viernes, febrero 01, 2002

Unique problems in designing and testing probiotic

FoodInfo Online Features -->31 January 2002
Food Research and Development Centre, Agriculture and Agri-Food Canada, St. Hyacinthe, Quebec, Canada
Microorganisms have always contaminated mankind's food. In most cases, this was considered detrimental, since bacterial contamination is often the cause of food spoilage. Over time, however, it became apparent that fermentation by microorganisms could produce desirable products such as bread and wine, and that the fermentation process could be used to preserve perishable foods such as milk and meat.
The research that identified, characterized and defined microorganisms established the science of microbiology, and created an interest in the many roles - both positive and negative - that microorganisms play.
Probiotic foods have been consumed in various parts of the world for many centuries. Foods that are fermented by bacteria and yeasts have many unique properties. Probiotic foods may also be functional foods, when they contain ingredients that are good for human health.
There are already many probiotic foods on the market, and food manufacturers are looking to develop more. However, because probiotic foods are produced by, and may contain, live microorganisms, the production, design, and testing of probiotic foods present many unique challenges, particularly when a food manufacturer wishes to make health claims about the probiotic food.
The human digestive system, from the mouth to the anus, is inhabited by a large number and a wide variety of microorganisms (Marteau et al., 1993; Tannock, 1995). Some of these microorganisms are beneficial to the host and some are detrimental (Gibson and Roberfroid, 1995).
Originally, research was carried out to find substances of microbial origin that could selectively kill unwanted microorganisms. These compounds were termed antibiotics. The definition has expanded to include substances of a non-microbial nature that have the same microbial killing properties.
The term probiotic was first used to describe substances that had the opposite effect of antibiotics.
Thus probiotics were originally 'substances secreted by one microorganism that stimulates the growth of another' (Lilley and Stillwell, 1965). The definition evolved to the more general definition of Sperti (1971) - 'tissue extracts which stimulate microbial growth' while Parker (1974) stressed the beneficial effects on the host when he defined probiotics as bacteria 'which contribute to intestinal microbial balance'.
However, Fuller felt that a probiotic preparation needed to contain viable microorganisms (Fuller, 1989,1992), and this led to him to define a probiotic as 'a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance'. This is perhaps the most widely quoted definition in the scientific literature. Recent advances have led to an even broader definition - 'a microbial preparation which contains live and/or dead cells including their metabolites which is intended to improve the microbial or enzymatic balance at mucosal surfaces or to stimulate immune mechanisms' (Reuter, 1997). Reuter's definition is more inclusive in that it does not require the probiotic to contain live microorganisms and it also broadens the possible beneficial effects to the host.
In the scientific and non-scientific literature, both bacteria and foods are referred to as probiotics. Probiotic foods interact with the intestinal microflora and bring about changes that benefit the health and metabolism of the host and therefore fit into the broader category of functional foods.
The type of microorganism that may be added to a probiotic food depends on the function that the microorganism is to play as it passes through the host.
From a more practical perspective, other criteria are also important in choosing potential probiotic microorganisms. Practical characteristics, safety aspects and efficacy are all important in the design and production of new probiotic products. Table 1 lists the criteria that various authors have suggested when judging the suitability of probiotic microorganisms for inclusion in foods and beverages. At the present time, the list of microorganisms that meet all or most of these criteria is not long, and some characteristics, such as resistance to low pH, have been shown to be strain specific (Berrada, et al., 1991; Clark et al., 1993). In spite of this, the number of probiotic products in markets around the world continues to grow. Lactobacillus spp., Bifidobacterium spp., and Streptococcus spp. appear most commonly in probiotic products.
Yeasts and fungi could also be included in probiotic products, but at the present time the majority of probiotic products contain only bacteria. Probiotics can contain one or more microorganisms. It is becoming evident that desirable characteristics may be unique to specific strains of microorganisms and so more emphasis is being placed on precisely defining the strain being used in a probiotic product. This need is being filled by new molecular genetic methods that allow definitive identification to the species level.
4. Probiotics - number of microorganisms required
It is generally believed that a probiotic product must contain high numbers of microorganisms after production, packaging and storage. To date, the majority of probiotic products have been milk-based. These products have low pH (~ pH 4.2) and have high concentrations of organic acids (lactic, acetic), both of which discourage growth and survival of possible probiotic bacteria. Yoghurt-type products often contain probiotic bacteria in addition to the bacteria that ferment the milk to produce the product. However, when two or more bacteria are incorporated into a single product, the production of bacteriocins and hydrogen peroxide by one bacteria can often reduce the viable numbers of other bacteria.
A two-step fermentation process has been proposed as a way of increasing the numbers of probiotic bacteria in the final product (Lankaputhra and Shah, 1997). The probiotic bacteria are added first, allowed to ferment and then the yoghurt producing bacteria are added. Using this strategy, probiotic bacteria (Bifidobacterium longum) counts were increased 4-5 times over those found after a single combined fermentation. Storage conditions also impact on the number of viable bacteria that reaches the consumer. Dave and Shah (1997) showed that the type of container used in the preparation and storage of yoghurt affected the numbers and survival of Lactobacillus acidophilus.
Many potential probiotic bacteria are sensitive to oxygen. Dave and Shah showed that the amount of dissolved oxygen in yoghurt stored in glass containers is lower than the same product stored in plastic containers, resulting in a better survival rate in glass bottles. The pH of the stored product also influences survival (Martin and Chou, 1992). The numbers of several species of Bifidobacterium declined (reductions of 2 log units or more) in yoghurt fermented to a final pH of 4.2 and stored for 56 days. Yoghurt fermented to a final pH of 5.5 showed slower drops in viable bacteria, and these losses were attributed to declines in the pH of the products while in storage. The strategy used by some food manufacturers is to add a high number of microorganisms to the product to compensate for losses during processing and storage, and to hope that the survival rate is high enough to benefit the consumer.
However, Hamilton-Miller et al. (1999) found that only 7 of 21 probiotic supplements purchased in Britain had bacterial counts quantitatively similar to values reported on the product labels. This may indicate manufacturing problems that impact on the final product, or there may be a lack of good microbiological methods to monitor product composition. As pointed out by Shah (1999), while selective media for counting a single bacterial species in pure culture are available, reliable protocols for counting populations of different species in a complex matrix such as yoghurt have not been established.
It may be for this reason that many manufacturers list only the type of organism contained in their product, rather than the type and number of organisms (Hamilton-Miller et al., 1999). Some countries, such as Japan, have set standards for viable probiotic bacteria cells per millilitre of fresh dairy product (107), but many other countries have not (Shah, 1999).
The answer to the question of how many bacteria need to be consumed to produce a positive effect on the metabolism and health of the host depends on the microorganism in question and also the desired effect.
It is evident that approved health claims for a product will be forthcoming only when such values are well established by sound scientific studies. For probiotic bacteria in general, the estimated number of organisms required ranges from 106 to 1011 cfu/day (Robinson, 1978; Sellers, 1991; Saxelin et al., 1991).
These values are based on the criterion that sufficient numbers of microorganisms must traverse the stomach and reach sites in the lower intestine to be effective. Various groups have measured the percentage of viable bacteria that survive the harsh conditions in the stomach in vivo (Table 2). The overall survival rate is generally low, and therefore the numbers of live bacteria in the probiotic product consumed must be large. Some bifidobacteria appear to be more capable of passing through the stomach than lactobacilli. This in part may be due to the fact that bacteria derived from humans may be more resilient to in vivo conditions than non-human bacteria. Saxelin et al. (1991) carried out a dose response experiment in which they fed humans 1.5x106 to 1.1 x1011 cfu Lactobacillus casei GG per day. This human strain had been shown to be both acid resistant and bile resistant and was therefore a good candidate as a probiotic. The test bacteria could not be found in subjects receiving 106 to 108 bacteria/day at any point in the 7 day feeding trial, but 100% colonisation was found in subjects receiving 1010 or 1011 cfu/day.
Lower dose rates might be effective if the bacteria to be used in probiotic products are coated or encapsulated to protect them until they reach the target area (usually the large intestine) in the gastrointestinal tract. However, Saxelin et al. (1995) reported that 1.6 x108 cfu Lactobacillus casei GG given in gelatine capsules was not sufficient to ensure colonisation in humans. Strategies such as encapsulation may be developed to protect microorganisms during gastric transit and thus widen the application of candidate microorganisms; however, even if colonisation occurs, the probiotic bacteria persist in the intestine for only a few days after the cessation of administration, even for strains of human origin (Saxelin et al., 1991).
Fuller (1989) in his definition of a probiotic emphasised the need for viable microorganisms to be consumed. This may not be a necessary requirement in all cases. If the active ingredient in a probiotic product is produced during the fermentation of the starting food (milk for example), then as long as this active ingredient is not destroyed during processing and storage, the viability of the microorganisms in the product when consumed is not important. Matar et al. (1996, 1997) showed that milk fermented by Lactobacillus helveticus L89 produced peptide fractions that had antimutagentic properties and that the amount of antimutagentic activity increased as the length of the fermentation increased.
They concluded that it was the products of proteolysis during fermentation and not the bacteria themselves that were the bioactive ingredients. Charteris et al. (1998) list a number of different bacteria that have been shown to produce exopolysaccharides when added to milk. Polysaccharides have applications in food science related to viscosity enhancement but may also be beneficial to human health. In at least one case (kefir) there is published data to show that the polysaccharide may have beneficial effects on cancer initiation and promotion (Murofushi et al., 1983, 1986) which again are independent of the bacteria that produced the polysaccharide.
The bacteria that make up kefir grains that are added to milk to produce kefir drink also produce a sphingomyelin that has been shown to enhance interferon-ß production in cell culture (Osada et al., 1994). It is evident that, based on these examples, the active ingredient is present in some probiotic products whether the microorganisms that produced them are alive or not. Restricting the definition of a probiotic to only foods or beverages that contain live microorganisms is therefore too restrictive.
Experiments designed to show the efficacy of probiotic products are more difficult to carry out than typical nutrition experiments or tests of pharmaceutical products. Probiotic products contain live microorganisms - the numbers and populations of which are dynamic - and therefore the test product must be analysed over the course of a feeding trial to ensure that the numbers and types of microorganisms are not changing. Fig. 1 shows how the numbers of different bacteria in a fermented milk product changed during storage, and emphasises the importance of proper handling and storage of probiotics in a feeding trial before consumption.
Double blind placebo controlled experiments testing the efficacy of probiotic products are particularly difficult to design because of the need for a proper control (Farnworth, 2000). When there is uncertainty about the active agent, whether it is a particular microorganism or a product of fermentation, the choice of a suitable control is even more difficult. If a microorganism is the active ingredient, the product containing the microorganism in a deactivated form would be the most appropriate control (Mainville et al., 2001). Creating a positive effect on the host is the principle on which all functional foods are based. Slowly, a scientific consensus is being reached about the beneficial health effects of some probiotics. Table 3 is a recent summary of beneficial effects of some probiotics based on at least two human feeding trials. Health regulatory bodies have indicated that approval to use health claims on product labels will only be granted when such claims are supported by sound scientific evidence (Anon., 2001).
The future of probiotic products lies in the identification of microorganisms - bacteria and fungi - that can produce a positive effect on human metabolism and health when they are used to produce, or are contained in, foods and beverages. We need a better understanding of the gastrointestinal tract microflora, what microorganisms are present and what affects their population numbers. Production and processing procedures need to be developed which ensure that large enough numbers of probiotic bacteria are contained in products when they are consumed and traverse the stomach to reach target areas in the intestines. Well designed human experiments need to be carried out to demonstrate the beneficial effects of probiotics.
Anon. 2001. Health Canada, Standards of Evidence for Evaluating Foods with Health Claims. Synopsis of the Consultation Document. Available at:
About the author Edward Farnworth is a senior scientist, section head of the bio-ingredients section and co-ordinator of the functional foods / nutraceuticals programme at the Food Research and Development Centre. His multidisciplinary team brings together expertise in nutrition, metabolism, microbiology and food science to study the effects of functional foods on human health. He is working with industrial partners to demonstrate the health benefits of fermented foods.

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