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Asia Pacific J Clin Nutr (1996) 5: 25-28

Safety of probiotic bacteria

DC Donohue, S Salminen

Key Centre for Applied and Nutritional Toxicology, RMIT, Melbourne, Australia

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In recent years interest has been renewed in health promotion and disease prevention by the incorporation of probiotic bacteria into foods to counteract harmful bacteria in the intestinal tract. There is considerable interest in extending the range of foods containing probiotic organisms from dairy foods to infant formulae, baby foods, fruit juice-based products, cereal-based products and pharmaceuticals. New and more specific strains of probiotic bacteria are being sought. Traditional probiotic dairy strains of lactic acid bacteria have a long history of safe use and most strains are considered commensal microorganisms with no pathogenic potential. It cannot be assumed that these novel probiotic organisms share the historical safety of the traditional strains. Before their incorporation into products new strains should be carefully assessed and tested for the safety and efficacy of their proposed use. As yet, no general guidelines exist for the safety testing of probiotics. Different aspects of the safety of probiotic bacteria can be assessed using a panel of in vitro methods, animal models and human subjects.

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Introduction

Probiotic bacteria are commonly defined as viable bacteria, in single or mixed culture, that have a beneficial effect on the health of the host.

In the dairy industry the most widely used probiotic bacteria belong to the group of lactic acid bacteria, though some bifidobacteria and yeasts are also utilised. The term ‘lactic acid bacteria’ (LAB) currently includes the genera Lactobacillus, Leuconostoc, Pediococcus and Lactococcus. Although some strains of Streptococcus and Enterococcus share the properties of LAB, Streptococcus thermophilus is the only strain currently used in fermented dairy products.

The use of LAB in foods has a long history and most strains are considered commensal microorganisms with no pathogenic potential. Their ubiquitous presence in intestinal epithelium and the human gastrointestinal tract, and their traditional use in fermented foods and dairy products without significant problems attest to their safety. Members of the genus Lactobacillus are most commonly given safe or generally recognised as safe (GRAS) status, whilst members of the genera Streptococcus and Enterococcus contain many opportunistic pathogens (Table 1).

The safety of probiotics has been questioned in recent reviews and clinical 1000 reports which have drawn attention to cases of human bacteraemia associated with the presence of LAB1-3. A variety of strains of probiotic organisms have been used in the clinical treatment of gastrointestinal disorders in both children and adults. These include conditions where mucosal integrity is impaired by antibiotics or radiotherapy, acute diarrhoea of bacterial or viral origin, and in prevention of gut colonisation by pathogens4. No evidence of opportunistic infection by probiotics was seen in these studies. Recent analyses by Saxelin et al5,6 of clinical isolates of lactobacilli from bacteraemic patients and comparison with both starter strains and strains used in pharmaceutical preparations has confirmed that these LAB are not involved in human infections.

Table 1. Classification of probiotic organisms and their safety status Organism Infection potential Lactobacillus Mainly non-pathogens, some opportunistic infections (usually in immunocompromised patients), Lactococcus Mainly non-pathogens Leuconostoc Mainly non-pathogens, some isolated cases of infection Streptococcus Oral streptococci mainly non-pathogens (including Streptococcus thermophilus); some may cause opportunistic infections Enterococcus Some strains are opportunistic pathogens with haemolytic activity and antibiotic resistance Bifidobacterium Mainly non-pathogens, some isolated cases of human infection Saccharomyces Mainly non-pathogens, some isolated cases of human infection

In addition to these clinical studies and animal studies showing an absence of infectivity, toxicity studies have also been carried out^7-9 confirming the a 1000 bsence of acute toxicity of the studied strains of probiotic bacteria. Although acute toxicity tests were originally designed for chemicals they also give an indication of any harmful effects associated with extremely high doses of freeze-dried bacteria.

Studies on safety of probiotic bacteria

Different aspects of the safety of probiotic bacteria can be studied using in vitro methods, animal models and human subjects. As yet no general guidelines exist for the safety testing of probiotics. However, some recommendations are given in the review of Donohue et al⁷ Many countries, including the European Community, are currently developing more detailed guidelines with respect to regulations for novel and functional foods and related probiotic preparations.

In vitro studies

One of the most important requirements for a probiotic organism is that it be non-invasive.

In vitro studies are an initial means of assessing whether a test organism alters the integrity of the intestinal mucosa and its ability to penetrate the intestinal cells. The local effects of LAB on the intestine are commonly measured by their in vitro ability to adhere to human intestinal cell lines and to degrade protective intestinal mucus. These tests provide an indirect measure of the potential of LAB to invade intestinal cells and to damage the protective glycoproteins of the intestinal mucus.

A large number of adhesion studies have been conducted with different strains of LAB using Caco-2 cells as the most common cell line. Most strains of LAB have shown no invasive properties in this test system, even though the selection of new probiotics has favoured those strains that are strongly adherent to human intestinal cell lines^10,11.

Degradation of intestinal mucus has also been used as a marker of toxicity. It is thought a stable gastrointestinal microflora with normal patterns of fermentation and colonisation resistance and low pH are important in protecting the mucosal layer from injury¹². Strains that do not degrade intestinal mucus or its glycoproteins are thought to be non-invasive. Strains which do not degrade intestinal mucosa are also thought to be therapeutic in the probiotic treatment of mucosal diseases such as pouchitis, ulcerative colitis and Crohn’s disease¹². In a recent study, commercial probiotic strains (Lactobacillus GG, Lacto-bacillus acidophilus, Bifidobacterium bifidum) were shown to be inactive in mucosal degradation¹². In earlier studies, some faecal Bifidobacteria were found to take part in mucus degradation^13,14.

Production of antimicrobial compounds and inhibition of pathogen growth by LAB has been assessed in vitro. The competitive exclusion of pathogens altering the balance of the intestinal microflora has been studied in the Caco-2 cell line. Data from these tests support the safety of LAB and indicate that many strains decrease intestinal pH and reduce the numbers of pathogenic bacteria in the intestinal tract, thus protecting the host^10,11. It has been shown by Australian researchers that LAB strains isolated from cases of infective endocarditis have some properties in common. These properties include platelet aggregation, binding of fibronectin and fibrinogen and the production of glycosidases and proteases which are postulated as factors in the pathogenesis of endocarditis^15,16. However, comparative studies are needed to determine whether these are also properties of the strains of LAB normally found in the oral cavity and the intestinal tract of healthy humans. < 1000 /font>

Animal studies

Acute toxicity studies have been conducted with several strains of LAB and for reference they have also included Bifidobacterium longum strains. In general, no acute toxicity has been observed with any of the tested strains as indicated in Table 2.

Recently, the association of LAB in germ free rodents has also been used as a criterion for safety. Ruseler-van Embden et al12 studied the association or colonisation of germ free rodents with several probiotic lactobacilli and detected no adverse effects in these animals. Studies by Goldin and Gorbach17 of the promotion and induction of colon cancer in laboratory animals have indicated that adherent lactobacilli appear to delay and slow down the development of dimethyl hydrazine (DMH)-induced colon tumours. The strains tested were Lactobacillus acidophilus and Lactobacillus casei. Clinical Studies A large amount of data from clinical trials or studies in human volunteers also attest to the safety of LAB. These studies have included short-term trials in normal volunteers; prevention and treatment of acute diarrhoea in premature infants18, infants19,20, children with diarrhoea20-22, studies on immune effects23 and studies in patients with severe intestinal infections24-26. A study using Lactobacillus acidophilus preparations in the effective prevention of intestinal side-effects during pelvic radiotherapy has also been reported27,28. Aso et al29 reported that the recurrence-free interval after resection of superficial bladder cancer in humans was extended by treatment with Lactobacillus casei Shirota strain. A summary from the literature of safety studies and reported effects for probiotic and yoghurt strains is shown in Table 3.

Table 2. Acute toxicity of probiotic bacteria (Adapted from 7) Probiotic strain LD50 (g/kg body weight) Streptococcus faecium AD1050a >6.6 Streptococcus equinusa >6.39 La 1000 ctobacillus fermentum AD002a >6.62 Lactobacillus salivarius AD0001a >6.47 Lactobacillus GG (ATCC 53103) >6.00 Lactobacillus helveticus >6.00 Lactobacillus bulgaricus >6.00 Bifidobacterium longum 25 a: Heat-treated nonviable preparations

All available data indicate that no harmful effects have been observed in controlled clinical studies with lactobacilli and bifidobacteria. To the contrary, during treatment of intestinal infections beneficial effects have been observed including stabilization of gut mucosal barrier, prevention of diarrhoea and amelioration of infant and antibiotic-associated diarrhoea.

Table 3. Safety studies and reported effects of current successful probiotic strains and yoghurt strains.

Probiotic	Reported effects	Safety studies
strain		In vitro	Animal studies	Human studies
Lactobacillus acidophilus NCFB 1748	Treatment of constipation, alleviation of radiotherapy related diarrhoea, lowering of faecal enzymes (4,6,12,27,28,31)	+	+	+
Lactobacillus casei Shirota	Balancing intestinal microflora, prevention of intestinal disturbances, treatment of superficial bladder cancer (4,30,31)	+	+	+
Lactobacillus GG (ATCC 53103)	Treatment of acute viral and bacterial diarrhoea in infants, prevention of antibiotic associated diarrhoea, immune enhancing, stabilisation of intestinal permeability (6,7,10,11,18,20,21,24,26)	+	+	+
Lactobacillus acidophilus LA1	Immune enhancing, vaccine adjuvant, balancing intestinal microflora (4,10,23)	+	+	+
Bifidobacterium bifidum	Prevention of rotavirus diarrhoea (4,12,22,31)	+	+	+

Epidemiological data

Case reports from the literature of LAB in association with clinical infection in humans have recently been analysed in reviews by Gasser² and Aguirre and Collins¹. Both reviews conclude that, considering their wide-spread consumption, LAB appear to have very low pathogenic potential. Two recent Finnish studies confirm that the number of infections associated with LAB is small. In the first study, genetic methods (16 SRNA) were used to characterise and identify LAB isolated from blood cultures of bacteraemic patients in Southern Finland⁵. The results showed that a newly introduced probiotic strain in fermented milks was not associated with infections and the total number of infections caused by lactobacilli was extremely low. In a further study, lactobacilli isolated from bacteraemic patients between 1989 and 1994 were compared to common dairy or pharmaceutical strains⁶. From a total of 5192 blood cultures 12 were positive for lactobacilli, an incidence of 0.23 per cent. None of the clinical cases could be related to lactobacilli strains used by the dairy industry. In both studies, patients with LAB bacteraemia had other severe underlying illnesses.

Safety of n 1000 ovel probiotics

Traditional probiotic dairy strains of LAB have a long history of safe use. In latter years interest has been renewed in preventing disease and promoting health by using probiotic bacteria to fight harmful bacteria in the intestinal tract³¹. There is considerable interest in extending the range of foods incorporating probiotic organisms from dairy foods to infant formulae, baby foods, fruit juice-based products, cereal-based products and pharma-ceuticals⁴. New and more specific strains of probiotic bacteria are being sought. It cannot be assumed that these novel probiotic organisms share the historical safety of traditional strains. Before their incorporation into products new strains should be carefully assessed and tested for the safety and efficacy of their proposed use. The following suggestions and recommendations have been proposed as suitable models and methods to test the safety of probiotic bacteria^3,7.

1. Determine the intrinsic properties of bacteria and strains selected for probiotic use eg adhesion factors, antibiotic resistance, plasmid transfer, enzyme profile.
2. Assess the effects of the metabolic products of the bacteria.
3. Assess the acute and subacute toxicity of ingestion of extremely large amounts of the bacteria.
4. Estimate the in vitro infective properties of probiotic bacteria using cell lines and human intestinal mucus degradation. Assess infectivity in animal models eg immunocompromised animals or lethally irradiated animals.
5. Determine the efficacy of ingested probiotic bacteria as measured by dose-response (minimum and maximum dose required, consequent health effects); assess the effect of massive probiotic doses on the composition of human intestinal microflora.
6. Carefully assess side-effects during human volunteer studies and clinical studies in various disease-specific states.
7. Epidemiological surveillance of people ingesting large amounts of newly introduced probiotic bacteria for infections.
8. The most rigorous safety testing along the above lines to be undertaken for genetically modified strains and strains derived from animals.

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Chinese abstract

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References

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Revised: January 19, 1999 .

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