Formulating with Probiotics

Probiotics are hot. From Jamie Lee Curtis gushing about the digestive benefits of Dannon's Activia® yogurt to nutritional bars touting their immune-boosting formulas, probiotics are carving a substantial place for themselves in the market. As beneficial as they are, there are several challenges when it comes to formulating with probiotics; manufacturers must take care to select and deliver the most applicable strain with substantiated health benefits.

Probiotics, typically beneficial bacteria found in gut flora and, less commonly, certain types of yeast, are a key to maintaining gut health, an effective digestive process and a strong immune system.

"Probiotics may be defined as food supplements containing concentrates of defined strains of living microorganisms that, on ingestion in certain doses, exert health benefits beyond basic nutrition. The concept of probiotic foods is based on the fact that the micro flora in the gastrointestinal (GI) tract has a significant role in the health status of an individual, which is influenced by the diet consisting of organisms," explained Luis Echeverria, new business development manager, Pharmacenter.

Strains of lactic acid bacteria, Lactobacillus and Bifidobacterium, are the most commonly-used probiotic bacteria in the nutraceuticals industry. "Across the globe, about 20 probiotic strains, singly or in combination, [are used], mainly Lactobacilli such as L. acidophilus, L. casei and L.reuteri," said Lakshmi Prakash, Ph.D., director of technical services, Sabinsa Corp. She added probiotic yeast culture Saccharomyces boulardii is popular for use in dietary supplements, functional foods or mainstream food products.

While probiotics may be making headlines today, they are not a new idea, according to Prakash. "Ancient physicians in the Near and Middle East prescribed soured milk [which contains lactic acid bacteria] for appetite stimulation, as well as in the management of diseases, including tuberculosis, GI disorders and liver troubles. Contemporary interest in probiotics can be partially attributed to [Russian microbiologist Ilya Ilyich Mechnikov's] theory of longevity, which associated prolonged youth and a healthy old age, common in Balkan peasants of those times, with the use of cultured milks in their diet."

Todays standard American diet doesnt generally deliver these types of fermented foods that can enhance probiotic colonization. The situation is further exacerbated by other factors, according to S.K. Dash, Ph.D., founder of UAS Laboratories. In his book A Consumers Guide to Probiotics (Freedom Press, 2005), he wrote: A number of factors are responsible for the lack of friendly cultures in our intestinal tract. Beneficial microflora are reduced by excessive use of antibiotics, chlorinated water, food preservatives, junk foods, and pollution in our environment. But their right probiotic recipe for good gut health is very specific. As Dash wrote: A probiotic supplement can only be effective if it contains the right strain(s), in the right number (potency), in the right condition (viable) and in the right formulation.

Health Benefits of Probiotics

Indeed, probiotics have been shown to have several health advantages, GI health being the most popular. "Receiving live 'bugs' to the gut will certainly create numerous health benefits, such as improved daily bowel habits, correcting constipation or diarrhea, improved skin conditions and overall better general health," explained Tony Serra, sales representative for Mitsui and Co.

Hui Zhu, Ph.D., laboratory manager, Specialty Enzymes and Biochemicals Co., echoed Serra's statements, noting, "These microorganisms benefit their host by maintaining intestinal homeostasis, competitive exclusion of pathogens, production of antimicrobial compounds, promotion of the intestinal barrier function, immune modulation and some very promising results in the area of inflammatory bowel disease."

In a 2008 review from Institut National de la Recherche Agronomique, Nouzilly (Tours), France, researchers found Saccharomyces boulardii has beneficial effects in the prevention and the treatment of intestinal infections, and in the management of inflammatory bowel disease (IBD).¹ All these diseases are characterized by acute diarrhea; administration of the yeast, either alone or in combination with an anti-biotherapy, was shown to significantly decrease the duration and the frequency of diarrhea. However, a similar review admitted that, while some clinical studies have shown the therapeutic effects of probiotics in the treatment of IBD or prevention of allergic disease, researchers have not yet determined their exact role.²

Probiotics have also been shown to play a protective role in colon cancer, according to a 2001 review from the Institute for Nutritional Physiology, Karlsruhe, Germany.³ Researchers found ingestion of viable probiotics was associated with anti-carcinogenic effects, one mechanism of which is the detoxification of genotoxins in the gut. They added, newer studies showed short-lived metabolite mixtures isolated from milk that was fermented with strains of L. bulgaricus and Streptococcus thermophilus are more effective in deactivating risk factors of colon carcinogenesis than are cellular components of microorganisms. In spite of this, a 2006 review from the University La Sapienza, Rome, stated there is not enough specific evidence that probiotics are useful against cancer.⁴ Researchers noted, "In our search of the literature, few and conflicting epidemiologic data regarding the impact of fermented dairy products consumption in humans have been gathered. There are no positive data from interventional studies so far. Therefore, even though an ample body of evidence supports the potential anti-carcinogenic action of probiotics on the basis of the results obtained in both in vitro and in vivo models, further evidence is very much needed."

Dash, however, noted in his book that probiotics do have a clear role to play in prevention of colon cancer. He wrote: We dont know precisely how probiotics exert their anti-cancer benefits, but believe that by enhancing populations of bifidobacteria, this may result in direct removal of procarcinogens, indirect removal of procarcinogens, or activation of the bodys immune system.

Behind the Scenes

So how, specifically, do probiotics work? "Since the definition of probiotics is fairly wide and includes various strains of both bacteria and yeast, it is not possible to give a simple answer. In general terms, a probiotic will balance or maintain your GI microflora through its presence and colonization," explained Niklas Larsson, R&D director, Probi AB, and Martin Antonsson, director of production and application, Probi AB. "When a probiotic bacteria adheres to the intestinal mucus, it will not only prevent 'bad' bacteria from binding to and overgrowing the intestines, but it will also, in specific cases, improve the uptake of certain nutrients, modulate the immune system and aid the digestion system."

More specifically, according to Prakash, probiotics produce metabolites, such as lactic acid and bacteriocins, antibiotic-like substances that suppress the growth of putrefactive microorganisms. "Their metabolic activities also help in the pre-digestion of food components and the production of B vitamins, and improve the bioavailability of minerals and other nutrients, for example, isoflavones from soy milk, as reported in a recent study.⁵ Additionally, their cell wall components and metabolic products provide immune support and anti-inflammatory action," Prakash added.

Gene Bruno, MS, MHS, Dean of Academics for Huntington College of Health Sciences, affirmed Prakash's statements, noting probiotics also break down bile acids, allowing them to exert an influence over the balance of bacteria; probiotics combine with nitrates, which help to inhibit the damage nitrates may cause; and they are useful in the reduction of ammonia.

Interestingly enough, probiotics aren't just relegated to the gut; they're also being used in oral care products. "Oral care probiotics are a novel approach of utilizing a well-established method of increasing the levels of good bacteria to restore a healthy balance to the mouth. ... In the case of dental cavity-causing pathogens, probiotic strains can crowd out the pathogens and significantly reduce their numbers on the teeth. In the case of periodontal disease-causing bacteria, the probiotic strains colonize close by and produce antibacterial agents, such as hydrogen peroxide, that can kill the key pathogens associated with this disease. An additional benefit associated with this decrease in the number of harmful bacteria associated with gum disease can be a noticeable freshening of the breath," said Jeffrey Hillman, DMD, Ph.D., chief scientific officer for ONI BioPharma.

Overcoming Formulation Obstacles

Although probiotics are powerful bacteria, they are perishable and can easily be made inefficient if they are not formulated and stored properly. Echeverria noted there are several components that must be met for a probiotic to be considered efficacious. "An effective probiotic should exert a beneficial effect on the host, be non-pathogenic and nontoxic, contain a large number of viable cells, be capable of surviving and metabolizing in the gut, remain viable during storage and use, have good sensory properties, and be isolated from the same species as its intended host."

One of the main challenges when formulating with probiotics, explained Armin Salmen, vice president of research and development and quality assurance for NextFoods, is maintaining the probiotic counts and activity throughout the shelf-life of the product without affecting the stability or sensory characteristics. "The key to an efficacious product is a thorough selection process of the probiotic for the specific food it is intended to be used in, followed by extensive shelf-life studies."

This also includes how the product is stored, said Tim Gamble, vice president of sales and marketing, Nutraceutix Inc. "The shelf-life of safe, natural probiotics varies depending on manufacturing techniques, delivery form, packaging and storage conditions. At the point of sale, its unrealistic to expect products to remain frozen or refrigerated due to the costs and complexities of doing so. After the sale, obviously, asking a consumer to comply with storage guidelines is hit or miss." Gamble explained this is why Nutraceutix, among others, have developed processing methods that help to extend the shelf-life of probiotic capsules, even at room temperature.

However, Salmen pointed out that dry products containing probiotics, such as cereal, supplements or bars, can typically tolerate storage at an "ambient temperature," but do benefit from refrigerated storage. "In general, dry products have the longest shelf-life, six months to a few years. ... Yogurts and drinks typically have a 40- to 80-day shelf-life from the date of production," Salmen said.

Hillman noted in order to assure consumers are receiving the most potent probiotic dose, product labels should specify the number of live bacteria in each freeze-dried probiotic serving, typically listed as "colony forming units" (CFU). "Depending on the processing steps used following fermentation, a shelf-life of a finished product containing a freeze-dried probiotic preparation may be from 12 to 24 months, when stored at room temperature, which means at the end of the listed shelf-life, each serving of the product must contain at least the number of live bacteria listed on the label." As Michael Shahani, director of operations, Nebraska Cultures, Inc. explained, "It is important to remember that the number of live organisms is how probiotic potency is measured. They are not measured by weight but by potency, so manufacturers must know that the same weight of different probiotics products may contain vastly different numbers of live organisms."

Dash further emphasized the importance of providing CFU details on product labels for consumers: If the [product] does not list viable cells, or does not list the amount in CFU form, it is not valid. Consumption of [products] with 2 billion to 5 billion CFU per day is necessary to have any chance of offering significant beneficial effects.

Another challenge is ensuring probiotics are unable to become oxidized and can survive the acidity levels found in the digestive tract and stomach, as these good bugs can often be destroyed in low pH. "Probiotics are live microorganisms which need to be handled gently in technical processes, Larsson and Antonsson explained. They are sensitive to high temperatures and chemical substances with anti-microbial properties, such as cleaning agents and preservatives.

As well as using very large counts of probiotics per serving to overcome this obstacle, many formulators use a variety of special technologies to ensure full potency. Zhu stated: "Techniques include coating or encapsulation techniques, such as enteric coating and microencapsulation, to actually avoid the stomachs acidic exposure. Another option is to utilize a spore forming probiotic like Lactobacillus sporogenes (also called Bacillus coagulans). Its natural spore formation protects it in the harsh environment of the stomach."

Entrapment of the bacteria in a polymer matrix, such as alginate, carboxymethyl cellulose (CMC), gelatine and pectin, is a popular manufacturing method for probiotics. Echeverria explained, "In general, cells are mixed with a solution of the polymer and dropped into a solution of a cross-linking ion. Given the correct conditions, spherical beads are formed. These beads are typically 1 to 2 mm in diameter and their dimensions can be controlled within certain limits."

According to Echeverria, entrapment of probiotic bacteria in a biodegradable polymer matrix has a number of advantages, as encapsulation tends to stabilize cells, potentially enhancing their viability and stability in the production, storage and handling of these probiotic cultures.

"The number of cells in each bead can be quantified allowing dosages to be readily controlled. Cryo- and osmo- protective components can be incorporated in the polymer matrix, enhancing survival of cells during processing and storage." Once the beads have been dried, a surface coating can be applied.

"This outer layer can be used to alter the aesthetic and sensory properties of the product, and may also be functional, providing an extra level of protection for the cells. In addition, the coating layer can have desirable dissolution properties which permit delayed release of the cells or release when actually required."

Mitsui and Co., which uses a similar entrapment method, employs triple-layer, seamless probiotic capsules with a physical stimulation protection layer and an acid-resistant shell to protect the core layer, which contains the probiotics. "Most other probiotic capsules or tablets are not shelf-stable and the bacteria is dead inside the capsule; it degrades quickly after production, especially without refrigeration.

The other biggest problem for probiotic supplements is the payload cannot survive the gastric juices of the stomach pH, so most of the bugs will be killed off, creating a product that provides minimal consumer benefit. The key [to keeping the probiotics potent] is producing a totally spherical, seamless, triple-layer capsule that will ensure delivery of live bugs to the gut," said Serra.

Freeze-drying is also commonly used when formulating with probiotics. "Freeze-drying, or lyophilization, with or without microencapsulation, is employed to produce a 'dry powder' for use in a tablet or capsule-type product, and to extend the shelf life of such a product; this freeze drying can eliminate the need for refrigeration during storage," Hillman said. "The freeze-drying process maintains the viability of the bacteria, so that, when activated by water, these bacteria are released from the delivery system in numbers equivalent to those found in refrigerated 'live' probiotic products."

He added, packaging is also important to protect freeze-dried formulations from moisture during storage, which could prematurely activate the bacteria. However, in the case of oral care probiotics, "there is no need to take additional protective steps, such as microencapsulating the probiotic strains, since their point of action is the mouth and so they do not need to be protected against the harsh pH conditions existing in the stomach as do probiotics intended for delivery to the colon."

While probiotics can make formulation difficult, their benefits far outweigh any disadvantages manufacturers might have. With the industry making way for probiotic-enhanced drinks, bars, dairy products, candies and even baby food, formulators would be remiss to not take advantage of this ever-growing trend.

References on the next page...

References for "Formulating with Probiotics"

1. Zanello G, et al. " Saccharomyces boulardii effects on gastrointestinal diseases." Curr Issues Mol Biol. 2008 Sep 8;11(1):47-58. [Epub ahead of print]

  2. Kamiya T, et al. [The efficacy of probiotics in gastrointestinal disease][Article in Japanese] Nippon Rinsho. 2008 Jul;66(7):1385-90.

  3. Wollowski I, et al. "Protective role of probiotics and prebiotics in colon cancer." Am J Clin Nutr. 2001 Feb;73(2 Suppl):451S-455S.

4. Capurso G, et al. "Probiotics and the incidence of colorectal cancer: when evidence is not evident." Dig Liver Dis. 2006 Dec;38 Suppl 2:S277-82.

  5. Pham TT, et al. "Biotransformation of isoflavone glycosides by Bifidobacterium animalis in soymilk supplemented with skim milk powder." J Food Sci. 2007 Oct;72(8):M316-24.