Bacteria banish fowl bugs

by Helen R. Pilchernews@nature.com
Publicado en: BioEd Online (http://www.BioEdOnline.org)

A probiotic diet makes chickens healthier and safer to eat.

Chickens could benefit from a daily dose of friendly bacteria, researchers say.

Probiotic bugs can destroy food-poisoning bacteria inside poultry, making the birds healthier and safer to eat.
The benefits to human health of probiotics are well known. The bacteria, found in yoghurt, are thought to out-compete other gut bacteria, including those that cause food poisoning.
Probiotics are thought to have similar benefits in animals, and are already included in some agricultural feeds. But such feeds contain a slew of bacteria, says Arjan Narbad from the Institute of Food Research, Norwich, so their effects are uncertain.

Narbad and colleagues tested a single probiotic dose in the lab. The good bacterium Lactobacillus johnsonii ousted the harmful Clostridium perfringens from chicks' guts, they report in Letters in Applied Microbiology 1 .

"We have used a single strain and shown that it can be targeted to eliminate a specific pathogen," says Narbad.
Clostridium can flare up chickens, making them sickly and thin. It's also one of the top five bacterial causes of food poisoning in humans. In the United Kingdom, the bug poisons about 200 people through undercooked chicken each year.

Health food
"We also have preliminary data suggesting that Lactobacillus may be effective against Campylobacter," Narbad says. This is a much nastier bug, causing about 63,000 UK cases of food poisoning each year. Lactobacillus also has a weak effect against the sometimes-deadly gut bacterium E. coli.

Probiotics could have other benefits too. They may also increase chicken growth rate, for example, says microbiologist Anne McCartney from the University of Reading.
The probiotic bacteria are easy to give to animals, as they can be put in animal feed or drinking water, says Narbad.
They should also help to reduce the use of antibiotics in animals. Farmers are being encouraged to cut their use of antibiotics to reduce the chance of bacteria evolving resistance.
The team is now seeking other probiotic species to help combat different pathogens. They plan to test the treatments on farms to see if results are as good as they are in the lab.

1 La Ragione, R.M., La Narbad, M.J., Gasson, M.J. & Woodward, M.J.. Letters in Applied Microbiology, 28, 197 - 205, (2004).

Microbiology: Gut reaction

Nature 427, 284 - 286 (22 January 2004); doi:10.1038/427284a

Consumers are stocking up on live yoghurts and fermented drinks that claim to improve health. But is there any science behind the marketing of these 'probiotic' products? Alison Abbott investigates.

Glenn Gibson's wife prefers him to tell dinner-party guests that he works as a painter and decorator. That's understandable, because if he talks about his real job as a researcher of gut bacteria at theUniversity of Reading, UK, the conversation all too easily turns to the source of his research material — human excrement.

For better or worse, faeces provide the best window into the microbial life of the human gut, a subject that is attracting more funding nowthan ever before. Partly in reaction to commercial claims that the bacteria in some yoghurts and other 'probiotic' products can boost health, the European Union (EU) has invested more than 15 million (US$19 million) since 1995 to research this poorly explored frontier.

As a result, a growing number of microbiologists are taking an interest in the ecology of the human gut. They are adapting tools previously developed for the study of microbes in oceans and soil to answer a range of questions. What lives in our gut? Do some natural gut microbes predispose us to diseases such as colon cancer? And can we change the make-up of our intestinal residents to improve our health?
Gibson has even built a collection of artificial guts to study our internal microbial ecology under controlled laboratory conditions (see 'Roboguts').

Hidden world
The average human intestine contains about 1.2 kilograms of bacteria plus a smattering of yeasts. So far, few of these microbes have been characterized or even identified. But this dearth of information hasn't kept companies from promoting the health value of probiotics, which contain living bacteria, and prebiotics — nutrients designed to boost populations of beneficial bacteria already living in the gut.

Probiotic dietary supplements are available in just about any form imaginable, from tubes of liquid to capsules. Some yoghurts and fermented milk drinks also promote their living contents. A typical online shop claims that its probiotic products can "strengthen the immune system, reverse the negative effects on the digestive tract of infections, antibiotics, alcohol ... treat symptoms of irritable bowel disease" and more.

Worldwide, the pro- and prebiotics market is now worth about US$6 billion.
But so far, the science behind these commercial boasts is rather limited. "There are a lot of bogus claims and vested interests," says Michael Blaut, head of gastrointestinal microbiology at the German Institute of Human Nutrition in Potsdam, and one of the researchers who helped to convince the EU to fund probiotics research.

Although some clinical trials of probiotics have suggested a benefit, Gibson adds, few of these have been sufficiently rigorous. And even when probiotics seem to work, he says, we know too little about the normal gut ecosystem to understand why.

Soon after it was established, the EU-funded network, which includes scientists from 16 countries, discovered that the gut ecosystem is much more diverse than previously thought. Microbiologists knew that their traditional techniques of isolating and cultivating individual microorganisms were not pulling out all of the species that we live with. Many gut bacteria are notoriously difficult to grow in culture — largely because they depend on the presence of other bacterial species. But few scientists had anticipated just how diverse the ecosystem would turn out to be.

To begin to quantify the diversity, gut researchers borrowed a method from soil and ocean microbiologists that relies on comparisons of the gene for a portion of the ribosome — the cellular machine that manufactures new proteins — known as 16S. The ribosome is so fundamental to the workings of the cell that it has changed little during evolution. That makes it easy to extract the 16S genes from all microorganisms in a single faecal sample using the DNA-amplifying polymerase chain reaction. By looking for subtle differences between the sequences of these genes, microbiologists can gauge the number of different species present in the sample.

One surprise was that no two people have quite the same complement of bacteria. In unpublished work, molecular biologist Joël Doré of the INRA, the French agricultural research agency in Jouy-en-Josas, near Paris, has so far analysed the faeces of more than a dozen healthy adults and found the contents of each to be quite different. Although thousands of microbes can live in the gut, each person has only about 100 different species. "There is remarkably little overlap in the gut bacterial species between individuals," he says.

This is partly because of the haphazard way in which the bacteria arrive. Our guts start off in the womb completely sterile, but they are rapidly colonized with vaginal and faecal bacteria during birth.
Microorganisms from food and other environmental sources contribute to the mix during the first months of life. By the age of two at the latest, the average human gut hosts its full complement of microbial species, mixed and matched from a dozen or so dominant groups of bacteria and a longer list of rarer bacteria and yeasts.

From this point on, little changes until old age — a person's microbial complement seems to remain stable throughout adulthood, Doré says.
But he has found that the faeces of people over 60 contain a much larger number of different bacteria than younger people. He suspects that the weakening barrier to new species may help explain why the elderly are more susceptible to gut infections and certain forms of cancer.

Colonic closed shop
The basis of the microbial stability that persists throughout most of our lives is still poorly understood, but is probably related to nutrient supply. By the time an infant is two years old, resident bacteria have monopolized every source of nutrients in the gut. They have also become interdependent, supplying nutrients to each other — one cell's waste is another's food. With all the nutrients accounted for, newcomers may find it hard to gain a toehold.

The stability of this ecosystem benefits not only the gut microbes, but also the human host. It prevents pathogenic bacteria, such as the various species of Salmonella that cause food poisoning, from taking up long-term residence. But it also means that probiotics cannot permanently change the make-up of the gut — they must be taken daily to have any effect.

What are the possible benefits? Individual species of bacteria are informally classified on a sliding scale of 'goodness' and 'badness'.
Collectively, gut bacteria aid digestion by breaking down tough fibres, enzymes and other proteins. In addition, 'good' bacteria, such as species of Lactobacillus, Bifidobacterium and Eubacterium, are involved in fermentation reactions that produce organic acids that can be absorbed into the body and used as an energy source. 'Bad' bacteria, such as some members of the genus Clostridium, generate as by-products compounds including nitrosamines and cresols, which are possible carcinogens.

Commercial probiotic strains are, of course, 'good' bacteria. The probiotic milk products, yoghurts and capsules on the market generally contain Lactobacillus and Bifidobacterium. Most studies of their efficacy have been poorly controlled and have produced contradictory results, says Gibson. But a handful of well-designed clinical trials indicates that some such bacteria may help ameliorate diarrhoea1-3 and some types
of inflammatory bowel disease4, 5.
Less well documented are the claims for beneficial stimulation of the immune system. The gut, with its massive blood supply, is the immune system's primary contact with the outside world, and gut bacteria seem to play a role in teaching the immune system to differentiate between dangerous invaders and non-hostile challenges.
Although some studies suggest that probiotics can affect features of the immune system, few have shown that these changes are beneficial to health.
A notable exception is a long-term study supported by the Finnish Academy of Sciences, in which pregnant women from families prone to allergies ate Lactobacillus rhamnosus daily. After delivery, the bacteria were given daily to the babies for the first six months of their lives.

The treated infants were much less prone to allergic reactions such as eczema than controls who did not get the bacteria6, 7. Erika Isolauri, an immunologist at th University of Turku who led the study, is now trying to determine how the treatment works. She suspects that the probiotics shift the balance between pro- and anti-inflammatory factors in the developing gut.

Friend or foe?
Other studies to assess the health benefits of probiotics are under way. With funding from the EU, for example, Doré is setting out to test a combination of Bifidobacterium animalis and a type of prebiotic sugar known as FOS on the gut ecosystems of young and old people in France, Germany, Sweden and Italy. "We are testing faecal samples to see whether the level of Bifidobacterium really does rise with this treatment, as would be expected," he says. His team will also assess how the levels of toxic and potentially carcinogenic compounds in the gut rise and fall with treatment by exposing cell cultures to extracts from the subjects' faeces. The researchers hope to determine whether suppressing 'bad' bacteria with pre- and probiotics might protect against colon cancer.

Francisco Guarner, a gastroenterologist at the Vall d'Hebron Hospital in Barcelona, Spain, is helping to organize an EU-backed clinical study involving 360 patients chronically suffering from one of two types of inflammatory bowel disease — ulcerative colitis or Crohn's disease — at centres in Ireland, Spain, Finland and France. The patients, all in remission, receive either Lactobacillus salivarius or Bifidobacterium infantis, two species that reduce gut inflammation in lab animals. The researchers then test the patients' saliva for marker molecules associated with inflammation. "Animal studies show that inflammatory disorders of the bowel may be helped by making the gut microbes less aggressive," says Guarner. He hopes that the probiotics will extend the patients' remission so they can reduce their reliance on immunosuppressive drugs, which have severe side effects.
Outside the EU network, Gibson is running trials at six British centres using Lactobacillus plantarum together with a second type of prebiotic sugar called GOS in various kinds of inflammatory bowel disease.

Gibson's hypothesis is that the yeast Candida causes the symptoms, and he hopes that the probiotics will outcompete its growth. In a separate study, he is testing his hypothesis that certain 'bad' bacteria contribute to ulcerative colitis by generating toxic sulphur compounds such hydrogen sulphide, which smells of rotten eggs. He is giving patients FOS and GOS to stimulate the growth of competing 'good' bacteria to see whether this eases the symptoms.

Microbes on trial
In the next few years, these and other studies will help to determine how beneficial probiotics actually are. In the meantime, other salient questions are being addressed. Can we, for example, assume that probiotics are safe? One potential problem is that many probiotic strains have genes that allow them to resist antibiotics, which they might pass on to pathogenic bacteria. To address these concerns, Herman Goossens of the University of Antwerp in Belgium has acquired more than 200 commercial probiotic strains — the world's largest collection. He is systematically analysing them for their potential to transfer antibiotic resistance genes, and is also testing for any direct toxic effects that they may have.

Some experts believe that another important step will be to read the genomic sequence of every species of microbe that can colonize the human gut. They argue that a complete genomic databank would make it much easier to select species for specific probiotic effects.

To that end, the Defense Advanced Research Projects Agency, a research arm of the US military, is sponsoring a project to read all the genomes in the gut ecosystem with the same 'shotgun' method used for the privately funded effort to sequence the human genome. This approach avoids the need to separate out individual organisms.

Instead, fragments of all the genomes are read off together. Computer algorithms then reassemble the fragments on the basis of overlapping sequences into complete genomes. "It's possible to conceive of doing this because the cost of sequencing has come right down," says Claire Fraser, director of The Institute for Genomic Research in Rockville, Maryland, where the work will be done.

Even if probiotics and prebiotics prove to have only modest health benefits, some scientists are considering the possibility of souping them up with genetic engineering. Many proponents of probiotics reject this idea, saying that it would be too hard to convince the public to eat live, genetically modified bacteria. But among the traits that would be useful to engineer are the ability to survive the acid environment of the stomach, a bit of 'stickiness' to help bacteria adhere to the gut lining, and so take residence for longer, and the ability to produce organic acids. Bacteria might even be engineered to deliver drugs, vitamins or vaccines8, 9.

There is already evidence that some bacteria can serve as efficient delivery vehicles. For example, Lothar Steidler of Ghent University in Belgium and his colleagues have shown that Lactococcus lactis genetically modified to secrete the anti -inflammatory molecule interleukin-10 can reduce colitis in mice10. A version for humans has also been developed that includes safety features to prevent the escape of the inserted gene into the environment11. A small clinical trial of this microbe is planned in Amsterdam, marking the first use of a genetically engineered bacterium as a therapeutic agent.

Unfortunately for the scientists studying probiotics, the only way forward is to delve into more human waste. Doré says he recently felt a pang of regret over that fact on a trip to visit some oceanographer friends in Marseille. "I looked out into the Mediterranean and thought:
'I'm obviously working on the wrong ecosystem'," he sighs. But the scientific challenges presented by gut bacteria are interesting enough to keep him going, he says. "It makes up for the unpleasantness."

ALISON ABBOTT
Alison Abbott is Nature's senior European correspondent.
References
1. Guandalini, S. et al. J. Pediatr. Gastroenterol. Nutr. 30, 54–60
(2000). Article PubMed ISI ChemPort
2. D'Souza, A. L., Rajkumar, C., Cooke, J. & Bulpitt, C. J. Br. Med. J. 324,
1361–1366 (2002). Article
3. Cremonini, F. et al. Aliment. Pharmacol. Ther. 16, 1461–1467
(2002). Article PubMed ISI ChemPort
4. Rembacken, B. J., Snelling, A. M., Hawkey, P. M. Chalmers, D. M. & Axon,
A. T. R. Lancet 354, 635–639 (1999). Article PubMed ISI ChemPort
5. Gionchetti, P. et al. Gastroenterology 119, 305–309
(2000). PubMed ISI ChemPort
6. Kalliomäki, M. et al. Lancet 357, 1076–1079
(2001). Article PubMed ISI ChemPort
7. Kalliomäki, M., Salminen, S., Poussa, T., Arvilommi, H. & Isolauri, E. Lancet
361, 1869–1871 (2003). Article PubMed ISI
8. Seegers, J. F. M. L. Trends Biotechnol. 20, 508–515
(2002). Article PubMed ISI ChemPort
9. Wood, B. J. B. & Warner, P. J. (eds) in The Lactic Acid Bacteria Vol. 3, 261–
290 (Kluwer Academic, New York, 2003).
10. Steidler, L. et al. Science 289, 1352–1355
(2000). Article PubMed ISI ChemPort
11. Steidler, L. et al. Nature Biotechnol. 21, 785–789
(2003). Article PubMed ISI ChemPort

Microflora in water kefir grains

Foodinfo Online FSTA Reports -->13 November 2003

Water kefir grains are a unique starter culture community commonly used to produce home-made sparkling acidic beverages with low-alcohol content.

The white to grey coloured, irregularly shaped grains comprise a backbone of a polysaccharide matrix such as dextran. The microflora of water kefir grains differs significantly from that of dairy kefir grains, which, unlike water kefir grains, have been widely studied. The grains can be simply inoculated under non-sterile conditions using sucrose-containing tap water in the presence of dried figs. They are also known as Tibi grains, sugary kefir grains, gingerbeer plants or Californian bees.

These different grains are all thought to have a similar complex microbial community comprising yeasts and lactic acid bacteria. The addition of figs is essential for the optimal fermentation and production of lactic acid and acetic acid. Figs also contain growth-promoting substances, which can be extracted with cold water. Water kefir grains utilize a nutritionally simple environment compared with the complex milk medium for dairy kefir grains. It is therefore of interest to compare the microflora of these two types of kefir grains.

In a study by Neve and Heller,1 the composition of the microflora of water kefir grains, obtained from a domestic household, was analysed. The diversity and distribution of the microbial population of the grains were also studied by scanning electron microscopy.

Water kefir grains were found to comprise a complex biofilm of yeasts and bacteria of various shapes on the grains surfaces embedded in the dextran matrix. There were high numbers of bacterial cocci in water kefir grains, compared with the scarcity reported in dairy kefir grains. Both types had a predominance of specific yeast populations.

---------------------------------------------------------------------------------

1 Neve, H; Heller, KJ (2003).

The microflora of water kefir: a glance by scanning electron microscopy.

Kieler Milchwirtschaftliche Forschungsberichte 54 (4) 337-349.

Página de WEB referencia del artículo

AN: 2003-07-Hs1624
TI:
The microflora of water kefir: a glance by scanning electron microscopy.
DA:
5-Jul-2003
DT:
Journal Article
AU:
Neve, H.; Heller, K. J.
PY:
2002
AD:
Inst. for Microbiol., Fed. Dairy Res. Cent., PO Box 6069, D-24121 Kiel, Germany
SO:
Kieler Milchwirtschaftliche Forschungsberichte 54 (4) 337–349
RF:
32 ref.
LA:
English with French, German summaries
SN:
0023-1347
AB:
Water kefir grains comprise a unique starter culture community used for production of home-made sparkling acidic beverages. Composition of water kefir grains obtained from a private household and preserved in the laboratory for several years was investigated by SEM. A complex and tightly packed biofilm was observed on the exterior of the grains, while the interior mainly comprised unstructured (dextran) material. Microflora was dominated by lemon-shaped or long filamentous yeast cells growing in close association with bacterial cocci and short or long rod shaped bacteria (lactobacilli, responsible for polymer production). Cocci grew preferentially on the surfaces of the yeast cells, while the rod-shaped bacteria were mainly found between the yeast cells. Results are discussed in relation to microflora of dairy kefir grains.
SC:
Alcoholic and non-alcoholic beverages
KW: BEVERAGES; ELECTRON MICROSCOPY; FERMENTED MILK; KEFIR GRAINS; MICROFLORA; MICROORGANISMS; SEM; STRUCTURE

Bacteria and us

FoodInfo Online Features -->18 July 2003
Svetlana Rodgers Senior Lecturer, School of Management, College of Law and Business, University of Western Sydney, Campbelltown campus, Building 17, Locked Bag 1797, Penrith South DC NSW 1797 Australia.

Hygiene and cleanliness are undoubtedly embedded in our consciousness.

Our supermarkets are full of antibacterial detergents, sanitisers, toothpastes, sprays, creams and lotions, while the food we eat is often over-processed and devoid of natural bacteria. Even salad vegetables are washed in a sanitiser.

Psychologically, the dislike of bacteria most likely originates from the fear of infection, which has followed humankind throughout its history.

In the Middle Ages, for example, the poor quality of the water meant that it was safer to drink wine. From a practical perspective, modern food companies follow strict hygiene rules so that they comply with food legislation requirements, with many resources being utilised so that they may adhere to such guidelines.

Our frequent preoccupation with hygiene and cleanliness carries a price not only to the environment, but also to our health. Throughout much of our evolution, humans have been bombarded with dirt and germs - removal of which from our surroundings also reduces stimulation of the human immune system, which, as a consequence, would function below optimum if man lived in such a barren environment.

Scientists believe that children raised in less hygienic conditions are less likely as adults to develop asthma, allergies, diabetes, rheumatoid arthritis and other immunity-linked disorders. In 1910, in his book, The Prolongation of Life, the founder of the theory of 'probiotic' or beneficial bacteria, Russian scientist and Nobel Prize Laureate Ilya Metchnikoff, said 'a reader who has little knowledge of such matters may be surprised by my recommendation to absorb large quantities of microbes, as the general belief is that microbes are all harmful. This belief, however, is erroneous'. Since then, it has been found that probiotic bacteria can reduce the risk of colon cancer, lower cholesterol, increase immunity, and can even offer protection from diarrhoea.

We carry about 8 kg of indigenous bacteria in our gut, the quality of which is important to our health. The addition of probiotic bacteria to fermented products has become common today.

In Russia, yoghurt made using the 'strong' bacteria taken from the guts of cosmonauts are a marketing attraction, while in the livestock industry, pathogen-fighting bacteria can replace the use of antibiotics.

Probiotic bacteria can even be added to women's hygiene products to exclude bacteria that can cause debilitating infections, premature births or infection of newborn infants.

Harmless bacteria can not only improve our health and immunity, but can also exclude food poisoning bacteria in foods. So-called lactic acid bacteria are present in high numbers in products such as yoghurt, fermented vegetables and sausages. This explains why fermented products have an excellent food safety record.

In 1995, a leading scientist in food microbiology, James Jay, wrote an article called 'Foods with low numbers of microorganisms may not be the safest foods'. He argued that, with improvements in cleaning/sanitising, not enough harmless background organisms were left to prevent proliferation of food poisoning bacteria. Surprisingly, there are very few commercial applications of so-called 'protective' cultures in non-fermented foods.

The Wisconsin process for cured bacon is one of these applications and employs addition of sucrose and lactic acid bacteria to prevent botulism in bacon, together with low concentrations of nitrite. Botulism is also a potential risk for refrigerated minimally processed foods, such as packaged cook-chill meals used in catering and retail. At the Centre for Advanced Food Research at the University of Western Sydney, we have developed a method using lactic acid bacteria for the prevention of botulinal toxin formation in cook-chill meals. As with the majority of microorganisms, these protective cultures are dormant when the product is stored under the correct temperature and 'switch on' to fight food poisoning bacteria only when temperature abuse takes place. Although the cultures may be visible in clear products, they go unnoticed in thick soups or casserole-style products.

In a recent trial, a sensory evaluation panel was unable to distinguish between seafood chowder, vegetable curry and chicken casserole with and without culture added. The biggest technological challenge now is to develop a method to protect these cultures during heating. If a product is cooked and then aseptically/hygienically packaged or stored under vacuum in Greenvac-type containers, the cultures can be incorporated directly. Extended shelf life cook-chill technology, however, is a modern development, which requires more sophisticated preservation methods.

In addition, the risks of food poisoning are increased through intensive farming practices, extensive transportation, the emergence of more resistant bacteria and an ageing population. Protective cultures offer temperature-responsive, natural preservation, and also bring some bacteria back into our lives.

For more information contact Svetlana Rodgers, senior lecturer, whose research led to the patent application "Preserving with Lactic Acid Bacteria" PCT/AU01/01549

La referencia en la página WEB

Homens e micróbios

Nobel Joshua Lederberg,

escreve no EXPRESSO On-Line

A PRIMEIRA grande notícia científica deste século foi a campanha de descodificação do genoma humano.
Devemos agora recordar que grande parte da composição biológica do nosso corpo consiste em genomas que não são humanos. Multidões de bactérias e vírus vivem na nossa pele, nas nossas membranas mucosas e na nossa região intestinal. Provavelmente, desempenham um papel muito mais importante no desenvolvimento e na resistência às doenças do que aquele que nós imaginamos. Compreender esta co-habitação de genomas dentro do corpo humano, a que chamo microbioma, é fundamental para perceber a dinâmica da saúde e da doença.

Depois de um intervalo de algumas décadas, os germes e as doenças ocupam de novo muito do nosso pensamento, em grande parte por causa do terrível impacto da sida em todo o mundo. Temos também uma nova consciência de que doenças globalmente espalhadas como a tuberculose e a malária permanecem flagelos históricos. Agora mais perto de casa, a notícia é que novos surtos de doenças como a SRA (síndrome respiratória aguda) se espalham a partir da China para o resto do mundo, com consequências imprevisíveis neste momento.

Ao longo de toda a história, as doenças infecto-contagiosas regularam as nossas vidas. Só no século XX, graças a medidas de higiene simples como lavar as mãos regularmente e separar a água de beber das águas dos esgotos, assumimos um papel mais importante, para o bem e para o mal, na tentativa de controlar o modo como os micróbios afectam a vida humana.

Uma criança nascida em 1900 nos Estados Unidos tinha uma esperança média de vida de 47 anos. Mas, no fim do século XX, devido principalmente às nossas conquistas relativamente às doenças infecto-contagiosas, essa esperança já era de 80 anos para a mulher e mais ou menos 75 para o homem.

Desde os finais da década de 1920, a metáfora que adoptámos de forma optimista no que se refere ao nosso relacionamento com os germes foi a da conquista dos «caçadores de micróbios» sobre doenças específicas. Na década de 1960, apoiados nos medicamentos e nas vacinas milagrosos do meio do século, muitos afirmavam que «as pragas serão banidas da face da Terra para todo o sempre» -apenas para serem humilhados com o trágico advento da epidemia da sida, que nos mostrou como estamos realmente longe desse objectivo. É óbvio que o excesso de confiança em relação ao contágio foi um efeito secundário da nossa campanha contra os micróbios. Agora, a síndrome respiratória aguda é o novo desafio dos nossos dias.

Em vez do excesso de confiança, da metáfora da conquista e do conceito de erradicação das doenças infecto-contagiosas, há uma lição mais subtil a aprender: o melhor a que podemos aspirar é a uma relação de coexistência simbiótica com as bactérias, vivendo com elas numa «trégua» mais do que numa vitória. Essa coexistência pode desenvolver-se num espectro que vai da terrível pandemia letal até à tolerância mútua.

CARACTERÍSTICAS DOS MICRÓBIOS RELATIVAMENTE AOS HUMANOS

Os micróbios abundam em populações com expoentes de 15 e 20. Digamos que existem na ordem dos ziliões. Trata-se de minúsculos organismos que crescem e se desenvolvem em ciclos de 20 minutos ou menos. Os indivíduos são inteiramente dispensáveis quando uma comunidade de um milhar de milhões de células pode ser substituída do dia para a noite a partir de um único gérmen. Dezenas de biliões de células podem ser cultivadas num simples tubo de ensaio.

Em contraste, a espécie humana tem uma população de menos de 10 mil milhões, muito modesta à escala microbiana. Cada organismo é multicelular e grande, com um ciclo de desenvolvimento longo e custoso. Cada um de nós como indivíduo seria o primeiro a resistir a oscilações violentas do número da população. Nem a sociedade humana poderia florescer sem o cuidado e a protecção da maioria dos indivíduos.

Num maior contraste com as capacidades biológicas dos micróbios, estes permutam facilmente genes no seio da espécie e com outras espécies. Eles não formam novas espécies biológicas nem se diferenciam em organismos isolados geneticamente como nós.
A verdade é que estes microrganismos praticam «uma transferência promíscua lateral de genes», fazendo do mundo microbiano uma espécie de «world wide web» com base no ADN, que partilha informação genética que pode ir de um micróbio para outro.

Quando, por exemplo, os antibióticos entram na nossa rede de esgotos e matam alguns micróbios, é o mutante resistente ocasional que sobrevive. Estes sobreviventes conseguem depois transferir a sua recém-descoberta imunidade aos genes de outros micróbios, incluindo espécies patogénicas que fomentam as doenças humanas.

Os seres humanos não obtêm qualquer vantagem biológica de inovações que evoluíram em aves, ratos ou macacos – excepto o facto de agora termos uma inteligência evoluída que consegue criar uma rede informativa para aquisição e partilha de informação ou de ideias.

Estes micróbios rapidamente evolutivos podem coligar-se nos seres humanos através de sinergias de organismos que provocam doenças ligeiras, mas que, quando juntos com outros, se tornam perigosas. Este pode ser o caso da SRA, que parece uma variante de um vírus da gripe comum.

Para além de estarmos isolados geneticamente das outras espécies, as células da linha de micróbios humanos estão isoladas nas nossas glândulas sexuais, protegidas da maioria das vicissitudes do corpo. Nada que esse corpo possa aprender para criar imunidade por exemplo, contra novo vírus pode ser transmitido através de um espermatozóide ou de um ovo para a geração seguinte. As novas gerações têm de aprender tudo outra vez, num novo ciclo.

Em resumo, as vantagens evolutivas competitivas parecem estar muito mais a favor dos micróbios. Vemos esta disparidade quando grandes pestes e epidemias varrem o mundo.
Tudo levaria a crer que a capacidade evolutiva dos micróbios nos devia ter derrotado há muito tempo.

Então porque não o fizeram? Porque é que estamos ainda aqui, partilhando o planeta com os micróbios? Eles não nos exterminaram porque têm interesse na domesticação e na sobrevivência do hospedeiro os seres humanos e outras criaturas multicelulares. Um micróbio que mata o seu hospedeiro é um micróbio sem futuro. Se for um conquistador vitorioso, extingue a sua vida tal como a nossa. Biologicamente falando, a razão por que ainda estamos aqui é que os parasitas necessitam de hospedeiros vivos para a sua própria sobrevivência.

AS REGRAS BÁSICAS

Esta realidade permite-nos identificar algumas das regras básicas do sucesso evolutivo no mundo dos microrganismos – as regras fundamentais do comportamento dos parasitas.

Até parece que leram a Bíblia e conhecem o Génesis: ide e disseminai-vos como primeira regra. Multiplicai-vos. Em seguida, de acordo com o malthusianismo ou o darwinismo, têm de ser os mais aptos para sobreviver a garantir a maior descendência possível. Depois confrontam-se com um dilema: se exterminarem o seu hospedeiro com demasiada rapidez, não conseguirão propagar-se. Mas, claro, também têm um imperativo de garantir um local de alojamento no hospedeiro, uma cabeça-de-ponte, combatendo as defesas locais e estabelecendo um reservatório para disseminação. É isto que se passa com a doença tal como é sentida pelos seres humanos: a criação de uma base de operações, de maneira que o hospedeiro servil lhe forneça comida quente e abrigo e seja domesticado ao serviço do parasita.

Os sintomas de doença que vemos são muitas vezes secundários para o nosso mecanismo de defesa, mas são explorados em nome da capacidade do parasita de se multiplicar.

Por exemplo, logo que um organismo como o da cólera entra no nosso intestino, provoca a diarreia mais intensa que se pode imaginar. Para dar origem à diarreia, a cólera segrega uma hormona que resulta na libertação de água no intestino. Enquanto o paciente entrar no jogo de uma re-hidratação intensa, é possível equilibrar a perda de líquido, sobreviver, mas também disseminar micróbios aos biliões.

A cólera não «quer» fazer-nos mal, mas a sua sobrevivência como espécie depende da poluição das fontes de abastecimento de água. A doença é então transmitida a outros hospedeiros. Se pudesse prosseguir sem nunca matar o hospedeiro seria o ideal.
Efectivamente, com hidratação adequada, a cólera não tem uma taxa de mortalidade muito elevada. Essa ideia escapou-nos durante 75 anos por não compreendermos que uma «hormona de secreção de água» era tudo o de que necessitávamos para entender como actua a cólera. Por isso é justo dizer que milhões de vidas foram reféns de uma filosofia errada da doença.

Por vezes, um micróbio poderá mesmo proteger o hospedeiro de outros elementos patogénicos concorrentes. Exemplo disso na investigação da sida é a descoberta de que o contágio com uma variante do vírus da hepatite C parece estar relacionado com uma considerável resistência ao progresso do VIH. Não surpreende que um vírus tente expulsar outro. Faz parte da sua estratégia para manter a vantagem competitiva.

A melhor estratégia de todas é fundir-se com o hospedeiro e tornar-se parte do seu genoma.

Após uma tão longa evolução, nós carregamos de facto cerca de 500 retrovírus diferentes integrados no nosso próprio genoma, que são testemunhos de uma história de experiências com parentes do vírus VIH. Após milhões de anos, os antigos vírus que encontramos agora desempenham funções indispensáveis de defesa para o hospedeiro.

CONTENÇÃO

Em resumo, os micróbios que co-habitam o nosso corpo dão mostras de uma contenção considerável ao moderarem a virulência da doença, especialmente em relações bem identificadas com hospedeiros animais. Elementos patogénicos sistémicos como os estafilococos e os estreptococos, que há muito invadiram e vivem dentro do nosso corpo, raramente segregam toxinas fatais. Em consequência, provavelmente um terço de nós anda por aí como portador saudável destes micróbios.

MICROBIOMA

Alargaria, portanto, os nossos horizontes filosóficos pensarmos um ser humano, um espaço corporal em qualquer humano, como mais do que um organismo. É um superorganismo com um genoma alargado, que inclui não apenas as próprias células mas também o conjunto de bactérias e vírus do genoma microbiano flutuante que partilham esse espaço corporal. Alguns destes antigos invasores passaram a viver em permanência nas nossas células, atravessando mesmo a fronteira e tornando-se parte do nosso genoma. Chamo a esse alargado conjunto de companheiros o microbioma e rezo por uma investigação mais profunda sobre o impacto que eles têm nas nossas vidas, para além dos acessos ou dos tropeções a que chamamos doença.

Compreender isto significa que vivemos num pacto de colaboração, «uma trégua» com esses micróbios que não nos matam.

IMPLICAÇÕES

As implicações do nosso novo entendimento são precisarmos de mais investigação, não apenas sobre a virulência das bactérias mas sobre como elas «contêm» a virulência e moderam os ataques. Precisamos de investigar como a nossa flora microbiana –aquela com que nós vivemos sempre -não causa doenças e, em vez disso, nos protege dos seus concorrentes.

Outra implicação é que, filosoficamente, temos de desconfiar sempre do conceito de erradicação, de espetar uma estaca no coração de uma infecção bacteriológica de uma vez por todas. Num mundo assim, não teríamos a experiência crua de alojar estímulos infecciosos e tornarmo-nos mais vulneráveis.

Vemos um exemplo disso, hoje, nos nossos dilemas a propósito da varíola. Pensávamos que a tínhamos erradicado e, efectivamente, tínhamos uma política mundial que pressupunha a sua total erradicação. Como resultado, baixámos totalmente as nossas guardas, tornámo-nos imunologicamente ingénuos e suspendemos toda a investigação sobre a melhoria das vacinas e dos medicamentos antivirais que poderiam ter atenuado uma recorrência acidental ou com intenção criminosa. Agora apressamo-nos a colmatar a lacuna.

Finalmente, temos de compreender que a higiene pode por vezes não ser uma coisa boa se for exagerada. Na tentativa de ter ambientes infinitamente puros podemos por vezes privar-nos dos estímulos de que o nosso corpo necessita para se tornar «inteligente na rua» e desenvolver defesas contra a contaminação.

JOSHUA LEDERBERG**

Joshua Lederberg ganhou o Prémio Nobel em 1958, aos 33 anos de idade, pelo seu trabalho pioneiro sobre a mutação genética das bactérias. Foi presidente da Universidade Rockefeller, em Nova Iorque»

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(c) 2003, Prémios Nobel,
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Tradução de AIDA MACEDO
12:47 15 Maio 2003

¿Política de salud, Multinacionales y Globalización?:

Para comprender un poco más la relación entre concepto de salud, intereses farmacéuticos y comerciales, es interesante referirse a un artículo publicado recientemente por The Ecologist para España y Latinoamérica el 1 de Enero del 2003.

¨La obsesión "higienizante" de la sociedad tecnocientífica es muy rentable para las grandes corporaciones farmacéuticas.

Ahora bien, salvo algunos éxitos concretos, esta enorme inversión no parece muy justificada.

Es más, algunos analistas señalan que el uso y abuso de antibióticos podría tener, a corto plazo, consecuencias devastadoras para toda la Humanidad.

Una vez más, el ciego afán de lucro de unos pocos pone en peligro la vida de millones de personas.

En la actualidad, no pasa ni una semana sin noticias sobre la guerra que la industria empezó hace años contra los microbios.

Entre los últimos acontecimientos destacados podemos citar: una compañía estadounidense especializada en el transporte de productos alimentarios comercializa a partir de ahora un tomate que sólo "ha sido tocado una vez"; la Food and Drug Administration (FDA) estadounidense lanza un programa de investigación sobre la seguridad sanitaria de quesos; un gran fabricante de productos de higiene bucal financia una campaña de promoción a gran escala para sus tiras antibacterianas del tamaño del pulgar, que se aplican sobre la lengua, añadiéndose a los 700 productos de cuidados bucales ya en el mercado... ¡Anotemos, por último, una publicidad en la televisión, que pasa a una hora de gran audiencia, de un nuevo champú antibacteriano pensado para eliminar los microbios que nuestros animales domésticos dejan en las alfombras y moquetas!

NADA EXCEPCIONAL

Estas batallas contra los microbios no tienen, sin embargo, nada excepcional.

Desde que el vínculo entre microbios y enfermedades contagiosas fue establecido hace un siglo, los hombres no han parado de intensificar la lucha contra los microbios, con la esperanza de que éstos pertenecerían un día definitivamente al pasado.

Las batallas ganadas tal como la victoria sobre la polio y la caída espectacular de la mortalidad infantil en Occidente, así como la erradicación de la viruela, han acelerado las cosas.

Pero los recientes fracasos están ahí para recordarnos que no hay que bajar nunca la guardia. En la guerra contra los microbios, nunca se va bastante lejos, diría la voz de la ¿sabiduría? comercial...Sin embargo, esta concepción de los microbios es muy reduccionista. Porque los microbios están en todas partes: en la tierra, en el agua y en el aire. Pueden vivir privados de oxígeno, en el ácido, en el petróleo y en el azufre. Son también capaces de colonizar casi todas las superficies artificiales.

Últimamente se estima la totalidad de la biomasa microbiana como muy superior a la de cualquier otra forma de vida (1). Los microbios constituyen una parte importante de nuestro propio cuerpo y son el producto de miles, incluso de millones de años de co-evolución.

Nos protegen de otros microbios más agresivos e interactúan también con nuestras propias células, de una manera sutil y crucial que empezamos justo a descubrir.A pesar de la opinión extendida, no pueden ser considerados como enteramente buenos o del todo malos, inofensivos o mortales.

Un microbio mortal para tal persona puede muy bien no revelarse perjudicial para otra; el mismo microbio inofensivo, un día, podría llegar a ser perjudicial al día siguiente para el mismo huésped.

Cualquier persona en buena salud es susceptible de llevar microbios que pueden causar úlceras de estómago, neumonías, fiebres, diarreas, envenenamientos de sangre, meningitis... la lista es infinita.

Dicho de otra manera, enfocamos habitualmente sólo un aspecto de los microbios: su carácter infeccioso, pero no queremos saber en qué les debemos también nuestra buena salud.

En efecto, para nosotros la infección por el microbio es sinónimo de enfermedad. Sin embargo, si fuera efectivamente el caso, estaríamos todos muertos en este momento. En una palabra, llevando una guerra sin remisión contra los microbios... nos estamos equivocando.

Al aseptizar nuestro entorno, ponemos en peligro relaciones vitales…

UNA REFLEXIÓN PROFUNDA

El gran misterio ha sido saber cómo el cuerpo puede tolerar esta cohabitación.

Después de todo, numerosos microbios que viven en el cuerpo tienen estrecha semejanza con patógenos conocidos en el entorno, y muchos provocan una reacción inmunitaria cuando emigran de una parte del cuerpo a otra. Muchos de entre ellos portan liposacáridos, moléculas de superficie que se cuentan entre los más potentes estimuladores de reacción inmunitaria de la actividad celular descubiertos hasta la fecha. En estos últimos años nuevas herramientas han permitido a los biólogos sondear interacciones celulares más complejas que las que conducen a la enfermedad, es decir las interacciones entre un huésped y sus simbiosis.

Han descubierto que tales relaciones entre las especies parecen ser de naturaleza química donde cada uno de los protagonistas envía señales que activan los genes del otro… …Un indicio clave vino de trabajos realizados sobre otra relación simbiótica.

Algunos investigadores han identificado docenas de genes que participan en los intercambios entre las células de la raíz de algunas leguminosas y su bacteria residente, fijando el nitrógeno. Cuando la bacteria coloniza el huésped, las células de la raíz se ponen a fabricar nódulos en los cuales la bacteria puede vivir (17). Otro indicio vino de las modificaciones conocidas en animales de laboratorio criados en incubadoras. Estos animales, pretendidamente sin microbios y que crecen sin ninguno de sus microbios habituales, son muy diferentes de sus congéneres, particularmente en lo que concierne a su morfología intestinal. Para los ratones, por ejemplo, las células que tapizan la pared del intestino ciego, bolsa situada al principio del intestino grueso, producen en un momento dado células de naturaleza diferente con funciones especializadas. En los ratones sin microbios esta diferencia no se produce y el tejido del intestino ciego conserva un aspecto fino y elástico.

De ello resulta que la materia fecal no se desplaza normalmente en el tubo digestivo, sino que, al contrario, se acumula hasta formar una hinchazón en la membrana cecal. El intestino ciego de un ratón sin microbios puede así llegar a ser 10 veces más grande que el de un ratón normal (18)… … Se hizo una primera prueba de la observación de la fragilidad de los ratones sin microbios, que sucumbían fácilmente a infecciones que no tenían efecto en otros ratones. Ello condujo a estudios que demostraban que los microbios residentes proporcionan una protección fuerte contra los patógenos exteriores (22 a 27). En uno de estos estudios, un ratón sin microbios murió después de haber ingerido dosis de Listeria monocytogenes que contenían solamente 100 células de este mismo microbio (28).

La manera que tienen los "buenos" microbios de proteger el cuerpo no está establecida claramente. Se sabe que los humanos adquieren ácidos grasos y vitaminas indispensables a través de los subproductos de los microbios residentes. Uno de estos subproductos, la vitamina K, es un elemento esencial en la coagulación de la sangre.Otra manera de actuar que tienen los residentes para protegernos remite a la lucha entre microbios para apropiarse de los primeros organismos multicelulares. En efecto, los virus que han evolucionado con los hombres tienen más facilidad para ocupar las celdas del cuerpo. Dicho de otra manera, simplemente ya no hay más sitio para los patógenos, acontecimiento de apariencia trivial, aunque es ya la primera fase de infección por microbios presentes en el medio. Este fenómeno se ha podido establecer gracias a experiencias recientes en cultivos de laboratorio que estimulan el ecosistema intestinal.

Desde el momento en que los grupos microbianos han alcanzado un equilibrio es muy difícil de introducir entre ellos nuevas especies. Mejor aún, se ha probado que los virus residentes juegan un papel activo en la primera línea de defensa del cuerpo. Esta defensa se ejerce gracias a la fabricación y a la producción de moléculas que, en los cultivos de laboratorio, inhiben el crecimiento de microbios potencialmente peligrosos, lo que llevaría a probar que los primeros luchan para defender el cuerpo… …Otros trabajos han demostrado que los estreptococos orales inhiben el desarrollo del estreptococo neumoniae, al origen de la neumonía y del estreptococo pyogenes, al origen del dolor de garganta.

Algunos comparan este fenómeno a una carrera de armas químicas entre el virus en el cuerpo y los virus que le rodean. "Es un mundo invisible del cual no sospechábamos la existencia antes de estos dos últimos años", dice Page Caufield, microbiólogo en la Universidad de Alabama.

SISTEMA INMUNITARIO

…Además, la presencia de microbios normales parece fortalecer el sistema inmunitario. Hay pruebas en las comparaciones entre ratones sin microbios y ratones normales.

En los primeros el sistema inmunitario subdesarrollado está caracterizado por la ausencia casi total de células inflamatorias de la lámina propia (una de las tres cepas que compone el tubo digestivo desde la boca hasta el ano), un número menor de células plasmáticas produciendo anticuerpos y menos placas de Peyer, órganos linfoides y secundarios repartidos en el intestino en el que las células inmunitarias interactúan (30), (31).

Animales sin microbios tardan más en elaborar una defensa inmunitaria y en cicatrizar después de la vacuna (32).

ANTIBIÓTICOS Y TRASTORNOS DEL EQUILIBRIO CORPORAL

Esta concepción de la enfermedad está ilustrada más adelante con lo que se produce en el cuerpo, acto seguido a la toma de antibióticos, que tiene sobre los ecosistemas microbianos el efecto de una bomba.

Desde que estos medicamentos "milagro" fueron introducidos, los médicos se han familiarizado con la letanía de problemas que surgen con su ingesta.

Uno de ellos es el Pseudomembranus colitis, desorden intestinal que atacó a personas de edad, sobre todo en los hospitales, durante los años setenta. Después de varias muertes, los investigadores acabaron por descubrir que su causa era el Clostridium difficile, un miembro de la microflora normal.

En cantidad escasa este organismo vive en paz en nuestro cuerpo. Pero si su virulencia no está contrarrestada por competidores se desarrolla y segrega una toxina potente que transforma la membrana intestinal en un montón de células muertas, afección mortal si no es tratada (33).

FACTORES ECOLÓGICOS DE LA ENFERMEDAD
Algunos investigadores intentan actualmente estudiar sobre la manera en que los cambios en los factores ecológicos pueden alterar la microflora, y exponer el cuerpo aún más a las enfermedades. En 1999, científicos holandeses (35) hicieron una serie de experimentos con ratones para ver cómo el régimen alimentario afecta a la flora indígena del animal y su sensibilidad a la salmonelosis.

Una colonización acrecentada de lactobacilos provocó un incremento en la absorción de fosfato de calcio. Estos animales no manifestaron ninguna señal de enfermedad cuando fueron contaminados por dosis de salmonela que enfermaron a otro grupo de ratones alimentados de manera diferente.

Las hormonas del estrés son quizás otro factor ecológico que contribuye a la integridad de los ecosistemas microbianos del cuerpo. Estos últimos años se ha probado que el estrés psicológico y la emoción pueden influir en la gravedad de la hemorragia gástrica, la diarrea crónica y otros desórdenes digestivos vinculados con patógenos en las personas. Los científicos saben también que los animales enferman cuando viven muchos confinados en pequeños espacios; esa situación estresante provoca la reacción de microbios que, antes de la cría intensiva de animales, habían sido siempre inofensivos.

La opinión compartida es que estos fenómenos son debidos a las hormonas del estrés que, o bien ahogan el sistema inmunitario, o bien dan a los microbios invasores una virulencia acrecentada. Recientemente, Michael Bailey, estudiante diplomado de la Universidad de Wisconsin en Madison, se preguntó si el problema no podría, al contrario, ser vinculado con la alteración de la ecología microbiana en reacción a niveles hormonales perturbados.

EXCESOS EN LA GUERRA CONTRA LOS MICROBIOS
Lo que temen Abigail Salyers, y tantos otros, es que este equilibrio se vea amenazado, ironía del destino, por la guerra contra los microbios.

Los hombres del mundo desarrollado son, desde luego, hoy en día más limpios de lo que nunca se ha sido jamás en la historia de nuestra especie. Los gusanos parásitos del intestino han sido erradicados de casi todos nosotros en el mundo desarrollado.

El contacto con los protozoos, grupo muy variado de microbios que comprenden la ameba y el paramecio, ha sido ya reducido gracias al tratamiento del agua y de los alimentos. La tendencia es a alegrarse y ver en ello una victoria en la guerra contra los microbios. Pero, por varias razones, mejor sería preocuparse.

Una de ellas es que esta coexistencia pacífica con algunos microbios depende de la duración de la exposición, puesto que los humanos permanecen menos vulnerables dependiendo de si han sido infectados al principio de su existencia. Ello podría, según algunos, explicar el misterioso aumento en el mundo desarrollado de la poliomielitis paralítica al principio del siglo XIX y de las úlceras de estómago hoy en día.

Se sabe que el virus de la poliomielitis (40) y HP (41) eran corrientemente difundidos e inofensivos en el pasado. Aunque una mejor higiene ha reducido la expansión de los microbios, un mayor número de personas se han visto infectadas más tarde en su vida. Esta higiene ha contribuido a modificar los efectos que estos microbios podían tener sobre el sistema inmunitario, transformando una cohabitación apacible en una cohabitación mortal.

Si es éste el caso, tales amenazas pueden provenir de los numerosos microbios que viven pacíficamente en tal población y concretizarse en el caso de una higiene demasiado meticulosa.

CÁNCERES
Otro problema es que los excesos en la guerra contra los microbios afectan la flora autóctona, al igual que los antibióticos, pero de manera más sutil y más duradera.

Los investigadores piensan que la erradicación de HP es el origen de nuevos problemas de úlceras, de reflejos gastrointestinales, que pueden conducir a cánceres.

Tanto es así que eliminar un miembro de la flora autóctona, problemático en condiciones anormales, es arriesgarse a provocar perturbaciones cuando se haya restablecido la situación. En 1995 se constató que las duchas vaginales provocaban una forma de vaginitis (inflamación de la mucosa vaginal) en la que unos lactobacilos son sustituidos por una variedad de organismos responsables de infecciones graves del aparato genital superior y de partos prematuros.

MICROBIOS Y ALERGIAS
Hacia 1970, se tuvo un esbozo de estos vínculos, al constatar que las alergias eran más escasas en las regiones infestadas con gusanos parásitos (48), (49). John Turton, inmunólogo en el Medical Research Council, puso al día un fenómeno que iba a figurar entre los más extraños en los anales de los descubrimientos científicos.

En efecto, había notado la ausencia de sus habituales rinitis del heno durante dos veranos con invasión de anquilostoma (gusano nematodo, parásito del intestino delgado), condiciones que experimentó luego en su casa, criando larvas del gusano (50).

El tema tomó más amplitud en 1989 cuando David Strachan, epidemiólogo en el London School of Higiene and Tropical Medecine, investigó sobre los expedientes de 14.000 ciudadanos británicos y descubrió que la frecuencia de las alergias era inversamente proporcional al tamaño de sus familias. Strachan sostuvo la hipótesis de que tener hermanos y hermanas mayores y estar expuesto a más microbios impedía la perturbación del sistema inmunitario y las reacciones alérgicas que de ahí se encadenan (51).

ARSENAL DE DEFENSAS
Esta hipótesis ha ganado credibilidad recientemente gracias a los adelantos de la inmunología.

Se sabe que el arsenal de las defensas inmunitarias se halla bajo el control de una red de señales químicas conocidas con el nombre de citocinas.

Algunas citocinas actúan durante las reacciones inmunitarias, en respuesta a la presencia de bacterias y de virus. Otras son parte de un proceso que determina un ataque alérgico.

Algunos elementos, que enseñan que estas dos tendencias se regulan, prueban que un sistema inmunitario sano y equilibrado puede depender de la exposición a cierto tipo de microbios (63 a 67).Sin embargo, no se sabe claramente cuáles son los microbios necesarios para una salud óptima. Según algunos, el mundo desarrollado no está suficientemente expuesto a los organismos que viven de la tierra (68). Para otros, hay que acusar a la perturbación de la flora autóctona corporal (69). Como quiera que sea, parece posible concluir que el mundo desarrollado no tiene los microbios suficientes. No se trata de preconizar un regreso a la peste bubónica, si no la aceptación del hecho de que los microbios constituyen una parte del cuerpo. "No digo que tendríamos que estar más sucios", señala Tore Medtvedt, microbiólogo del Instituto Karolinska de Estocolmo y el mayor experto en flora autóctona, "digo que tendríamos que estar menos ‘higienizados’". Con respecto a esta nueva necesidad de microbios, René Dubos escribió hace 40 años: "El verdadero problema no es la aplicación o la mejora de los procedimientos de control que ya conocemos, sino más bien la búsqueda de un saber cualitativamente diferente". Hoy en día Medtvedt y otros hacen suyo este argumento. Dicen que se está a tiempo de aplazar la guerra contra los microbios en una perspectiva que refleje mejor la ecología de las enfermedades infecciosas (70), (71). Ello no quiere decir que los hombres deberían revolcarse en sus propios excrementos, beber agua contaminada o vivir entre las ratas, las pulgas u otros vectores potenciales de enfermedades, o también que tendríamos que abandonar totalmente las armas de lucha antimicrobiana utilizadas en el curso de este siglo. Pero debemos cesar de aseptizar nuestras casas y mantener un grado de limpieza dentro de lo razonable.

Deberemos practicar un modo de crianza de ganado y técnicas de preservación alimentaria que respeten las realidades ecológicas de un mundo lleno de microbios; deberíamos hacer uso de antibióticos y vacunas sólo cuando se revelen indispensables y, antes que nada, definir de nuevo la infección y las enfermedades infecciosas. "Hay que encontrar los mecanismos que hacen que algunas personas estén enfermas", dice Medtvedt, y "eliminar la enfermedad sin erradicar el virus"….

COMPRENSIÓN MÁS PROFUNDA
Este enfoque necesita una comprensión más profunda del número de componentes corporales que interactúan con el mundo microbiano.

En la realidad, los microbios que viven en los intestinos ni siquiera tienen nombre.

Haríamos mal en creer que tal conocimiento está fuera de nuestro alcance. En efecto, parece que la ciencia moderna ya ha tomado este camino.Citemos a este respecto el interés reciente en la investigación de los probióticos que utilizan bacterias para preservar la salud y tratar la enfermedad.

En un reciente encuentro, organizado por la British Association of Paedriatic Surgeons, científicos japoneses testificaron acerca de la utilización de bacterias vivientes para eliminar las endotoxinas del suero (precursores potenciales de una infección sistémica mortal) en nueve bebés, después de una operación. Estos investigadores sugieren que tal enfoque podría ser más seguro que los antibióticos, porque protege a los pacientes de las infecciones post-operatorias peligrosas (72 a 75). …

MEDICINA ORGÁNICA
Para mostrar hasta dónde se ha podido llegar, se puede citar a Joel Weinstock, profesor de medicina orgánica en la Universidad de Iowa, que el año anterior llevó a cabo un estudio clínico en el cual seis pacientes que padecían de la enfermedad de Crohn fueron tratados con una dosis de gusanos parásitos (77).

En cinco de entre seis casos, la enfermedad desapareció y el sexto vio sus dolores disminuir claramente. Los resultados han conducido a estudios más extendidos; en uno, por ejemplo, una paciente está siendo curada exitosamente con la ayuda de estos gusanos.A pesar de estas victorias, los cazadores de microbios imponen aún su ley. En efecto, una teoría sobre los microbios, de hace cien años, ha dado lugar a una descendencia antimicrobiana impresionante. Junto con los asaltos publicitarios de firmas que vierten en el mercado un abanico vertiginoso de productos antibacterianos; asimismo la industria privada y el medio universitario hacen una guerra sin precedentes contra los microbios.

Dentro del número creciente de programas de vacuna, algunos apuntan a los microbios corporales, no solamente HP sino también S. mutans, C. difficile, S. aureus, S. epidermis y Porphyromonas gingivalis, uno de los microbios que vive en la boca vinculado con una enfermedad bucal específica.Desafortunadamente, la idea de que las enfermedades cardiovasculares y la arteriosclerosis pueden ser causadas por los microbios ha suscitado un fuerte apasionamiento, puesto que significaría que las enfermedades crónicas de la Humanidad estarían causadas por microbios y se revelarían curables.

La lista de los candidatos potenciales es larga: el cáncer, la enfermedad de Alzheimer, la esclerosis múltiple, la sarcoidosis, la enfermedad de la inflamación del intestino, la artritis reumatoide, el lupus, la enfermedad de Kawasaki, la tiroiditis de Hashimoto, la mayor parte de las enfermedades psiquiátricas, la parálisis cerebral, la enfermedad ovárica poliquística, la obesidad y la anorexia (78), todo ello demuestra que la ilusoria teoría microbiana de la enfermedad está aún profundamente arraigada en los espíritus.

La medicina occidental está en un cruce definitivo en cuanto a la salvación de vidas de la enfermedad infecciosa.

El problema de la resistencia a los antibióticos y la emergencia de nuevas amenazas microbianas exigen una réplica, que podría ser la intensificación de la guerra contra los microbios.

Otra será aceptar la realidad: “Ya no vivimos en una burbuja”, dice Stuart Levy, “provenimos de y hemos evolucionado en el mundo bacteriano; deshacerse de las bacterias sería como tratar de deshacerse del mundo”.¨

Extractos seleccionados por Rumifilo, del artículo publicado por The Ecologist y escrito por Garry Hamilton periodista científico independiente. Colabora habitualmente en New Scientist.
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Ginger-beer "plant"

Marriage of equals
Botanist Harry Ward discovered that the production of ginger beer

depended on two crucial microorganisms that coexisted in the ginger-beer plant

Summer was once the time to quaff ginger beer, served up in brown stone bottles. All over the British Isles people relished its frothy, fizzy gingery tang, enhanced by an alcohol content that temperance campaigners warned could rival that of strong London stout. Best of all it was virtually free: you could make it at home with just a bit of sugar, ginger, water and a ginger-beer "plant".

No wonder, then, that this plant was a family heirloom, passed from mother to daughter and father to son. But it wasn't your typical green, leafy kind of plant. This was a sloppy mess of whitish, gelatinous lumps that typically lived in a jam jar. Exactly what this stuff was, nobody had a due. It worked, and that was enough.
But in 1887, a 33-year-old botanist called Harry Marshall Ward became curious. When a famous friend at the Royal Botanic Gardens in Kew, London, gave him a specimen, he was hooked. Unwittingly, he had embarked on a Herculean labour. "Had I known how long and difficult a task I had set myself," he later remarked, "the attempt would possibly have been abandoned at an early date."
EVERYONE knew that Harry Ward could never resist a challenge. On a visit to his old mentor, the director of Kew Gardens, Ward couldn't help but notice the bottle of ginger-beer plant, perched on a shelf in the director's study. "There is a thing you have to worry out," suggested William Thistleton Dyer, knowing all too well of Ward's penchant for botanical mysteries.

From now on Ward devoted every hour he could snatch from his job -teaching young men about to enter the in the Indian Forest Service to his hunt for the mysterious agent that transformed sweet, gingery water into a tasty and potent pint.
Ward had always been passionate about botany. While attending the revolutionary courses run by Darwin's champion, Thomas Henry Huxley, he had famously fainted at his microscope from sheer over-excitement. After his time with Huxley in London, Ward won a place to read natural sciences at Cambridge, and blossomed.
He went on to become a brilliant exponent of the "new botany". Radical ideas were spreading from mainland Europe, and he and his friends wanted to learn about how plants worked, not just how they were classified. He went on to become one of the great names of the day. Before he died aged just 52, reputedly of overwork, he pioneered the study of both symbiosis and pathology, investigating how plants and microorganisms live together as friend as well as foe.

Ward's first major study, as botanist to the colonial government of Ceylon, is now a classic of plant pathology. In 1879, the coffee plantations of Ceylon were threatened with extinction by a leaf disease. The disease was coffee rust, and for the next two years Ward worked out the life cycle of the rust fungus and showed how leaving belts of natural forest between the coffee plantations could prevent the spread of its spores. This was a brilliant piece of scientific detection, but it came too late. As the epidemic wiped out vast monocultures of coffee across the British colonies, the "mother country" quietly returned to drinking tea.
And ginger beer of course. Back in England and inspired by the "plant" from Kew, Ward set out to amass a comprehensive collection of specimens. Soon his laboratory shelves were crowded with jars of ginger-beer plants from all over the country, and even from North America. To this day, no one has ever worked out where the first ginger-beer plants came from. Rumour had it that soldiers had returned from the Crimean War with the stuff, but Ward said that was sheer speculation. "The whole question as to whence it was first derived, in fact, is enshrouded in mystery," he concluded. But he did solve the ultimate mystery, that of the plant's real nature. His meticulous analyses revealed it to be a fascinating alliance of cooperating microorganisms.

Everything turned on his scrupulous technique. Over the years, he had established nearly 2000 separate cultures, some of which he had to keep going for months or even years, as he struggled to separate and cultivate each microorganism in a pure state. To avoid contamination, he first ensured that every flask, beaker tube, funnel, watch glass and microscope slide was absolutely sterile. All apparatus was baked or boiled for several hours. Next, he concocted an extensive menu of nutrient broths to cater for every taste. The fussiest fungi dined on best bouillon made from lean beefsteak, finely chopped and soaked overnight in distilled water, then filtered and boiled. Even then, some microorganisms failed to thrive or resisted purification, and for these cases Ward perfected a way of isolating a single yeast cell in a "hanging drop" secured to a microscope slide, thus guaranteeing the culture's purity while he tracked down its identity.
His diligence paid off, for when he published his results in 1892 in the Philosophical Transactions of the Royal Society, no one questioned his astonishing announcement. Buried in this scholarly text is a biological bombshell. The ginger-beer plant, Ward proclaimed, was a new kind of organism - a "composite body", consisting of dozens of microorganisms living amicably together in a symbiotic lump. Not all of these microbes helped in making the beer. The majority Ward regarded as opportunistic interlopers. They turned up by chance, and hung around for the free lunch. But two organisms were present in every plant sampled, and seemed to be vital to the production of ginger beer.
One was a fungus, a new species of yeast he called Saccharomycespyriformis. The other was a bacterium, which he named Bacterium vermiforme, and is now called Brevibacterium vermiforme.
Ward reckoned that these two microbes had developed a symbiotic relationship, to their mutual benefit. He couldn't be sure of the biochemical details, but he guessed that the bacterium consumed the yeast's waste products, while the yeast benefited from their removal. Together, the two produced the essential ingredients of traditional ginger beer: carbon dioxide and alcohol. The conclusive proof came when Ward made perfect ginger beer in his laboratory, using his own plant, reconstituted from his pure cultures of the right yeast and bacterium.

So the ginger-beer plant was a bona fide "dual organism", rather like lichens. Everything pointed to a true symbiosis. For instance, when Ward tried to feed the bacteria with dead or feeble yeast cells, the experiments failed. The plant emerged only from a marriage of equals, which needed time: it took several days for the partners to find and embrace one another. No one could have predicted that the crude home brew of country folk would reveal a phenomenon new to science - what Ward called "symbiotic fermentation".
It was landmark research. Yet as the study of symbiosis fell out of fashion, Ward's work sank into obscurity. Vindication of a sort came half a century later, when a research team decided to investigate kefir. Ward had also been interested in this yogurt-like drink, made from fermented milk, and popular in the Caucasus mountains of southern Russia and Georgia, and he had begun to investigate its secrets. Legend has it that the Prophet Muhammad first gave kefir curds to Christians living near Mount Elbrus with strict instructions never to give them away. All the same, kefir curds did eventually turn up in a laboratory where, just as Ward had predicted, investigators identified a symbiotic collaboration between yeast and bacteria.
Years after Ward's pioneering work, Soviet researchers discovered a further instance of symbiotic fermentation. A yeast and a bacterium apparently cooperate to form the "tea fungus" or kambucha that thrives on sweetened tea. After a few days, the liquid acquires a pleasant acidity and a peculiar fruity taste that eastern Europeans once regarded as ideal for gastric upsets.

Indeed, not so long ago, even ordinary bread owed its distinctive taste and consistency to microbial liaisons. The traditional baker's yeast or "barm" passed from baker to baker was found in the 1950s to consist not only of the conventional baker's yeast Saccharomyces cerevisiae but at least one other yeast, as well as one or more bacterial species. By cooperating, this microbial syndicate fermented a greater number of carbohydrates than any of the various microbial components alone. The bread that resulted was surely like nothing you can buy today.

Today's commercial ginger beer is also much altered, purged of both its alcohol and its symbiotic liaisons. It is possible that Ward's own lovingly reconstituted ginger-beer plant survived into the 1940s. Max Walters, now 82, says he made and drank the stuff in the Botany School at Cambridge just after the Second World War. But no one knows what happened to it after that. • Gail Vines

28 September 2002 Gail Vines Magazine issue 2362 New Scientist

Exopolysaccharides Produced by Lactic Acid Bacteria of Kefir Grains

Ginka I. Frengovaa, Emilina D. Simovaa,*, Dora M. Beshkovaa and Zhelyasko I. Simovb a Laboratory of Applied Microbiology, Institute of Microbiology, Bulgarian Academy of Sciences…

Introduction

Exopolysaccharides produced by lactic acid bacteria have generated increasing attention among researchers for the last few years. The lactic acid bacteria are food-grade organisms, and the exopolysaccharides that they produce contribute to the specific rheology and texture of fermented milk products and may have application in nondairy foods. When added to food products, polysaccharides function as thickeners, stabilizers, emulsifiers, gelling agents, and water binding agents (Giraffa,1994; Crescenzi, 1995). Kefir Ð a unique product among the cultured milk varieties Ð is produced with an original native starter (kefir grains). Kefir is defined as the yogurt of the 21st century (Gorski,1994). The kefir grains consist of slimy materials in which yeast and bacterial cells are firmly embedded.
The polysaccharide matrix, forming the structure of the kefir grain, is kefiran, identified by a number of researchers (La Riviere et al., 1967; Neve, 1992; Pintado et al., 1996). The lactic acid bacteria, yeast and polysaccharide “kefiran” that make up the kefir grains have been described as a symbiotic community that impart unique properties to kefir (Margulis, 1996).
Investigations into the active producers of kefiran are controversial. Although La Riviere et al. (1967) reported that Lactobacillus brevis, now regarded as Lactobacillus kefir, was responsible for kefiran production, Kandler and Kunath (1983) concluded that Lactobacillus kefir was not a kefiran producer. According to other authors, the principal producer of the kefiran polymer in kefir grains is Lactobacillus kefiranofaciens and several other unidentified species of Lactobacillus (Mitsue et al., 1998, 1999; Yokoi et al., 1990; Toba et al., 1987). Thus it remains undecided which microorganism is responsible for kefiran production in kefir grains. Exopolysaccharide production is an important feature of lactic acid bacteria characterization in forming starter cultures for fermented milk products with suitable texture and specific rheology.
The present paper reports on kefiran production by lactic acid bacteria, isolated from kefir grains, and selection of an active producer of kefiran with view of including it in a kefir starter. There is no information available about production of exopolysaccharides by single strain cultures and kefir starter cultures during kefir fermentation and storage.

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From spoilage to probiotic: the new role of yeast in dairy products

Foodinfo Online FSTA Reports -->19 March 2002

Probiotic microorganisms are increasingly added to foods to promote the maintenance of a healthy balance of gastrointestinal microflora. The most frequently used microorganisms are lactobacilli and bifidobacteria, which are often added to fermented dairy products, particularly yoghurts. However, the potential role of yeasts as probiotic agents has not been fully investigated, despite the fact that yeasts form an integral part of the microflora of many dairy-related products. Starter cultures containing yeasts and bacteria are used in the preparation of some fermented milk products, including kefir, koumiss and laban, and several antagonistic reactions have been observed between yeasts such as Saccharomyces cerevisiae and enteric pathogens, such as Escherichia coli and Salmonella and Shigella species. However, yeasts are frequently associated with the spoilage of the final product, causing alcoholic fermentation and gas formation in yoghurts containing added fruit and sucrose. The yeast Saccharomyces boulardii was first isolated from lychees in the 1950s and has since been used in the prevention and treatment of diarrhoeal diseases. A study by Laurens-Hattingh et al.1 reports on the ability of S. boulardii to grow in bio-yoghurt, UHT-treated yoghurt and UHT-treated milk, in order to determine at a later stage the effect on survival of Lactobacillus acidophilus and Bifidobacterium bifidum during shelf life. Previous studies have shown that survival of L. acidophilus and B. bifidum is poor in yoghurts, due to their low acid tolerance. Since yeasts are able to utilize organic acids and increase the pH of the environment, addition of yeasts to bio-yoghurts may enhance the stability of their probiotic bacteria.

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1Lourens-Hattingh A; Viljoen BC (2001). Growth and survival of a probiotic yeast in dairy products.

Food Research International 34 (9) 791-796.

An abstract of this paper can be found in Food Science and Technology Abstracts, citation reference 2001-12-Pl1955.

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Unique problems in designing and testing probiotic


FoodInfo Online Features -->31 January 2002

http://www.foodsciencecentral.com/fsc/ixid3803

Edward R. Farnworth

Food Research and Development Centre, Agriculture and Agri-Food Canada, St. Hyacinthe, Quebec, Canada

1. Introduction

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.

2. The changing definition of a probiotic

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.

3. Required characteristics of probiotics

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).

5. Probiotics - number of microorganisms required to affect metabolism/health

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).

6. Probiotics - live bacteria or active ingredient?

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.

7. Probiotics - efficacy testing

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).

8. Probiotics - the future

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.

9. References

Anon. 2001. Health Canada, Standards of Evidence for Evaluating Foods with Health Claims. Synopsis of the Consultation Document. Available at:

http://www.hc-sc.gc.ca/food-aliment/english/subjects/health_claims/soe_synopsis_consultation.html

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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