Gut reaction

The concept of "good bacteria" in the gut has been accepted by a large number of consumers. So much so, that the probiotic drinks segment has been one of the fastest growing areas in functional foods in the past few years. To remain successful, however, it is essential that consumers trust probiotics to actually deliver beneficial effects. But this trust can only grow if individual consumers experience the claimed benefits and if claims are based on solid evidence.Probiotic strains have been shown to have beneficial effects on health. However, the effects vary among strains, which is why a tailor-made approach is required in the research with a specific strain.

Under the EU Nutrition and Health Claims Regulation, regulators make a distinction between article 14 health claims, which refer to reduction of disease risk or children's health and development, and article 13 claims, which cover nutrient function, psychological and weight management claims based on 'generally accepted scientific evidence', and are now being evaluated by the European Food Safety Authority (EFSA).

In the preliminary list compiled by European trade associations, a whopping 74 claims relate to health benefits of probiotic strains. However, given that an important criterion for success is that claim support should be based on good quality human intervention studies, it seems unlikely that all 74 will make the grade.

Although EFSA has published guidance on substantiating article 14 claims and claims based on emerging science (article 13.5), this does not reveal which outcome/effect markers are considered acceptable to establish the claimed effect. This issue has been addressed by European scientists collaborating in the PASSCLAIM project, and for most of the health benefits claimed for probiotics in the preliminary article 13 list, markers have been described. However, the claimed effect should be relevant for human health - not easy to quantify for all target functions.

What does 'gut health' mean?​

Parameters of bowel habit are relatively well defined. PASSCLAIM tried to answer the question: "What would constitute a significant beneficial change in bowel habit?", and concluded that for most European populations an increase in mean daily stool weight of 25% might be described as significant, 50% as beneficial. This is based on observations that mean daily stool weight is about 100g/day, and should be above 150g/day to obtain any protection from colorectal cancer. For transit time, any dietary change that would bring transit time into the normal range of 1-4 days is beneficial.

To assess an effect on the composition of the gut microbiota, changes can be assayed in faeces, with a range of molecular techniques. Assuming a typical faecal lactobacilli count of 105/g faeces, and 106/g for bifidobacteria, a one-log increase in response to a functional product may be expected, but the relative response depends on the base level.

Overall competence of mucosal barrier function can be assessed non-invasively in intestinal permeability studies in human subjects. Urinary excretion of orally administered test substances, mostly sugars of different molecular size (lactulose and mannitol), is measured so that transcellular and paracellular permeability pathways can be measured at the same time. Improved function of the mucosal barrier is shown by a more or less similar urinary excretion of the paracellularly transported component (lactulose) and increased excretion of the transcellular marker (mannitol), resulting in a decreased lactulose/mannitol ratio. However, this marker is only applicable to the barrier of the small intestine, not the colon.

Measuring immune system response​

The immune system is a complex structure with large individual variations. There is no single marker for either its status or functional capacity. In vivo measures of immune competence are considered superior when predicting host resistance to infections. The ILSI Europe Task Force on Nutrition and Immunity in Man defined a set of relevant markers of immune function to measure enhanced immune function in response to a nutritional intervention and recommend the use of a combination of markers, including: vaccine-specific serum antibody production, delayed-type hypersensitivity response, vaccine-specific or total secretory IgA in saliva (all three representing adaptive immune function) and the response to attenuated pathogens (combination of innate and adaptive immune function). However, measuring a change in susceptibility to infectious disease is the most significant outcome.

Human studies with probiotics​

Specific probiotic strains have been shown to be effective in decreasing the severity and duration of acute diarrhoea through rotavirus infections in children as well as travellers' diarrhoea in adults. Meta-analyses show that probiotics significantly reduce antibiotic-associated diarrhoea in children and adults.

Several controlled human trials have also addressed the effect of probiotics on the common cold and other respiratory tract and gastrointestinal infections. Probiotic bacteria were found to shorten common cold episodes and reduce severity of symptoms, to reduce the frequency of sick-leave in a working population and to decrease the number of days with fever, clinic visits, child care absences and antibiotic prescriptions. Results for the specific strains tested should be confirmed in other studies to strengthen the evidence.

Probiotics are also used as nutritional intervention to fight allergies, with the evidence appearing to be stronger for prevention of atopic disease than for treatment of atopic dermatitis.

Inflammatory bowel disease (IBD) can involve problems in the development of the gut's immune response. Gut microbiota play a role in the inflammatory responses in Crohn's disease and ulcerative colitis. Modifying the intestinal environment through probiotics may be helpful in the treatment of IBD. A recent review by Helman concludes that the human trial evidence of clinical benefit in IBD from treatment with probiotics is mixed. Outcomes are predominantly positive for ulcerative colitis, but for Crohn's disease most studies found no effect.

Consumption of certain probiotic bacteria may also result in reduced lactose intolerance by preventing symptoms of intolerance in the large intestine in addition to or rather than by improving lactose digestion in the small intestine.

Probiotics and health claims​

Some of the probiotic strains currently on the market have been extensively studied. Examples are Bifidobacterium lactis BB-12​ and Lactobacillus acidophilus LA-5​ (Chr Hansen); L. casei Shirota​ (Yakult); L. rhamnosus GG​ (Valio); L. casei F19​ (Arla Foods); L. casei DN-114001​ and B. animalis DN-173010​ (Danone). In the Netherlands, three products containing probiotics have passed the test for scientific substantiation: Yakult, Danone and Campina have submitted a dossier to substantiate a health claim for their respective probiotic strains to an expert panel, according to a voluntary "Code of Practice". The experts judged that there is enough scientific evidence for Danone Activia yoghurt containing B. animalis DN-173010​ to show that consumption stimulates the intestinal transit in subjects with slow transit times and increases the number of bifidobacteria in the large intestine, which may support well-balanced gut microbiota.

Yakult may claim that its fermented milk product containing L. casei strain Shirota​ (LcS), may improve bowel habit in subjects susceptible to constipation and support a well-balanced gut microbiota through an increased number of lactobacilli.

The approved health benefit of Campina's Vifit with LGG (L. rhamnosus Goldin & Gorbach)​ is that it supports the barrier function of the intestine.

How do we know they work?​

The efficacy of probiotics can also be dependent on the ability of the bacteria to reach the target site alive. However, the ability to study what is going on in the intestines of healthy people is fairly limited. One option is to test for the presence of probiotics in faeces. A more sophisticated approach is to use a validated, dynamic, computer-controlled in vitro model of the human gastrointestinal tract such as the 'TIM' model gut systems developed by TNO, which allow detailed study of probiotic survival at different sites and time periods.

In order to rationally design the most appropriate human study, you have to know which health effects are induced in the host by a specific strain. The more insight into the mechanisms you have, the more able you are to select the most appropriate markers.

Probiotic strains can exert their functional effects through various mechanisms. Screening assays can be applied to identify which mechanism is associated with a particular strain.

For example, you can screen for inhibition of pathogenic organisms using models of the gut. This allows you to identify changes in the composition of the microbiota and the activity of pathogens. TNO recently developed a multi-channel micro-gut system in which the intestinal microbiota can be cultured as well as kept in a stable condition.

This system allows the simultaneous measurement of the effects of hundreds of food components - or combinations of them - on the composition and activity of the intestinal microbiota. The technique is based on the analysis of bacterial DNA and is independent of information from genome databases. The DNA profile is the basis for a customised microbiota micro-array, the Intestinal-Chip, or I-Chip.

After such a pre-screening, selected compounds, strains or combinations can then be evaluated in TIM-2, which closely mimics the conditions in the colon, for instance by the removal of microbial metabolites, which prevents accumulation of these metabolites, something which would otherwise lead to the arrest of metabolic activity of the microbiota.

The TIM-2 system has been used to evaluate the fate and effect of a number of dietary carbohydrates and probiotics. Products can be screened for increased production of butyrate, the major energy source of colonic epithelial cells, and a metabolite considered to be beneficial to health. Newest developments include the use of stable-isotope labelled substrates to trace the label into metabolites produced and into microbial biomass.

To test for an effect on epithelial barrier function, so-called Ussing chamber experiments with intestinal biopsies are the most commonly used in vitro method. An Ussing chamber consists of two compartments that are separated by a biopsy of tissue which is supposed to have a function in blocking, selecting or transporting materials or in communicating with the external world. Transwell models of epithelium under stress can also be used to study effects on permeability. Time and cost-effective screening models are now being developed that look at upregulation of tight junction proteins to select food components that may strengthen epithelial barrier function.

Immunomodulation by probiotics can be investigated by using either in vitro assays or, often in a later stage, in vivo animal models. Dendritic cells (DC) and regulatory T-cells (Treg cells) play a pivotal role in some of the more recently developed assays. DC are important in driving the differentiation of naïve T-helper (TH) cells into either TH1, TH2, or Treg cells (Kalinski, 1999). They are able to discriminate between different pathogenic and non-pathogenic compounds through the expression of various pattern-recognition receptors. Besides the toll-like receptors (TLR) these include the C-type lectin (CTL) receptors, which recognize carbohydrate structures.

It is speculated that certain probiotic bacteria are able to prime DCs for the capacity to drive the development of Treg cells, which may play a role in the prevention or alleviation of excessive inflammation, allergy or autoimmune diseases. The use of receptor binding assays and induction of Treg cells may predict efficacy, further to be tested in animal models and/or human trials.

In vivo models should be selected based on how appropriate they are to test specific functionalities.

Examples of such models are in vivo rodent models of food allergy, which may also be used to study the effect of probiotic bacteria in the prevention of allergen sensitization. Intestinal immune homeostasis is probably dependent on a delicate balance between triggering of the innate immune system via TLR and tolerisation via CTL; perturbation of this balance may result in chronic inflammation of the gut. To evaluate the immunomodulatory effects of food ingredients and probiotics on inflammation, a chronic colitis model was developed using BALB/c mice. In a preliminary study, administration of L.plantarum​ resulted in significant inhibition of various aspects of colitis, including changes in serum levels of specific cytokines and numbers of T-cell subsets in the mucosa of the colon. Similar immune-modulatory effects have been reported for the consumption of fermented milk with probiotic bacteria.

The future​

Although the number of probiotic products carrying an approved health claim is limited, there is an increasing body of evidence supporting various health benefits.

Evidence from human intervention studies is essential, addressing specific strains, dosage and matrix effects and making use of appropriate study populations, control treatment and effect markers or endpoints. Application of innovative, predictive in vitro and in vivo models in the preclinical phase can help to find the health relationship that is most likely to be substantiated in subsequent human trials.

Health claims for probiotics related to bowel habit and composition of gut microbiota have been approved already. The evidence that certain probiotics may prevent rotavirus diarrhoea is quite convincing. Other health benefits will need additional confirmation to convince authorities that a health claim is warranted, but the final proof seems to be within reach for some of the strains that are on the market now.

This was written by Alwine Kardinaal, Coby Eelderink and Koen Venema from TNO Quality of Life, Zeist, The Netherlands. TNO is Europe's largest independent research company operating in a variety of fields from food research to defence. References are available upon request. http://www.tno.nl​​

Main markers​

Bowel habit​

Measured by:

• Frequency of defecation

• Stool consistency and form

• Stool weight

• Transit time

Composition and activity of gut microbiota​

Measured by:

• Plating faecal microorganisms

• Molecular techniques

• Model fermenter systems only for mechanistic research

Mucosal barrier​

Measured by:

• Bacterial translocation (in vivo)

• Intestinal permeability (in vivo)

• Surface hydrofobicity (ex vivo)

• Ussing chamber (ex vivo)

Functional capacity of immune system​

Measured by:

• Responses to challenges (antibody response, secretory IgA

measured by response, DTH response)

• Incidence and severity of infections

• Measuring specific cell functions ex vivo

Reduction of disease risk - acute diarrhoeal disorders, inflammatory bowel disease, allergy​

Measured by

• Prevalence of the condition after appropriate follow-up

Source: Cummings et al, 2004​

Probiotic health claims​

As submitted for approval under article 13 of the EU Nutrition and Health Claims Regulation​

Digestive health

• Promotes/maintains intestinal health

• Helps to reduce gastrointestinal discomfort or complaints

• Decreases harmful substances in the gut

• Protects the intestinal tract

• For good digestive health/ normal/good digestion/ maintains/supports a healthy digestive system

• Promotes/stimulates/supports good bowel movement/normal bowel regularity

• Helps against slow transit/to improve your natural digestive transit

• Improves stool frequency

Intestinal flora

• Supports healthy gastrointestinal flora/maintains healthy gut floraBifidogenic properties

• Balances/beneficially affects the intestinal microflora

Natural defence/immune system

• Stimulates natural resistance in the digestive tract

• Supports/strengthens/activates/increases natural defence

• Stimulates/actively supports immune system/helps to restore immune balance

• Increases resistance of organism against bacterial contamination

• Helps regulate the body's immune response

Urogenital tract/vaginal health

• Supports/promotes/maintains a healthy vaginal microflora

• Restores and maintains normal vaginal microflora

• Helps during treatment of urogenital disorders

Skin health

• Helps preserve skin health

• Helps fight against UV damage

• Reinforces skin defences altered by UV

• Helps reinforce skin barrier function

• Helps reduce reactivity/sensitivity