Probiotics: strains matter

The tendency to generalise about 'probiotic' effects is widespread. Underlying such generalisations is the erroneous assumption that the body of research on specific probiotic strains can be applied to any product marketed as a probiotic. Mary Ellen Sanders, PhD, explains why not just any probiotic will do for your next product launch

Probiotics are live microbes that, when administered in adequate amounts, confer a health benefit on the host.1 There is mounting awareness in the United States about probiotics, spurred in part by a growing number of high-profile probiotic-containing products, such as Dannon's successful Activa yoghurt. The potential of this product category seems evident, especially when considering the success of such products in Europe and Asia. One report estimated that more than 100 companies in the United States market probiotic supplements and approximately two million US adults use them.2

Furthermore, there are many people who stand to benefit from efficacious probiotic products. Studies documenting effects on a variety of gastrointestinal disorders including irritable bowel syndrome (IBS), vaginal infections and immune enhancement resulting in getting sick less often are compelling reasons for including probiotics as part of a healthy diet.3,4,5

Probiotic complexities
When considering this product area, a few guidelines can help provide context for understanding the complexities of the science and use of probiotics.

Although a consensus scientific definition has been advanced, no legal definition of the term 'probiotic' exists. The term unfortunately can be (and is) used on products that do not meet the minimum criteria that the probiotic be alive, delivered in adequate dose (through the end of shelf life); and shown to be efficacious in controlled human studies.6 The 'truthful and not misleading' FDA standard for content and support of structure/function label claims on products is not, in practice, enforced by the FDA. Therefore, it is incumbent on the industry to maintain integrity in formulation and labelling of these products so that consumers can be confident in this product category.

There is little scientific evidence that in order to qualify as probiotics, these beneficial bacteria must demonstrate specific physiological attributes such as be of human origin, adhere to intestinal cells or produce bacteriocins. Although many have used these criteria as a basis for selection of strains 'appropriate' for use as probiotics, no studies have compared isogenic strains (ie, strains that are identical genetically except with altered capacity for one specific attribute) with and without these traits in humans to determine their significance. Until such studies are conducted, it is more productive to focus on proof of their ability to improve human health, regardless of mechanism. Notably, one research group did conduct a study of colitis in mice comparing physiological effects of isogenic strains of L. crispatus which differed only in their ability to auto-aggregate (or clump).7 They found that the aggregating strain, but not the non-aggregating mutant or heat-killed aggregating strain, reduced severity of colitis.

Another attribute that is sometimes questioned is the requirement that probiotics be alive. It is true that some research shows beneficial effects from cells killed by heat or radiation.8 By definition, however, these substances do not qualify as bona fide probiotics. Studies have demonstrated superior activity of live compared to killed probiotics, in in vitro and in human studies.9,10,11 Even though not all studies show an advantage to viability;12,13 probiotics by definition must be administered alive.

Strain-specific effects
There is one particularly important consideration for probiotics: strain-dependent effects. Just as different breeds of dogs have attributes that are distinguishing, so too different strains of even the same species of bacteria may have different probiotic functions.

The scientific rationale that effects must be considered strain-specific is based mostly on in vitro and animal data where strain differences are evident. Attributes such as acid tolerance, sensitivity to therapeutic antibiotics, bile resistance, lactase activity, hydrogen-peroxide production, growth on prebiotics, genetic accessibility, production of antimicrobial compounds and stability in product have all been tested for a variety of strains in vitro.14,15,16,17,18,19 Among tested strains, differences are clear. Frequently such testing compares strains of different species, but less commonly comparison of multiple strains of the same species has been conducted in in vitro tests.

In animal models, differences in responses evoked in tests of immune function are apparent. When one strain of each of Lactobacillus salivarius Ls-33 and Lactobacillus rhamnosus were tested via oral administration in a mouse model of colitis, researchers observed significant reduction in inflammation. However, one strain each of Lactobacillus acidophilus, Lactococcus lactis and Streptococcus gordonii showed no improvement.20 The importance of testing specific strains for effects is further emphasised in a study that documented that a strain of L. paracasei isolated from an endocarditis patient actually worsened colitis in an animal model of severe inflammation.21

It is possible to visualize differences among strains of the same species at the DNA level as well. The chart below illustrates such differences among several strains of L. crispatus. Such results are typical among strains of lactobacillus species. Interestingly, findings with commercial bifidobacterium strains suggest more genetic similarity among strains of the same species.

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In one recent assessment, researchers found that among 39 independent isolates of B. animalis subspecies lactis strains from commercial products, only four different types were identified, based on pulsed field gel electrophoresis (PFGE). 22 This finding may reflect a de facto greater similarity among strains of the same bifidobacterium species, that commercial products contain the same strains, or that differences are not evident from this type of chromosomal analysis. Interestingly, large strain-specific differences in immunopotential were observed among different strains of the same bifidobacterium species. 23

Strain-specifics: human data
Head-to-head comparisons of different strains in human studies are rare. For example, B. lactis BB-12 was compared to L. reuteri SD2112 and to a placebo in a study determining the impact of supplementing infant formula with one of these strains at identical doses on incidence, symptoms and absences due to intestinal or respiratory infections in infants in day-care centers.24 Both strains showed statistically significant improvements over the placebo control; however, the L. reuteri group outperformed both BB-12 and the placebo control, with significant decreases in number of days with fever, clinic visits, child-care absences and antibiotic prescriptions. The rate and duration of respiratory illnesses did not differ significantly among groups.

In another clinical study, B. infantis 35624 was compared to L. salivarius UCC4331 for its ability to reduce symptoms of irritable bowel syndrome.25B. infantis 35624 improved symptoms, whereas L. salivarius UCC4331 did not.

What these studies do not tell us is if two strains of the same species would have performed equivalently. However, it cannot be presumed that they will.

The implications of the strain-specificity of effects are:

  1. Documentation of health effects must be conducted on the specific strain being sold.
  2. Review articles that discuss the many studies done on specific strains are not sufficient evidence to support health effects of an untested strain.
  3. Studies that document efficacy of specific strains at a specific dose are not sufficient evidence to support health effects at a lower dose (see 'Formulators' sidebar, below).
  4. The role of carrier in delivering functional benefits is not well understood (see 'R&D' sidebar, below).

This issue is complicated by the fact that the mechanisms that lead to specific health effects are often not known. When these are better understood, it may be possible to predict functionality in vivo. Certainly there are some physiological characteristics that are present in essentially all strains of given species. Such physiological similarities contribute to their grouping into the same species. For example: reuterin production by L. reuteri, although levels produced vary by strain;26 high in vivo lactase activity in strains of S. thermophilus;27 lactate production by all lactobacilli and acetate production by bifidobacteria. However, how such physiological or metabolic characteristics are expressed in vivo among different strains drives the need to confirm functionality in the target host.

Taken from a different perspective, the current body of published literature suggests that similar effects are observed for a variety of different strains. Effects on diarrhoeal illnesses, enhanced immune responses and improvement of symptoms of lactose intolerance are associated with more than one strain. Therefore, multiple strains of the same species may in fact have functional traits in common. But they may not. So again, studies on the specific strains are still needed.

Be pro-active
The tendency to generalise about 'probiotic' effects is widespread. Underlying such generalisations is the erroneous assumption that the body of research on specific probiotic strains can be applied to any product marketed as a probiotic. Although it is cumbersome to always need to define what strains at what doses are known to lead to health effects, it is essential to do so to prevent misrepresentation of a product.

In addition, commercial products that are marketed with no specific human studies documenting effects should not be marketed as probiotics, but perhaps as 'potentially beneficial cultures.' The term 'probiotic' should be used only for products composed of microbes that are alive, delivered in adequate dose (through the end of shelf life), and shown to be efficacious in controlled human studies (see box, below). As mechanisms leading to probiotic effects are better understood, perhaps extrapolation of results from certain strains to strains possessing the pertinent biological and physiological traits will be possible. Certainly, genomic sequencing efforts will grease the wheels for such advances.28

For more information, see and

What levels of probiotics need to be delivered in products?
Probiotic levels used in product offerings must be based on levels found to be efficacious in human studies. Although it is tempting to offer a general recommendation for a minimum amount of probiotic that is needed to be effective, the reality is that such generalisations cannot be accurate.

This is because efficacious doses vary widely in documented human studies. For example, several studies of L. reuteri SD2112 and of B. infantis 35264 have documented that 1x108 (100 million)/day is an adequate dose for several different health targets. However, the product VSL#3 is recommended at 1.8x1012 (1.8 trillion)/day for management of recurrence of certain inflammatory bowel conditions. This is over a four-log cycle difference in recommended dose.

It is likely that the required dose is dependent on a variety of factors, including physiological characteristics of strains being used, types of clinical endpoints being tracked, whether the endpoint is prophylactic or therapeutic, length of time of administration of probiotic, and if other bioactive ingredients are used in conjunction with the probiotic.

Unlike other food/nutrient issues, where a company can get away with putting in one-fourth the daily recommended amount with the understanding/hope that a consumer will get nutrient sources from places other than the specific functional food, there are not many other natural food sources to attain probiotics.

Food manufacturers should therefore include the required efficacious amount in each serving. Finally, probiotic product labels should indicate the levels of each strain in the product — through the end of shelf life. This information should be tied directly to scientific publications that document that this formulation is efficacious. Labelling of levels 'at time of manufacture' is inadequate and has been shown to be linked to products less likely to meet label claims (



Human data

Tested as single strains

L. rhamnosus GG

Immune enhancement, infectious diarrhoea in children, primary prevention of atopic dermatitis

B. lactis BB-12

Immune enhancement, diarrhoea in children

L. reuteri SD2112

Reduced absences from work, diarrhoea, immune function

B. infantis 35624

Irritable bowel syndrome (IBS)

L. casei DN114-001

Immune enhancement

B. longum BB536

Allergy symptoms, intestinal micro-ecology

L. acidophilus NCFM

Symptoms of lactose intolerance, reduced small-bowel bacterial overgrowth

B. lactis HN019 (DR10)

Immune enhancement, especially in elderly

B. animalis DN173-010

Normalizes intestinal transit time

L. plantarum 299V

IBS, post-surgical gut nutrition

Lactobacillus casei Shirota YIT9029

Superficial bladder-cancer recurrence, intestinal microbiota, immune enhancement

L. salivarius UCC118

Inflammatory bowel disease

L. johnsonii La1 (Lj1)

Immune function, Helicobacter pylori eradication

Escherichia coli Nissle 1917

Immune function, intestinal health

Saccharomyces cerevisiae (boulardii) lyo

Antibiotic-associated diarrhoea, Clostridium difficile infections

S. thermophilus (most strains)

Symptoms of lactose intolerance

Tested as blends

L. rhamnosus GR-1 + L. reuteri RC-14

Oral consumption leads to colonization of vaginal tract and improved therapeutic outcome for women being treated for bacterial vaginosis

VSL#3 (8 strain blend of S. thermophilus, four strains of lactobacillus and three strains of bifidobacterium)

Inflammatory bowel conditions

L. acidophilus (CUL60)
B. bifidum (CUL 20)

Reduction of C. difficile toxin in feces

L. acidophilus (CUL60)
B. bifidum (CUL 20)

Reduction of C. difficile toxin in feces

L. helveticus R0052
L. rhamnosus R0011

H. pylori eradication, diarrhoea in children

Select suppliers
Probiotics and prebiotics are growing quickly
Biogaia: Reuteri culture comes in three different, producer-friendly forms: freeze-dried powder, freeze- dried DVS (Direct Vat Set) granules, and frozen pellets.

Chr Hansen: The nu-trish brand probiotic culture range consists of Probio-Tec, Yo-Fast and other nu-trish culture blends with well-defined viscosity profile that ferment quickly.

Danisco: Cultures division produces, develops and markets starter cultures, media, coagulants and enzymes for cheese, fresh dairy and other food products, and also supplies probiotic cultures for foods and supplements as well as natural food protectants.

DSM: Lafti line of probiotics are formulated for stability, survivability and concentration, and contains L. acidophilus (Lafti L10), L. casei (Lafti L26), and bifidobacterium (Lafti B94).

GTC Nutrition: NutraFlora short-chain fructo-oligosaccharides (scFOS) are a cane- or beet sugar-derived natural prebiotic fibre.

Jintan: Custom-makes triple-layered, enteric, seamless capsules specifically for probiotic supplements.

Lallemand: Canadian supplier delivers novel, high-quality probiotics and biosupplements to the nutraceuticals, functional-foods and pharmaceuticals industries.

National Starch: Hi-Maize brand corn-based resistant starch has multiple benefits, among them it acts as a prebiotic for digestive health.

Nutraceutix: Bio-tract protects probiotic tablets from acids, LiveBac extends shelf life of probiotics, and 20 strains are offered by the company, which controls manufacturing processes from start to finish.

Orafti: BeneoSynergy1 is the unique, patented oligofructose-enriched inulin prebiotic used in the landmark SynCan project on synbiotics and colon cancer.

Probi: Biotech company develops and patents probiotic strains, among them L. plantarum 299v and L. rhamnosus 271. L. plantarum 299 has not yet been commercialised, but it is in the out-licensing phase.

Roquette: Nutriose is a range of soluble fibres that are resistant corn dextrin with 85 per cent fibre content (dry substance), which studies show matches up well with probiotics Streptococcus thermophilus and Lactobacillus bulgaricus.

Sensus: Frutafit inulin and Frutalose fructo-oligosaccharides (FOS) are soluble dietary fibres with bifidogenic/prebiotic properties, suitable for a variety of food systems to enrich fibre, reduce calories, and replace sugars and fats.

Valio: Lactobacillus rhamnosus GG probiotic is the most researched in the world and was recently licensed to Dannon for the US yoghurt market. The Gefilus family containing LGG is marketed worldwide.

1. FAO/WHO. 2001. Health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria.
2. Trueman S. Are you pro-biotic? Bugs are finding their way into our food—and that's a good thing. Nerac Analyst, March 2007., accessed April 16, 2007.
3. Floch MH, et al. Recommendations for probiotic use. J Clin Gastroenterol 2006 Mar;40(3):275-8.
4. Marteau P, et al. Cellular and physiological effects of probiotics and prebiotics. Mini-Rev Medicinal Chem 2004;4:889-896.
5. Sanders ME. Probiotics: considerations for human health. Nutr Rev 2003;61:91-99.
6. FAO 2002. Guidelines for the evaluation of probiotics in food.
7. Castagliuolo I, et al. Beneficial effect of auto-aggregating Lactobacillus crispatus on experimentally induced colitis in mice. FEMS Immunol Med Microbiol 2005 Feb 1;43(2):197-204.
8. Simakachorn N, et al. Clinical evaluation of the addition of lyophilized, heat-killed Lactobacillus acidophilus LB to oral rehydration therapy in the treatment of acute diarrhea in children. J Ped Gastroenterol Nutr 2000;30:68-72.
9. Zhang L, et al. Alive and dead Lactobacillus rhamnosus GG decrease tumor necrosis factor-alpha-induced interleukin-8 production in Caco-2 cells. J Nutr 2005 Jul;135(7):1752-6.
10. Cruchet S, et al. Effect of the ingestion of a dietary product containing Lactobacillus johnsonii La1 on Helicobacter pylori colonization in children. Nutrition 2003 Sep;19(9):716-21.
11. Gotteland M, et al. Effect of Lactobacillus ingestion on the gastrointestinal mucosal barrier alterations induced by indometacin in humans. Aliment Pharmacol Ther 2001 Jan;15(1):11-7.
12. Peng GC, Hsu CH. The efficacy and safety of heat-killed Lactobacillus paracasei for treatment of perennial allergic rhinitis induced by house-dust mite. Pediatr Allergy Immunol 2005 Aug;16(5):433-8.
13. Rachmilewitz D, et al. Toll-like receptor 9 signaling mediates the anti-inflammatory effects of probiotics in murine experimental colitis. Gastroenterology 2004 Feb;126(2):520-8.
14. Asahara T, et al. Probiotic bifidobacteria protect mice from lethal infection with Shiga toxin-producing Escherichia coli O157:H7. Infect Immun 2004, 72(4):2240-7.
15. Tallon R, et al. Strain- and matrix-dependent adhesion of Lactobacillus plantarum is mediated by proteinaceous bacterial compounds. J Appl Microbiol. 2007 Feb;102(2):442-51.
16. Foligne B, et al. Correlation between in vitro and in vivo immunomodulatory properties of lactic acid bacteria. World J Gastroenterol 2007; 14;13(2):236-43.
17. D'Aimmo MR, et al. Antibiotic resistance of lactic acid bacteria and Bifidobacterium spp. isolated from dairy and pharmaceutical products. Int J Food Microbiol 2007 Apr 1;115(1):35-42. Epub 2007 Jan 2.
18. Sanders ME, et al. Performance of commercial cultures in fluid milk applications. J Dairy Sci 1996 Jun;79(6):943-55.
19. Olivares M, et al. Antimicrobial potential of four Lactobacillus strains isolated from breast milk. J Appl Microbiol 2006 Jul;101(1):72-9.
20. Foligne B, et al. Probiotics in IBD: mucosal and systemic routes of administration may promote similar effects. Gut 2005 May;54(5):727-8.
21. Daniel C, et al. Selecting lactic acid bacteria for their safety and functionality by use of a mouse colitis model. Appl Environ Microbiol 2006 Sep;72(9):5799-805.
22. Masco L, et al. Culture-dependent and culture-independent qualitative analysis of probiotic products claimed to contain bifidobacteria. Int J Food Microbiol. 2005 Jul 15;102(2):221-30.
23. Pot B. Personal communication.
24. Weizman Z, et al. Effect of a probiotic infant formula on infections in child care centers: comparison of two probiotic agents. Pediatrics 2005 Jan;115(1):5-9.
25. O'Mahony L, et al. Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterology 2005 Mar;128(3):541-51.
26. Talarico TL, et al. Production and isolation of reuterin, a growth inhibitor produced by Lactobacillus reuteri. Antimicrob Agents Chemother. 1988 Dec;32(12):1854-8.
27. Guarner F, et al. Should yoghurt cultures be considered probiotic? Br J Nutr. 2005 Jun;93(6):783-6.
28. Klaenhammer TR, et al. Genomic features of lactic acid bacteria effecting bioprocessing and health. FEMS Microbiol Rev. 2005 Aug;29(3):393-409.)

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