefit on the host. Through mechanisms including antagonism (the production of antimicrobial substances) and competition for epithelial adhesion and for nutrients, the intestinal microbiota acts as a barrier for pathogens. Final products of carbohydrate metabolism are mostly SCFAs, namely: acetic acid, butyric acid, and propionic acid, which are subsequently used by the host as a source of energy [151]. As a result of the fermentation of carbohydrates, Bifidobacterium or Lactobacillus may produce some compounds inhibiting the development of gastrointestinal pathogens, as well as cause a reduction in the intestinal pH [152]. Moreover, Bifidobacterium genus bacteria demonstrate tolerance to the produced SCFAs and reduced pH. Therefore, due to their favourable effect on the development of beneficial intestinal bacteria, the administration of prebiotics may participate in the inhibition of the development of pathogens.
There are very few documented study results regarding the inhibition of the development of pathogens by prebiotics. In 1997 and 2003, Bovee-Oudenhoven et al. studied the use of lactulose in the prevention of Salmonella Enteritidis infections on a rat model. Their results indicated that the acidification of the intestine occurring as a result of lactulose fermentation caused the reduced development of pathogens and increased translocation of pathogens from the bowel [153]. It was also demonstrated that the administration of prebiotics increases the absorption of minerals, mostly of magnesium and calcium [154,155].

Prebiotics for Humans
The presence of prebiotics in the diet may lead to numerous health benefits. Studies on colorectal carcinoma demonstrated that the disease occurs less commonly in people who often eat vegetables and fruit. This effect is attributed mostly to inulin and oligofructose [156]. Among the advantages of those prebiotics, one may also mention the reduction of the blood LDL (low-density lipoprotein) level, stimulation of the immunological system, increased absorbability of calcium, maintenance of correct intestinal pH value, low caloric value, and alleviation of symptoms of peptic ulcers and vaginal mycosis [157]. Other effects of inulin and oligofructose on human health are: the prevention of carcinogenesis, as well as the support of lactose intolerance or dental caries treatment [131]. Rat studies demonstrated that administration of inulin for five weeks caused a significant reduction of blood triacylglycerol levels [156]. Human studies demonstrated that the daily use of 12 g of inulin for one month led to the reduction of blood VLDL (very low-density lipoprotein) levels (the reduction of triacylglycerols by 27%, and of cholesterol by 5%). This effect is associated with the effect of the prebiotic on hepatic metabolism and the inhibition of acetyl-CoA carboxylase and of glukose-6-phosphate dehydrogenase. It is also supposed that oligofructose accelerates lipid catabolism [157]. Asahara et al. (2001) demonstrated a protective effect of galactooligosaccharides (GOS) in the prevention of Salmonella Typhimurium infections in a murine model [158]. Buddington et al. (2002) confirmed a positive effect of fructooligosaccharides (FOS) on protection against Salmonella Typhimurium and Listeria monocytogenes infections [159]. Moreover, prebiotics are helpful in combating pathogenic microorganisms, such as Salmonella Enteritidis and Escherichia coli, and reduce odour compounds [160]. There are many reports regarding the positive effect of prebiotics on the carcinogenesis process. Results of rat studies proved that a prebiotic-enriched diet leads to significantly reduced indexes of carcinogenesis. Scientific research demonstrated that butyric acid may be a chemopreventive factor in carcinogenesis [161], or an agent protecting against the development of colorectal carcinoma through the promotion of cell differentiation [162]. Besides butyric acid, propionic acid also may possess anti-inflammatory properties in relation to colorectal carcinoma cells. In vitro studies on human L97 and HT29 cell lines (representing early and late stages of colorectal carcinoma) demonstrated that inulin fractions in plasma supernatant caused a significant inhibition of growth and induction of apoptosis in human colorectal carcinoma [163]. According to scientific reports, the administration of inulin and oligofructose to rats caused the inhibition of azoxymethane-induced colorectal carcinoma at the growth stage [164]. The supplementation of inulin and oligofructose at the dose of 5%-15% had also an effect on reduced occurrence of breast cancer in rats and of metastases to lungs [165]. However, those results have to be confirmed in humans. Table 5 lists the results of studies focusing on the effect of prebiotics on human health. There are examples of clinical trials during which the prebiotics group received the prebiotic prophylactically or in addition to the standard therapy. Table 5. Examples of clinical trials regarding the effect of prebiotics on human health.

Subjects Prebiotic Time of Administration Main Outcome
Obesity [166] 48 healthy adults with a body mass index (in kg/m 2 ) >25

OFS 12 weeks
There was a reduction in body weight of 1.03 ± 0.43 kg with oligofructose supplementation, whereas the control group experienced an increase in body weight of 0.45 ± 0.31 kg over 12 weeks (p = 0.01). Glucose decreased in the oligofructose group and increased in the control group between the initial and final tests (p ≤ 0.05). Insulin concentrations mirrored this pattern (p ≤ 0.05). Oligofructose supplementation did not affect plasma active glucagon-like peptide 1 secretion. According to a visual analogue scale designed to assess side effects, oligofructose was well tolerated.
Insulin resistance syndrome [167] 10 patients with type 2 diabetes FOS 4 weeks (double repetition) The plasma glucose response to a fixed exogenous insulin bolus did not differ at the end of the two periods. FOS had no effect on glucose and lipid metabolism in type 2 diabetics.

Type 2 diabetes
[168] 15 subjects with type 2 diabetes AX 5 weeks (double repetition) A supplement of 15 g/day of AX-rich fibre can significantly improve glycaemic control in people with type 2 diabetes. [169] 11 patients with impaired glucose tolerance

AX 6 weeks
No effects of arabinoxylan were observed for insulin, adiponectin, leptin, or resistin as well as for apolipoprotein B, and unesterified fatty acids. In conclusion, the consumption of AX in subjects with impaired glucose tolerance improved fasting serum glucose and triglycerides. However, this beneficial effect was not accompanied by changes in fasting adipokine concentrations.

OFS 8 weeks
Compared to placebo, OFS significantly decreased serum aminotransferases, aspartate aminotransferase after 8 weeks, and insulin level after 4 weeks, but this could not be related to a significant effect on plasma lipids. Prebiotic formula well tolerated, normal growth trend toward a higher percentage of Bifidobacterium and a lower percentage of E. coli in stool, suppresses Clostridium in stool. [173] 215 healthy infants GOS, FOS 27 weeks The concentration of secretory IgA was higher in the prebiotic group than the control; also, Bifidobacterium percentage was higher than the control and Clostridium was lower.
[174] 24 patients with chronic pouchitis inulin 3 weeks Inulin treatment resulted in decreased endoscopic and histological inflammation. This effect was associated with increased intestinal butyrate, lowered pH, and significantly decreased numbers of Bacteroides fragilis.
Atopic dermatitis [176] 259 infants at risk for atopy GOS, FOS 6 months Significant reduction of frequency of AD. [177] 259 healthy term infants with a parental history of atopy

Synbiotics
Synbiotics are used not only for the improved survival of beneficial microorganisms added to food or feed, but also for the stimulation of the proliferation of specific native bacterial strains present in the gastrointestinal tract [179]. The effect of synbiotics on metabolic health remains unclear. It should be mentioned that the health effect of synbiotics is probably associated with the individual combination of a probiotic and prebiotic [180]. Considering a huge number of possible combinations, the application of synbiotics for the modulation of intestinal microbiota in humans seems promising [181].

Synbiotic Selection Criteria
The first aspect to be taken into account when composing a synbiotic formula should be a selection of an appropriate probiotic and prebiotic, exerting a positive effect on the host's health when used separately. The determination of specific properties to be possessed by a prebiotic to have a favourable effect on the probiotic seems to be the most appropriate approach. A prebiotic should selectively stimulate the growth of microorganisms, having a beneficial effect on health, with simultaneous absent (or limited) stimulation of other microorganisms.

Synbiotics in Use
Previous sections discussed probiotic microorganisms and prebiotic substances most commonly used in human nutrition. A combination of Bifidobacterium or Lactobacillus genus bacteria with fructooligosaccharides in synbiotic products seems to be the most popular. Table 4 presents the most commonly used combinations of probiotics and prebiotics.

Mechanism of Action of Synbiotics
Considering the fact that a probiotic is essentially active in the small and large intestine, and the effect of a prebiotic is observed mainly in the large intestine, the combination of the two may have a synergistic effect [182]. Prebiotics are used mostly as a selective medium for the growth of a probiotic strain, fermentation, and intestinal passage. There are indications in the literature that, due to the use of prebiotics, probiotic microorganisms acquire higher tolerance to environmental conditions, including: oxygenation, pH, and temperature in the intestine of a particular organism [183]. However, the mechanism of action of an extra energy source that provides higher tolerance to these factors is not sufficiently explained. That combination of components leads to the creation of viable microbiological dietary supplements, and ensuring an appropriate environment allows a positive impact on the host's health. Two modes of synbiotic action are known [184]: (1) Action through the improved viability of probiotic microorganisms; (2) Action through the provision of specific health effects.
The stimulation of probiotics with prebiotics results in the modulation of the metabolic activity in the intestine with the maintenance of the intestinal biostructure, development of beneficial microbiota, and inhibition of potential pathogens present in the gastrointestinal tract [180]. Synbiotics result in reduced concentrations of undesirable metabolites, as well as the inactivation of nitrosamines and cancerogenic substances. Their use leads to a significant increase of levels of short-chain fatty acids, ketones, carbon disulphides, and methyl acetates, which potentially results in a positive effect on the host's health [184]. As for their therapeutic efficacy, the desirable properties of synbiotics include antibacterial, anticancerogenic, and anti-allergic effects. They also counteract decay processes in the intestine and prevent constipation and diarrhoea. It turns out that synbiotics may be highly efficient in the prevention of osteoporosis, reduction of blood fat and sugar levels, regulation of the immunological system, and treatment of brain disorders associated with abnormal hepatic function [185]. The concept of mechanisms of synbiotic action, based on the modification of intestinal microbiota with probiotic microorganisms and appropriately selected prebiotics as their substrates, is presented in Figure 1.

Synbiotics for Humans
Synbiotics have the following beneficial effects on humans [186]: (1) Increased Lactobacillus and Bifidobacterium genus count and maintenance of balance of the intestinal microbiota; (2) Improved hepatic function in patients suffering from cirrhosis; (3) Improved immunomodulative abilities; (4) Prevention of bacterial translocation and reduced incidence of nosocomial infections in patients' post-surgical procedures and similar interventions.
The translocation of bacterial metabolism products, such as lipopolysaccharides (LPSs), ethanol, and short-chain fatty acids (SFCAs), leads to their penetration of the liver. SCFAs also stimulate the synthesis and storage of hepatic triacylglycerols. Those processes may intensify the mechanisms of hepatic detoxication, which may result in hepatic storage of triacylglycerol (IHTG), and intensify steatosis of the organ. A randomised trial on the use of a synbiotic containing five probiotics (Lactobacillus plantarum, Lactobacillus delbrueckii spp. bulgaricus, Lactobacillus acidophilus, Lactobacillus rhamnosus, Bifidobacterium bifidum) and inulin as a prebiotic in adult subjects with NASH (non-alcoholic steatohepatisis) demonstrated a significant reduction of IHTG (intrahepatic triacylglycerol) within six months [187]. It is also known that LPSs induce proinflammatory cytokines, such as the tumour necrosis factor alpha (TNF-α), playing a crucial role in insulin resistance and inflammatory cell uptake in NAFLD (non-alcoholic fatty liver disease). In the study on the effect of the synbiotic product containing a blend of probiotics (Lactobacillus casei, Lactobacillus rhamnosus, Streptococcus thermophilus, Bifidobacterium breve, Lactobacillus acidophilus, Bifidobacterium longum, Lactobacillus bulgaricus) and fructooligosccharides, 52 adults participated for 28 weeks. It was found that supplementation with the synbiotic resulted in the inhibition of NF-κB (nuclear factor κB) and reduced production of TNF-α (tumour necrosis factor α) [188].
In rat studies, an increased level of intestinal IgA was found, following the introduction of the synbiotic product containing Lactobacillus rhamnosus and Bifidobacterium lactis, and inulin and oligofructose as prebiotics to the diet. Synbiotics lead to reduced blood cholesterol levels and lower blood pressure [157]. Moreover, synbiotics are used in the treatment of hepatic conditions [189] and improve the absorption of calcium, magnesium, and phosphorus [190]. Danq et al. (2013), in a meta-analysis, evaluated published studies on pro/prebiotics for eczema prevention, investigating bacterial strain efficacy and changes to the allergy status of the children involved. This meta-analysis found that probiotics or synbiotics may reduce the incidence of eczema in infants aged <2 years. Systemic sensitization did not change following probiotic administration [191].
Studies carried out within the framework of the SYNCAN project funded by the European Union verified the anti-carcinogenic properties of synbiotics. The effect of fructooligosaccharides (SYN1) combined with two probiotic strains (Lactobacillus rhamnosus GG and Bifidobacterium animalis subsp. lactis Bb12) on the health of patients at risk of colorectal cancer was studied. As a result, a change of biomarkers (genotoxicity, labelling index, labelled cells/crypt, transepithelial resistance, necrosis, interleukin 2, interferon γ) indicating the development of the disease in cancer patients, and in patients post polyp excision, was observed [192]. It was concluded that the application of the studied synbiotic may reduce the risk of colorectal carcinoma. A lower level of DNA damage was also observed, as well as a lower colonocyte proliferation ratio [147]. Table 6 lists the results of studies focusing on the effect of synbiotics on human health. There are examples of clinical trials during which the synbiotics group received the synbiotic prophylactically or in addition to the standard therapy.   Consumption of the probiotic mixture improved the gastrointestinal performance associated with lactose load in subjects with LI. Symptoms were additionally reduced by the addition of prebiotics. The supplementation was safe and well tolerated, with no significant adverse effect observed.
Different types of cancer and side effects associated with cancer [192] 43 polypeptomized and 37 colon cancer patients L. rhamnosus GG, B. lactis Bb12, inulin 12 weeks Increased L. rhamnosus and B. lactis in faeces, reduction in C. perfringens, prevents increased secretion of IL-2 in polypectomized patients, increased production of interferon-γ in cancer patients.

Summary
Probiotic organisms are crucial for the maintenance of balance of human intestinal microbiota. Numerous scientific reports confirm their positive effect in the host's health. Probiotic microorganisms are attributed a high therapeutic potential in, e.g., obesity, insulin resistance syndrome, type 2 diabetes, and non-alcohol hepatic steatosis [207]. It seems also that probiotics may be helpful in the treatment of irritable bowel syndrome, enteritis, bacterial infections, and various gastrointestinal disorders and diarrhoeas. Probiotic microorganisms are also effective in the alleviation of lactose intolerance and the treatment of atopic dermatitis. A positive effect of probiotics in the course of various neoplastic diseases and side effects associated with anti-cancer therapies is also worth noting. Prebiotics may be used as an alternative to probiotics, or as an additional support for them. It turns out that the development of bio-therapeutic formulas containing both appropriate microbial strains and synergistic prebiotics may lead to the enhancement of the probiotic effect in the small intestine and the colon. Those "enhanced" probiotic products may be even more effective, and their protective and stimulatory effect superior to their components administered separately [208]. It seems that we will see further studies on combinations of probiotics and prebiotics, and further development of synbiotics. Future studies may explain the mechanisms of actions of those components, which may confer a beneficial effect on human health.