Clinical Applications of Probiotic Bacteria

PII : S0958-6946(98)00077-6

Int. Dairy Journal 8 (1998) 563—572 ( 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0958-6946/98/$19.00#0.00

Clinical Applications of Probiotic Bacteria S. Salminena *, A. C. Ouwehandb and E. Isolauric a Department of Biochemistry and Food Chemistry, University of Turku, Finland and Key Centre for Applied and Nutritional Toxicology, RMIT, Melbourne, Australia b Centre for Biotechnology, University of Turku and As bo Akademi University, 20014 Turku, Finland c Department of Pediatrics, The Medical School, University of Turku, 20014 Turku, Finland ABSTRACT Probiotic bacteria are applied to balance disturbed intestinal microflora and related dysfunctions of the gastrointestinal tract. Current clinical applications include well-documented areas such as treatment of acute rotavirus diarrhoea, lactose maldigestion, constipation, colonic disorders and side-effects of pelvic radiotherapy, and more recently, food allergy including milk hypersensitivity and changes associated with colon cancer development. Many novel probiotics appear to be promising candidates for the treatment of clinical conditions related to disease. They may be used as ingredients for functional foods and foods for specific disease requirements provided that basic demands for identifying and following the strains and, consequently, good clinical studies are carefully followed. Current data indicate that specific probiotic strains have useful properties for defined clinical conditions mainly involving the gastrointestinal tract. Some natural components can be used to enhance these probiotic properties. Future challenges include the incorporation of one or more probiotics together or in combination with suitable prebiotic substrates to enhance the efficacy of the preparations for clinical use. ( 1998 Elsevier Science Ltd. All rights reserved

INTRODUCTION

but different probiotic strains are needed for specific applications. The mechanisms behind the specific health benefits are related to gut microflora modification, the strengthening of the gut mucosal barrier, e.g. adherence of probiotics to intestinal mucosa with capacity to prevent pathogen adherence, pathogen inactivation, modification of dietary proteins by intestinal microflora, modification of bacterial enzyme activity, and influence on gut mucosal permeability, and regulation of the immune system. The intact intestinal epithelium with the normal intestinal microflora represents a barrier to the movement of pathogenic bacteria, antigens and other noxious substances from the gut lumen. In healthy subjects this barrier is stable protecting the host and facilitating normal intestinal function. When either the normal microflora or the epithelial cells are disturbed by triggers such as dietary antigens, pathogens, chemicals or radiation, defects in the barrier mechanisms become evident. Disturbed intestinal microflora may lead to diarrhoea, mucosal inflammation, a permeability disorder or activation of carcinogens in the intestinal contents (Salminen et al., 1996, 1998a). For future research and the development of new food and clinical applications of probiotic bacteria a thorough understanding of the mechanisms of this barrier system as well as the mechanisms of probiotic action is essential.

Probiotics are ‘live microbial feed supplements which beneficially affect the host animal by improving its intestinal microbial balance’ (Fuller, 1991). This definition has been slightly redefined later (Salminen et al., 1998a). The bulk of evidence on probiotic cultures and foods is based on anecdotal reports and poorly controlled studies making the work inconclusive and recommendations difficult. However, evidence is currently accumulating from welldesigned, randomized and placebo-controlled doubleblind studies indicating that a few well characterised lactic acid bacteria strains have documented probiotic health promoting effects when defined doses are administered (Lee and Salminen, 1995; Salminen et al., 1998a, b). Increasing numbers of colonization and dose-response studies defining the required doses have been published (Saxelin et al., 1995; Wolf et al., 1995). It is known that the human gastointestinal tract and the intestinal microflora are involved in a series of disease states. Especially the large intestine is a major colonization site for bacterial, viral and parasitic pathogens. The result of an overgrowth of these pathogens may be acute diarrhoeal disease. Major foodborne bacterial pathogens include campylobacters, salmonellae, listeriae and certain strains of Escherichia coli. There are, however, other more chronic forms of gut disease such as inflammatory bowel disease (e.g. ulcerative colitis, Crohn’s disease), colon cancer and pseudomembranous colitis. Each of these disorders may have a bacterial aetiology (Gibson and Roberfroid, 1995). Probiotics can be used to balance or prevent such disturbances,

Properties of probiotics The most important properties for current and future probiotics include the acid and bile tolerance, adherence to human intestinal mucosa, temporary colonization of the human gastrointestinal tract, production of antimicrobial substances and inhibition of pathogen growth

* Corresponding author. Tel.: 00#358-2-3336880; fax: 00#3582-3336 860. 563

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S. Salminen et al. Table 1. Desirable Properties of Probiotic Bacteria

Probiotic strain characteristics

Functional properties

Human origin, if intended for human use

Species-dependent health effects and maintained viability; applicability to functional and clinical foods Survival in the intestine, maintaining adhesiveness and other properties Immune modulation, competitive exclusion of pathogens

Acid and bile stability Adherence to human intestinal cells and intestinal mucus glycoproteins (mucin) Competitive exclusion and colonization of the human intestinal tract Production of antimicrobial substances Antagonism agains cariogenic and pathogenic bacteria Safety in food and clinical use Clinically validated and documented health effects

Multiplication in the intestinal tract, competitive exclusion of pathogens, stimulation of beneficial microflora, immune modulation by contact with gut associated lymphoid tissue Pathogen inactivation in the intestine, normalization of gut flora Pathogen exclusion, prevention of pathogen adhesion, normalization of gut flora, normalization of oral microflora Accurate strain identification (genus, species, strain) and characterization, documented safety Dose—response data for minimum effective dosage in different products

(Lee and Salminen, 1995). It is also important that the strains used are of human origin if intended for human use since many of the properties may be species dependent (Table 1). Adhesion of lactic acid bacteria to intestinal cells is the first step of colonization or temporary colonization. Adhesion can be non-specific, based on physico-chemical factors, or specific, involving adhesin molecules on the surfaces of adherent bacteria and receptor molecules on epithelial cells. Variability of adhesion properties tested using human intestinal cells has become a standard procedure for selecting new probiotic strains. The adhesion properties of a strain are considered species-specific, but often there appears to be adhesion on non-host species. In general, common dairy strains are not among the most adhesive ones, whereas probiotic strains appear to have strong adhesion. Based on adhesion, some lactic acid bacteria of human origin and some of dairy origin show moderate to good adhesion properties in human cell lines (Lehto and Salminen, 1997). The mechanisms of probiotics to be assessed prior to and during clinical studies are summarized in Table 2.

Table 2. Important Studies for the Clinical Assessment of Probiotics Type of property studied

Clinical factor to be assessed

Intrinsic properties of lactic acid bacteria

Adhesion factors, antibiotic resistance, existence of plasmids and plasmid transfer potential, enzyme profile Concentrations, antimicrobial effects on intestinal microflora, safety Adhesion to intestinal cells and mucus, intestinal mucus degradation, stabilisation effect on intestinal permeability Dose—response studies by oral administration in volunteers Potential for side-effects, diseasespecific effects, nutritional studies, intestinal microflora effects Surveillance of large populations following introduction of new strains and products for both health effects and safety

Metabolic products

Mucosal effects

Dose—response effects Clinical assessment

Epidemiological studies

Safety of probiotics The safety of lactic acid bacteria used in clinical and functional food is of great importance. In general, lactic acid bacteria have a good record of safety, and no major problems have occurred. Cases of infection have been reported with several strains, most commonly with the ones that are naturally most abundant in the human intestinal mucosa (Gasser, 1994; Aguirre and Collins, 1993). Studies on safety have been documented on dairy strains (Saxelin et al., 1996a, b) and a review of current knowledge on safety of probiotic bacteria has been reported by Donohue and Salminen (1996). It is most important for probiotic lactic acid bacteria that their safety has been assured and that they confirm to all regulations. A proposed scheme for safety assessment has been presented earlier (Donohue and Salminen, 1996). Most stringent studies have to be completed for genetically modified strains intended to human consumption.

INTESTINAL MICROFLORA DYSFUNCTION Major dysfunctions of the gastrointestinal tract are thought to be related to disturbances of intestinal microflora. Examples of such disturbances are also targets for probiotic action and indicate further areas of research for clinical applications. Lactose intolerance Among the physiological factors that influence the amount of lactose digested and its tolerance are gastrointestinal transit, intestinal lactase activity, the composition of intestinal and especially colonic microflora and in some cases, visceral sensitivity (Mayer and Gebhart, 1994). Lactose intolerance is generally related to the deficiency of the enzyme b-galactosidase in the intestinal

Clinical applications of probiotic bacteria

mucosa. Incompletely absorbed lactose then causes watery diarrhoea and large amounts of lactose may lead to dysfunctions of intestinal microflora. Many lactose-intolerant subjects may tolerate small amounts of lactose due to the capacity of intestinal microbes to metabolise lactose. Constipation Traditionally, constipation is defined in terms of bowel regularity varying from 3 to 1 times weekly or less. The main symptom in constipation is straining of defaecation and additionally discomfort, distention, meteorism, abdominal bloating and incomplete rectal emptying are all considered part of the condition. The total gut transit time is generally prolonged in constipated subjects. An inappropriate diet is one of the main causes of constipation and high risk diets include low fibre diets, gluten-free diets, low residue diets and enteral feeding. Low residue diets or diets low in fibre or non-starch polysaccharides may well cause an altered colonic flora. Many treatment regimens for constipation increase the metabolic activity of colonic microflora and lower the pH in the colon. In connection with improved diet with prebiotics and probiotic bacteria, intestinal microflora appears to play a major role in constipation and probiotic lactic acid bacteria may offer one means of treating and preventing constipation (Cummings, 1994). Acute gastroenteritis Rotavirus is perhaps the most common cause of acute childhood diarrhoea world-wide (Claeson and Merson, 1990). Rotaviruses invade the differentiated absorptive columnar cells of the small intestinal epithelium where they replicate. The result is partial disruption of the intestinal mucosa with loss of microvilli and a decrease in the villus/crypt ratio. Rotavirus infection has been shown to be associated with increased intestinal permeability (Jalonen et al., 1991). Significantly higher levels of immune complexes containing dietary b-lactoglobulin have been reported in the serum of patients with rotavirus diarrhoea when compared to nondiarrhoeal patients. Macromolecular absorption has also been reported to be increased in rotavirus gastoenteritis (Jalonen et al., 1991; Heyman et al., 1987; Uhnoo et al., 1990; Isolauri et al., 1993a). The intestinal microflora affects gut permeability, so that in the absence of intestinal microflora, disturbance in intestinal absorption of macromolecules is more severe than in its presence. Several studies by different groups and in different conditions have proven without doubt that ¸actobacillus GG (ATCC 53103) significantly shortens the duration of rotavirus diarrhoea in developed countries (Isolauri et al., 1991; Kaila et al., 1992; Isolauri et al., 1994; Majamaa et al., 1995; Guarino et al., 1998) and also in developing countries (Raza et al., 1995; Pant et al., 1996). ¸actobacillus reuteri has had a similar clinical effect on the duration of rotavirus diarrhoea without an increase in immune response in one clinical study (Shornikova et al., 1997) and Bifidobacterium bifidum has been reported to prevent rotavirus diarrhoea (Saavedra et al., 1995). Food hypersensitivity and allergy Food allergy is defined as an immunologically mediated adverse reaction against dietary antigens. The

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immaturity of the immune system and the gastrointestinal barrier may explain the peak prevalence of food allergies in infancy (Isolauri and Turjamaa, 1996). In food allergy, intestinal inflammation (Majamaa et al., 1995) and disturbances in intestinal permeability (Jalonen, 1991) and antigen transfer (Heyman et al., 1988) occur when an allergen comes into contact with the intestinal mucosa. During dietary elimination of the antigen, the barrier and transfer functions of the mucosa are normal (Majamaa et al., 1995; Jalonen, 1991; Heyman et al., 1988). It has therefore been concluded that impairment of the intestinal function is secondary to an abnormal intestinal immune response to the offending antigens. Atopic dermatitis Atopic dermatitis is a common chronically relapsing skin disorder of infancy and childhood. Hereditary predisposition is an important denominator of atopic dermatitis, and hypersensitivity reactions contribute the expression of this predisposition (Sampson and McCaskill, 1985). The relationship between environmental allergens and exacerbation of atopic dermatitis is particularly apparent in infancy so that dietary antigens predominate and allergic reactions to foods are common (Isolauri and Turjanmaa, 1996). In a recent study (Majamaa and Isolauri, 1996), macromolecular absorption across the intestinal mucosa was assessed in children (aged 0.5—8 yr) with atopic dermatitis. In these patients, the offending foods were identified and eliminated, and the intestinal mucosa was not challenged in vitro nor in vivo. Significantly increased absorption of protein, in intact and degraded form, was found in the atopic dermatitis patients compared to controls. The result may reflect a primarily altered antigen transfer in atopic dermatitis. Aberrant antigen absorption could partly explain why patients with atopic dermatitis frequently show hightened immune responses to common environmental antigens, including dietary antigens. The expression of the receptors of the constant (Fc) region of IgG (FccRI, FccRII, FccRIII) and that for the complement fragments C3b and C3bi (CR1 and CR3) in neutrophils and monocytes of children with atopic dermatitis have a distinct profile which indicates a role of the immune activation and phagocytes in atopic dermatitis (Isolauri et al., 1997). Crohn’s disease Crohn’s disease is a chronic and idiopathic inflammation of the gastrointestinal tract with characteristic patchy transmural lesions containing granulomas. The outbreak of Crohn’s disease is thought to require genetic predisposition, immunological disturbance and the influence of intraluminal triggering agent(s), for example bacteria or viruses. Crohn’s disease is associated with impairment of the barrier function. In an in vitro study (Isolauri, 1995), a rise in macromolecular absorption in uninvolved parts of the intestine was detected in patients with clinically moderate or severe Crohn’s disease. An interplay between the immune effector cells and the intestinal vascular endothelium has been suggested to result in disrupted vasculature, cell-mediated immunity with lymphokine production and a vigorous IgG response and finally dysfunction of the mucosa (Podolsky, 1991; MacDonald, 1993).

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Rheumatoid arthritis Dysfunction of the gut mucosal barrier as a consequence of abnormal intestinal microflora, intestinal inflammation due to diet or medication may be the key connecting the gut and joint in rheumatoid arthritis (Midtvedt, 1987). Evidence for this suggestion is afforded by observations that patients with rheumatoid arthiritis experience relief of clinical symptoms by fasting or by embarking on specific dietary regimens. In a recent study (Malin et al., 1996a, 1997), the enzyme activities in faeces were investigated in patients with juvenile chronic arthritis (JCA) and controls. Specifically, the activity of urease but not the activities of b-glucosidase and b-glucuronidase in faeces was increased in JCA patients. Urease catalyses the hydrolysis of urea to yield ammonia. Urease and ammonia may contribute to tissue damage, and elevated urease activity may indicate a disturbance in the population of anaerobic bacteria in JCA. Pelvic radiotherapy Radiation treatment has a profound effect on the intestinal microflora and mucosa. Radiation alters the intestinal microflora, vascular permeability of the mucosa and the intestinal motility (Friberg, 1980; Silva, 1993). The villi are shortened and flattened and there is decreased mitosis in the intestinal crypts with necrosis. Heavy infiltration of the lamina propria with plasma cells is seen with polymorphonuclear leukocytosis (Friberg, 1980; Silva, 1993). Overgrowth of pathogenic microorganisms has been proposed as a factor enhancing the severity of radiation enteritis (Danielsson et al., 1991). Bacteria can penetrate the damaged villi leading to bacteremia in extreme cases (Friberg, 1980; Silva, 1993). The changes leading to radiation enteropathy in man include both damage to intestinal mucosa, changes in the intestinal microflora and impaired immune response. Clinically, the primary reactions start during the first and second weeks of treatment giving such symptoms as nausea, vomiting and diarrhoea with peritonitis resulting from the necrotizing effects of radiation. The late secondary reaction, e.g. fibrosis and obstruction of the intestine, may give clinical symptoms years after the treatment. The relationship between early and late reactions is not clear, although some studies have indicated that the severe early reactions precede serious late effects (Silva, 1993; Dahl et al., 1994). Intestinal inflammation and chemical exposure Long-term inflammatory changes, changes in gut microflora, alterations in drug and carcinogen metabolism, nutrition of the intestinal epithelial cells, and genetic factors may alter the ability of the intestinal tract to handle drugs and carcinogens. It has been reported that nutritional factors, such as increasing fat intake, can alter the incidence and number of chemically induced tumours in animals. These changes have been suggested to be related to intestinal microflora, intestinal microbial enzyme activities, intestinal immune response and intestinal mutagen production (Goldin et al., 1996). Subjects with intestinal inflammatory conditions, such as ulcerative colitis and Crohn’s disease, may have an increased risk for colon cancer with the risk increasing with the duration and extent of disease (Tytgat et al., 1995). In a study

conducted in the United Kingdom, cancer risk was compared in two groups of patients with extensive ulcerative colitis and equally extensive colonic Crohn’s disease. Both the relative risk and the cumulative incidences of cancer were the same (Gillen et al., 1994). In the colitic mucosa there may be a spectrum of changes occurring with cycles of inflammation that may contribute to harmful alterations in the mucosa (Tytgat et al., 1995). Inflammatory alterations may be potentiated by the disturbed microflora and its metabolic activities. The intestinal microflora influences the handling of drugs and carcinogens in a healthy subject by assisting the removal of these substances as well as products of the enterohepatic circulation (Goldin and Gorbach, 1984; Goldin et al., 1992). These functions may be enhanced by oral administration of suitable lactic acid bacteria either in fermented milks or in other foods. PROBIOTIC TREATMENT OF HUMAN INTESTINAL BARRIER DYSFUNCTIONS Lactose intolerance Frequently, lactase-deficient people may be symptomfree when they consume only limited amounts of lactose except for a proportion of these subjects who are severely lactose intolerant. The beneficial effect of some probiotics on lactose digestion has been demonstrated (Sanders, 1993). In particular, in studies comparing yoghurt and milk consumption it was shown that yoghurt consumption enhances lactose digestion in lactase deficient subjects (Kolars et al., 1984) and increased the oro-caecal transit time (Marteau et al., 1990). Lactose maldigesters appear to tolerate lactose in yoghurt better than an equivalent quantity of lactose in milk (Kolars et al., 1984; Dewit et al., 1988; Marteau et al., 1990; Rosado et al., 1992). This has been attributed to both the concentration of b-galactosidase enzyme in probiotic bacteria and the suitable modification of colonic microflora to better utilise lactose (Lerebours et al., 1989; Marteau et al., 1990, 1997; Vesa et al., 1996). Also, the bacteria with b-galactosidase may lyse in the small intestine releasing their b-galactosidase activity. Acute gastroenteritis ¸actobacillus GG has been proven effective in the treatment of rotavirus diarrhoea. It repeatedly reduces the duration of diarrhoea to about half in children with rotavirus diarrhoea. It has also been proven effective in watery diarrhoea in several studies in Asia (Raza et al., 1995). One such study has been reported for ¸actobacillus casei Shirota strain and one prevention study for Bifidobacterium animalis (Sugita and Togawa, 1994; Saavedra et al., 1994). Different lactic acid bacteria were compared for their effects on the outcome and the immune response to rotavirus in children with acute rotavirus gastroenteritis (Kaila et al., 1992; Majamaa et al., 1995). The results indicated that ¸actobacillus strain GG had a significant effect on the duration of diarrhoea while no such effect was observed with the other strains tested. Serum antibodies to rotavirus, total number of immunoglobulinsecreting cells (ISC) and specific antibody-secreting cells

Clinical applications of probiotic bacteria

(sASC) to rotavirus were measured at the acute stage and at convalescence. The treatment with ¸actobacillus GG was associated with an enhancement of IgA sASC to rotavirus and serum IgA antibody level at convalescence. It was therefore suggested that certain strains of lactic acid bacteria, particularly ¸actobacillus GG, promote systemic and local immune response to rotavirus, which may be of importance for protective immunity against reinfections (Kaila et al., 1992). Next, a study was made to compare the immunological effects of viable and heat inactivated lactic acid bacteria (Majamaa et al., 1995). ¸actobacillus GG administered as a viable preparation during acute rotavirus gastroenteritis resulted in a significant rotavirus specific IgA response at convalescence. The heat-inactivated ¸actobacillus GG was clinically as efficient, but the IgA response was not detected. This result suggests that viability of the strain is critical in determining the capacity of lactic acid bacteria to induce immune stimulation. Also, in a study with different preparations of lactic acid bacteria in the treatment of rotavirus diarrhoea it was shown that ¸actobacillus GG was most effective whilst a preparation containing Streptococcus thermophilus and ¸actobacillus bulgaricus or a ¸actobacillus rhamnosus strain did not have any effect on the duration of diarrhoea (Majamaa et al., 1995). In a Japanese study, 1.5]109~10 cfu of Biolactis powder (¸actobacillus casei Shirota) was administered to children with rotavirus diarrhoea (n"17) along with the normal Japanese treatment using lactase and albumin tannate. The controls (n"15) received lactase and albumin tannate only (Sugita and Togawa, 1994). The calculated days until improvement were 3.8 in the ¸actobacillus group and 5.3 in the control group (p(0.05). While the result is positive, further studies are required to assess the efficacy using standard biomarkers such as the duration of diarrhoea. Food allergy The mechanisms of the immune enhancing effect of ¸actobacillus GG are not entirely understood, these may relate to the antigen transport in the intestinal mucosa. Therefore, the effect of ¸actobacillus GG on the gut mucosal barrier was investigated in a suckling rat model (Isolauri et al., 1993b). Rat pups were divided into three experimental feeding groups to receive a daily gavage of cow milk, or ¸actobacillus GG with cow milk, while controls were gavaged with water. At 21 d, the absorption of horse radish peroxidase across patch-free jejunal segments and segments containing Peyer’s patches was studied in Ussing chambers. Gut immune response was indirectly monitored by the ELISPOT method of sASC to b-lactoglobulin. Prolonged cow milk challenge increased macromolecular absorption, whereas ¸actobacillus GG stabilized the mucosal barrier with a concomitant enhancement of antigen-specific immune defense and proportional transport across Peyer’s patches. These results indicate that there exists a link between stabilization of non-specific antigen absorption and enhancement of the antigen-specific immune response. They further suggest that the route of antigen absorption is an important determinant of the subsequent immune response to the antigen (Isolauri et al., 1993b). Recently, also the use of probiotic preparations has been discussed in adults with milk hypersensitivity (Pelto et al., 1996, 1998).

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Atopic dermatitis The capacity of ¸actobacillus GG to degrade food antigens, and thereby modify their immunoactivity was investigated (Su¨tas et al., 1996a). For this purpose, the immunoactivity of caseins and ¸actobacillus GG-degraded caseins was assessed in lymphocyte transformation tests in healthy adults. Casein, a -casein and 41 b-casein suppressed the lymphocyte proliferation capacity, whereas i-casein induced it. Degradation of casein, a -casein and b-casein by ¸actobacillus GG enhanced 41 the suppressive effect of these caseins on the lymphocyte proliferation capacity. Degradation of i-casein by ¸actobacillus GG reversed its inducive effect to profound suppression. These results indicate that ¸actobacillus GG can degrade food antigens such as bovine casein, and down-regulate T-cell responses. The results were identical in healthy children and in children with atopic dermatitis (Su¨tas et al., 1996b). Moreover, in cultures of atopic children, casein was found to increase the IL-4 production capacity while ¸actobacillus GG-degraded casein was found to reduce the IL-4 generation. These results suggest that probiotic bacteria may have the capacity to release tolerogens from allergens. Crohn’s disease The effect of a ten-day oral bacteriotherapy with ¸actobacillus GG in Crohn’s disease was investigated (Malin et al., 1996b). Irrespective of the activity of Crohn’s disease, an increase in IgA sASC to dietary b-lactoglobulin and casein was detected. The result indicates that probiotic bacteria may have the potential to increase gut IgA and thereby promote the gut immunological barrier. Rheumatoid arthritis In JCA patients, ten days of oral bacteriotherapy reduced elevated urease activity in faeces suggesting an effect to counteract imbalanced microflora in JCA (Malin et al., 1996a, 1997). Thus, the dysfunction of the intestinal microflora which produces increased concentrations of urease may be normalised by effective probiotic strains. Pelvic radiotherapy In a randomised study, it was reported that patients receiving pelvic radiotherapy and a fermented milk containing viable ¸actobacillus acidophilus NCFB 1748 had a significantly lower incidence of diarrhoea (Salminen et al., 1988). In a five-year follow up study there was a trend to less late serious intestinal complications (Salminen et al., 1995). It has been shown that ¸actobacillus acidophilus (NCFB 1748) has ability to colonise human colon mucosa in vitro even though adhesion to Caco-2 cells is relatively small. In a Swedish study by Henriksson and coworkers (1995), fermented milk intake decreased the severity of late effects caused by pelvic radiotherapy. Also earlier studies have shown that some ¸actobacillus acidophilus and Bifidobacterium preparations may have potential in controlling diarrhoea and intestinal side effects of radiation. However, the strains utilized are not accurately described. The recent results indicate an important role for lactic acid bacteria in the intestinal tract following radiotherapy, but further studies are required in this area.

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Intestinal inflammation, chemical exposure and colon cancer related parameters There is evidence that lactic acid bacteria influence the mutagenicity of intestinal contents and the levels of faecal microbial enzymes, such as b-glucuronidase, b-glucosidase, nitroreductrase and urease. ¸actobacillus acidophilus (NCFB 1748) has been shown to significantly decrease faecal mutagenicity and urinary mutagenicity in healthy volunteers consuming fried ground beef. The same strain decreased faecal E. coli levels in colon cancer patients and reduced the faecal b-glucuronidase levels (Lidbeck et al., 1991). Similar results have been reported for ¸actobacillus GG, ¸actobacillus acidophilus, ¸actobacillus casei Shirota strain and other strains (Ling et al., 1994; Goldin and Gorbach, 1984; Goldin et al., 1992; Morotomi, 1996; Pedrosa et al., 1995). It has also been demonstrated with the ¸actobacillus casei Shirota strain that there is potential in the area of ‘dietary prevention’ of cancer. Parenteral administration of the strain has had antitumour and immunostimulating activities on experimentally implanted tumours (Morotomi, 1996). Similar effects have been verified by oral administration (Sawamura et al., 1994). In a recent study ¸actobacillus GG was shown to cause a dramatic reduction in DMH induced tumour formation in rats. It was reported that ¸actobacillus GG inhibited the initiation and early phase promotion on the tumorigenesis process (Goldin et al., 1996). However, it was reported that the organism was not able to prevent the growth of tumours once they had been established (Goldin et al., 1996). The fact that ¸actobacillus GG is an organisms of human origin capable of surviving in the gastrointestinal tract and that it inhibits tumour initiation or early promotion in the colon provides a basis for further studies of these factors in humans. In a similar manner, ¸actobacillus casei Shirota strain has been shown to have inhibitory properties on chemically in-

duced tumours in animals (Kato et al., 1994; Morotomi, 1996). In a clinical study, prophylactic effects of oral administration of ¸actobacillus casei Shirota on the recurrence of superficial bladder cancer have been reported in two Japanese studies (Aso et al., 1992, 1995). Lactic acid bacteria have also been indicated in colonic fermentation producing butyrate or butyric acid in the colon. This alteration in intestinal metabolism may be due to changes in the intestinal microecology following probiotic intake or the direct metabolism of slowly absorbable components by orally administered colonising lactic acid bacteria. Butyrate has been shown in in vitro studies to slow the growth of cultured colon cancer cells. Young (1996) has proposed that butyrate may be a diet-regulated, natural anti-tumour compound at least partly responsible for the antitumour effect of dietary components and also dietary probiotics. When these studies are related to animal data on several ¸actobacillus strains and colon cancer related parameters (Table 3), it is important to assess the potential of probiotic lactobacilli for cancer chemoprevention. Successful probiotic strains Probiotic bacteria with these properties and documented clinical effects include ¸actobacillus acidophilus (NCFB 1478), ¸actobacillus casei Shirota strain, ¸actobacillus GG (ATCC 53103), ¸actobacillus johnsonii L1, ¸actobacillus reuteri and Bifidobacterium bifidum (Lee and Salminen, 1995). A large number of published studies exists on each preparation documenting their health effects. All of these are currently further tested for different intestinal problems and they offer alternatives for dietary treatment of intestinal disorders (Table 3). In the future, it is likely that we shall see more specific clinical targets for probiotic therapy and then the abovementioned strains are likely to play an important role in new products. New strains are isolated and are likely to be included in our diet.

Table 3. Documented Clinical Effects of Probiotics (Marteau and Rambaud 1993; Lee and Salminen 1995; Salminen et al., 1996) Type of problem

Reported effects in clinical studies

Major references

Rotavirus diarrhoea

Several studies showing reduced duration of diarrhoea with ¸actobacillus strain GG, one study with ¸actobacillus reuteri, and one on prevention of diarrhoea Bifidobacterium animalis

Lactose maldigestion

Improved lactose digestion in fermented milks with various lactase containing starters Improved intestinal transit time with fermented milks with several lactic acid bacteria especially with added substrates such as lactulose and lactitol Several studies showing and enhanced immune response with ¸actobacillus johnsonii in normal subjects, enhanced immune response in rotavirus diarrhoea with ¸actobacillus GG

Isolauri et al. (1991), Kaila et al. (1992), Shornikova et al. (1997) Saavedra et al. (1994), Saavedra (1995) Vesa et al. (1996)

Constipation

Enhanced immune response

Pelvic radiotherapy side effects Faecal mutagenicity Traveller’s diarrhoea

Decreased side-effects and prevention of radiotherapy-related diarrhoea with ¸actobacillus acidophilus or Streptococcus lactis fermented milk Decreasing faecal mutagenicity, treatment of constipation Prevention of traveller’s diarrhoea, treatment of traveller’s diarrhoea

Salminen and Salminen (1997), Rajala et al. (1988) Kaila et al. (1992), Bernet et al. (1993 & 1994) Schiffrin et al. (1997), Marteau et al. (1997) Salminen et al. (1988, 1995), Henriksson et al. (1995) Lidbeck et al. (1991), Salminen et al. (1988, 1995) Oksanen et al. (1990), Hilton et al. (1997)

Clinical applications of probiotic bacteria

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Table 4. Established Health Effects of Probiotics Reasonably established

Future challenges

Reduction of the duration of rotavirus diarrhoea, reduction of the duration of antibiotic associated diarrhoea, reduction of the duration of traveller’s diarrhoea, reduction of the symptoms of lactose intolerance, prevention of rotavirus diarrhoea Studies on treatment of food allergy, colon cancer and bladder cancer prevention and treatment, studies on irritable bowel disease and irritable bowel syndrome, Crohn’s disease Clostridium difficile diarrhoea, antibiotic associated diarrhoea

FOOD AND CLINICAL APPLICATIONS OF PROBIOTICS Probiotic bacteria (e.g. Bifidobacterium animalis and ¸actobacillus GG, ¸actobacillus casei Shirota) have beneficial effects on the clinical course of rotavirus diarrhoea, including prevention and treatment, and the enhanced immune response thereafter (Isolauri et al., 1991; Kaila et al., 1992; Saavedra et al., 1994). In a similar manner, ¸actobacillus acidophilus preparations and ¸actobacillus casei (powder prepared from heat killed ¸actobacillus casei Shirota) preparations have been beneficial in the prevention of radiation enterophathy (Salminen et al., 1988, 1995). ¸actobacillus acidophilus and some other ¸actobacillus strains have enhancing effects on the immune system in connection with oral vaccines and ¸actobacillus reuteri on the duration of acute diarrhoea in children (Shornikova et al., 1997). Among the possible mechanisms responsible for the favourable clinical response is promotion of the immunologic and nonimmunologic defence barrier in the gut. The areas for established scientific results and future challenges are summarized in Table 4. Oral introduction of ¸actobacillus johnsonii L1 and ¸actobacillus GG has been associated with alleviation of intestinal inflammation and ¸actobacillus GG with normalization of increased intestinal permeability (Isolauri et al., 1993b) and gut microflora (Isolauri et al., 1994). Such studies have not been reported for the other successful probiotic strains. Another explanation for the gut stabilizing effect of ¸actobacillus GG could be improvement of the intestine’s immunologic barrier, particularly intestinal IgA responses (Isolauri et al., 1993b). The immune effects have been reported in many studies and can be concluded for ¸actobacillus GG as follows. Milk hypersensitivity is characterized by an unfocussed immune response induced by milk antigens. Acute infections mount a non-specific immune response such as phagocytosis (Isolauri et al., 1997). The effect of probiotics depends on the disease state and the immune reactivity of a person. In other words, probiotics aid in focussing an unfocussed immune response and alleviate exaggerated immune responses during infection. In healthy subjects, only minor variations are induced (Kaila and Isolauri, unpublished observations). Thus, the regulation of the immune response can be beneficially influenced by probiotic bacteria (Fig. 1). The above-mentioned health effects and known mechanisms offer a basis for both applications of these strains in functional food products and, in specific disease states,

Fig. 1. Suggested regulation of immune response by ¸actobacillus GG during diarrhoea, in normal healthy subjects, patients with acute gastroenteritis, and in milk hypersensitive subjects (Petto et al., 1998).

Table 5. Requirements for Good Clinical Studies of Demonstration of Probiotic Properties for Functional and Clinical Use Each strain documented and tested independently, on its own merit Extrapolation of data from closely related strains not acceptable Well-defined probiotic strains, well-defined study preparations Double-blind, placebo controlled human studies Randomized human studies Results confirmed by several independent research groups Publication in peer-reviewed journals

also in clinical foods and medical foods. Due to differences even between closely related strains, all strains should be tested on their own merit with good clinical study protocols as outlined in Table 5. Types of clinical food applications Management in food allergy includes exclusion of responsible food from the diet. In attempts to eliminate cow milk allergens from the diet, the most common approach is predigestion of bovine casein or whey to create formulae that provide nitrogen as mixtures of peptides and amino acids (Isolauri et al., 1995). The data taken together suggest that probiotic bacteria downregulate hypersensitivity reactions and promote endogenous barrier mechanisms. The use of probiotic bacteria supplemented formula can further reduce antigenicity of substitute formulae and consequently have an important role in the development of specific therapy for patients who have food allergy. Management of acute diarrhoea requires infant formulae with efficient lactic acid bacteria with potential to up-regulate the immune system. Another alternative is to supply oral rehydration solutions with similar bacteria. In clinical nutrition, elemental diets are used for the treatment of intestinal inflammation, radiation enteritis and inflammatory bowel disease. Probiotic lactic acid bacteria do have a role in alleviating these disturbances and thus, the introduction of such strains into clinical foods and special dietary foods is likely. A new area is opened with the possibility of influencing food allergy and a variety of products could be developed for this area using selected strains.

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Another promising area for future research is the prevention and adjuvant treatment of cancer of the colon or bladder. Human studies using ¸actobacillus casei Shirota strain appear promising in both areas. Other strains, such as ¸actobacillus GG and ¸actobacillus acidophilus NFCB 1748 have been beneficial in animal studies with colon carcinogens and in human studies using faecal bacterial enzymes as biomarkers.

CONCLUSION Probiotic bacteria have some well-documented beneficial effects on gastrointestinal disturbances including some forms of diarrhoea and related diseases associated with intestinal dysbiosis. Probiotic bacteria offer new dietary alternatives for the management of such conditions through stabilization of the intestinal microflora, promotion of colonization resistance, regulation of the immune response and preservation of intestinal integrity. The immunomodulatory properties of probiotics indicate that they may also have a role in the dietary management of food allergy. The applications described indicate that there are common dysfunctions and microflora disturbances which facilitate effective use of probiotic bacteria. Probiotic bacteria have potential in the treatment of clinical conditions with altered gut mucosal barrier functions. Probiotic bacteria offer new dietary alternatives for the stabilization of the intestinal microflora. They can be used for immunotherapy to counteract local immunological dysfunctions and to stabilize the natural gut mucosal barrier mechanisms. The probiotic properties of each strain should be demonstrated in carefully planned and controlled human studies.

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