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Science, Technology and Medicine open access publisher.Publish, read and share novel research. Lactic Acid Bacteria as Probiotics:Characteristics, Selection Criteria and Role in Immunomodulation of Human GI Muccosal BarrierDaoud Harzallah and Hani Belhadj[1] Laboratory of Applied Microbiology, Faculty of Natural and Life Sciences, University Ferhat Abbas, Setif, Algeria1. The definition of probiotics has evolved from a live active culture which improves the balance of the gut microbiota composition to specific effects, in particular, the immunomodulatory potential of clearly defined strains.
The defense systems at the level of the intestine may be divided into three lines of defense: the intestinal flora, the mucosa, the intestinal epithelium and the related immune system. In addition to the main physiological role in digestion and absorption of nutrients, the gastrointestinal tract and its associated immune system act as a barrier between the internal environment and the antigens, such as food and microorganisms from the external environment.
The integrity of the defense of the intestinal mucosa depends on a number of factors, both at mucosal and at the intestinal lumen level. Boosts immunity of the host by acting on cells involved in natural immunity and specific immunity. Evolving in the same environment with bacteria, man has come to establish a symbiotic relationship with some of them, thus developing gut microbiota.
There is experimental evidence supporting the influence of ingested probiotic strains on the composition and metabolic activity of gut microbiota in healthy individuals.
As a result of the concentration in the lumen, they may contribute to the transit of the ecology of the micro-flora, at least in the period in which they are consumed. Significant changes were observed in the number of bacteria in human faeces after eating specific probiotic strains, resulting in overall increased number of species that promote health (Lactobacillus and Bifidobacterium) and a low number of potentially harmful strains (some strains Clostridium and Enterococcus). The bifidobacteria, in addition to other sanogenetic actions (the improvement of vitamin and protidic metabolism) have antibacterial action especially on pathogenic species (coli, Staphylococcus aureus, Shigella, Salmonella, etc.) through the production of short chain volatile fatty acids and substances with antibiotic action. The lactobacilli produce themselves organic acids, hydrogen peroxide and antibacterial peptides (lactocidin, acidophilin, lactacin B, etc.).
By reducing the pH of the intestinal tract, the probiotics may be able to interfere with the enzymatic activity of intestinal flora. Goldin and collaborators have shown that probiotic strains reduce the activity of pro-carcinogenic enzymes produced by the intestinal flora. The probiotics compete with noncommensal bacteria and favor their elimination through the secretion of antimicrobial factors, the increased production of antibodies and activation of macrophages and participates in modulating nutrition by producing certain vitamins and fragmentation of undigested molecules. There is epidemiological evidence, which showed that the incidence of autoimmune diseases has increased over the past three decades in industrialized countries, together with a decrease in infectious diseases, which was attributable to antibiotics, vaccines and to the improvement of general hygiene conditions.
Studies in vitro and in vivo have shown that certain species and bacterial strains produce extracellular glycosidase which degrades the glycoproteins or intestinal mucins and that others are able to stimulate the secretion of mucus, although specific data on probiotics are relatively sporadic.
The biological role of changes in the expression of complex glycoconjugates and mucus is not yet established. Intestinal microbial changes following dietary changes may affect the mucosal immune system. The increase of the specific immune response translates into an activation of T and B lymphocytes which causes an increase in the level of interleukins and circulating antibodies (immunoglobulin M and immunoglobulin G).
It is known that phagocytosis is accomplished mainly by macrophages and this is the main nonspecific defense mechanism of the body upon penetration of foreign substances. When intestinal epithelial cells (IEC) are exposed to pathogen microorganisms they produce pro-inflammatory mediators such as IL8 and TNFα. The probiotics may determine and direct the differentiation of DCreg, followed by the induction of regulatory T cells (Treg). The probiotics may determine the reduction of this production and the increase of the production of IL10. The evidence of the immune-stimulating effect of fermented milk products was emphasized by Perdington in 1995. Mice who consumed probiotic yogurt were subjected to a translocation of the lactobacillus in the mesenteric nodules, with stimulation of the ganglionic lymphocyte.
A study made in China by Leyer in 2009, showed that daily supplementation with probiotics is useful in preventing respiratory virus diseases during winter in healthy small children.
It is interesting to relate recent French research conducted on children with Bifidobacterium longum, anaerobic bacteria (Gram positive) and that is a normal inhabitant of the intestine of children, adults and animals. Specific strains of gut microbiota contribute to the processing of dietary antigens in the intestine to reduce their immunogenicity in vivo and in vitro. However, the exact role of the modulation of the immune response for clinical response to probiotics is still unelucidated. This is an open access article distributed under the Creative Commons Attribution License (CC BY 3.0). Prof.ssa Tiziana Montalcini Ricercatrice in Scienze e Tecniche Dietetiche Applicate Specialista in Medicina Interna UOC Nutrizione Clinica Responsabile.
Alimento funzionale Alimento convenzionale che, oltre al valore nutrizionale di base, contiene sostanze che agiscono regolando alcune funzioni in grado di recare benefici.
Per nutraceutici, si intendono quei prodotti per uso orale contenenti alte concentrazioni di principi attivi derivati da alimenti funzionali, e che forniscono benefici sulla salute anche in senso preventivo e terapeutico.
Gli studi Studio su 66 donne in post-menopausa condotto presso la Universita di Illinois (Erdman and Potter, 1997) ha dimostrato che 40 g di proteine isolate della soia (ISP) al giorno (contenenti 90 mg totali isoflavonoidi) incrementano (approssimativamente del 2%) il contenuto minerale osseo alla colonna dopo 6 mesi. Flavonoli: Quercetina Nel vino rosso Scarso assorbimento intestinale Esistono altri fenoli della frutta a grappolo…. Resveratrolo da Uva che permette di aumentarne di 2000 volte la quantita contenuta nella buccia dell'Uva rossa, ottenendo il massimo dosaggio di Resveratrolo da Uva finora possibile, cioe 8,2 mg per capsula. 1) availability and easy handling for high-throughput testing.2) retention of tissue characteristics to support interpretation of results for the in vivo situation. In spite of the fact that primary cells, isolated from human or animal tissue ressemble direct comparability, they survive only few days in cell culture and are difficult to grow (reviewed by Marian, 2002).So far, human colon tumorigenic cell lines CaCo-2, T84 and HT-29 have been widely used for attachment assays and mechanistic studies, but first they do not derive from the small intestine, second they have tumorigenic phenotype distinguished from the normal gut epithelia and third, they express modified surface glycoconjugates.
IntroductionAs it was reported by Chow (2002), the notion that food could serve as medicine was first conceived thousands of years ago by the Greek philosopher and father of medicine, Hippocrates, who once wrote: 'Let food be thy medicine, and let medicine be thy food'.
Epithelial cells lining the gastrointestinal tract are able to respond to infection by initiating either nonspecific or specific host-defence response (Kagnoff and Eckmann 1997, Strober 1998).
The strains with beneficial properties, potential sources of probiotics, most frequently belong to the genera Bifidobacterium and Lactobacillus, and some of these strains exhibit powerful anti-inflammatory properties.
Recent data have shown that probiotics can initiate intestinal barrier repair after injury.
The survival of probiotics ingested in different segments of the digestive tract varies from one strain to another. This specific alteration can be observed a few days after the beginning of the consumption of probiotic formula, depending on the ability of the respective strain to modulate the functioning of the gastrointestinal tract. However, the studies only reflect the bacteriological situation of the faeces and do not provide an exact situation of the existing conditions in different parts of the digestive tract or the intestinal mucosa. Most probiotics are producing lactic acid, which lowers the local pH and thus prevents the growth of sensitive bacteria in acid and renders permeable the outer membrane Gram-negative bacteria. This effect has been shown in detail in animal models and human studies that measured the activity of certain bacterial enzymes (involved in colonic carcinogenesis) in faeces after ingestion of specific probiotic strains. All these features argue in favor of a symbiotic relationship between the human organism and probiotics. The bacteria of the digestive tract form a protective barrier that prevents colonization by pathogenic organisms.
In contact with antigens present in the digestive content, the immunoglobulin A is very important in the digestive tract, representing a first defense against infection. The generation and maintenance of the gut barrier and the anti-inflammatory tone of the immune regulation in the gut by probiotics. These ones exercise an anti-inflammatory function by controlling cell Th1, Th2 and probably Th17. The intestine usually produces small quantities of IL12, but probiotics have the potential to increase this production dependant on the local environment. After the ingestion of lactic acid bacteria species by laboratory animals a significant increase in IgA was noticed and the direct yogurt injection into the peritoneum caused the production of a cytokine, type I interferon.
For example, the casein degraded by enzymes derived from probiotics has been shown to modulate cytokine production by CD3, in infants with allergy to cow's milk proteins.
Uptake and transport of macromolecules by the intestine: possible role in clinical disorders(an update). Ouwehand PhD Department of Biochemistry and Food Chemistry, University of Turku, Turku, Finland.
Strong antimicrobial activity of Lactobacillusrhamnosus GG against Salmonella typhimurium is due to accumulation ofl acticacid. Alterations in faecal microflora enzymes related to diet, age, Lactobacillus supplements and dimethylhydrazine.
Immediate effect of Lactobacillus acidophilus on the intestinal flora and faecal enzymes of rats and the in vitro inhibition of Escherichia coli coculture.
Competitive exclusion of diarrheagenic Escherichia coli from human enterocyte-like Caco-2 cells by heat killed Lactobacillus. Inability of Lactobacillus casei strain GG, Lactobacillus acidophilus and Bifidobacterium bifidum to degrade intestinal mucus glycoproteins. Lactobacillus acidophilus LA1 binds to cultured human intestinal cell lines and inhibits cell attachment and cell invasion by enterovirulent bacteria.
Expression of MUC1, MUC2, MUC3 and MUC4 mucin mRNAs in human pancreatic and intestinal tumor cell lines. Saccharomyces boulardii preserves the barrier function and modulates the signal transduction pathway induced in enteropathogenic Ecoli infected T84 cells. Production of interferon induced by Streptococcus thermophilus:role of CD4+ and CD8+ lymphocytes. Immunomodulation of human blood cells following the ingestion of lactic acid bacteria .J Dairy Sci.
Modulation of specific humoral imunne response and changes in intestinal flora mediated through fermented milk intake.
Downregulation of antiCD3antibody-induced IL-4 production by bovine caseinshydrolysed with Lactobacillus GG-derived enzymes.
There is a growing body of evidence documenting the immune-modulatory ability of probiotic bacteria, it is therefore reasonable to suggest that this is potentiated via a combination of prebiotics and probiotics as a symbiotic mix. Probiotics, Prebiotics and Immunomodulation of Gut Mucosal Defences: Homeostasis and Immunopathology. Non sono considerate al momento farmaci, scarsi sono gli studi controllati che ne valutano la reale efficacia. In uno studio 60 gr di ISP al giorno per 3 mesi riducono le vampate del 45% in 104 donne in postmenopausa (Albertazzi et al., 1998).
Tra l'altro, a differenza di altre fonti di Resveratrolo, Stilvid ® non contiene antrachinoni o altre sostanze potenzialmente nocive per l'organismo. The inability to effectively improve this situation in spite of the very significant resources spent is a matter of major concern, calling for novel studies on pathogens to obtain new information and means for their elimination in European food products.
In addition, in experimental models, they are mostly cultivated as monolayers on plastic surfaces, where the establishment of functional, epithelial character is not defined.Recently, some laboratories started to use Caco-2 cells grown on the microporous membranes to establish functionality in studies of pathogen translocation (McCormick, 2003). Potential and established health benefits associated with the usage of probiotics (Leroy et al., 2008).
However, during recent times, the concept of food having medicinal value has been reborn as 'functional foods'. Bacterial adhesion to the host cell or recognition by the host cell is often an essential first stage in the disease process. Escherichia coli Nissle 1917 (EcN 1917) not only prevents mucosal barrier damage, but restores also the mucosal integrity in T84 epithelial cells. Oral administration of probiotics was associated with a decrease in the occurrence of autoimmune processes, especially inflammatory intestinal disease and allergies. Some strains are rapidly inactivated in the stomach, while specific strains of lactic acid bacteria can pass through the entire gastrointestinal tract in large quantities. Although many studies have been conducted on animals, the effects of lactic acid bacteria on human subjects remain controversial. This hypothesis would explain in part why administration of probiotics reduces the incidence and severity of autoimmune diseases. The evidence from studies both on animals and on people shows that the probiotics can restore damaged epithelial permeability and also support the existence of such a mechanism.
This barrier can be affected in various pathological circumstances or ill-judged antibiotic treatments and it is considered that it can be restored by ingestion of live bacteria, even if they temporarily colonize the intestine (which requires their regular consumption). They are produced by the plasma cells of its own lamina, transported through the epithelium and secreted into the intestinal lumen as secretor IgA in combination with a secretor compound. And in healthy children, who ate dishes prepared with bifidobacteria, there was also an increase in the total IgA and those of the antipoliovirus. The recent experiments and studies have shown that specific strains of probiotics inhibit the T cell proliferation and reduce both the secretion of Th1 and Th2 lymphocytes inducing the development of T cell populations producing TGF-β (transforming growth factor) and IL10. Feeding of Bifidobacterium bifidum and Streptococcust hermophilus to infants in hospital for prevention of diarrhoea and shedding of rotavirus. The need for probiotic formulations has been appreciated for the health benefits in “topping up your good bacteria” or indeed in an attempt to normalise the dysbiotic microbiota associated with immunopathology.
Stilvid ®, oltre al Resveratrolo da Uva, contiene anche procianidine, antocianosidi, flavonoidi e altri stilbeni che lavorano in sinergia, tanto che una capsula di Revidox apporta i benefici contenuti in 45 Kg di Uva. 1994) Significativa riduzione (58%) incidenza di batteriuria con piuria comparati al gruppo di controllo dopo 6 mesi. Functional cell models have been successfully used in studies of transepithelial crossing and biological activity of biomolecules mimicing uterine events (Cencic & La Bonnardiere, 2002; Cencic et al. Trypan blue uptake by chinese hamster ovary cultured epithelial cells: a cellular model to study Escherichia coli STb enterotoxin. Key and desirable criteria for the selection of probiotics in commercial applications (Vasiljevic and Shah, 2008). The list of health benefits accredited to functional food continues to increase, and the gut is an obvious target for the development of functional foods, because it acts as an interface between the diet and all other body functions. A wide range of gastrointestinal cell surface constituents, such as several glygoconjucates, can serve as receptors for bacterial adherence (Servin and Coconnier 2003, Pretzer et al., 2005).
The application of probiotics in pediatric practice currently lies in enhancing these barrier functions in the gut and reducing the risk of diseases associated with their dysfunction.
Since the pH of the colon cannot be measured, it is difficult to evaluate in vivo the effects of acid production by lactic acid bacteria on carcinogenesis.
The modifications specific to the mucus layer might be beneficial (by destroying receptors for certain pathogens) or harmful (by exposing receptors covered by mucus layer, providing a place to anchor pathogens in the intestinal wall). In addition to the barrier effect, several metabolites of lactic bacteria can inhibit the growth of pathogen germs. Immediately after birth, there can be implemented types belonging to the indigenous normal micro-flora. This review will focus on the immunomodulatory role of probiotics and prebiotics on the cells, molecules and immune responses in the gut mucosae, from epithelial barrier to priming of adaptive responses by antigen presenting cells: immune fate decision—tolerance or activation? Moreover, consumers are more and more demanding to eat healthy food and being aware of the animal welfare. One of the most promising areas for the development of functional food components lies in the use of probiotics and prebiotics which scientific researches have demonstrated therapeutic evidence. Furthermore, epithelial cells express constitutively host pattern recognition receptors (PRRS), such as Toll-like receptors (TLR). The most fully documented probiotic intervention is the treatment and prevention of acute infectious diarrhea. Because of the complexity of developing colon tumors in humans, it is impossible to formulate, on the basis of this result, a benefit in preventing colon cancer.


Modulation of normal homeostatic mechanisms, coupled with findings from probiotic and prebiotic delivery in pathological studies, will highlight the role for these xenobiotics in dysbiosis associated with immunopathology in the context of inflammatory bowel disease, colorectal cancer and hypersensitivity. Nowadays, consumers are aware of the link among lifestyle, diet and good health, which explains the emerging demand for products that are able to enhance health beyond providing basic nutrition.
However, the role of probiotics in animal health and diseases, is one of the most promising areas of development regarding functional foods.
In spite of the fact that most of the research has been focused on the gastrointestinal tract, probiotics can increase the overall animal health performance.“Probiotics, microorganisms that have a favourable influence on physiologic and pathological processes of thehost by their effect on the intestinal flora, may play a role in improving animal health”.
TLRs are also found on innate immune cells, such as dendritic cells and macrophages (Vinderola et al., 2005).
They can be found in fermented products as meat, milk products, vegetables, beverages and bakery products. First possible mechanism may be interference with virus attachment or entry into the cells, perhaps by steric hindrance (short term pre-incubation).It was further analysed whether the inhibition of VSV infectivity was due to the adsorption of the virus to probiotic bacteria. They are part of the microbiota on mucous membranes, such as the intestines, mouth, skin, urinary and genital organs of both humans and animals, and may have a beneficial influence on these ecosystems.
Other known recognition receptors are nucleotide-binding oligomerization domain proteins, which recognize both gram-positive and gram-negative bacteria. LAB that grow as the adventitious microflora of foods or that are added to foods as cultures are generally considered to be harmless or even an advantage for human health.
Antibodies are large protein molecules capable of binding and neutralizing an invading organism.
IJFM, submitted.Canil C, Rosenshine I, Ruchkowski S, Donnenberg MS, Kaper JB, Finlay BB, Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarised epithelial monolayers. Since their discovery, LAB has been gained mush interest in various applications, as starter cultures in food and feed fermentations, pharmaceuticals, probiotics and as biological control agents.
Increased epithelial barrier permeability is frequently associated with gastrointestinal disorders contributing to both disease onset and persistence (Lu and Walker 2001, Berkes 2003).
High doses of probiotic bacteria administered by the oral route may allow beneficial effects through the transient colonization of more proximal parts of the gut. In food industry, LAB are widely used as starters to achieve favorable changes in texture, aroma, flavor and acidity (Leory and De Vuyst, 2004).
The gatekeeper of the paracellular pathway is the tight junction, which is an apically located cell-cell junction between epithelial cells. T cells that specifically recognize pathogens can help antibodies to clear the infection or directly kill the invader themselves.
As the gut translocation of probiotic microorganisms is probably negligible, the effect of bacterial compounds released in the intestinal lumen and potentially absorbed by the gut epithelium, as well as the direct modulation of the epithelial cell in contact with bacteria have to be considered as possible mechanisms for the development of local or systemic effects of probiotic microorganisms agains pathogenic bacteria and viruses.
The tight junction permits the passage of small molecules, such as ions, while restricting the movement of large molecules, such as antigens and microorganisms, which can cause inflammation. Furthermore, possible mehanisms that shold not be overmissed is also direct contact of probiotic bacteria with pathogen by trapping them before they reach attachment sit on cells of the host (Heyman et Menard, 2002).Understanding the role of probiotic bacteria in activation of macrophages and stimulation of proinflammatory cytokine production in early virus infection was studied additionaly in pig alveolar macrophage cell line. Du to their antimicrobial and antioxidant activities some LAB strains are used in food biopreservation. A special risk group for hepatitis E infection: Turkish agricultural workers who use untreated waste water fro irrigation.
Origine and safety of probiotics An old dogma of probiotic selection has been that the probiotic strains should be of “human origin”.
Enterocytes are separated among themselves by tight junctions and form tight epithelial barrier. Many of the indications for probiotic activity have been obtained from effects observed in various clinical situations.
One may argue that from evolutionary point of view, describing bacteria to be of human origin does not make much sense at all.
The requirement for probiotics to be of human origin relates actually to the isolation of the strain rather than the “origin” itself. Like the thymus, it plays a role in generating antigen reactive lymphoid cells which will become specific effectors upon further maturation and on the other hand the mucosal lymphatic tissues are responsible for inducing tolerance to antigens that are commonly experienced in the enteric canal.
Further studies in functional cell models may help to dissect the detail mechanisms involved in the antiviral potential of these friendly bacteria.Figure 3.
Usually, the strains claimed to be “of human origin” have been isolated from faecal samples of healthy human subjects, and have therefore been considered to be “part of normal healthy human gut microbiota”. The mucosal lymphatic tissues are nonencapsulated submucosal lymphoid nodules and diffuse lymphocytic infiltrates in the submucosa of intestinal and respiratory tracts. Overview of probioticsThe most tried and tested manner in which the gut microbiota composition may be influenced is through the use of live microbial dietary additions, as probiotics. In reality the recovery of a strain from a faecal sample does not necessarily mean that this strain is part of the normal microbiota of this individual, since microbes passing the GI tract transiently can also be recovered from the faecal samples (Forssten et al., 2011).
Following incubation, cells were carefully rinsed with PBS to remove excess of the bacteria and the plates were further incubated for 24h before VSV challenge.
In practice it is impossible to know the actual origin of the probiotic strains, regardless of whether they have been isolated from faecal samples, fermented dairy products or any other source for that matter.
The dome epithelium covering each follicle is composed of cuboidal absorptive epithelial cells interrupted by delicate membraneous cells which have luminal microfolds instead of microvillus borders. Isolation of a strain from faeces of a healthy individual is also not a guarantee of the safety of the strain—such a sample will also always contain commensal microbes which can act as opportunistic pathogens, or even low levels of true pathogens, which are present in the individual at sub-clinical levels.
However, at the beginning of this century probiotics were first put onto a scientific basis by the work of Metchnikoff (1908). Antigen is deposited into lymphocytes, mononuclear phagocytes and dendritic cells immediately beneath M-cells above the B-cell mantle of Peyer’s patch germinal follicles. He hypothesised that the normal gut microflora could exert adverse effects on the host and that consumption of ‘soured milks’ reversed this effect.
Minute quantities of intact antigen and products of digestion are transported to the lamina propria and lacteals by ordinary absorptive epithelial cells anywhere in the small bowel. The microflora of thegastrointestinal tract is essential for mucosal protection, for immune education and for metabolism of fecal residue.
However, many species of the genera Lactobacillus, Leuconostoc, Pediococcus, Enterococcus, and Bifidobacterium were isolated frequently from various types of infective lesions.
It is important to point out that these products enter interfollicular areas and do not have access to the dome area. Creating and maintaining the gastroinstestinal ecosystem: What we know and need to know from gnotobiology?
Between the dome epithelium and the follicles there is a thin region of reticulum, containing a delicate plexus of blood vessels and plasma cells (Fig.1). Therapeutic activity of probiotic bacteria can be due to competition with pathogens for nutrients and mucosal adherence, production of antimicrobial substances, and modulation of mucosal immune functions.
The origin of the first use can be traced back to Kollath (1953), who used it to describe the restoration of the health of malnourished patients by different organic and inorganic supplements. Observed beneficial effect of these friendly bacteria were mainly been studied in In vivo trials on diverse animal models were results implicate on their ability of immunomodulation and protection against several emerging animal pathogens as Salmonella thyphimurium, Listeria and Campylobacter.Results of our study furthermore implicate that probiotics can be used in protection against several virus caused infections in animals.
Later, Vergin (1954) proposed that the microbial imbalance in the body caused by antibiotic treatment could have been restored by a probiotic rich diet; a suggestion cited by many as the first reference to probiotics as they are defined nowadays.
The prototypical Mucosal Lymphatic Tissue is the Peyer’s patch which has a unique dome epithelium that is specialized to sample environmental antigens. Similarly, Kolb recognized detrimental effects of antibiotic therapy and proposed the prevention by probiotics (Vasiljevic and Shah, 2008) Later on, Lilly and Stillwell (1965) defined probiotics as “…microorganisms promoting the growth of other microorganisms”.
Peyer’s patches contain lymphoid compartments that are analogous to the deep cortex and follicles of lymph nodes, but there are no afferent lymphatics and no medullary cords for local accumulation of plasma cells. Each Peyer’s patch contains multiple individual B-cell follicles separated by diffuse lymphoid tissue in interfollicular areas (Parrot, 1976).FOLLICLES.
The idea of health-promoting effects of LAB is by no means new, as Metchnikoff proposed that lactobacilli may fight against intestinal putrefaction and contribute to long life. Peyer’s patch follicles, located beneath the dome epithelium, have a mantle of small B-cells surrounding germinal centers that is thicker facing the dome.
The germinal centers contain large and intermediate sized B lymphoblasts, follicular dendritic cells, macrophages and rare T-cells. Cytokine effects on maturation of the phagosomes containing Mycobacteria avium subspecies paratuberculosis in J774 cells. Although minor side effects of the use of probiotics have been reported, infections with probiotic bacteria occur and invariably only in immunocompromised patients or those with intestinal bleeding (Leroy et al., 2008). Other definitions advanced through the years have been restrictive by specification of mechanisms, site of action, delivery format, method, or host. An issue of concern regarding the use of probiotics is the presence of chromosomal, transposon, or plasmid-located antibiotic resistance genes amongst the probiotic microorganisms. At this moment, insufficient information is available on situations in which these genetic elements could be mobilised, and it is not known if situations could arise where this would become a clinical problem (Leroy et al., 2008). The mechanism of action of probiotics (e.g, having an impact on the intestinal microbiota or enhancing immune function) was dropped from the definition to encompass health effects due to novel mechanisms and to allow application of the term before the mechanism is confirmed. Secondly, isolated IEL exhibit in vitro effector functions which identify them as natural killer-like or Cytotoxic cells that are spontaneous or “natural” but without the identifying characteristics of NK cells (Fig.1). In mucosae, cytotoxicity directed toward virus-infected or otherwise parasitized epithelial cells would be prophylactic or protective because the lysed cell would be rapidly eliminated from the body in the mucosal stream. Furthermore, certain mechanisms of action (such as delivery of certain enzymes to the intestine) may not require live cells. In vitro safety screenings of probiotics may include, among others, antibiotic resistance assays, screenings for virulence factors, resistance to host defence mechanisms and induction of haemolysis. If IgA is also present, it would prevent reinfection of mucosal epithelium.Host-pathogen interactionsAfter passing the food chain, food-borne pathogens invade a mammalian body via interactions with the host intestinal tract.
Reaching intestinal tract, food-borne pathogens not only interact with the host intestinal barrier but also with commensal microorganisms that colonise intestinal epithelia.
In relation to food, probiotics are considered as “viable preparations in foods or dietary supplements to improve the health of humans and animals”. Most food-borne pathogens must cross intestinal epithelial barrier to exert their physiopathological effects and to interact with mucosa-associated lymphoid tissue (MALT) (Kerneis et al, 1997).
According to these definitions, an impressive number of microbial species are considered as probiotics. Listeria monocytogenes and Mycobacterium paratuberculosis translocate through M-cells or disrupted tight-epithelia and use host inflammatory response to reorganise enterocytic cytoskeleton and open the paracellular pathway to enhance entrance to the MALT.Recently, a new mechanism for bacterial uptake in the mucosa has been identified. It has been shown that dendritic cells open the tight-junctions between epithelial cells in order to send dendrites outside the epithelium to directly sample bacteria (reviewed by McCormick, 2003).
ConclusionThe individual diversity of the intestinal microflora underscores the difficulty of identifying the entire human microbiota and poses barriers to this ?eld of research. Selection of probioticsMany in vitro tests are performed when screening for potential probiotic strains. It has also been shown that pathogens may specifically interact with the intestinal epithelium, resulting in trans-cellular signaling cascades to subepithelial neutrophils.Not only gram positive pathogens can alter the epithelial barrier.
The first step in the selection of a probiotic LAB strain is the determination of its taxonomic classification, which may give an indication of the origin, habitat and physiology of the strain. It is also apparent that even a single strain of probiotic may exert its actions via multiple, concomitant pathways. It has been shown that enteropathogenic E.coli infection leads to significant decrease in barrier function, loss of microvilli and altered distribution of tight junctional protein ZO-1. All these characteristics have important consequences on the selection of the novel strains (Morelli, 2007). Probiotics have long been used as an alternative to traditional medicine with the goal of maintaining enteric homeostasis and preventing disease. Drop in barrier function is accompanied by an increase in flux of paracellular fluid markers (Canil et al., 1993). Specificity of two tests for the early diagnosis of bovine paratuberculosis based on cellmediated immunity: the Johnin skin test and the gamma interferon assay. This conclusion was brought forward due to uncertainty of the origin of the human intestinal microflora since the infants are borne with virtually sterile intestine. Clinical trials have shown that probiotic treatment can reduce the risk of some diseases, especially antibiotic-associated diarrhea, but conclusive evidence is impeded owing to the wide range of doses and strains of bacteria used. For the attachment to the host epithelium glycan expression plays a pivotal role (reviewed by Syzmanski et al., 2003). However, the panel also underlined a need for improvement of in vitro tests to predict the performance of probiotics in humans. While many probiotics meet criteria such as acid and bile resistance and survival during gastrointestinal transit, an ideal probiotic strain remains to be identified for any given indication. Many studies, as discussed above, have shown that probiotics increase barrier function in terms of increased mucus, antimicrobial peptides, and sIgA production, competitive adherence for pathogens, and increased TJ integrity of epithelial cells. Conversion by Peyer’s patch lymphocytes of human enterocytes into M cells that transport bacteria. Furthermore, it seems unlikely that a single probiotic will be equally suited to all indications; selection of strains for disease-specific indications will be required (Shanahan, 2003).
Current investigation into the mechanism of action of speci?c probiotics has focused on probiotic-induced changes in the innate immune functions involvingTLRs and its downstream systems Like NF-?B, and other pathways (Yoon and Sun, 2011). The mechanisms of host responses are still not well understood, but it is believed that humoral response occurs in the intestine (reviewed by Wassenaar and Blaser, 1999).Very few food related studies have been carried out for a human pathogen such as TBEV.
Although the immunomodulatory effects of probiotics have been demonstrated in experimental animal models of allergy, autoimmunity, and IBD, information from clinical trials in humans is scarce.
Although TBEV is an arbovirus, it is an emerging pathogen, as it was found that approximately 10% of infections were associated with consumption of goat, sheep and cow raw milk and raw milk products (Gritsun et al, 2003; Juceviciene, A. The ability to adhere to the intestinal mucosa is one of the more important selection criteria for probiotics because adhesion to the intestinal mucosa is considered to be a prerequisite for colonization (Tuomola et al., 2001).
The table below (Table 2) indicates key creteria for sellecting probiotic candidat for commercial application, and figure 1 presents major and cardinal steps for sellecting probiotic candidats.It is of high importance that the probiotic strain can survive the location where it is presumed to be active.
Therefore, more research, especially in the form of well-designed clinical trials, is needed to evaluate the ef?cacy and safety of probiotics (Ezendam and Van Loveren, 2008). For a longer and perhaps higher activity, it is necessary that the strain can proliferate and colonise at this specific location. Generally the best protection against virus diseases is vaccination (Barrett et al, 2003), however the prophylactic use of probiotics may be a valuable complement to vaccination in endemic TBEV areas, where consumption of raw milk from goats and sheep is common.With the new lifestyle trends in Europe (more ecological or organic food production, processing and consumption) a potential for much higher incidence of food borne infections by TBEV.
Probably only host-specific microbial strains are able to compete with the indigenous microflora and to colonise the niches.
Generally the best protection against virus diseases is vaccination (Barrett et al, 2003) however the prophylactic use of probiotics may be a valuable complement to vaccination in endemic TBEV areas, where consumption of raw milk from goats and sheep is common.
Besides, the probiotic strain must be tolerated by the immune system and not provoke the formation of antibodies against the probiotic strain. It is the causative agent of hepatitis E in many developing countries in Asia, Africa and Latin America where hepatitis E is an important public health concern. On the other hand, the probiotic strain can act as an adjuvant and stimulate the immune system against pathogenic microorganisms. HEV is transmitted primarily by the faecal-oral route and waterborne epidemics are characteristic of hepatitis E.Recent studies have documented that sporadic acute hepatitis E also occurs in industrialised countries with no history of travel to areas endemic for HEV leading to suggestions that HEV may be endemic at low levels in developed countries.
Basic initial characterization of strain identity and taxonomy should be conducted, followed by evaluation with validated assays both in studies of animal models and in controlled studies in the target host. Moreover, cases of acute human hepatitis linked to novel HEV variants have been reported in Europe and Japan (Worm et al., 2002). In vitro assays are frequently conducted that have not been proved to be predictive of in vivo function.


Technological robustness must also be determined, such as the strain’s ability to be grown to high numbers, concentrated, stabilized, and incorporated into a ?nal product with good sensory properties, if applicable, and to be stable, both physiologically and genetically, through the end of the shelf life of the product and at the active site in the host. Assessment of stability can also be a challenge, since factors such as chain length and injury may challenge the typical assessment of colony-forming units, as well as in vivo function (Sanders, 2008).
Dose levels of probiotics should be based on levels found to be ef?cacious in human studies.
Moreover, it is likely that some foods like shellfish can act as vehicles for transmission of HEV (Smith, 2001).
Thus the possibility that farm animal species could represent reservoirs for human contamination through food and meat has to be considered seriously. Furthermore, the impact of product format on Figure 1.Scheme of the Guidelines for the Evaluation of Probiotics for Food Use. Cases of acute hepatites of novel HEV variants have been reported in humans in Europe, Japan thus showing that HEV is not limited and geographically distributed but being endemic (Worm et al., 2002).
The common quality-control parameter of colony-forming units per gram may not be the only parameter indicative of the ef?cacy of the ?nal product.
There are results showing 43,5% sewage samples positive in Barcelona (Spain), 20% in Washington (USA) and 25% in Nancy (France) that HEV may be more prevalent that previously considered (Clemente-Casares et al., 2003). Other factors, such as probiotic growth during product manufacture, coating, preservation technology, metabolic state of the probiotic, and the presence of other functional ingredients in the ?nal product, may play a role in the effectiveness of a product. There has also been an increasing number of reports from Europe and USA of sporadic hepatitis attributable to HEV, leading to suggestions that HEV may be endemic at low levels in developed countries. Potential mechanisms of action of probioticsA wide variety of potential beneficial health effects have been attributed to probiotics (Table 3).
It is also of importance that HEV-RNA was found to be present in colostrums of breast-fed mothers that were HEV infected (Chibber et al., 2003).
Claimed effects range from the alleviation of constipation to the prevention of major life-threatening diseases such as inflammatory bowel disease, cancer, and cardiovascular incidents. Animal reservoirs like the occurrence of HEV in swine native to industrialised countries should be considered a potential public health hazard and food related studies initiated (Worm et al. MTP Press, Lancaster.Rastall RA, Gibson GR, Harsharnjit SG, Guarner F, Klaenhammer TR, Pot B, Reid G, Rowland IR, Sanders ME.
Some of these claims, such as the effects of probiotics on the shortening of intestinal transit time or the relief from lactose maldigestion, are considered well-established, while others, such as cancer prevention or the effect on blood cholesterol levels, need further scientific backup (Leroy et al., 2008). The mechanisms of action may vary from one probiotic strain to another and are, in most cases, probably a combination of activities, thus making the investigation of the responsible mechanisms a very difficult and complex task. What do beneficial bacteria (probiotics) do to benefit to the animal health?Animals form a dynamic, functionally stable and complex ecosystem with microbial community in their gut. Modulation of the microbial ecology of the human colon by probiotics, prebiotics and sybiotics to enhance human health: An overview of enabling science and potential applications. In general, three levels of action can be distinguished: probiotics can influence human Probiotic organisms can provide a beneficial effect on intestinal epithelial cells in numerous ways. Approximately 400 species of commensal microbial habitants colonise the surface of complex gut epithelium.
In fact, the body contains more bacteria than the number of people who have ever lived on the planet. At least the intestine should contain 85% friendly bacteria to prevent the over colonization of disease causing micro-organisms like E. Therefore, the commensal microorganisms are essential for the functionality of the host and appreciable in host immune defence. Immune effects of probiotics in man include the stimulation of cell-mediated immune effector functions with enhanced secretion of IFN-gamma by blood cells, enhanced phagocytosis and cell activation (reviewed by Clancy, 2003).It is important to elucidate the actions of probiotics in their hosts as it was shown that a combination therapy with mixture of probiotics led to inhibition of stimulatory effects of certain species (reviewed by Clancy, 2003). Probiotics can inhibit the growth of harmful bacteria that cause digestive stress, improve digestion of food and absorption of vitamins, stimulate the body’s natural defence mechanism, help to make vitamins needed by the body,…Why are probiotics supplements necessary for animal health?Formerly, the intake of probiotic bacteria was high because one of the main methods to preserve the food, was fermentation. Probiotics prevent the intestinal tract infections (bacteria or virus induced) and help to reduce the diarrhea.
Probiotics also play a pivotal role in the regulation of gut motility, improvement of the immune system, reduction of catabolic products eliminated by kidney and liver and improved wellbeing of the animal.Probiotics and the intestinal barrierThe intestine has a mucosa which works as a selective barrier allowing the passage of useful substances and preventing the penetration of undesirable agents in the bloodstream. Cluster of cases of acute hepatitis associated with hepatitis E virus infection acquired in the Netherlands. Probiotic bacteria stimulate the formation of epithelial cells and decrease the inflammation in the intestinal mucosa. The gut barrier function, which protects against the constant exposure to foreign antigens from food and the environment, can be stabilised by probiotic administration.This is thought to arise from stimulation of production of secretory IgA and mucus and by attenuating pro-inflammatory responses such as IL-8, MCP1, MIP1 and RANTES, pro-inflammatory cytokines (TNF-a, GM-CSF, IL-a and IL-1b) and prostaglandins and leukotrienes induced by pathogens. Probiotics have been administered safely to individuals with immuno-inflammatory disorders such as atopy and Crohn_s disease as well as those with HIV and immunosuppression. Gut microbiotaThe human gastrointestinal tract is inhabited by a complex and dynamic population of around 500-1000 of different microbial species which remain in a complex equilibrium. It has been estimated that bacteria account for 35–50% of the volume content of the human colon. Administration of probiotic strains causes a range of non-specific and specific host immune responses in diseased and healthy subjects. These include Bacteroides, Lactobacillus, Clostridium, Fusobacterium, Bifidobacterium, Eubacterium, Peptococcus, Peptostreptococcus, Escherichia and Veillonella. These include, for example, the enhancement of phagocytic activity of peripheral blood leukocytes and natural killer cell activity. The bacterial strains with identified beneficial properties include mainly Bifidobacterium and Lactobacillus species. Additionally, stimulation of both non-specific secretory IgA and specific antibody responses, especially mucosal IgA, to mucosal vaccines such as rotavirus, polio and Salmonella typhi and enteric pathogens such as rotavirus has been seen.
The dominant microbial composition of the intestine have been shown to be stable over time during adulthood, and the microbial patterns are unique for each individual. Increased cytokine production in vivo (IFN-c, IFN-a, IL-2) and by peripheral blood mononuclear cells ex vivo (IL-1b, TNF-a, IL-6, IL-10, IFNa, IFN-c) have been reported following appropriate probiotic stimulation.The question is can the reactions be predicted for a given subject, and can they be effectively directed? However, there are numerous external factors that have potential to influence the microbial composition in the gut as host genetics, birth delivery mode, diet, age, antibiotic treatments and also, other microorganisms as probiotics. Few studies have examined the anti-infection effects and host immune responses in the same subjects followingadministration of probiotics.
The intestine is one of the main surfaces of contact with exogenous agents (viruses, bacteria, allergens) in the human body. It has a primary role in the host defense against external aggressions by means of the intestinal mucosa, the local immune system, and the interactions with the intestinal microbiota (resident and in transitbacteria). Listeria).There is also a scientific critisism as animal and human models failed to develop a mechanistic framework.
Gut microbiota influences human health through an impact on the gut defense barrier, immune function, nutrient utilization and potentially by direct signaling with the gastrointestinal epithelium (Collado et al., 2009).
Results obtained in such models were difficult to reproduce, were not clearly linked to clinical outcomes and were outside any physiological framework of host (reviewed by Clancy, 2003). Although the explosive growth in the field genomics and proteomics with technological innovations in recent years will give new data, in vitro models are still essential tools in biological mechanistic studies such as for the accession of cytokine production it is necessary to localise the protein and mRNA. In healthy adults, 80% of phylotypes belong to four major phylogenetic groups, which are the Clostiridium leptum, Clostridium coccoides, Bacteroides and Bifidobacteria groups. Localisation of protein alone may identify not only producing cells but also target cells and cells which have taken up the protein by endocytosis. Also, studies have found that mucosal microbiota is stable along the distal gastrointestinal tract from ileum to rectum, but mucosa-associated microbiota is different from fecal microbiota.
The number of bacterial cells present in the mammalian gut shows a continuum that goes from 101 to 103 bacteria per gram of contents in the stomach and duodenum, progressing to 104 to 107 bacteria per gram in the jejunum and ileum and culminating in 1011 to 1012 cells per gram in the colon (Figure 3a). In addition to the longitudinal heterogeneity displayed by the intestinal microbiota, there is also a great deal of latitudinal variation in the microbiota composition (Figure 3b). The intestinal epithelium is separated from the lumen by a thick and physicochemically complex mucus layer. The microbiota present in the intestinal lumen differs significantly from the microbiota attached and embedded in this mucus layer as well as the microbiota present in the immediate a: variations in microbial numbers and composition across the length of the gastrointestinal tract. For instance, Bacteroides, Bifidobacterium, Streptococcus, members of Enterobacteriacea, Enterococcus, Clostridium, Lactobacillus, and Ruminococcus were all found in feces, whereas only Clostridium, Lactobacillus, and Enterococcus were detected in the mucus layer and epithelial crypts of the small intestine (Sekirov et al., 2010). Upon passage through the birth canal, infants are exposed to a complex microbial population. After the initial establishment of the intestinal microbiota and during the first year of life, the microbial composition of the mammalian intestine is relatively simple and varies widely between different individuals and also with time. Survival and antagonism effects of probiotics in the gutThe intestinal epithelium is the largest mucosal surface in the human body, provides an interface between the external environment and the host.
The gut epithelium is constantly exposed to foreign microbes and antigens derived from digested foods. Thus, the gut epithelium acts as a physical barrier against microbial invaders and is equipped with various elements of the innate defense system. In the gut, two key elements govern the interplay between environmental triggers and the host: intestinal permeability and intestinal mucosal defense.
Resident bacteria can interact with pathogenic microorganisms and external antigens to protect the gut using various strategies.According to the generally accepted de?nition of a probiotic, the probiotic microorganism should be viable at the time of ingestion to confer a health bene?t.
Although not explicitly stated, this de?nition implies that a probiotic should survive GI tract passage and, colonize the host epithelium.
A variety of traits are believed to be relevant for surviving GI tract passage, the most important of which is tolerance both to the highly acidic conditions present in the stomach and to concentrations of bile salts found in the small intestine. These properties have consequently become important selection criteria for new probiotic functionality.
One of the mechanisms by which the gut ?ora resists colonization by pathogenic bacteria is by the production of a physiologically restrictive environment, with respect to pH, redox potential, and hydrogen sul?de production. Probiotic bacteria decrease the luminal pH, as has been demonstrated in patients with ulcerative colitis (UC) following ingestion of the probiotic preparation VSL#3. Several bacteriocins produced by different species from the genus Lactobacillus have been described. The inhibitory activity of these bacteriocins varies; some inhibit taxonomically related Gram-positive bacteria, and some are active against a much wider range of Gram-positive and Gram-negative bacteria as well as yeasts and molds. Lacticin 3147, a broad-spectrum bacteriocin produced by Lactococcus lactis, inhibits a range of genetically distinct Clostridium dif?cile isolates from healthy subjects and patients with IBD.
A further example is the antimicrobial effect of Lactobacillus species on Helicobacter pylori infection of gastric mucosa, achieved by the release of bacteriocins and the ability to decrease adherence of this pathogen to epithelial cells (Gotteland et al., 2006).
The pretreatment of intestinal (T84) cells with lactic acid-producing bacteria reduced the ability of pathogenic E.
Adhesion and invasion of an intestinal epithelial cell line (Intestine 407) by adherent invasive E.
Probiotics and the mucous layerMost mucosal surfaces are covered by a hydrated gel formed by mucins.
Mucins are secreted by specialized epithelial cells, such as gastric foveolar mucous cells and intestinal goblet cells, Goblet cells are found along the entire length of the intestinal tract, as well as other mucosal surfaces. Of the 18 mucin-type glycoproteins expressed by humans, MUC2 is the predominant glycoprotein found in the small and large bowel mucus. The NH2- and COOH-termini are not glycosylated to the same extent, but are rich in cysteine residues that form intra- and inter-molecular disul?de bonds.
These glycan groups confer proteolytic resistance and hydrophilicity to the mucins, whereas the disul?de linkages form a matrix of glycoproteins that is the backbone of the mucous layer (Ohland and MacNaughton, 2010). Although small molecules pass through the heavily glycosylated mucus layer with relative ease, bulk fluid flow is limited and thereby contributes to the development of an unstirred layer of fluid at the epithelial cell surface. As the unstirred layer is protected from convective mixing forces, the diffusion of ions and small solutes is slowed (Turner, 2009). This gel layer provides protection by shielding the epithelium from potentially harmful antigens and molecules including bacteria from directly contacting the epithelial cell layer, while acting as a lubricant for intestinal motility.
Mucins can also bind the epithelial cell surface carbohydrates and form the bottom layer, which is ?rmly attached to the mucosa, whereas the upper layer is loosely adherent. The mucus is the ?rst barrier that intestinal bacteria meet, and pathogens must penetrate it to reach the epithelial cells during infection (Ohland and MacNaughton, 2010).Probiotics may promote mucus secretion as one mechanism to improve barrier function and exclusion of pathogens.
In support of this concept, probiotics have been shown to increase mucin expression in vitro, contributing to barrier function and exclusion of pathogens. Several studies showed that increased mucin expression in the human intestinal cell lines Caco-2 (MUC2) and HT29 (MUC2 and 3), thus blocking pathogenic E.
However, healthy rats did not display increased colonic TFF3 expression after stimulation by VSL#3 probiotics (Caballero-Franco et al., 2007). Furthermore, mice treated with 1% dextran sodium sulfate (DSS) to induce chronic colitis did not exhibit increased TFF3 expression or wound healing when subsequently treated with VSL#3. This observation indicates that probiotics do not enhance barrier function by up-regulation of TFF3, nor are they effective at healing established in?ammation. Therefore, use of current probiotics is likely to be effective only in preventing in?ammation as shown by studies in animal models (Ohland and MacNaughton, 2010).5.
Interaction of probiotic bacteria with gut epitheliumThe composition of the commensal gut microbiota is probably influenced by the combination of food practices and other factors like the geographical localization, various levels of hygiene or various climates. The establishment of a normal microbiota provides the most substantial antigenic challenge to the immune system, thus helping the gut associated lymphoid tissus (GALT) maturation.
The intestinal microbiota contributes to the anti-inflammatory character of the intestinal immune system. Several immunoregulatory mechanisms, including regulatory cells, cytokines, apoptosis among others, participate in the control of immune responses by preventing the pathological processes associated with excessive reactivity. An interesting premise for probiotic physiological action is their capacity to modulate the immune system.
Consequently, many studies have focused on the effects of probiotics on diverse aspects of the immune response. Following consumption of probiotic products, the interaction of these bacteria with intestinal enterocytes initiates a host response, since intestinal cells produce various immunomodulatory molecules when stimulated by bacteria (Delcenseri et al., 2009). Furthermore, The indigenous microbiota is a natural resistance factor against potential pathogenic microorganisms and provides colonization resistance, also known as gut barrier, by controlling the growth of opportunistic microorganisms.
It has been suggested that commensal bacteria protect their host against microbial pathogens by interfering with their adhesion and toxic effects (Myllyluoma, 2007).A fraction of ingested probiotics are able to interact with intestinal epithelial cells (IECs) and dendritic cells (DCs), depending on the presence of a dynamic mucus layer.
Probiotics can occasionally encounter DCs through two routes: DCs residing in the lamina propria sample luminal bacterial antigens by passing their dendrites between IECs into the gut lumen, and DCs can also interact directly with bacteria that have gained access to the dome region of the gut-associated lymphoid tissue (GALT) through specialized epithelial cells, termed microfold or M cells. The interaction of the host cells with microorganism-associated molecular patterns (MAMPs) that are present on the surface macromolecules of probiotic bacteria will induce a certain molecular response. The host pattern recognition receptors (PRRs) that can perceive probiotic signals include Toll-like receptors (TLRs) and the C type lectin DC-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN).
Some molecular responses of IECs depend on the subtype of cell, for example, Paneth cells produce defensins and goblet cells produce mucus.
Important responses of DCs against probiotics include the production of cytokines, major histocompatibility complex molecules for antigen presentation, and co-stimulatory molecules that polarize T cells into T helper or CD4+CD25+ regulatory T cells in the mesenteric lymph nodes (MLNs) or subepithelial dome of the GALT.
Host interactions of probiotic bacterial surface molecules: comparison with commensals and pathogens.



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