Canadian Food Business
Natallia V. Varankovich | Michael T. Nickerson | Darren R. Korber*
The microbiota of the human gastrointestinal tract (GIT) is extremely complex, consisting of bacteria, archaea, some protozoa, anaerobic fungi and different bacteriophages and viruses, with more than 1,000 species and up to 5×1011 (100 billion) bacterial cells per gram of intestinal contents1. According to American Society for Microbiology (2008), bacteria living on and within our bodies outnumber human cells by a factor of 10. Surely, such an enormous number of microorganisms would be expected to have a major impact on the functioning of the human organism. In fact, clinical studies have shown that the human microbiota play a variety of key roles, ranging from anti-pathogen protection, digestion of essential nutrients and immune-system training to affecting our weight and mood.2-4 Furthermore, experiments on germ-free mice have shown that microbiota-negative mice are unable to maintain a normally functioning intestinal epithelium, effective nutrient digestion, or a normal level of immunological activity.5 Hence, without indigenous microorganisms, a number of bodily functions would be impacted, including the ability to provide sufficient protection from pathogens.
Our way of life, and especially our dietary habits, has a major impact on the composition of the GIT microbiota. Factors such as antibiotic therapy, increased amount of sugar and fat consumption and contamination of food by pathogens bring considerable alterations to the gut microbial balance, generally causing infections or even more serious disorders, such as acute gastroenteritis or irritable bowel syndrome.6 Antibiotic therapy is usually used to treat GIT disorders; however, the search for more natural and less intrusive alternatives is ongoing. This article reviews some of the potential health benefits of probiotics, along with the challenges industry faces in terms of processing and efficacy surrounding some probiotic health claims.
Probiotics and their health benefits
One of the most promising directions of human microbiota research which targets a number of aspects of human health is the applied use of probiotics – bacterial supplements that have beneficial effects on gut microbiota. The Food and Agriculture and World Health Organizations (FAO and WHO) define probiotics as “live microorganisms which when administered in adequate amounts confer a health benefit on the host”.7 Probiotic benefits are usually species – and even strain-specific and include, but are not limited to, inhibiting the growth and spread of pathogens in the GIT, stimulating the immune response towards pathogens and producing antimicrobial factors.8,9 The popularity of probiotics has dramatically increased in recent years as has the public’s general interest in healthier diets and lifestyles, functional foods and food supplements. However, it’s not just advertising that has shifted consumer behaviour – the scientific community worldwide has invested considerable amounts of time and funds in finding a reliable and “natural” medication for gut disorders. A literature survey for publications featuring the keyword ‘probiotic’ in the NIH PubMed database reveals an exponential growth in probiotic research: only 173 articles were published from 1990 to 2000, whereas this amount increased by more than ten times (1916) over the next decade. In the following 5 years (from 2010 to January 15th, 2015), 1,434 probiotics articles were published, with 10 of them being released in the first 2 weeks of 2015.
The expanding field of probiotic research includes studies of many bacterial and several yeast species, each possessing potential beneficial properties. However, the majority of published papers in this area focus on the two most popular probiotic genera – Lactobacilli and Bifidobacteria. Lactobacilli or Lactic acid bacteria are Gram-positive, non-spore forming cocci, coccobacilli or rods, which ferment glucose primarily to lactic acid, grow anaerobically, but can tolerate the presence of O2. Unlike Bifidobacteria, which are mostly present in lower parts of the colon, Lactobacilli can be found in the upper GIT. They are also part of the normal human microflora of the oral cavity, the small intestine and the vaginal epithelium, where they are thought to play beneficial roles. In the human GIT, Lactobacilli improve digestion, absorption and availability of nutrients, influence energy homeostasis, particularly in obese patients, and produce B-group vitamins.10 It has also been shown that Lactobacilli can inhibit or kill Helicobacter pylori, a major causative agent of gastritis and peptic ulcers.11 The most famous species of the genus are Lactobacillus acidophilus and Lactobacillus rhamnosus GG due to their declared beneficial properties and wide use in probiotic products.
The second major group of potential probiotics are Bifidobacteria, also widely present in human GIT microbiota. They are Gram-positive, non-motile, often-branched anaerobes, producing acetic and lactic acids. In the human gut, Bifidobacteria are involved in the utilization of complex carbohydrates such as plant-derived dietary fibre that cannot be digested solely by human-derived enzymes or processes. Bifidobacterial strains have also proven to be effective in the prevention or alleviation of infectious diarrhoea and the improvement of inflammatory bowel disease symptoms.12 Well-known species of this genus, widely-used as probiotic supplements, include Bifidobacterium bifidum, Bifidobacterium animalis and Bifidobacterium longum.
Though Bifidobacteria and Lactobacilli are the most popular subjects of probiotic research, other genera of bacteria and yeasts, such as Saccharomyces spp., Bacillus spp. and Streptococcus spp., have also been studied for probiotic potential.
Challenges for probiotics in the food industry
In 2002, a joint FAO/WHO working group elaborated a list of specific requirements that have to be met in order to grant a strain a “probiotic” status.13 These guidelines include:
i. complete identification of the strain (genus, species, strain);
ii. in vitro tests on probiotic potential (e.g. resistance to low pH, bile salts, digestive enzymes);
iii. safety assessment: the final product has to be safe for human consumption and not contaminated with other microorganisms; and
iv. in vivo studies on both animal models and human clinical trials, wherein health benefits are proven.
Most strains with probiotic properties are isolated from healthy human gut microbiota or food products, such as milk, which potentially makes them safe for use as food supplements. However, each strain has to be tested individually, even if it belongs to a species that is ‘Generally Recognised as Safe’ before it can be used in an actual functional food product or medication. Additional requirements include determination of antibiotic resistance patterns and possible side-effects during human studies.13 The last requirement from the list (demonstration of in vivo efficacy) above is usually the most challenging for most potential probiotics, since most of them are sensitive to handling during manufacturing (especially anaerobes) and, when consumed, to the harsh conditions of the stomach, such as low pH and pepsin treatment. A probiotic product must contain at least 106 – 107 viable bacterial cells per gram, where upon continued consumption, the probiotics are able to exert beneficial effects on human health.13 However, delivering such high numbers of viable cells is rarely the case. Probiotic products, such as milk and yogurts, usually do not to meet this requirement, and consequently may fail to elicit the desired health benefits in the host. It is therefore desirable to use techniques to protect the probiotic-containing food product so that the numbers of viable bacteria remain high until they reach the colon where they may provide a functional effect or benefit.
Protection of probiotics during storage and passage through the upper gastrointestinal tract
In general, probiotic bacteria that are administered as a food supplement are in a dried or freeze-dried form, which improves preservation and extend shelf-life. However, this approach leaves bacteria unprotected from the highly-acidic conditions of the stomach. Alternatively, encapsulation technology has been shown to help circumvent this issue by coating the probiotics using single or multiple biopolymer layers. The effectiveness of these carrier matrices can be optimized by altering the biopolymers present, concentration and viable payloads.14 The structural integrity of capsules is key to the survival of bacteria; however, capsules must be insoluble at the acidic pH of the stomach but dissolve under the alkaline pH of the intestine to time the release of the entrapped microorganisms. Capsules must also ensure the stability of viable bacterial counts during manufacturing (particularly during drying or freeze-drying of the product) and storage. Materials that meet these requirements must be non-toxic and thus typically are based on polysaccharides (sodium alginate, carrageenans, gums) and proteins (pea, whey). These substances have been widely-tested as potentially useful for encapsulation of probiotics. In order to enhance protection of immobilized bacteria, polysaccharides may be mixed with proteins to produce capsule wall material with desired characteristics (Figures 1 and 2). The most common polysaccharide used in probiotic microencapsulation studies is sodium alginate due to its ability to form a gel in the presence of divalent cations, a feature that makes the production of bacteria-containing capsules a fast and easy process. While microencapsulated probiotics have not yet gained popularity over dried and freeze-dried microorganisms in the health-food market, they have proven to be an effective delivery tool for bacteria used for the treatment of GIT disorders in human clinical trials.15,16
Figure 1. Fresh iota-carrageenan – pea protein capsules containing immobilized probiotics. |
Figure 2. Confocal laser scanning microscopy image of the surface of an iota-carrageenan-pea protein capsule containing immobilized probiotics. Bacteria may be observed as small green or red fluorescently stained objects whereas pea protein emits a yellow autofluorescence. Scale bar – 100 µm. |
Questions surrounding health claims and the efficacy of probiotics
Health Canada is responsible for developing the proper use of terminology and allowable health claims as it relates to probiotics. Terms and phrases used on food packages, such as ‘probiotics’, ‘…with beneficial probiotic cultures’, or ‘…contains bacteria that are essential to a healthy system’ are all allowable only when they can be validated for the specific probiotic strain present within the food. Allowable claims must be supported by scientifically proven physiological effects associated with maintaining or supporting good body health and performance (e.g., promotes regularity, improves mineral absorption and helps with digestion). However, more general statements, such as ‘promotes gut health’ or ‘supports immune function’ are not allowed. Validation of these claims in Canada are dealt with by the Food and Drugs Act and involves a systematic review of all of the scientific evidence collected by individuals/companies that implies an effect by the consumption of each individual probiotic strain taken at set doses associated with that claim.
The majority of probiotic products available on the market are generally considered beneficial for human health; however, there remains a lack of evidence as to their efficacy in treatment of specific gastrointestinal disorders. In order to be considered a medication, a product must undergo clinical trials where it must be proven equally, or more effective, than a standard treatment for a specific condition.13 Currently, most probiotic research is at the in vitro study stage, where putatively-beneficial bacteria are tested in the lab for their ability to inhibit the growth of pathogens, produce antimicrobials and vitamins and withstand simulated conditions of the upper GIT. Strains demonstrating probiotic potential are then tested in animal models against a specific gastrointestinal disorder and, if found effective and safe, in human clinical trials. Results of multiple trials involving humans suggest that certain bacterial strains are indeed effective in the treatment and prevention of antibiotic-associated diarrhea (Saccharomyces cerevisiae, Lactobacillus rhamnosus GG), irritable bowel syndrome (Lactobacillus acidophilus) and viral gastroenteritis (Bifidobacterium longum).15,17-18 VSL#3, a mixture of several bacterial species (4 Lactobacilli strains, 3 Bifidobacteria strains, and one strain of Streptococcus spp.), has been shown to be effective in treatment of pouchitis according to the results of three trials.20 However, in order for these probiotics to enter the market, the findings of human clinical trials must be highly reproducible and on a much larger scale. Additionally, the strain with claimed probiotic properties must be well characterized, and the mechanism of providing the specific health benefit must be explained in detail. Due to failure to meet these requirements, many potential probiotic products are being rejected as inappropriate for food market as they lack sufficient “scientific substantiation of the claim” or because “the cause and effect relationship has not been established” between the consumption of a probiotic product and the health benefit it claims to provide.21,22 Other important considerations are the long shelf-life of a probiotic-containing product and its ability to provide sufficient protection for bacterial cells during transit through the human GIT in order to preserve the probiotic effect.
The integration of new techniques for bacterial cell protection, such as microencapsulation, into probiotic research and industrial application will lead to the emergence of novel forms of functional food products that would ensure the effective targeted delivery of selected probiotics in sufficient amounts to have their benefit on the host organism.
References
[1] Tuohy, K.M. et al. (2012). J Agric Food Chem 60:8776. [2] Doré, J. & Corthier, G. (2010). Gastroentérol Clin Biolog 34:S7. [3] Delzenne, N.M. et al. (2011). Microb Cell Fact 10(1):1475. [4] Naseribafrouei, A. et al. (2014). Motil 26:1155. [5] Aureli, P. et al. (2011). Pharmacol Res 63:366. [6] Kotzampassi, K. & Giamarellos-Bourboulis, E.J. (2012).Int J Antimicrob Agents 40:288. [7] Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria (October 2001). Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria. Food and Agriculture Organization of the United Nations, World Health Organization. [8] Perdigon, G. et al. (2002). Eur J Clin Nutr 56(4):21. [9] Collado, M.C. et al. (2007). Lett Appl Microbiol 45:454. [10] Wallace, T.C. et al. (2011). Nutr Rev 69:392. [11] Hamilton-Miller, J.M.T. (2003). Int J Antimicrob Agents 22:360. [12] Sanz, Y. (2007). Int Dairy J 17:1284. [13] FAO/WHO. Guidelines for the evaluation of probiotics in food. London, Ontario: Food and Agriculture Organization of the United Nations and World Health Organization Working Group Report (2002) 1–11. [14] Tripathi, M.K. & Giri, S.K. (2014). J Funct Foods 9:225. [15] Sinn, D.H. et al. (2008). Digest Dis Sci 53:2714. [16] Omar, J. M. et al. (2013). J Funct Foods 5:116. [17] Lee, D.K. et al. (2014). Clin Res Hepatol Gastroenterol. In Press.
doi:10.1016/j.clinre.2014.09.006. [18] Videlock, E.J. & Cremonini, F. (2012). Aliment Pharmacol Ther 35:1355. [19] Vanderhoof, J.A. et al. (1999). J Pediatr 135:564. [20] Saad, N. et al. (2013) LWT – Food Sci Technol. 50:1. [21] European Food Safety Authority (EFSA), 2012. Scientific Opinion on the substantiation of health claims related to Lactobacillus casei DG CNCM I-1572 and decreasing potentially pathogenic gastro-intestinal microorganisms (ID 2949, 3061, further assessment) pursuant to Article 13(1) of Regulation (EC) No 1924/20061. Available at: http://www.efsa.europa.eu/en/efsajournal/doc/2723.pdf [22] European Food Safety Authority (EFSA), 2014. Scientific Opinion on the substantiation of a health claim related to “Lactobacillus plantarum TENSIA® in the semi-hard Edam-type ”heart cheese” of Harmony™” and maintenance of normal blood pressure pursuant to Article 13(5) of Regulation (EC)
No 1924/20061. Available at: http://www.efsa.europa.eu/en/efsajournal/doc/3842.pdf
Department of Food and Bioproduct Sciences, University of Saskatchewan, Saskatoon, SK
(*Corresponding author email: Darren.Korber@usask.ca)
