Probiotics and Their Influence on Health and Gut Microbiota
Food Science and Nutrition
Probiotics is a term generally used for living microorganisms that upon ingestion exert beneficial health effects. Probiotics consist of one or several strains, mostly from species of the bacterial genera Lactobacillus, Lactococcus or Bifidobacterium. The yeast Saccharomyces is also used. With the exception of Bifidobacterium, these are microorganisms which spontaneously multiply to high numbers in traditional fermented foods. When live bacteria are eaten, they will come into contact with the gastro-intestinal (GI) tract, where they can target different functions from the mouth to the anus: the immune system can be targeted through different receptors in the mucosa. Physiological events can be initiated, for example, by stimulating the epithelial cells of the mucosa to produce more mucin (Mack et al.,1999; Mack et al., 2003). Probiotics can interfere with food components, e.g. by fermenting dietary fibres in the colon to carboxylic acids, or splitting up tannins to flavonoids (Osawa et al., 2000; Vaquero et al., 2004) and phenolic acids (Barthelmebs et al., 2000).
Probiotics have by tradition mostly been linked to gut health, and especially well proven for several different probiotics are the beneficial effects against antibiotic associated diarrhoea, and diarrhoea caused by Clostridium difficile, which make up the main part of the antibiotic associated diarrhoeas (meta-analysis, D'Souza et al., 2002). This agrees with the traditional explanation of the beneficial health effects of probiotics, i.e. that probiotics maintain the microbial balance in the gut (fig. 1). Thus, probiotics support beneficial components of the microbiota and suppress adverse ones, e.g. C. difficile. In contrast, probiotics, and particularly Lactobacillus rhamnosus GG, have been shown to suppress symptoms of acute, nonbacterial diarrhoea in small children, primarily rotavirus diarrhoea, where the probiotics appear to activate the immune defence in a supportive fashion (meta-analysis, Huang et al., 2002).
Different probiotics have also shown convincing effects against irritable bowel syndrome (IBS; Camilleri, 2006), e.g.
Lactobacillus plantarum 299v (Nobaek et al., 2000; Niedzielin et al., 2001), Bifidobacterium infantis 35624 (O'Mahony et al., 2005) and Bifidobacterium animalis DN-173 010 (Guyonnet et al., 2007). The alleviated IBS-symptoms by B. infantis 35624 were associated with normalisation of the cytokine-ratio IL-10/IL-12, suggesting an immune-modulating role for this organism in IBS (O'Mahony et al., 2005).
A Systemic Error in Modern Diet?
Diseases with a background of dysfunctional immune reactions are increasing in urban societies, but also so are diseases linked to the metabolic syndrome and characterised by a low-grade systemic inflammation. The increase in incidence is often blamed on urban life-style and diet. The fact that bacterial consumption and the resident gut microbiota can influence immune functions has often been neglected. Has urban living lead to an adversely altered bacterial flora in the gut, and can this change become a driving force for an over-reacting, dysfunctional immune system?
Humans have from the start of existence consumed large amounts of live bacteria by eating fermented foods as this is the simplest way to preserve sensitive raw-material for food. Depending on how the fermentation is carried out and what raw materials are being fermented, the product will contain varying concentrations of carboxylic acids (mainly lactic acid) and ethanol. Fermentation occurs spontaneously if food materials are stored with limited access to air and at temperatures above refrigeration. Certain types of bacteria, but also fungi such as baker's yeast, will grow in the product and convert some of the carbohydrates of the raw material to lactic acid, and often also to ethanol, acetic acid and carbon dioxide. The pH will go down to 3.5-4.0. The low pH together with organic acids and the high level of live bacteria keep away harmful microorganisms and protect from lipidoxidation. Due to the production of lactic acid, these types of bacteria which are dominant in lactic acid fermented foods are called lactic acid bacteria (LAB) which, hence, refers to a functional group of organisms, and not to a group of phylogenetic relationships.
When fermented products are consumed without heating, a large amount of living microorganisms are consumed. This, presumably, more or less daily supply of LAB that our ancestors have been exposed to through the centuries, has successively declined in industrialised countries. The food industry has preferred other means to process and secure the shelf-life of foods. Even several of the still remaining lactic acid fermented food products that today can be found in the shops are heat treated after the fermentation and, hence, do not contain any live LAB. LAB dominating the bacterial flora in fermented plant-based products as, for example sauerkraut, olives and capers are, typically, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus pentosus and Pediococcus species. In milk-based products, however, Lactobacillus paracasei and Lactobacillus rhamnosus, together with different species of Lactococcus and milk-streptococci, are often found after spontaneous fermentation while Lactococcus lactis, Streptococcus thermophilus and Lactobacillus delbrueckii often are used as starter cultures in dairy products.
The altered expression-profiles in the intestinal duodenal mucosa were analysed in humans after eating live or heat-killed Lactobacillus plantarum WCFS1 (van Baarlena et al., 2009). Both living and dead cells caused mucosal gene expression, but the expression profiles displayed differences in modulation of NF-κΒ-dependent pathways. Consumption of living L. plantarum induced a gene expression pattern that correlated with the establishment of immune tolerance (van Baarlena et al., 2009).
The urban consumer of today eats to a large extent either food that is free from living bacteria or food where bacteria have multiplied during storage in refrigeration, and these bacteria are primarily not LAB, but Gram-negatives, e.g. Pseudomonas or related genera and the genera of the family Enterobacteriaceae. Gram-negatives have lipopolysaccharides (LPS) in the cell-wall and LPS is a strongly pro-inflammatory compound which give a different message to the immune system. By designing probiotic food, the food industry in a way re-creates a more ancient diet, a diet lost somewhere in the Industrial revolution, a deficiency evolved by the requirements of long shelf-life and strictly standardised eating quality.
The Bacterial Heritage
It can be hypothesised that (i) infants inherit the mother's bacterial flora, (ii) the mucosa associated bacterial flora of the GI-tract is important for tuning of the immune system, (iii) urban individuals frequently have a disturbed bacterial flora in the GI-tract, with an increased proportion of pro-inflammatory bacteria, and (iv) a high proportion of pro-inflammatory bacteria in the GI-tract increases the risk for inflammation and a dysfunctional immune system. Under these presumptions, the bacterial flora of the gut becomes an important factor for the health status and may be especially important in infants.
The first bacteria a baby will meet are probably those present in the vagina of the mother, and interestingly a healthy vagina is dominated by lactobacilli (Vásquez et al., 2002). Unfortunately the incidence of bacterial vaginosis (BV) is high (20-30%) in urban women (Allsworth and Peipert, 2007). BV is a syndrome linked to increased risk for urinary tract infection, miscarriage and preterm delivery. BV is defined by loss of lactobacilli-domination in the vagina and foul smell (Carey et al., 2000; Onderdonk et al., 2003; Genc et al., 2004). Instead of lactobacilli the vagina is over-grown with, for example, Atopobium, Eggerthella, Gardnerella, Megasphaera, Leptotrichia and Sneathia (Thies et al., 2007; Ferris et al., 2007; Fredricks et al., 2007). Megasphaera and Fusobacteria (includes Leptotrichia and Sneathia) can normally be part of the dominating flora in the colon (Wang et al., 2005). Other prominent fractions of the colonic flora with tendencies to be spread to the vagina are Enterobacteriaceae and Bacteroides. Both have LPS in the cell wall, and so have Gardnerella, Megasphaera and Fusobacteria. Naturally, the baby will also be contaminated by faeces from the mother during birth.
Consequently, a baby carried by a mother with BV or with a disturbed colonic flora is, from the start, exposed to high proportions of pro-inflammatory bacteria and this is instead of meeting high doses of lactobacilli which, in provoked animal models, has shown to possess anti-inflammatory effects (Mao et al., 1996; Osman et al., 2004). Obviously there is a risk that the immune system of that baby will misinterpret the message of the environment and overreact, for example with a subclinical inflammation, allergy or even autoimmunity. Interestingly, oral administration of Lactobacillus acidophilus, or a mixture of Lactobacillus rhamnosus GR-1 and Lactobacillus fermentum RC-14 for two months, resulted in the cure of BV (Falagas et al., 2007).
Aggravated Immune Response
Rat dams that during pregnancy and suckling, had an increased proportion of Escherichia coli in the gut had off-spring that at the time of weaning showed increased levels of haptoglobin in the blood, i.e. the off-spring had a subclinical systemic inflammation (Fåk et al., 2008a). Off-spring from dams that received L. plantarum 299v did not get elevated haptoglobin levels, instead the gut growth was stimulated and the gut barrier function was improved (Fåk et al., 2008b). Human infants can occasionally be overgrown with E. coli (Wang et al., 2004). This means that the baby is being exposed to (i) high levels of LPS-containing bacteria, and (ii) a bacterial flora of low diversity. It has been shown that babies who at one week of age have a faecal flora of low bacterial diversity were likely to exhibit atopic eczema at the age of 18 months (Wang et al., 2008). In contrast, oral administration of Lactobacillus paracasei F19 to babies decreased the incidence of atopic eczema (West, 2008).
Diabetes type 2, non-alcoholic fatty-liver disease (NAFLD) and obesity are examples of diseases that are characterised by a low-grade systemic inflammation. It has been shown in mice that LPS in the blood (endotoxemia) is a starting point for insulin resistance and obesity (Cani et al., 2007). In humans it has been shown that a high-fat meal can induce endotoxemia, which is suggested to be the reason for an observed post-pandrial inflammation (Erridge et al., 2007). In mice, it was depicted that a high-fat diet increased LPS-containing organisms in the gut and increased transfer of LPS out into the blood which resulted in increased systemic inflammation (Cani et al., 2008).
In adult humans, orally administrated L. plantarum 299v has been shown to lower different inflammatory markers in the blood. In men with somewhat increased cholesterol levels, the cholesterol decreased after administration and so did the fibrinogen levels (Bukowska et al.,1998). In smoking men, the systolic blood pressure was decreased together with the levels of leptin, fibrinogen and IL-6 in the blood (Naruszewics et al., 2002). Also the adhesion of monocytes to non-stimulated or stimulated human endothel-cells was decreased (Naruszewics et al., 2002). In a prospective randomised trial of L. plantarum 299v to critically ill patients the concentrations of IL-6 was decreased in the blood after probiotic administration (McNaught et al., 2005).
The liver is a crucial organ for immune and metabolic control. Inflammation in the liver will, amongst other things, result in increased levels of inflammatory mediators not only in the liver but also in the blood. In an open study where different categories of patients with liver problems (NAFLD, alcoholic liver cirrhosis and hepatitis C virus, respectively) were given orally a mixture of strains of the species Lactobacillus casei, L. plantarum, L. acidophilus, Lactobacillus delbrueckii subsp. bulgaricus, Bifidobacterium longum, Bifidobacterium brevis, Bifidobacterium infantis and Streptococcus thermophilu for three months, it was shown that aspartate aminotransferase, alanine aminotransferase and bilirubin (markers for liver function) decreased in all patients, markers for lipid peroxidation (oxidative stress) decreased in the patients with NAFLD and alcoholic liver cirrhosis, and TNF-alfa and IL-6 decreased while IL-10 increased in patients with alcoholic liver cirrhosis (Loguercio et al., 2005). The increasing occurrence of obesity and limited physical activity have led to an increase in the prevalence of NAFLD. NAFLD is currently considered as one component of the metabolic syndrome (Federico et al., 2006).
The Gut Barrier
The mucosa of the GI-tract makes up a substantial surface and the status of this surface is in many aspects crucial for the well-being of the individual. A leaky mucosa will lead to translocation of pathological agents, for example LPS, and the consequence will be inflammation which characterises several diseases (fig. 2). In acute phases, mass-translocation can come about, leading to abscesses, sepsis and organ failure. In a variety of animal models, probiotic administration has been shown to suppress translocation, e.g. in liver injury (Adawi et al., 1997; Osman et al., 2007), colitis (Mao et al.,1996; Osman et al., 2004 ), and pancreatitis (Mangiante et al., 2001; Rychter, 2009). There is even some tentative evidence in humans: for example, L. plantarum 299 decreased the permeability of the gut mucosa in critically ill patients (Klarin et al., 2008). A mixture of two strains of L. rhamnosus and Lactobacillus reuteri given to children with atopic dermatitis, resulted in a decrease of the intestinal permeability and alleviated the symptoms of dermatitis (Rosenfeldt et al., 2004).
Several explanations for how the probiotics improve the barrier effect of the mucosa can be valid, but the consequence that permeability of the mucosa is decreased is important in many different settings, and probably one of the more important out-comes of probiotic administration.
A disturbed bacterial flora of the GI-tract might be one factor contributing to the increasing incidence of diseases based on dysfunction in the immune system, such as diabetes 1 and allergy, and diseases characterised by subclinical inflammation such as diabetes type 2, NAFLD and obesity. Daily intake of live, harmless bacteria might be an overlooked dietary component of importance for the well-being. Health claims for probiotics have so far mostly been associated with gut health, but interesting new fields to explore are the effects against systemic inflammation and immune-modulation, perhaps especially important in early life.
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