The Science Behind Probiotics and Prebiotics

You’ve got to hand it to Dannon. At a time when E. coli scares put product safety under the microscope, the yogurt giant has convinced notoriously squeamish Americans to spend as much as twice the going rate on yogurt that’s literally infested with bugs.

How did they do it? Not by exploiting some latent affection for microbes, but by shining the steely eye of science on both the safety and the outright advantages of the bacteria in question.

That bacteria would be Bifidus animalis DN-173010—trade name Bifidus Regularis—and the product it inhabits is Activia, Dannon’s blockbuster and a bellwether of probiotic formulation to come. Activia is a bona fide phenomenon, posting nearly $2 billion in worldwide sales in 2006, ranking it among the most-successful product launches in U.S. food-biz history, according to BusinessWeek.

Dannon deliberately pitched its probiotics as capable of surviving the stomach into the large intestine, where they modify intestinal transit to alleviate uncomfortable bloating and aches. As a result, gut health is a topic for polite conversation. And that’s no mean feat. As Terri Rexroat, global product manager, lactic cultures, Cargill Texturizing Solutions, Minneapolis, points out, “Increasing consumer awareness and education was the first real step toward removing the U.S. societal stigma associated with talking about intestinal and digestive health.”

Gut feelings

Probiotics are not your average bacteria. In 2002, an expert panel of the Food and Agriculture Organization of the United Nations (FAO) defined probiotics as “live microorganisms administered in adequate amounts which confer a beneficial health effect on the host.” By definition, these bugs are good for us. And their benefits begin in the gut.

The lumenal surface of the large intestine is the front line in our immune defense, the prime interface between outside and in. Its ability to carry out its duties owes greatly to its microfloral makeup. The colon contains thousands of bacterial species numbering as high as 1011 to 1012 cells per gram of contents. Many of these bugs shore up the barrier function of our intestinal lining, guarding against infections and allergies in the process. Some help metabolize indigestible dietary components—fiber and, in the case of the lactose-intolerant, lactose. Some produce vitamins that we can’t produce on our own. And others metabolically “defuse” dietary carcinogens, mutagens and toxins.

Stressing the strain

For an intestinal troop to rank as a full-fledged probiotic, it’s got to be the right caliber. While all probiotics are friendly organisms, “not all friendly organisms are considered to be probiotics,” Rexroat says.

That’s why, in addition to the FAO definition, the joint FAO/World Health Organization (WHO) “Guidelines for the Evaluation of Probiotics in Food” set out further criteria, starting with the requirement that probiotic bacteria display documented benefits not merely at the genus or species levels, but at the level of the strain. Beyond that, the probiotic must reside in an internationally recognized culture bank to allow for replication of current and future research; must have undergone sufficient and appropriate in vitro and animal trials to support its benefits; must maintain viability at required levels during a product’s normal shelf life to achieve benefits in the host; must be subject to further studies in the host; and—above all—must be safe.

By these lights, most probiotic strains fall into the genera Lactobacillus and Bifidobacterium, plus some Streptococcus thermophilus, Escherichia, Enterococcus and Bacillus strains, and one strain of Saccharomyces cerevisiae yeast, called S. boulardii. Their presence promotes a healthy gut environment through their production of lactic and acetic acids, which drive off pathogens by lowering gut pH. More remarkable are the hundreds of published clinical and observational studies detailing their effects not only on the body’s normal functions, but on specific diseases.

The website USProbiotics.org gives a comprehensive rundown of these benefits, and the state of the science it lists hints at possible roles in everything from allergy prevention to alleviating symptoms of irritable bowel syndrome and inflammatory bowel diseases to preventing kidney-stone formation. Studies have investigated probiotics’ ability to decrease risk for certain cancers, lower blood pressure and cholesterol, reduce incidence of dental caries, and even battle Helicobacter pylori.

No matter the benefit, experts stress the importance of strain. As Peggy Steele, global business director, probiotics, Danisco Cultures, Madison, WI, says: “All probiotic health benefits are demonstrated via scientific studies using a specific strain or blend of strains for which structure/function claims can be made. Benefits documented with one strain cannot necessarily be transferred to other strains of the same species, as results are believed to be strain-specific.”

So, outside their general ability to confer health benefits, probiotics transcend generalization. You can no more attribute the effects of one strain to another than you can credit the benefits of vitamin D to vitamin C. And, just as vitamin D may be great for bone strength but do little for carbohydrate metabolism, a probiotic strain that can shorten the duration of diarrheal illness may have no effect on the common cold.

The sound of science

In making sense of these causes and effects, the sound science behind probiotic benefits proves its worth. While FDA has yet to bless a health claim for probiotics, structure/function claims are common. These don’t receive the same scrutiny as formal health claims, but still merit substantiating evidence.

“What we’re seeing in the market is that there’s becoming almost an assumed health benefit of probiotics,” says Mike Bush, vice president, business development, Ganeden Biotech, Mayfield Heights, OH. “But, in order to make a claim on a product, you have to do good clinical trials with the finished product. We always encourage our customers to do as much science as they can possibly do. Once you’ve got the clinical work, then you can say things like ‘proven to boost the immune system,’ or whatever the trial indicated.”

Mary Ellen Sanders, a consultant in probiotic microbiology with Dairy & Food Culture Technologies, Centennial, CO, and executive director of the International Scientific Association for Probiotics and Prebiotics (ISAPP), Davis, CA, is encouraged by the rigorous inquiry of probiotic research. “Many of these companies have piggybacked on research that’s been done painstakingly in different research labs over the course of years,” she says. “So, it’s not as if these claims were just picked out of the air.” Yet, when asked which claims deserve the most confidence, she says, “it becomes a little difficult to be very quantitative about that.”

Frankly, we’ve still got a lot to learn. Even the bedrock assumption that probiotics regulate intestinal flora isn’t as solid as it seems. “Not that it’s not important,” Sanders says. “It is important. But the situation we’re in today, if you want to have a really strong science-based perspective, we’re just now starting to characterize fully what populations of microbes are even there, and how they communicate with the host, how the host communicates with them, what roles they play in terms of driving our health, and to what extent consuming probiotics really influence that. Less is known about that than you might think.”

Stressing that she’s not a clinician, Sanders feels safe saying that, when looking at the science as a whole, “there are probably more publications and more evidence for antibiotic-associated diarrhea and infectious diarrhea in children than any other specific topic.” Gut and immune health also receive close attention, with supporting studies that “are really focused on very specific endpoints,” she adds.

Steele notes that probiotics are showing up in “‘cosmetofoods’ in skincare formulations,” and that manufacturers are targeting benefits to specific audiences—children, women, seniors—with specific needs. Her company recently presented a study on children younger than five that documents the effects of its probiotics on reduced incidence and duration of cold and flu symptoms. The formulation, she says, “reduced the number of sick days by almost half, and the number of antibiotic prescriptions by 80%.”

These boots-on-the-ground results excite Sanders. While it’s all well and good to link probiotic consumption to, say, enhanced expression of certain immune biomarkers, “having said that, what does that mean?” she asks. “At the end of the day, does that make me healthier? Those animal studies and biomarker studies in humans are very important for understanding mechanisms of effect, but to me, the more-convincing studies are the ones that note fewer absences from work, reductions in how many colds you get, other respiratory illnesses, diarrheal illnesses”—real-world improvements.

Thus, Sanders’ message is: “Let’s look at the endpoint health effects.” The best advice she can give, “at least for what we understand right now, comes from recommending products that have studies that have shown a real effect. That way, you know what dose to use, what subjects were studied, what degree of effect was seen and what might be expected in the consumer.”

Such standards apply especially to dosage. “Just as the strain that is used to claim a specific benefit is important, so is the dosage or the amount of living probiotic cells that is ingested or applied,” Steele says. “It is key that the amount of probiotic cells at the end of a food product’s shelf life should be identical to the dosage used in the scientific studies.”

Doses range anywhere from several hundred million colony-forming units, or CFUs, per serving to tens of billions. Determination is elementary. “Say you want a product that’s a shot-type beverage,” Bush explains. “Ultimately, you want to make immune claims. So you’re going to run a study that shows your product’s ability to bump the immune system; our clinical trial showed that a billion cells per day every day for 30 days had some effect on the immune system. So, because it’s designed to be a one-shot-a-day product, you would dose the shot so that, at the end of shelf life, you had a billion cells. If you’re going to have something that you may consume in multiple, maybe four, servings throughout the course of a day—let’s say it’s a tea—and you want to deliver the same effect, you just knock down the dose so you have 250 million cells per serving.”

Live and active

Bush believes transparency in labeling doses and the strains delivered not only empowers consumers, but benefits the category by buttressing its credibility. “We’re strong believers in saying what’s in there—the specific strain, the dose. And we’re not yet seeing a lot of companies saying how many cells are there in food. But, eventually, people should be saying that there are X number of cells at the end of shelf life.”

That assumes, of course, that X number of cells actually survive processing and digestion. “Probiotics are generally very sensitive to heat, pressure, shear, pH, water activity and a variety of other conditions found throughout many stages of a manufacturing process,” says Sean Farmer, founder and chief scientific officer, Ganeden Biotech. In addition, stomach acid, bile and digestive enzymes do a number on even healthy cells, let alone those weakened by processing or long residence in a dairy case.

But, notes Bush, “people who are in the business of probiotics know what the overage parameters are.” For a product with 10 billion cells at the end of shelf life, for example, “they may put 30 billion in at the beginning,” he says.

The take-home lesson according to Farmer is that “quality is more important than quantity. If the cells are not able to survive to colonize the host, even huge doses are not going to make any difference.”

That colonization needn’t occur in the colon. “If your target is to prevent dental caries,” Sanders says, “then no, they don’t have to survive stomach acid.” Probiotics that act on H. pylori don’t need to inhabit the colon either. “If you have a microbe that can be delivered to the stomach and do what it needs to do there,” she says, “do you need to isolate that from the feces? You don’t.”

Super bugs

Traditional formulations linked probiotics to dairy applications partly because dairy buffered the bugs’ ride through the stomach, and because refrigerated storage prolonged survival. But other reasons suit probiotics to dairy delivery, as well, including dairy’s healthful image; consumers’ comfort at seeing live and active cultures in dairy foods; and the fact that many of the same microbes that ferment dairy also colonize us, and many of those are probiotic. And, says Rexroat, “dairy products are often consumed on a daily basis, so they fit well with the daily-dose concept,” as the popular probiotic-shot concept exploits.

As probiotic suppliers tinker with strains and their manufacturing techniques, they open doors to “many new market areas, such as chocolate confectionery, fruit juices, breakfast cereals and more,” Steele says. Whereas water-activity restraints, pH limitations and the presence of antimicrobial agents once excluded probiotics from wider applications, companies are “developing advanced stabilization systems such as encapsulation or specific processing technologies to help handle some of these application issues,” she says.

It’s still a work in progress. Steele notes that “in evaluating multiple commercially available encapsulation technologies, no stability improvement has been noted to date in dry to intermediate-moisture systems.” Alginate systems, on the other hand, have provided some reported stability benefit in acid products, such as fermented yogurts. And Danisco, working with technology from the University of Wisconsin, has developed a freeze-drying process that “can provide specific strains with a 2-year shelf life at room temperature,” she says.

Sometimes, the bacterial strain itself improves its shelf life and viability. For example GanedenBC³⁰ (Bacillus coagulans GBI-30, 6086), a spore-forming probiotic, produces a protective layer that helps it survive manufacturing and human digestion to colonize the gut. Enclosed in its spore, the dormant bacteria withstand the rigors of processing, the supermarket and the consumer’s grocery bag. Once in the gut—their ideal growing environment—“they germinate and produce L+ lactic acid, which is a great form of lactic acid that helps lower gut pH,” Bush says.

This expands application options. While ready-to-eat cereal manufacturers once sprayed a topical probiotic solution onto their finished puffs, spore-forming probiotics allow you “to put the bacteria right into a dry food mix, mix it with your wet ingredients, run it through your dies to extrude it, and then stick it on the shelf and test its stability,” Bush says. “We’re able to grow the cells and enumerate the bacteria and get good survival through the process”—more than 85% viable cells post-processing in HTST milk, for example, and more than 80% in a hot-mixed granola bar.

This spore-forming probiotic is actually better suited to dry applications than to wet, too, to prevent premature germination. “Our bacteria allows us to do things that you can’t ordinarily do—dried products and baked products and products that involve reconstitution by the consumer,” Bush says. “We work fine and dandy in a hot tea made from a teabag.”

When live and active, the probiotic can survive a pH range of 3 to 12 and tolerate high-salt media—20% sodium chloride according to Farmer. “It’s very hardy as a vegetative cell,” he says. “The spores, obviously, are much tougher.”

Bug food

To keep probiotics vital, we shouldn’t overlook a factor perhaps even more obvious than salt levels, pH and processing conditions: the probiotics’ nutrition. Prebiotics—nondigestible dietary carbohydrates that selectively stimulate the growth or activity of beneficial colonic bacteria—are probiotics’ favorite foods.

When we eat a prebiotic, usually some form of fiber, it resists digestive breakdown and ends up in the large intestine, where it gives the microflora, particularly probiotic lactobacilli and bifidobacteria, something to eat. Proven-effective prebiotics include inulin and fructooligosaccharides, polydextrose, lactose derivatives such as lactulose and lactitol, granular RS2 resistant starch, and hydrocolloids like xanthan gum and pectin.

Proven effectiveness is important when choosing prebiotics. “Every fiber is not prebiotic,” notes Lisa Sanders, nutrition scientist, Tate & Lyle, Decatur, IL. “The fibers that tend to be prebiotic are the ones that are fermentable.” Similarly, all prebiotics do not stimulate all probiotic bacteria. As an example, she says, “resistant starch has a very large particle size, so it can only be fermented by particular bugs. But the metabolites that those bugs produce from fermenting the resistant starch can then be used as food by other bacteria in the colon. So there’s this cross-feeding that can occur.”

Putting prebiotics to the test

Manufacturers are devoting increased effort to identifying probiotic-prebiotic combinations that generate the best effects. “It is not clear which prebiotic carbohydrates are the most-suitable substrates for selective growth of specific strains,” says Donna Brooks, regional director, Danisco Texturants & Sweeteners, Elmsford, NY. “But, for the time being, in vitro tests have been conducted to determine the functional activity of various prebiotic carbohydrates combined with some probiotic strains. The results indicate that some associations, such as combinations of galactooligosaccharides and some Lactobacillus acidophilus or Bifidobacterium lactis, are best-suited for a synergistic effect.” And, she notes “encouraging results” from a clinical study, conducted at Danisco’s health and nutrition facilities in Finland, on the synergistic effect between the company’s Dophilus probiotic and the prebiotic sweetener lactitol.

In explaining her company’s approach to evaluating prebiotic efficacy, Sanders says, “we started with in vitro tests looking at specific bugs in the culture system and feeding them our fiber to see if they grow when fed that particular prebiotic.” The results showed “some very prebiotic effects” for the company’s resistant starch and soluble corn fiber, which led them to the next step: human studies. “We would feed humans the resistant starch or soluble corn fiber, and we would measure in the fecal material the different bugs that were promoted as a result of feeding the resistant starch and soluble corn fiber,” she says. Preliminary dosages to produce a prebiotic response worked out to about 10 to 12 grams per day of resistant starch or soluble corn fiber. However, “the dosing part is still under investigation,” she says. “Whether the response will increase the more that you give, we don’t know yet. But we do have some studies underway that are looking at higher doses.”

Prebiotic inulin and oligofructose has also come in for investigation, according to Joe O’Neill, executive vice president of sales and marketing, Beneo-Orafti, Morris Plains, NJ. “In vitro tests have found that Orafti inulin and oligofructose are excellent selective growth media and energy substrates for healthy bifidobacteria,” he says. He singles out their effect on bifidobacteria in particular as a key advantage. “Bifidobacteria prevent colonization of the gut by pathogens by creating a barrier effect, and also produce a range of short-chain fatty acids that lower the overall pH in the digestive system,” he says. “This lowering of the pH of the colon has been shown to facilitate increased calcium and magnesium absorption in the body.” According to human intervention studies, regular intake of this inulin and oligofructose increases the gut’s population of beneficial bifidobacteria five- to tenfold, while reducing the level of harmful organisms like clostridia.

Banking on synergism

Egged on by prebiotics’ stimulative effects on probiotics, manufacturers have aimed to parlay the relationship into a whole new category of functional food: the “synbiotic.” While no official definition for such products yet exists, “when both probiotic cultures and prebiotics are added to foods, the two work in synergy and are referred to as synbiotic,” O’Neill says. “The concept of synbiotic foods developed in Europe and was used as an opportunity to differentiate and market cultured dairy products.”

The category is still in its infancy, both in terms of public awareness and scientific acceptance. The main question remains whether or not the interaction between probiotics and prebiotics in a single food is truly synergistic—as in more than the sum of its parts.

Mary Ellen Sanders isn’t so sure. “I have not seen strong evidence of that,” she says. “What we see sometimes are additive effects—you have a probiotic alone, a prebiotic alone, you put them together, and you get more than each by itself. But you don’t necessarily get more than the sum total.”

Researchers continue to look for the synergies, however, and O’Neill is bullish on their prospects. “For instance, studies have shown that prebiotics exert a stabilizing and protective impact on probiotics in products from manufacturing through shelf life all the way to the digestive tract,” he says. In one study, researchers supplemented Lactobacillus rhamnosus or Lactobacillus casei yogurts with 3% oligofructose in two forms. They tested the products at 10-week intervals for culture viability during shelf life, while also using a model in vitro digestive system to determine viability after consumption. Results showed that, at 10 weeks, both forms of oligofructose displayed a protective effect on the probiotics with no change in viability. The prebiotic yogurt processed through in vitro digestion showed no significant difference in L. rhamnosus cell counts, compared with a 36% decrease in the control, and the prebiotic L. casei yogurt saw a 5% decrease in culture viability after in vitro digestion, vs. a 19% decrease in the control. The conclusion, O’Neill says, is that the prebiotics “protect probiotics during both storage and digestion.”

Perhaps more intriguing is how synbiotic foods protect us. Puzzling out those benefits is the goal of the SYNCAN Program, an EU-funded research project that brings together scientists from six different countries to test the hypothesis that prebitoics and probiotics in combination protect the gut from the DNA changes that trigger colon cancer. One study looked at patients treated for colon cancer, as well as healthy subjects who had intestinal polyps removed. The subjects received either a placebo or a daily synbiotic supplement combining an Orafti prebiotic with two probiotic cultures. After 12 weeks, the subjects in the synbiotic group saw reductions in colon-cancer risk markers, normalization of cell turnover, and a 60% decrease in mucosal DNA damage, with risk reduction particularly strong in the polyp patients. “The dramatic risk reduction seen in the synbiotic group coincided with changes to the composition of gut bacteria that favored protective species, such as Bifidobacterium and Lactobacillus,” O’Neill adds. “These changes happened within a few weeks, showing that synbiotics can deliver immediate, as well as long-term, benefits.”

Formulation fundamentals

Though the jury may be out on synbiotics’ staying power, manufacturers are working on formulations while they wait for a consensus. The key points to keep in mind are that the synbiotic combination should suit the application, the dosage should be effective and tolerable, and the whole getup should stay stable during and after processing.

Brooks suggests working with prebiotic ingredients that don’t get in the way of color, texture or flavor. A product like Danisco’s refined polydextrose has “virtually no taste and is colorless in solutions,” she says, making it easy to incorporate into a range of systems. “Lactitol, a polyol, is equally easily incorporated into food matrices, although some local legislation may limit its use in beverages,” she adds.

Being soluble, soluble corn fiber gives beverage makers an opportunity to get on the bandwagon, Sanders says. “It can be put into a water and you can’t even tell that it’s in there.”

Inulin and oligofructose are also formulation-friendly, showing up in everything from refrigerated orange juice to nutritional bars to cereal. “Technical benefits include bulking, sugar reduction, calorie reduction and the masking of high-intensity sweeteners,” O’Neill notes. “Lately, Orafti has introduced liquid syrups for ease of use and as a low-cost fiber-delivery option.”

As far as dosing is concerned, you want enough prebiotic to stimulate the bugs, but not so much that it overloads consumers with fiber. Brooks says that human intervention studies have determined a comfortably low, effective, prebiotic dose of 4 grams per day for her company’s fiber. “The tolerance for lactitol is on par with other prebiotics, such as fructooligosaccharides and inulin,” she adds.

Fortunately for all involved, most prebiotic fibers are reliably stable in formulation. Inulin and oligofructose can break down in acidic conditions at room temperature, but generally do well under refrigeration. “Prebiotics are usually stable during product shelf life and easily incorporated into any food application,” Brooks says. “Any limitations are likely to depend on the application of the probiotics.”

That’s why, as the experts have said all along, the proof is ultimately in the product. Case-by-case assessments of pre- and probiotic composition and functionality—after production, during storage, even after consumption—are the only proof we have to convince the public to sign on. “Again, that’s why it’s so important to do the studies,” says Sanders. “At the end of the day, that’s the sum total of all of these factors: the host factors, the survival factors, the microbial factors. And at the end of the day, does it ultimately have an effect?”

Kimberly J. Decker, a California-based technical writer, has a B.S. in Consumer Food Science with a minor in English from the University of California, Davis. She lives in the San Francisco Bay area, where she enjoys eating and writing about food. You can reach her at [email protected].

Probiotic Products Multiplying

The friendly bug boom is predicted to continue as research increases and health-conscious consumers become more aware of probiotics’ benefits. According to a 2008 report by Global Industry Analysts, Inc., San Jose, CA, “Probiotics: A Global Strategic Business Report,” the global probiotics market is forecast to reach $20 billion by 2010.

The company points out that Asians and Europeans lead the way in embracing these products, but the rest of the world is catching up quickly. In fact, Europe has the largest, fastest-growing market, with revenues of approximately $5.⁷ billion in 2007. Germany represents the largest market, and the United Kingdom has the fastest growing market, increasing at a compound annual growth rate (CAGR) of around 14%. The second-largest market is the United States, with sales expected to reach $4.⁶ billion by 2010.

The report identifies two potential barriers to growth: consumer concerns about GMO cultures, and companies that put forth health claims that are misleading and lack scientific evidence.

Digestive Health: Not Just for People

Humans aren’t the only ones who benefit from enhanced intestinal flora. Animals can, too.

Agricultural Research Service (ARS) scientists at the Poultry Production and Product Safety Research Unit in Fayetteville, AR, identified probiotics with the potential to protect live chickens from Salmonella, Campylobacter and other pathogens that cause foodborne illness, plus allow the birds to grow more efficiently.

Using competitive exclusion―where the “good” bacteria successfully compete with the “bad”—the scientists fed probiotics to newly hatched poults, when they are most susceptible to infection. These probiotics occupy sites in the young birds’ intestinal tracts and reduce the opportunity for pathogenic bacteria to become established. The team has screened more than 4 million intestinal isolates to identify several promising probiotic combinations. The University of Arkansas and ARS have filed a patent on the selection techniques.

ARS researchers found oligosaccharides provide prebiotic advantages when used as feed additives for pigs and chickens. They found the compounds aid probiotic organisms in unlocking minerals, vitamins and other nutrients, as well as making the animal’s colon less hospitable to the pathogens, such as Salmonella and Escherichia coli, that can contaminate meat products and cause human illness.

Chemist Greg Cote, with ARS Bioproducts and Biocatalysis Research Unit, Peoria, IL, co-developed these oligosaccharides with Scott Holt, an associate professor with Western Illinois University’s Department of Biological Sciences, using a microbial enzyme, alternansucrase, to catalyze a series of reactions that convert sugars into different kinds of oligosaccharides. Some of the resulting oligosaccharides encouraged growth of Bifidobacterium, Lactobacillus, Bacteroides and some enterococci bacteria, but not of pathogens such as Salmonella, E. coli or Clostridium perfringens. ARS patented the technology in 2007.

— Lynn A. Kuntz