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Effect of Fermented Milk (Yogurt) Containing Lactobacillus Acidophilus L1 on Serum Cholesterol in Hypercholesterolemic Humans
James W. Anderson, MD and Stanley E. Gilliland, PhD Metabolic Research Group, VA Medical Center, University of Kentucky, Lexington, and Department of Animal Science, Oklahoma State University, Stillwater, Oklahoma Key words: lactobacilli, fermented milk, yogurt, cholesterol, hypercholesterolemia
Objective: Two controlled clinical studies were performed to examine effects of consumption of one daily serving of fermented milk (FM) (yogurt) on serum lipids. Methods: In the first study, subjects were randomly allocated to FM containing Lactobacillus acidophilus L1 of human origin or to FM containing L. acidophilus ATCC 43211 of swine origin. In this single-blind study, subjects consumed one 200 ml serving of FM daily for 3 weeks. The second study was a double-blind, placebo-controlled, cross-over study. Subjects completed a 4-week first treatment, had a 2-week washout, and completed a second 4-week treatment. In the second study subjects consumed FM containing L. acidophilus L1 or placebo FM over 4 weeks. Results: In the first study, FM containing L. acidophilus L1 was accompanied by a 2.4% (p 0.05) reduction of serum cholesterol concentration. In the second study, strain L1 reduced serum cholesterol concentration by 3.2% (p 0.05) in the first treatment period. In the second treatment period there were no significant changes in serum cholesterol concentration. Combined analysis of the two L1 treatment studies demonstrated a 2.9% (p 0.01) reduction in serum cholesterol concentration. Conclusion: Since every 1% reduction in serum cholesterol concentration is associated with an estimated 2% to 3% reduction in risk for coronary heart disease, regular intake of FM containing an appropriate strain of L. acidophilus has the potential of reducing risk for coronary heart disease by 6 to 10%.
INTRODUCTION
Since the early studies of Mann and Spoerry [1] there has been an increasing interest in the cholesterol-lowering properties of fermented milk (FM) (yogurt) products, and numerous studies have focused on the potential hypocholesterolemic activity of FMs in humans [2 8]. From these seven clinical studies, six reported a reduction in serum cholesterol concentration associated with FM intake. The following median reductions in serum cholesterol concentration were obtained: 1 week, 5.4%; 2 weeks, 6.6%; 3 weeks, 6.9%; and 4 weeks, 6.4%. These findings of cholesterol-lowering activity of FM products were also confirmed in experimental animals [9 12]. These and other studies strongly suggest that FMs have an important cholesterol-lowering potential. However, as was already demonstrated from the clinical study of Jaspers and
colleagues [4], different strains of lactic acid bacteria may have different effects on serum cholesterol concentrations. Although the underlying cholesterol-lowering mechanism(s) has not yet been sufficiently elucidated, the metabolism of cholesterol and/or bile salts by the bacteria incorporated in the FM product may be responsible for the cholesterol-lowering effect. Gilliland and colleagues [1316] made in vitro comparisons of isolates of intestinal L. acidophilus to assess their indigenous capacities to tolerate bile, to deconjugate bile salts, and to assimilate cholesterol. Furthermore, when an isolate of L. acidophilus from a pig (strain ATCC 43121) with high in vitro activity (i.e., bile resistance, bile salt hydrolysis and cholesterol assimilation) was administered to cholesterolfed pigs, serum cholesterol values were significantly lower than values of controls [16]. No such cholesterol reduction was observed upon feeding a L. acidophilus strain with poor bile
Address reprint requests to: James W. Anderson, MD, Chief Endocrine-Metabolic Section, VA Medical Center, Cooper Drive Division (111C), Lexington, KY 40511.
Journal of the American College of Nutrition, Vol. 18, No. 1, 4350 (1999) Published by the American College of Nutrition 43
Number of subjects 14 15 21 19
M/F*
Five subjects withdrew voluntarily because of personal reasons such as schedule conflicts. The remaining subjects in both studies, whose medical history, medications and serum lipids were consistent with inclusion and exclusion criteria, were divided into two experimental groups (Table 1). The initial subject characteristics did not significantly differ between the two groups (Table 1) for both studies.
Study Design
Study I. Study I was a single-blind, random-allocation, parallel study to compare serum lipid effects of two different FM products over a 3-week period; FM containing human L. acidophilus L1 (L1 FM) was compared with swine L. acidophilus strain ATCC 43121 (ATCC FM). Subjects, randomly allocated to one of two groups, received either 200 g of L1 FM or ATCC FM after each evening meal. Prior to the first FM consumption period, two fasting lipid measurements were made at baseline. Fasting serum lipid measurements were made at weeks 2 and 3, and subjects had weekly body weight measurements. At these weekly visits, subjects received fresh FM and adherence to fermented products was assessed. Subjects were further questioned about any symptoms or medication changes since their last visit. Study II. Study II was a double-blind, random-allocation, placebo-controlled cross-over study of 4-weeks duration with a 2-week wash-out period, to compare the effects on serum lipids of FM containing the L. acidophilus L1 strain (L1 FM) with a FM not containing these active bacteria (placebo FM). Subjects were randomly allocated to one of two groups receiving either 200 g placebo FM or L1 FM product after each evening meal for 4 weeks. In this study after the 2-week washout period, subjects received the other FM product for the final 4 weeks. Prior to the first FM consumption period, three fasting serum lipid measurements were made at baseline and two fasting lipid measurements were done before starting the second FM treatment. Fasting serum lipid measurements were made at week 2, 3 and 4 of each FM treatment period, and subjects had weekly body weight measurements. At these weekly visits, subjects received fresh FM and the adherence to fermented products
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Measurements
Body weight was determined by the use of a calibrated balance. Blood was drawn after an overnight fast for measurement of serum cholesterol [18], HDL-cholesterol [19], and triglyceride concentrations [20]. Serum LDL-cholesterol levels were calculated as previously described [21]. Nutrient intake was estimated by analysis of three-day diet records using a computerized nutrient data base (Nutritionist IV, 1994, NSquared Computing, San Bruno, CA). The concentrations of viable L. acidophilus cells for both strains in the FMs were determined on the first and seventh day of each weekly FM batch by plating serial dilutions prepared in physiological saline with Rogosa agar (Difco). The viable counts of L. acidophilus determined after 72 h of anaerobic incubation, were expressed as CFU/g FM product.
Diet Instructions
All subjects were instructed to maintain usual dietary habits throughout the study and to complete 3-day diet records, which were to be returned at each baseline and after each FM treatment period. While on FM treatment, subjects were asked not to use sweet Acidophilus milk and not to incorporate high fiber items such as preserves into FM.
Statistics
Change scores (treatment values minus baseline) among different treatments were compared by a one way analysis of variance (ANOVA). Change scores between groups also were compared using two-tailed T-tests assuming equal variance. Baseline and treatment values within groups were compared using paired T-tests. A p value of 0.05 was considered to be statistically significant.
RESULTS
Study I: Serum Lipid Changes
Serum Cholesterol. Table 2 presents serum lipid and body weight responses to a daily intake of 200 g of L1 FM or ATCC FM. Serum cholesterol concentrations in the L1 FM group decreased significantly by 2.4% after treatment (p 0.05). There was a small, but nonsignificant, reduction of serum cholesterol concentration in the ATCC FM group after treatment.
Table 2. Serum Lipida (mmol/L) and Body Weighta (kg), Responses in Hypercholesterolemic Subjects to Fermented Milk Containing Human L. acidophilus L1 (L1 (L1 FM) and to Fermented Milk Containing Swine L. acidophilus ATCC 43121 (ATCC FM)
L1 FMb Measurement Baseline Cholesterol LDL-cholesterol HDL-cholesterol Triglycerides Body weight
a b
ATCC FMc Change, % 2.4 (1.1) 2.6 (2.2) 3.9 (1.6) 3.2 (4.6) 0.6 (0.2) Baseline 6.38 (0.23) 4.21 (0.16) 1.50 (0.14) 1.47 (0.14) 72.1 (3.2) Treatment 6.30 (0.21) 4.15 (0.17) 1.40 (0.12)** 1.64 (0.20) 71.8 (3.1) Change, % 0.9 (1.7) 1.1 (2.6) 5.9 (1.7) 16.5 (9.6) 0.9 (0.2)
Treatment 6.12 (0.20)* 4.06 (0.19) 1.12 (0.08)* 2.06 (0.26) 75.7 (4.8)
6.27 (0.19) 4.18 (0.19) 1.17 (0.08) 2.12 (0.24) 75.7 (4.8)
Values represent mean (SEM) of two measurements. n 14. c n 15. P vs. baseline, * 0.05; ** 0.01.
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Table 3. Serum Lipida (mmol/L) and Body Weighta (kg) Responses to Fermented Milk Containing L. acidophilus L1 (L1 FM) and Placebo FM Product
Group Ib Total cholesterol LDL cholesterol HDL cholesterol Triglycerides Body weight Group IIc Total cholesterol LDL cholesterol HDL cholesterol Triglycerides Body weight
a b
Baseline 1 6.30 (0.14) 4.30 (0.12) 1.18 (0.07) 1.90 (0.24) 74.0 (3.8) Baseline 1 6.53 (0.17) 4.41 (0.17) 1.25 (0.09) 2.07 (0.24) 77.0 (3.7)
Placebo FM 6.38 (0.14) 4.38 (0.12) 1.17 (0.07) 1.81 (0.18) 73.7 (3.9) L1 FM 6.31 (0.16)** 4.22 (0.12)* 1.21 (0.09)** 2.01 (0.27) 77.4 (3.6)
Change (%) 1.4 (1.4) 1.8 (1.6) 0.8 (0.17) 3.7 (6.3) 0.4 (0.8) Change (%) 3.2 (1.1)** 4.1 (2.0)* 3.2 (0.8) 3.4 (4.8) 0.5 (0.3)
Baseline 2 6.45 (0.16) 4.53 (0.14) 1.18 (0.06) 1.63 (0.14) 74.7 (3.7) Baseline 2 6.42 (0.16) 4.29 (0.16) 1.27 (0.10) 1.91 (0.22) 77.3 (3.6)
L1 FM 6.60 (0.20) 4.52 (0.17) 1.23 (0.08) 1.75 (0.23) 74.7 (3.7) Placebo FM 6.43 (0.17) 4.29 (0.14) 1.28 (0.10) 1.90 (0.19) 77.5 (3.6)
Change (%) 2.3 (0.6) 0.2 (2.0) 2.5 (2.5) 11.7 (10.4) 0.3 (0.3) Change (%) 0.3 (0.0) 0.2 (1.9) 0.8 (1.6) 0.5 (3.7) 0.3 (0.1)
Values represent the mean (SEM) of three (Baseline 1, Placebo FM, L1 FM) or two measurements (Baseline 2). n 19. c n 21. Significant differences are indicated: treatment values are compared to baseline; change scores are compared to placebo; * p 0.05; ** p 0.01.
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Study I and II
Body Weight. No significant differences in body weight were found upon ANOVA analysis among all the groups in both studies. Dietary Intake. The average dietary intakes of study I and II are given in Tables 4 and 5, respectively. Both information during baseline periods and FM treatments are presented, including the information of group-I and -II subjects during baseline periods and FM treatments in the second study. There were no significant differences in nutrient intake between all experimental groups and no significant changes are seen. Combined Serum Cholesterol. Since design of the first study and the first wave of the second study were almost identical and both used L. acidophilus L1, we have evaluated the serum cholesterol results combining these two studies. Table 6 summarizes these results. Swine L. acidophilus ATCC 43121 showed a consistent reduction, which was not statistically significant. Placebo FM was associated with no distinct trends. The response to human L. acidophilus L1 showed remarkable similarities in time course for the two studies. ANOVA testing indicated that values differed between the three groups (L1-1st study, L1-2nd study, and placebo) at 2 weeks (p 0.03) and for overall treatment (p 0.011). When compared to baseline values, serum cholesterol values were significantly lower at 2 weeks (p 0.0015), 4 weeks (p 0.02), and overall treatment (p 0.001). Compared to placebo, serum cholesterol values were significantly lower at week 2 (p 0.008), and week 3 (p 0.022), and at overall treatment (p 0.0045). Human L. acidophilus L1 showed a net 4.3% reduction of serum cholesterol values for overall treatment, compared with the placebo FM (p 0.005).
DISCUSSION
In study I we compared a fairly newly isolated strain of L. acidophilus (L1) from human origin with a strain of pig origin (ATCC 43121). Effects on serum cholesterol concentrations of neither strains had been previously studied in human subjects. This first study showed that the swine strain had a very small and statistically insignificant lowering effect on serum cholesterol. However, the human strain had a significant lowering effect on total serum and LDL cholesterol. Study II was a placebo-controlled study to examine effects of human L. acidophilus L1. To increase power of the study a cross-over design was used. While a significant reduction of the serum cholesterol level was observed in the first treatment period, this was not seen in the second treatment period. Since both studies used the same L. acidophilus L1, presented in the same fermented vehicle and prepared in the same manner, we think it is appropriate to look at the combined responses. This also addresses the problem recognized at the outset that these studies did not have adequate numbers of subjects to provide optimal statistical power to address these questions. The combined results indicate that this yogurt product has a potential to lower serum cholesterol by 3 to 4% in hypercholesterolemic individuals. During the second treatment period of study II there were no significant changes in serum lipid concentrations for subjects receiving L1 FM. Several interacting factors may have contributed to the different response of these subjects. It is noteworthy
Bacterial Counts
Study I. L. acidophilus L1 content averaged 3.5 107 CFU/g at day 1 and 3.9 106 CFU/g at day 7. L. acidophilus
Table 4. Daily Dietary Intakesa of Group L1 FM and Group ATCC FM During Baseline and Treatment Periods. Values Were Obtained by Means of 3-day Diet Records
L1 FMb Baseline Energy (kcal/day) Cholesterol (mg/day) Proteins (% energy) Carbohydrates (% energy) Fatty acids (% energy) Alcohol (% energy)
a
ATCC FMc Treatment 1578.5 (121.6) 184.2 (22.6) 18.3 (1.4) 49.9 (2.5) 30.6 (2.3) 1.1 (0.4) Baseline 2084.7 (167.4) 199.8 (31.2) 16.5 (0.9) 51.5 (2.6) 28.6 (1.9) 3.5 (1.3) Treatment 1978.9 (161.3) 228.6 (30.1) 17.3 (0.8) 52.7 (2.1) 27.7 (1.9) 2.3 (0.7)
1845.9 (138.7) 207.8 (27.2) 17.7 (1.2) 50.7 (2.5) 30.5 (2.1) 1.0 (0.6)
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Baseline 1 2029.5 (140.4) 246.1 (26.9) 24.0 (2.1) 16.9 (0.8) 51.6 (1.3) 30.2 (1.1) 1.2 (0.5) Baseline 1 1865.0 (138.3) 221.1 (23.7) 19.0 (2.4) 16.4 (1.1) 50.8 (2.7) 30.9 (2.7) 1.9 (0.7)
Placebo FM 1776.8 (125.1) 198.9 (23.5) 19.9 (1.7) 18.2 (1.5) 50.1 (1.7) 30.3 (1.4) 1.0 (0.5) L1 FM 1814.4 (128.0) 228.8 (31.6) 21.2 (2.7) 17.8 (1.2) 48.2 (2.4) 30.8 (1.9) 2.8 (1.1)
Baseline 2 2013.2 (165.2) 267.0 (26.9) 27.1 (2.8) 15.9 (0.6) 48.4 (1.5) 34.1 (1.3) 1.6 (0.7) Baseline 2 1801.1 (125.0) 231.2 (22.2) 21.0 (2.1) 18.0 (1.1) 49.9 (1.4) 29.6 (1.1) 2.5 (0.9)
L1 FM 1751.6 (103.4) 200.9 (21.2) 22.2 (1.9) 15.8 (0.6) 50.3 (1.7) 32.1 (1.3) 1.8 (1.3) Placebo FM 1858.5 (157.3) 214.1 (32.4) 21.7 (2.3) 16.9 (1.1) 49.6 (1.5) 31.9 (1.1) 1.5 (0.7)
that group I did show a significant rise during the whole study, both serum cholesterol total and LDL-cholesterol concentrations increasing about 5%. After careful analyses of dietary compliances, adherence to fermented milk products, preparation techniques used for FM and bacterial counts, we can not explain these differences. Since the initial observations of Mann and Spoerry [1] in 1974, a number of investigators have examined effects of intake of FM or yogurt on serum cholesterol concentrations in humans. Harrison and Peat [23] evaluated the effects of introducing L. acidophilus into formula of newborn infants; infants receiving formula containing L. acidophilus had lower serum cholesterol values than infants fed control formula. These early observations suggested that introduction of L. acidophilus into human intestine might have a hypocholesterolemic effect. Since these early observations, at least nine clinical studies have examined the effects of FM or yogurt intake on serum cholesterol concentrations in humans. Early studies prior to 1984 did not control for fat intake on the different diets and are difficult to interpret. Only recent studies have assessed bacterial counts of tested yogurt products. The studies of Payens [7], Rossouw [8] Hepner [3] and Bazzarre [2] are difficult to interpret because fat contents of the various test diets were not controlled. The types of bacteria provided in yogurt are not specified in the studies of Massey [5] and McNamara [6]. Nevertheless, the last five studies [4 6,24,25] appear to be well-controlled studies. Massey and colleagues [5] prepared their yogurt in batches for use over 4 weeks of study. The decrease in cholesterol-lowering effects observed at 4 weeks may have been related to a lower bacterial count at 4 or more weeks after preparation of the yogurt.
Two recent studies [24,25] appear to have been well controlled and both reported a significant reduction in serum cholesterol concentrations with yogurt consumption. Agerbaek et al [24] found a significant 3.5% reduction in serum cholesterol concentration in the experimental group after 3 weeks daily consumption of a FM containing an Enterococcus faecium and two strains of S. thermophilus. After 6 weeks, a 6% reduction was found. In a study of Schaafsma et al [25] a significant 4.5% reduction of serum cholesterol concentration was seen with daily consumption of a yogurt containing a L. acidophilus during 3 weeks. This yogurt also contained 2.5% fructo-oligosaccharides which additionally may have affected serum cholesterol concentrations [26]. The mechanisms responsible for the hypocholesterolemic effects of FM are still under investigation. Gilliland and colleagues [1316,27,28] suggested that bacterial metabolism of cholesterol and bile acids contribute to the hypocholesterolemic effects of L. acidophilus. The early study of Harrison and Peat [23] suggested that introduction of live cultures of L. acidophilus into intestine of humans may have hypocholesterolemic effects. The recent studies of Gilliland and colleagues [16,27,28,33] have shown that some strains of L. acidophilus when growing under anaerobic conditions and in the presence of bile (as would be encountered in the small intestine) assimilate cholesterol, part of which is incorporated into their cellular membrane. Strains of L. acidophilus studied in their laboratories also deconjugate bile acids which can interrupt the enterohepatic circulation of bile acids. Free bile acids can be precipitated in presence of Ca2 , which would lead to a higher elimination of bile salts in the feces. To maintain the steady state the amount of excreted bile salts must be replaced by new
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% Chg
% Chg
Week 4 baseline
Table 6. Combined Study Results for Serum Cholesterol Changes; Values in mmol/L, with Means (SEM)
Week 3 baseline
Week 3
% Chg
Week 4
n.d. n.d.
n.d. n.d.
n.d. n.d.
Treatment average
Change
0.0045 0.011
ACKNOWLEDGMENTS
We appreciate and thank the following: Dr. C. Hicks for use of the facilities to prepare the yogurt; F. Elbers, Dr. J. Schlatmann and R. Vermin of Campina Melkunie for providing and preparing the yogurt for the study; T. Colson, J. Blake and L. Nichols of the Metabolic Research Group who performed study procedures with subjects; and R. Tijssens for helpful discussions. This study was supported by Campina Melkunie.
% Chg
0.26 (0.10) 0.10 (0.10) 21 6.53 (0.17) 6.27 (0.18) 19 6.30 (0.14) 6.40 (0.13)
Week 2 baseline
Week 2
0.24 (0.07)
REFERENCES
1. Mann GV, Spoerry A: Studies of a surfactant and cholesterolemia in the Maasai. Am J Clin Nutr 27:464469, 1974. 2. Bazarre TL, Wu SL, Yuhas JA: Total and HDL cholesterol concentrations following yogurt and calcium supplementation. Nutr Rept Int 28:12251232, 1983. 3. Hepner G, Fried R, StJeor S, Fusetti L, Morin R: Hypocholesterolemic effect of yogurt and milk. Am J Clin Nutr 32:1924, 1979. 4. Jaspers DA, Massey LK, Luedecke LO: Effect of consuming yogurt prepared with three culture strains on human serum lipoproteins. J Food Sci 49:11781181, 1984.
No.
Baseline average
1st Study L1 FM ATCC FM 2nd Study L1 FM Placebo Combined L1 FM P vs. baseline P vs. placebo ANOVA
n.d. no data.
Group
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