To select a probiotic bacteria that would reduce serum lipids in hypercholesterolaemic volunteers.
A strain of lactobacillus was selected for its ability to metabolise cholesterol under varying conditions in vitro. Freeze-dried Lactobacillus acidophilus or placebo were then given in a double-blind randomised crossover study to volunteers with high cholesterols.
A total of 80 volunteers with elevated cholesterols.
Volunteers were randomly allocated to receive either two capsules containing freeze-dried L. acidophilus 3 × 1010 CFU or placebo three times a day for 6 weeks. After a 6-week washout period, volunteers were crossed over to another 6 weeks of capsules. Serum lipids were measured at the beginning and end of each interventional period.
L. acidophilus was able to reduce cholesterol and survive in an acid and bile environment. No changes in anthropomorphic measurements or in dietary records were seen between the baseline and final records or between the two sets of baseline records. There were no changes in serum lipids seen throughout the study.
Despite the ability in vitro for L. acidophilus to reduce cholesterol, no effect was seen in volunteers.
Life-Care Technologies Ltd, Worcestershire.
Influx of cholesterol into the walls of large- and middle-sized arteries is a prime initiating event in development of atherosclerosis. Clinical studies have shown that reduction in serum cholesterol reduces ‘all cause’ mortality as well as cardiovascular events both in primary and secondary prophylaxis.
The use of fermented milks to treat gastrointestinal complaints dates from antiquity. Fermented dairy products such as yoghurts have traditionally been made using mixed bacterial cultures. One of the bacterial species commonly found in this bacterial mixture are lactobacilli. Lactobacilli are also present in the normal human intestinal flora, having been acquired at an early age through oral ingestion. Lactobacillus acidophilus is commonly used in fermented dairy products. There is some evidence that lactobacilli may reduce serum cholesterol. Gilliland et al (1985) fed a strain of lactobacillus (strain RP32) to pigs with a high-fat diet, which inhibited any rise in serum cholesterol. Possible mechanisms include bacterial assimilation of cholesterol (Gilliland et al, 1985), or through the deconjugating of bile salts (Klaver & Meer, 1993; De Smet et al, 1998). Short-chain fatty acids produced by probiotic bacteria may also inhibit hepatic cholesterol synthesis and/or redistribution of cholesterol from plasma to the liver (Pereira & Gibson, 2002). Human studies using various probiotic preparations have given mixed results (Hepner et al, 1979; Rossouw et al, 1981; Thompson et al, 1982; Massey, 1984; Lin et al, 1989; Agerbaek et al, 1995; Richelsen et al, 1996; Schaafsma et al, 1998). In some cases the results of the studies may have been complicated by the influence of dairy products on serum lipids (Mann, 1977; Anderson & Gilliland, 1999). In many of these animal and human studies, the strains of lactobacilli used were not selected for their ability to metabolise cholesterol.
We isolated bacteria from commercially available yoghurts (these bacteria are not under patent) with the assumption that safety issues with regard to consumption had already been addressed by the manufacturers. The selected bacteria were assessed for their ability to metabolise cholesterol and survive in bile and an acid pH. Using the most promising bacterial strain, we randomly allocated volunteers in a double-blind study to receive L. acidophilus or placebo capsules. Lipid profiles were measured at the start and finish of each intervention.
Selection of lactobacilli strain
Five lactobacilli were isolated from commercially available yoghurts (strains B-F), the RP-32 strain used by Gilliland et al (1985) was used as a positive control and a strain of Streptococcus thermophilus as a negative control. All experiments were conducted in duplicate, with the mean result presented. Cultures were passaged in MRS+0.4% Oxgall at 37°C in 5% CO2. Pure cultures were confirmed as containing lactobacilli by Gram stain, fermentation profile (API 50 CHL kit) and antibiotgram profiles (MASTRING-S discs).
The cholesterol-reducing characteristics were measured by incubating 1 × 107 CFU in MRS broth supplemented with 0.4% Oxgall and 10% cholesterol-rich lipids (Oxoid) for 48 h at 37°C in 5% CO2. We then passaged the bacteria to see if the ability to metabolise cholesterol could be induced and if so was it permanent. The cholesterol concentration of the media was determined using a commercial diagnostic kit (Sigma 352-30). The two most promising strains were tested for their ability to survive in MRS broth at different pH levels for 1 and 5 h.
Production of freeze-dried capsules
A master culture was created of strain B (having undergone a characterisation profile: Gram stain, fermentation profile using API 50 CHL kit, antibiotgram profiles (MASTRING-S discs), and microbiological contamination clearance cultures) and aliquots were stored in liquid nitrogen. Working cultures were then produced from the master culture, tested as above, then used to seed the fermenter. We used an MBR Bioreactor (Switzerland) fully automatic fermenter. The resulting product was tested again as above for purity before being aseptically recovered and centrifuged. The centrifugate was then freeze-dried (after mixing with a cryoprotectant, maltodextrin) under sterile conditions. Freeze-dried samples underwent further quality control tests as above, in addition were also tested as before for cholesterol-utilising ability. Capsules were produced and stored at 4°C before use. Freeze-dried capsules were quality control tested as above just prior to having been used in the trial. The freeze-dried capsules were quantitatively cultured and tested for their cholesterol-utilising ability before and at the end of the trial.
Volunteers aged between 20 and 65 y old were recruited by word of mouth and poster. Volunteers were omnivorous, nonsmokers, had no significant past medical history or family history of hypercholesterolaemia, not taking any medications nor had taken any antibiotics within the previous month. Volunteers gave written informed consent before starting the study. The volunteers confirmed that they are not in a high-risk group for acquiring HIV or were immunosuppressed in any way.
At the start of the study, volunteers fasting serum was analysed for total cholesterol, high-density lipoprotein cholesterol and triglycerides. Volunteers with cholesterol values greater than 5.0 mmol/l were entered into the study.
Volunteers were weighed and measured to calculate their body mass index. Four-day dietary records (two week days and two weekend days) were done.
The volunteers were randomly allocated to one of two groups. Group A received placebo capsules (maltodextran)(2 t.i.d.) and group B capsules (2 t.i.d.) containing freeze-dried L. acidophilus. Volunteers took the capsules for 6 weeks. After a 6-week washout period, volunteers took the other intervention. The baseline investigations were repeated at the beginning and end of each intervention. Any remaining capsules were collected and compliance calculated.
Subjects were asked to write down the type and amount of food eaten, using scales or household measures to gauge portion sizes where possible. Where insufficient information was given, the subjects were contacted by telephone for a fuller description. Volunteers were encouraged to keep their diets, alcohol intake and exercise patterns unchanged during the experiment. The records were analysed for individual nutrients (total energy, total dietary fibre (Southgate), insoluble nonstarch polysaccharide (NSP), soluble NSP, total NSP, total fat, saturated fat, polyunsaturated fat, protein, carbohydrate, extrinsic sugar and alcohol) using a computer program-based on McCance and Widdowson's The Composition of Foods (Paul & Southgate, 1978) and on published values for NSP (Englyst et al, 1988, 1989).
In a previous study that we have conducted using volunteers in a similar age group, the baseline cholesterol was 6.14 s.d. 1.03 mmol/l. Using these values, power calculations indicate that a sample size of 91 is needed to detect a 5% or greater reduction in cholesterol (power 0.8, α=0.05). Thus, a study size of 80 gives reasonable power (5.2% or greater) to detect a clinically significant reduction in cholesterol.
Data will be assessed as parametrically or nonparametrically distributed using histograms and Ryan–Joiner tests. Changes in serum concentrations will be analysed as appropriate using two-tailed Student's t and Wilcoxon rank sum tests.
Five lactobacillus species were isolated, the percentage reduction of cholesterol from supplemented MRS broth is given in Figure 1. The two most promising strains (RP-32 and B) were tested for their ability to survive in MRS broth at different pH levels (Table 1). We selected the most promising strain, strain B (we decided not to use the positive control RP-32 strain as it had not been used in humans before) to be grown up in batch culture and capsules containing about 3 × 1010 CFU of freeze-dried culture were produced.
We found no evidence of contamination of the freeze-dried strain B. There was no reduction in bacterial count or loss of ability to utilise cholesterol at the end of the trial.
A total of 80 volunteers were randomised, of whom one withdrew before starting the first phase and two before starting the second phase of the study. No changes anthropomorphic measurements (Table 2) or in dietary records (Table 3) were seen between the baseline and final records or between the two sets of baseline records.
There were no changes in serum lipids seen throughout the study (Table 4). The mean baseline cholesterol was 6.69 s.d. 0.81 mmol/l, thus the study had the power (0.8, α=0.05) of detecting a 3.2% or greater reduction in cholesterol between the baseline and active measurements. Compliance with medication calculated from the number of capsules returned at the end of each study period was 99.3% s.d. 1.2%.
We selected the strain B (L. acidophilus LA-1) as it reduced cholesterol in vitro, and it was stable in acid and bile. Feeding 6 × 1010 CFU of strain B three times a day to volunteers with elevated cholesterol did not lead to a reduction in their serum lipids. No changes in volunteers BMI, waist–hip ratio or dietary intake were seen to account for the apparent lack of effect. It would appear that our in vitro observations and previous animal studies cannot be applied to humans for the strain of lactobacilli that we used.
L. acidophilus clearly can reduce cholesterol in vitro and in animal models. Why we were unable to detect any reduction in serum cholesterol is difficult to explain. Previous studies have used fermented dairy products containing lactobacilli, in which the bacteria were presumably metabolically active when ingested. Considering that small intestinal transit is relatively short, it is possible that insufficient time was available for the freeze-dried bacteria in our study to become sufficiently metabolically active before being flushed into the colon. Unfortunately, we do not have data on the ability of strain B to colonise the small bowel. It is possible that our assumptions from the in vitro experiments such as the ability to metabolise cholesterol in MRS broth are not transferable to the in vivo environment of the small bowel. It is likely that the concentration of lactobacilli in the small bowel is much less than that used in the in vitro and gnotobiotic animal studies. In one human study, volunteers received 106–107 of mixed lactobacilli strains in a fermented oatmeal soup, which led to an increase from 3.0 to 4.0 log10 CFU/g of lactobacilli in the jejunal mucosal. If we assume a similar increase (1 log) was seen in our study, then this may not be enough to make a significant impact on luminal cholesterol concentrations. In some previous studies, probiotic bacteria have been taken with milk, calcium, fibre and fructo-oligosaccharides, which could complicate the interpretation of the results seen as many of these ‘additives’ also have lower serum lipids. Other studies have been able to detect transient alterations in lipids (3–4 weeks) even with the placebo used which were not apparent later on in the studies (Hepner et al, 1979; Richelsen et al, 1996; De Smet et al, 1998). Serum cholesterol concentration is determined by many factors. The relative importance of dietary cholesterol vs dietary fats to the concentration of serum cholesterol may be small. Unfortunately, we did not determine the effectiveness of the lactobacilli strain used to metabolise different types of fats. Thus, it is not possible to clearly state that probiotic bacteria can reduce serum lipids in humans without further unequivocal trials being done. Our study was able to detect a greater than 3% decrease in serum cholesterol, it is debatable if any smaller effect is of any clinical significance.
There is much theoretical and experimental data to suggest that probiotic bacteria can favourably alter serum lipids. Human studies examining the benefits of probiotic bacteria on serum lipids have shown conflicting results. Several studies have potentially been complicated by an effect of the placebo used on serum lipids. We have not shown any benefit on serum lipids from freeze-dried L. acidophilus strain B. Further studies are needed using different bacterial strains and using different delivery systems (fermented dairy product or freeze dried bacteria) to unequivocally establish the potential role for probiotic bacteria in the management of hyperlipidaemia.
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We thank all the volunteers who took part in the study. We also thank Mr B Holden and Ms C Day of Life-Care Technologies Ltd, Worcestershire for their help and support with the study.
Guarantors: SJ Lewis and S Burmeister.
Contributors: SJL and SB researched and wrote the review.
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Lewis, S., Burmeister, S. A double-blind placebo-controlled study of the effects of Lactobacillus acidophilus on plasma lipids. Eur J Clin Nutr 59, 776–780 (2005). https://doi.org/10.1038/sj.ejcn.1602139
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