Lactobacillus Clinical Trials

PREFACE

The bacterial strain, Lactobacillus plantarum 299v (=DSM 9843), is included in the “functional food” products ProViva® (fruitdrink, yoghurt and recoverydrink), produced and marketed by Såne Dairies (Malmö). The active component in ProViva is a lactic acid fermented oatmeal gruel that has been fermented with Lb. plantarum 299v (resulting in about 1 x 109 colony forming units [CFU] per ml). This fermented oatmeal formula was originally brought forward as a new concept for enteral feeding (nasogastric feeding). The process of production is patented (Molin et al. 1991a). The lactic acid fermented oatmeal gruel is an integral part of ProViva where 5% oatmeal gruel is mixed with drink of rose hip, strawberry, blueberry, black currant or tropical fruits (about 5 x 107 CFU/ml). In a drink intended to improve the recovery after exhaustion, the fermented oatmeal gruel is mixed with antioxidants, carbohydrates and whey protein (ProViva Active).
The strain, Lactobacillus plantarum 299v, that has been isolated from healthy human intestinal mucosa (Molin et al. 1993), is patented (Molin et al. 1991b; Molin et al. 1991c; Molin et al. 1995; Molin et al. 1998; Bengmark et al. 1995; Bengmark et al. 1996; Bengmark et al. 1997). Several reviews of Lb. plantarum 299v and its effects have been published (Molin 1995; Molin & Ahrné 1999; Molin 2001). All licences for Lb. plantarum 299v (and 299) are owned by Probi AB (Lund), which also produces the strain as starter culture or as fermented oatmeal gruel.

BACKGROUND

It is a well established fact that high numbers of lactobacilli counteract many pathogenic and potential pathogenic bacteria, regardless if the system is lactic acid fermented foods or the human intestine (De Vuyst & Vandamme 1994a; De Vuyst & Vandamme 1994b). However, should lactobacilli be able to perform in an optimal way in the intestine, a condition is that they also are present in high numbers in the mucosa. It is the bacteria in the mucosa that are the ones in direct contact with the body (Hentges 1992; Salminen et al. 1993). Even if there have been reports of Lactobacillus strains able to survive the passage through the gastro-intestinal tract, it has been difficult to find strains that are able to colonize the human intestine after oral administration; even a short-time establishment has often been difficult to achieve (Kurman & Rasic 1991; Bouhnik et al. 1992; Fuller 1992; Roberfroid & Gibbson 1994; Teuber 1995; Jacobsen et al. 1999; Klaenhammer & Kullen 1999). Short-time establishment of Lactobacillus strains in the intestinal mucosa after oral administration have only been proved for a few strains (showed for the first time by Johansson et al. 1993). It is especially two, genetically, closely related strains of Lactobacillus plantarum (strain 299v [=DSM 9843] and strain 299 [=DSM 6595]) that possess this ability. These two strains are included in a genetic subgroup within the species Lb. plantarum (Johansson et al. 1995) where the members mostly are originating from the intestinal mucosa (Molin et al. 1993; Johansson et al. 1995). The strains of this subgroup have been shown to have a pronounced (and unique) ability to attach to human mucosa cells in vitro; the adhesion is dependant on a mannose-binding adhesin (Adlerberth et al. 1995; Adlerberth et al. 1996a; Adlerberth et al. 1996b). Moreover, Lb. plantarum strains of this particular type are frequently dominating the total Lactobacillus flora of healthy individual, both in rectal and in oral mucosa (Ahrné et al., 1998).
The ability of Lb. plantarum 299v when administrated in ProViva (i) to survive the passage through the human gastro-intestinal tract (Johansson et al. 1998), and (ii) to establish itself for at least a shorter period of time in the intestine after consumption (Johansson et al. 1998), has been confirmed (Nobaek et al. 2000).

The level of lactobacilli decreases in an inflamed mucous membrane (Fabia et al. 1993a). But also a more normal, occasional stress is likely to affect the composition of the bacterial flora of the intestine (Alm 1991; Raibaud 1992). Administration of Lb. plantarum 299/299v can increase the total load of lactobacilli in the intestine and to some degree suppress bacterial groups with adverse effects (Johansson et al. 1993). Lb. plantarum was, in a study on healthy individuals requited from the Malmö-Lund region, found to be the predominating Lactobacillus species in the mucosa, both in the oral one and in the intestinal mucosa (Ahrné et al. 1998).

PROBIOTIC EFFECTS IN HEALTHY SUBJECTS

Lactobacillus plantarum 299 and 299v have been shown to possess anti-microbial activity against strains of potentially pathogenic species as Listeria monocytogenes, Bacillus cereus, Escherichia coli, Yersinia enterocolitica, Citrobacter freundii, Enterobacter cloacae and Enterococcus faecalis, in vitro (Jacobsen et al. 1999). When healthy volunteers consumed a mixture of lactobacilli strains, including Lactobacillus plantarum 299/299v, and the level of lactobacilli in the intestinal mucosa increased, there was also a decrease in the level of Gram-negative anaerobes (Johansson et al. 1993), Enterobacteriaceae (Johansson et al. 1993) and sulphite-reducing clostridia (Johansson et al. 1993).
Gram-negative anaerobes are noxious from the viewpoint that they often are involved in secondary infections after surgical abdominal surgery (Nichols 1980; Offenbartl & Bengmark 1990; Wittman 1991). Furthermore, Gram-negative bacteria always contain endotoxins and they initiate, even in small amounts, violent inflammatory reactions. Gram-negative anaerobes are also pointed out as responsible for the production of carcinogenic substances in the intestine (Rowland 1992; Roberfroid & Gibson 1994).
The group sulphite-reducing clostridia can contain subgroups that produce toxin; sulphite-reducing clostridia are also producing hydrogen sulphide that has a general toxicity. Furthermore, clostridia can produce carcinogenic substances in the intestine (Rowland 1992).
Enterobacteriaceae is a genetically close family including many pathogenic taxa, and even normally none-pathogenic taxa have a pathogenic potential in situations where the immunological defence of the host is failing. Lb. plantarum 299v have been shown to inhibit entero-pathogenic Escherichia coli adherence in vitro to HT-29 intestinal epithelial cells by inducing intestinal mucin gene expression in the cells, i.e. epithelial cells produce more mucin and the slime protects the cells from the entero-pathogenic E. coli (Mack et al. 1999).

Decreased presence of entero-pathogens: In a study in Tanzania, Lactobacillus plantarum 299v had been used as starter culture for producing a cereal based lactic acid fermented beverage called Togwa (Lactobacillus plantarum 299v was used for producing about 50% of the material; the other 50% was made by traditional back-sloping). The product was given to children (<5 years) once a day for 13 consecutive days and the presence of faecal entero-pathogens such as Campylobacter, entero-pathogenic Escherichia coli, Salmonella and Shigella was evaluated. The proportion of children with isolated faecal entero-pathogens decreased significantly (P<0.001) during the study period (Kingamkono et al. 1999). Spontaneously fermented togwa is frequently dominated by Lactobacillus plantarum (Mugula 2001).

Increased concentrations of carboxylic acids: Lb. plantarum 299v administrated in ProViva has, in a double blind placebo controlled study, been shown to increase the total level of carboxylic acids in faeces of health volunteers (mainly acetic acid and propionic acid; Johansson et al. 1998). Furthermore, the subjects administrated ProViva experienced a decreased flatulence during the treatment period (Johansson et al. 1998).
The carboxylic acids in the colon content are a major energy source to the human mucosa cells, i.e. an increased level of short-chain fatty acids in the lumen is beneficial for the condition of the mucosa. Moreover, absorbed short-chain fatty acids can have positive effects on the lipid metabolism in the liver (short-chain fatty acids that are absorbed from the colon are going directly to the liver).
The observed increase in the level of short-chain fatty acids (Johansson et al. 1998) can be due to a changed composition of the colonic microflora, i.e. Lb. plantarum supported an increased number of bacterial taxa able to produce acetic acid and propionic acid. A supplementary explanation might be that Lb. plantarum 299v increased the amount of mucin in the colon, i.e. Lb. plantarum 299v has been shown to stimulate the epithelial mucin production in vitro (Mack et al. 1999). Thus, the increased amount of mucin leads to a higher amount of fermentable material in the colon; material that can be converted to short-chain fatty acids.

CLINICAL TRIALS

Irritable bowel syndrome: Lb. plantarum 299v in ProViva (rose hip) has been administrated to patients with irritable bowel syndrome (IBS) in two, double blind, placebo controlled studies, one in Poland (Niedzielin et al., 2001) and one in Sweden (Nobaek et al., 2000). In both studies the patients were divided into two groups, one was given ProViva and the other a similar rose hip drink without Lb. plantarum 299v (placebo). The result of the Polish study was that the magnitude of several of the IBS-symptoms decreased in the ProViva group, and a higher proportion of the patients were cured from their symptoms in the ProViva group than in the placebo (Niedzielin et al., 2001).

In the Swedish study, ProViva significantly decreased the subjective experienced bloating during the treatment. The pain was also significantly reduced in both the treatment-group and in the placebo group, even if the decrease was more rapid and more pronounced in the ProViva-group. Twelve months after the treatment, the patients given ProViva in the study still experienced a better overall gastrointestinal function than the patients of the placebo group (Nobaek et al., 2000).

Cardiovascular risk factors: Lb. plantarum 299v administrated in ProViva for six weeks has shown to significantly decrease the fibrinogen and LDL-cholesterol levels in serum of Polish men with moderately elevated cholesterol levels (Bukowska et al., 1998 – no. 10). The study was performed in 30 subjects, randomly divided into two groups in a double-blind study with placebo. The placebo was a rose hip drink without Lb. plantarum 299v. Fibrinogen is an acute phase protein produced in the liver during inflammation, and suspected to be an independent risk-factor for thrombosis.

In another placebo controlled randomised double blind study, in thirty-eight healthy smokers, it was shown that 400 ml ProViva daily for six weeks decreased systolic blood pressure, and the levels of LDL-cholesterol, insulin and leptin (Naruszewicz et al. 2001 - 19). Also a simultaneous decrease in fibrinogen, F2-isoprostans and Il-6 was recorded (Naruszewicz et al. 2001 - 19).

Immune response in HIV positive children: Children congenitally exposed to human immunodeficiecy virus (HIV) have in a pilot-study received Lb. plantarum 299v in the fermented oatmeal gruel (freeze dried). The results suggested that Lb. plantarum 299v may be given safely to the immunocomprimised host and may have positive effect on immune response (Cunningham-Rundles et al. 2000; Cunningham-Rundles et al. 2002).

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