- Email: [email protected]
Hypocholesterolaemic action of Lactobacillus casei F0822 in rats fed a cholesterol-enriched diet
International Dairy Journal 32 (2013) 144e149
Contents lists available at SciVerse ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Hypocholesterolaemic action of Lactobacillus casei F0822 in rats fed a cholesterol-enriched diet C.F. Guo*, J.Y. Li School of Food Science and Engineering, Northwest A & F University, Yangling 712100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 December 2012 Received in revised form 22 March 2013 Accepted 2 April 2013
Elevated serum cholesterol is a major risk factor for coronary artery disease. Probiotics may manage elevated cholesterol. The hypocholesterolaemic effects of Lactobacillus casei F0822 and its functional mechanisms were investigated in rats. The serum total cholesterol, low-density lipoprotein cholesterol and liver total cholesterol levels significantly decreased in rats fed a high-cholesterol diet plus drinking water supplemented with viable cells of strain F0822; however, there was no significant difference in the serum high-density lipoprotein cholesterol levels among the treatment groups. The hypocholesterolaemic mechanisms of strain F0822 were attributed to its ability to suppress the reabsorption of bile acids into the enterohepatic circulation though hydrolysis of conjugated bile acids in the small intestine, binding of deoxycholic acid and hyodeoxycholic acid in the large intestine, and increase in propionate:acetate ratio in the large intestine in rats. Strain F0822 may be a promising probiotic culture with potential hypocholesterolaemic action in human. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction Results of several studies have shown that elevated total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels in serum are associated with an increased risk of developing coronary heart disease (CHD) (Lipid Research Clinics Program, 1984; Rosengren, Hagman, Wedel, & Wilhelmsen, 1997). Efforts to reduce TC and LDL-C levels by diet play an important role in the prevention of CHD (Bhupathiraju & Tucker, 2011). Thus, much attention has thus been drawn to different dietary ways of reducing the serum TC and LDL-C levels. The reduction of serum cholesterol levels could be effected by consumption of appropriate food containing low cholesterol, dietary fibre (Nijjar, Burke, Bloesch, & Rader, 2010), soy protein (Jenkins et al., 2010), plant sterols (Gupta, Savopoulos, Ahuja, & Hatzitolios, 2011), or lactic acid bacteria (LAB) (Ooi, Ahmad, Yuen, & Liong, 2011). LAB, especially lactobacilli, have recently attracted more focus as potential cholesterol lowering agents. It has been shown that fermented milk containing certain strains of lactobacilli could reduce serum cholesterol levels in humans (Anderson & Gilliland, 1999) and animals (Ramchandran & Shah, 2011). However, the cholesterol-lowering mechanisms of lactobacilli are not yet completely understood. The mechanisms that have been proposed involve inhibition of exogenous cholesterol absorption from small intestine by the binding or assimilation of cholesterol (Lye, * Corresponding author. Tel.: þ86 29 87091917. E-mail address: [email protected] (C.F. Guo).
Rahmat-Ali, & Liong, 2010), inhibition of deoxycholic acid (DCA) resorption from large intestine by the binding of DCA, downregulation of NiemannePick C1-Like 1(NPC1L1) gene expression (Huang & Zheng, 2010), as well as suppressing bile acids resorption by deconjugation of bile acids as a function of the bacterial bile salt hydrolase (BSH) activity (Kumar, Grover, & Batish, 2011). Lactobacillus casei is an indigenous and dominant Lactobacillus species that is present in the gastrointestinal tract of most healthy adults (Haarman & Knol, 2006). It has been recognised as a potentially beneficial microorganism for health in the human gastrointestinal tract. In our previous study it was reported that L. casei F0822 exhibited greater tolerance to artificial gastric juice and bile, better adhesion to epithelial cells, greater BSH activity and increased DCA removal ability than others of the 150 LAB strains of human origin tested (Guo et al., 2011, 2012). Although strain F0822 expressed better probiotic properties and stronger cholesterollowering potential in vitro, it is not yet understood if it can exert cholesterol-lowering activity in vivo. The objective of this study was to evaluate hypocholesterolaemic activity of strain F0822 and study its functional mechanisms in rats. 2. Materials and methods 2.1. Source and maintenance of cultures L. casei strain F0822 used in this study was isolated from a human faecal sample by spreading the faecal material onto LAMVAB
0958-6946/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.idairyj.2013.04.001
C.F. Guo, J.Y. Li / International Dairy Journal 32 (2013) 144e149
agar plates, and identified to the species level by partially sequencing the 16S rDNA gene. The culture was maintained by subculture in MRS broth (Oxoid Ltd., Basingstoke, Hampshire, UK) supplemented with 0.05% (w/v) L-cysteine using a 1% inoculum and 18 h of anaerobic incubation at 37 C. The culture was serially transferred three times in the MRSC broth before experimental use. Unless otherwise indicated, all reagents and materials used in this work were obtained from SigmaeAldrich (St. Louis, MO, USA). 2.2. Animal feeding and grouping Twenty four male Wistar rats were purchased from Laboratory Animal Center, Harbin Medical University, China, at the body weight (BW) of 180e200 g. The rats were individually housed in metal cages in a temperature-controlled room (22 2 C) with a 12 h light/dark cycle and humidity 55 5%. After a 5-day adaptation period, the rats were divided into four groups of six each. Group 1 (negative control) received cholesterol-free diet plus drinking water, group 2 (high cholesterol control) received cholesterol-enriched diet plus drinking water, group 3 (low-dose F0822) received cholesterol-enriched diet plus drinking water supplemented with 1 108 cfu mL1 viable cells of strain F0822, and group 4 (high-dose F0822) received cholesterol-enriched diet plus drinking water supplemented with 1 109 cfu mL1 viable cells of strain F0822. During the experimental period lasting 3 weeks, food and water were available ad libitum for all rats. Food and water consumption was monitored daily, and BW was recorded at the beginning and end of the study. The composition of the cholesterol-free and cholesterol-enriched diets was designed based on AIN 93M recommendation (Reeves, 1997), with protein content of 14% (Table 1). Casein, sucrose, soybean oil, cellulose, cornstarch and dextrinised cornstarch were obtained from Aladdin Reagents (Shanghai, China). Choline bitartrate, Lcystine, t-butylhydroquinone, cholesterol and sodium cholate were purchased from SigmaeAldrich (St. Louis, MO, USA). Mineral mixture (AIN-93M mineral mixture) and vitamin mixture (AIN-93M vitamin mixture) were obtained from ICN Biochemicals (Aurora, OH, USA).
145
2.4. Assay for liver lipids, relative organ weight and bacterial translocation After an animal had been killed, the viscera was opened, and the liver, spleen and kidneys were removed, rinsed with sterile physiological saline solution, blotted dry with sterile filter paper, and weighed quickly. Relative organ weight was calculated as the ratio of the absolute organ weight to body weight. Bacterial translocation to the above organs was determined by the method described by Shu et al. (1999), and liver TC contents were analysed by the direct saponification-gas chromatographic method (Fletouris, Botsoglou, Psomas, & Mantis, 1998). 2.5. Assay for faecal steroids Faecal samples were collected for the last 3 d of the experimental period, freeze-dried, weighed, and then stored at 80 C until analysis. Faecal neutral sterols (cholesterol, coprostanol and coprostanone) were analysed by a simplified micro-method based on gas chromatography using 5a-cholestane as internal standard (Czubayko, Beumers, Lammsfuss, Lütjohann, & von Bergmann, 1991). Faecal bile acids were extracted using the method described by Crowell and Macdonald (1980), then individual bile acids were analysed by high performance liquid chromatographye electrospray tandem mass spectrometry (HPLCeESI-MS/MS, model 4000 Q-Trap, Applied Biosystems, Foster City, CA, USA) (Hagio, Matsumoto, Fukushima, Hara, & Ishizuka, 2009), and total bile acids (TBA) were quantified enzymatically with a commercial kit (BioSino Bio-technology and Science Inc., Beijing, China). 2.6. Assay for caecal pH and short-chain fatty acid content After an animal had been killed, caecal contents (about 2 g) were removed rapidly, centrifuged at 20,000 g for 10 min, and supernatants were used to determine pH using a compact pH meter (Model C-1, Horiba, Tokyo, Japan) and short chain fatty acids (SCFAs; acetate, butyrate and propionate) using an Agilent 1100 HPLC system (Agilent Technologies, Wilmington, DE, USA) (Matsumoto et al., 2010).
2.3. Assay for serum lipids 2.7. Statistical analyses At the end of experiment, the rats were deprived of food overnight (16 h), and then anesthetised with an intraperitoneal injection of sodium pentobarbital at 50 mg kg1 BW. Blood samples were collected from the femoral artery, and serum was separated from the blood by centrifugation at 3000 g for 10 min. Serum TC, LDL-C and high-density lipoprotein cholesterol (HDL-C) were measured enzymatically with commercial kits (BioSino Bio-technology and Science Inc., Beijing, China). Atherogenic index was calculated by the formula: (TC HDL-C)/HDL-C (Matsubara, Maruoka, & Katayose, 2002).
The difference in the numbers of animals with bacterial translocation was tested by a contingency table using StatView 5.0 software (SAS Institute Inc., Cary, NC, USA). Other data are expressed as mean standard deviation (SD), and statistical analysis was performed by one-way analysis of variance (ANVOA) followed by Tukey’s multiple comparison tests using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). A difference was considered statistically significant when P < 0.05.
Table 1 Composition of the experimental diets (g kg1).
3. Results
Ingredient
Cholesterol-free diet
Cholesterol-enriched diet
Cornstarch Dextrinised cornstarch Casein Sucrose Soybean oil Cellulose Choline bitartrate L-Cystine t-Butylhydroquinone Mineral Vitamin Cholesterol Sodium cholate
465.692 155 140 100 40 50 2.5 1.8 0.008 35 10 e e
459.442 155 140 100 40 50 2.5 1.8 0.008 35 10 5 1.25
3.1. BW gain, food intake, feeding efficiency, relative organ weight and bacterial translocation The administered strain F0822 did not result in any significant (P > 0.05) change in food intake, body gain, feeding efficiency of rats, or the relative organ weight or bacterial translocation for liver, kidneys and spleen (data not shown). 3.2. Serum lipids Serum lipid levels of rats fed different diets are shown in Table 2. At the end of the experimental period of 3 weeks, high
146
C.F. Guo, J.Y. Li / International Dairy Journal 32 (2013) 144e149
Table 2 Changes in serum cholesterol levels of rats fed different diets.a
80.2 245.4 180.0 149.2
)
6.3c 26.4a 16.5b 20.8b
HDL-C (mg dL 48.0 52.7 48.0 54.5
4.0a 5.7a 5.6a 4.5a
)
a
1400 1
LDL-C (mg dL 15.1 125.4 89.1 58.7
)
2.1d 11.4a 5.3b 14.2c
Means in the same column with different superscript letters are significantly different (P < 0.05). Abbreviations are: TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. a Results are expressed as means standard deviation of means (n ¼ 6).
cholesterol control rats showed significantly (P < 0.05) higher serum cholesterol levels when compared with negative control rats. This indicated that the high cholesterol diet-induced experimental hypercholesterolaemic rat model had been established. There was no significant difference (P > 0.05) in serum HDL-C levels between low or high-dose F0822 fed rats and high cholesterol control rats; however, both low and high-dose F0822 fed rats had significantly (P < 0.05) lower levels of serum TC and LDL-C when compared with high cholesterol control rats. The serum TC and LDL-C levels were 25.7% and 28.9% lower in the low-dose F0822 fed rats than in the high cholesterol control rats. The serum TC and LDL-C levels were 39.2% and 63.2% lower in the high-dose F0822 fed rats than in the high cholesterol control rats. There were no significant differences (P > 0.05) in serum TC and HDL-C levels between high-dose F0822 fed rats and low-dose F0822 fed rats. However, the high-dose F0822 fed rats showed significantly (P < 0.05) levels of serum LDL-C when compared with the low-dose F0822 fed rats. The serum LDL-C levels were 34.1% lower in the high-dose F0822 fed rats than in the low-dose F0822 fed rats. 3.3. Arteriosclerotic index Arteriosclerotic index of rats fed different diets is shown in Fig. 1. There was no significant difference (P > 0.05) in arteriosclerotic index between the low-dose F0822 fed rats and high cholesterol control rats. However, the high-dose F0822 fed rats showed significantly (P < 0.05) lower levels in arteriosclerotic index when compared with the high cholesterol control rats. The
1200 -1
TC (mg dL
1
Liver TC (mg 100 g )
Group Negative control High cholesterol control Low-dose F0822 High-dose F0822
1
b
1000
b
800 600 400
c
200 0
NC
HCC
LD
HD
Fig. 2. Changes in liver total cholesterol (TC) level of rats fed different diets: NC, negative control; HCC, high cholesterol control; LD, low dose F0822; HD, high dose F0822. Results are expressed as means standard deviation of means (n ¼ 6). Different letters indicate significantly different means (P < 0.05).
arteriosclerotic index was 47.8% lower in the high-dose F0822 fed rats than in the high cholesterol control rats. Moreover, there was no significant difference (P > 0.05) in arteriosclerotic index between the low-dose F0822 fed rats and high-dose F0822 fed rats. 3.4. Liver lipids Liver TC levels of rats fed different diets are shown in Fig. 2. High cholesterol control rats showed significantly (P < 0.05) higher liver TC levels than did negative control rats. This resulted from oral intake of cholesterol of high cholesterol control rats. Both low and high-dose F0822 fed rats showed significantly (P < 0.05) lower levels in liver TC than the high cholesterol control rats. The liver TC levels were 25.0% and 41.0% respectively lower in the low-dose F0822 fed rats and high-dose F0822 fed rats than in the high cholesterol control rats. However, there was no significant difference (P > 0.05) in liver TC levels between the high-dose F0822 fed rats and low-dose F0822 fed rats. 3.5. Faecal neutral steroid excretion
4.5
a
Arteriosclerotic index
4.0 3.5 ab
3.0 b
2.5 2.0 1.5 1.0
Daily faecal excretion levels of neutral steroids of rats fed different diets are shown in Table 3. The high cholesterol control rats showed significantly (P < 0.05) more increased levels of daily faecal excretion of cholesterol, coprostanol, cholestanol, and total neutral steroids (TNS) when compared with the negative control rats. This also resulted from administration of cholesterol in the high cholesterol control rats. However, there were no significant differences (P > 0.05) in daily faecal excretion levels of cholesterol, Table 3 Changes in daily faecal excretion levels of neutral steroids of rats fed different diets.a
c
Group
0.5 0.0
NC
HCC
LD
HD
Fig. 1. Changes in arteriosclerotic index of rats fed different diets: NC, negative control; HCC, high cholesterol control; LD, low dose F0822; HD, high dose F0822. The atherogenic index was calculated as (total cholesterol high-density lipoprotein cholesterol)/high-density lipoprotein cholesterol. Results are expressed as means standard deviation of means (n ¼ 6). Different letters indicate significantly different means (P < 0.05).
Negative control High cholesterol control Low-dose F0822 High-dose F0822
Cholesterol a
Coprostanol a
Cholestanol a
TNS
5.2 0.9 42.9 7.9b
4.6 1.0 30.8 6.6b
1.4 0.3 8.6 1.7b
11.2 2.1a 82.3 11.4b
42.3 10.8b 49.3 9.3b
39.1 11.8b 42.1 6.3b
11.6 2.2b 9.5 3.3b
93.0 10.5b 100.9 10.9b
Means in the same column with different superscript letters are significantly different (P < 0.05). TNS (total neutral steroids) ¼ cholesterol þ coprostanol þ cholestanol. a Results, in milligrams, are expressed as means standard deviation of means (n ¼ 6).
C.F. Guo, J.Y. Li / International Dairy Journal 32 (2013) 144e149
147
Table 4 Changes in daily faecal excretion levels of bile acids of rats fed different diets.a Group
Total bile acids
Individual bile acids CA
Negative control High cholesterol control Low-dose F0822 High-dose F0822
7.1 13.6 18.2 26.9
d
0.3 1.1 1.2 1.4
0.5 1.3c 2.4b 3.2a
DCA
b
0.1 0.3a 0.3a 0.4a
1.5 4.7 7.5 9.4
LCA d
0.3 0.6c 0.4b 1.0a
0.7 0.8 1.6 2.0
b-MCA
HDCA
b
0.3 0.2b 0.4a 0.3a
2.2 2.7 5.6 8.2
b
0.4 0.5b 0.7a 1.1a
1.0 1.5 1.3 1.7
u-MCA a
0.3 0.4a 0.5a 0.7a
1.5 1.7 1.2 1.7
0.4a 0.3a 0.4a 0.5a
Means in the same column with different superscript letters are significantly different (P < 0.05). Total bile acids were determined enzymatically; individual bile acids were determined by high performance liquid chromatographyeelectrospray tandem mass spectrometry. Abbreviations are: CA, cholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; HDCA, hyodeoxycholic acid; b-MCA, b-muricholic acid; u-MCA, u-muricholic acid. a Results (in mmol) are expressed as means standard deviation of means (n ¼ 6).
Table 5 Changes in caecal pH and short-chain fatty acid (SCFA) concentrations of rats fed different diets.a Group Negative control High cholesterol control Low-dose F0822 High-dose F0822
Acetate (mmol L1)
pH 7.0 7.1 6.9 6.8
a
0.2 0.2a 0.1a 0.2a
60.6 55.2 61.3 65.3
a
6.2 6.7a 8.0a 7.0a
Propionate (mmol L1) 21.4 20.8 32.5 37.2
b
2.5 3.8b 4.5a 4.1a
Butyrate (mmol L1) 20.8 20.5 27.0 24.0
a
4.8 2.8a 5.3a 4.2a
Propionate:acetate 0.36 0.38 0.53 0.57
b
0.04 0.03b 0.04a 0.07a
Total SCFA (mmol L1) 102.9 96.5 120.8 126.5
10.1bc 12.9c 17.7ab 12.0a
Means in the same column with different superscript letters are significantly different (P < 0.05). Total SCFA is acetate þ propionate þ butyrate. a Results are expressed as means standard deviation of means (n ¼ 6).
coprostanol, cholestanol and TNS between the low or high-dose F0822 fed rats and high cholesterol control rats. 3.6. Faecal bile acid excretion Daily faecal bile acid excretion levels of rats fed different diets are shown in Table 4. The high cholesterol control rats showed significantly (P < 0.05) more increased daily faecal TBA excretion levels when compared with the negative control rats. This resulted from oral administration of bile acid (sodium cholate) in the high cholesterol control rats. Both the low and high-dose F0822 fed rats showed significantly (P < 0.05) more increased levels of daily faecal TBA excretion when compared with the high cholesterol control rats. Daily faecal TBA excretion levels were 33.8% and 97.8% higher, respectively, in the low-dose F0822 fed rats and high-dose F0822 fed rats than in the high cholesterol control rats. Moreover, the high-dose F0822 fed rats showed significantly (P < 0.05) more increased levels of daily faecal TBA excretion than did the lowdose F0822 fed rats. Daily faecal TBA excretion levels were 47.8% higher in the high-dose F0822 fed rats than in the low-dose F0822 fed rats. There was a significantly (P < 0.05) negative correlation between the daily faecal TBA excretion levels and serum or liver TC levels among the 18 rats fed the cholesterol-enriched diets. The Pearson’s correlation coefficients (r) were 0.83 and 0.85 respectively between serum TC levels and daily faecal TBA excretion levels and between liver TC levels and daily faecal TBA excretion levels. The results from the HPLCeESI-MS/MS analysis showed that the major faecal bile acids in rats consisted of cholic acid (CA), DCA, lithocholic acid (LCA), hyodeoxycholic acid (HDCA), b-muricholic acid (b-MCA), and u-muricholic acid (u-MCA). These bile acids accounted for more than 90% of faecal TBA. Both the low and highdose F0822 fed rats showed significantly (P < 0.05) more increased levels of daily faecal excretion of DCA, LCA and HDCA when compared with the high cholesterol control rats. Daily faecal excretion levels of DCA, LCA and HDCA were 59.6%, 100.0% and 107.4% higher respectively in the low-dose F0822 fed rats than in the high cholesterol control rats. Daily faecal excretion levels of DCA, LCA and HDCA were 100.0%, 150.0% and 203.7% higher respectively in the high-dose F0822 fed rats than in the high
cholesterol control rats. However, there were no significant differences (P > 0.05) in daily faecal excretion levels of CA, b-MCA and u-MCA between the low or high-dose F0822 fed rats and high cholesterol control rats. 3.7. Caecal pH and short chain fatty acid concentrations Caecal pH, SCFA concentrations and propionate:acetate ratio of rats fed different diets are shown in Table 5. There were no significant differences (P > 0.05) in caecal pH between rats fed different diets. However, both the low and high-dose F0822 fed rats showed significantly (P < 0.05) higher levels of caecal propionate and total SCFAs, and propionate:acetate ratio when compared with the high cholesterol control rats. Caecal propionate and total SCFA concentrations and propionate:acetate ratio were 25.2%, 56.3% and 28.3% higher, respectively, in the low-dose F0822 fed rats than in the high cholesterol control rats. Caecal propionate and total SCFA concentrations and propionate:acetate ratio were 50.0%, 78.8% and 31.1% higher respectively in the high-dose F0822 fed rats than in the high cholesterol control rats. However, no significant differences (P > 0.05) were found in caecal acetate and butyrate concentrations between the low or high-dose F0822 fed rats and high cholesterol control rats. 4. Discussion The present study showed that strain F0822 significantly reduced serum TC and LDL-C levels of rats fed high cholesterol diets, but had no significant effect on serum HDL-C levels of rats. This is in agreement with earlier observations on the hypocholesterolaemic activity of lactobacilli (Ooi et al., 2011) and bifidobacteria (Al-Sheraji et al., 2012) in rats. The strain F0822 resulted in a reduction in serum TC and LDL-C levels of rats by 39.2% and 63.2% respectively at a dose of 1 109 cfu mL1 drinking water. It was well-matched in the hypocholesterolaemic effects to Lactobacillus gasseri SBT0270 from Snow Brand Milk Products Co. Ltd. that resulted a reduction in serum TC and LDL-C levels of rats by 37.4% and 65.1% respectively at a dose of 2 109 cfu mL1 drinking water (Usman & Hosono, 2001).
148
C.F. Guo, J.Y. Li / International Dairy Journal 32 (2013) 144e149
The primary hypothesis of the mechanism responsible for the cholesterol-lowering effect of lactobacilli and bifidobacteria is an increased excretion of cholesterol and/or bile acids. The digestive balance of cholesterol may be altered by an inhibition of its absorption in the small intestine, since sterols reaching the large intestine are considered as non reabsorbable. Cholesterol can be converted by intestinal bacteria to corresponding coprostane and ketonic derivatives during intestinal transit in both human and rats. Cholesterol and these derivatives are called faecal neutral steroids. Although faecal neutral steroid pattern is highly complex, about 95% of the faecal neutral steroids consist of cholesterol, coprostanol and coprostanone in human and rats (McNamara, Proia, & Miettinen, 1981). No significant differences were found in the daily faecal excretion of the individual neutral steroids and TNS between the low or high-dose F0822 fed rats and high cholesterol control rats. This demonstrated that hypocholesterolaemic mechanisms of strain F0822 were unrelated with increase in daily faecal excretion of TNS. Bile acids are amphipathic molecules synthesised from cholesterol in the liver. Bile acid synthesis is a major pathway for endogenous cholesterol metabolism in human and other animals (Li & Chiang, 2009). Enhancement of faecal bile acid excretion is an important manner for reducing serum cholesterol levels (Charach, Rabinovich, Argov, Weintraub, & Rabinovich, 2012). In the present study, it was found that strain F0822 significantly increased dairy faecal TBA excretion levels. This is agreement with previous observations on effect of BSH-active lactobacilli on faecal TBA excretion of experimental animals (Kumar et al., 2011; Usman & Hosono, 2000; Wang et al., 2012). The presence of the significantly negative correlation between the daily faecal TBA excretion levels and serum TC levels demonstrated that the hypocholesterolaemic activity of strain F0822 in rats resulted from enhancement of daily faecal TBA excretion levels of rats. Bile acids are efficiently conserved under normal physiological conditions by enterohepatic recirculation process. Conjugated and free bile acids are absorbed by active transport in the terminal ileum and by passive diffusion along the entire intestine (Ridlon, Kang, & Hylemon, 2006). In human small intestine, bile acids are mainly presented in conjugated form. Ileal bile salt transport is highly efficient, but approximately 5% (300e600 mg) of bile acid escapes the enterohepatic circulation daily and enters large intestine (Kumar et al., 2012). The strain F0822 showed stronger BSH activity against human bile acids in our previous study (Guo et al., 2011). This suggested that it has potential to hydrolyse the conjugated bile acids in human ileum and produce free bile acid. Thus, administration of strain F0822 is likely to increase amounts of free bile acids that enter the human large intestine. The free bile acids that enter the large intestine are rapidly modified by indigenous bacteria, and the most abundant converted products consist of DCA and LCA in human (Hamilton et al., 2007). DCA can be absorbed from the large intestine in significant amounts (Samuel, Saypoi, Meilman, Mosbach, & Chafizadeh, 1968), whereas LCA is poorly absorbed from the large intestine duo to its lower solubility under physiological conditions of the large intestine (Martinez-Augustin & de Medina, 2008). It was found in our previous study that strain F0822 was able to remove DCA from a laboratory medium simulated conditions in the human large intestine through binding of S-layer protein of strain F0822 (Guo et al., 2012). DCA removal from the large intestine by cells of strain F0822 can further increase faecal TBA excretion. This could reduce the quantities of bile acids recycled to the liver via the portal vein, and thus to maintain the necessary levels of bile acids for the enterohepatic circulation, the excreted bile salts have to be replaced by synthesis of new ones in the body from cholesterol, thus providing the potential to reduce the pool of cholesterol in the body (DeRodas, Gilliland, & Maxwell, 1996).
Free bile acid patterns in the large intestine in rats are different from that in human. In the large intestine of rats, the abundant bile acids consists of CA, DCA, HDCA, b-MCA and u-MCA and LCA (Hagio et al., 2009); however, in the large intestine of human, it consists of only DCA and LCA (Hamilton et al., 2007). The three free bile acids (HDCA, b-MCA and u-MCA) are not present in the human body. Strain F0822 had no significant effect on daily faecal excretion levels of the trihydroxy bile acids (CA and b-MCA and u-MCA), but significantly increased daily faecal excretion levels of the monohydroxy bile acid (LCA) and dihydroxy bile acids (DCA and HDCA). Enhancement of daily faecal excretion levels of LCA resulted from its lower solubility in the large intestine, whereas enhancement of daily faecal excretion levels of DCA and HDCA could at least partially be attributed to their binding by cells of strains F0822 based our previous in vitro observation (Guo et al., 2012). Strain F0822 is impossible to remove HDCA from the large intestine in human. However, DCA concentration in the human large intestine is higher than in the large intestine of rats (Hagio et al., 2009; Hamilton et al., 2007), thus strain F0822 is likely to remove more DCA from the large intestine in human than in rats. Specific SCFAs in the large intestine may reduce the risk of developing CHD. Butyrate has been shown to decrease the transformation of primary to secondary bile acids as a result of colonic acidification (Thornton, 1981). Acetate is the principal SCFA in the large intestine, and after absorption it has been shown to increase cholesterol synthesis, however, propionate has been shown to inhibit cholesterol synthesis (Wolever, Spadafora, & Eshuis, 1991). Thus, substrates that can increase the propionate:acetate ratio may reduce serum lipid levels and possibly CHD risk (Wong & Jenkins, 2007). In this study, oral administered strain F0822 significantly increased the propionate:acetate ratio. This should be one of hypocholesterolaemic mechanisms of strain F0822 in rats. Lactobacilli do not produce SCFAs with more than two carbon atoms (Siigur et al., 1996), thus this elevated propionate concentration is most likely not the direct effect of the oral administrated strain F0822. The major product of fermentation in strain F0822 is lactic acid, which, in turn, is a substrate for many other intestinal bacteria that ferment it to propionate. The drop in large intestinal pH can decrease the solubility of free bile acids, which may suppress their reabsorption from the intestinal lumen and thereby increase their faecal excretion levels (Hofmann & Mysels, 1992). However, although strain F0822 significantly increased caecal SCFA concentrations, it did not significantly affect caecal pH of rats since the large intestinal juice was a buffer system. The development of any therapy, even those using biological products, requires the methodological research of the potential side effects. Organ:BW ratio is an indication of organ swelling, atrophy or hypertrophy (Amresh, Singh, & Rao, 2008). The lack of significant effect on the liver-, spleen- and kidney:BW ratios suggested that the strain F0822 at the two tested dose levels did not cause any swelling, atrophy and hypertrophy of the organs investigated in this study. Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal tract through the mucosal epithelium to other sites, such as the mesenteric lymph nodes, liver, spleen, kidney, and blood (Berg, 1995). There were several cases of translocation of lactobacilli (Ma, Deitch, Specian, Steffen, & Berg, 1990) and indigenous intestinal bacteria (Berg & Garlington, 1979) from the gut lumen into the extra-intestinal tissues. For this reason, bacterial translocation to the liver, spleen or kidneys was studied after the 21st day of feeding with strain F0822. No significant differences were observed in the number of animals with bacterial translocation to the liver, spleen or kidneys between rats fed different diets. This indicated that the strain F0822 neither translocated nor resulted in translocation of other intestinal bacteria from the intestinal lumen into the extra-intestinal tissues.
C.F. Guo, J.Y. Li / International Dairy Journal 32 (2013) 144e149
5. Conclusions The present study showed that L. casei F0822 exerted hypocholesterolaemic effect in rats fed a cholesterol-enriched diet through hydrolysis of conjugated bile acids in the small intestine, binding of free bile acids (DCA and HDCA) in the large intestine, and increase in propionate:acetate ratio in the large intestine. Because strains for most effective use as dietary adjuncts or probiotic for human should likely originate from human intestines, strain F0822 may be a promising candidate for use as an adjunct culture in fermented dairy products with hypocholesterolaemic potential on consumption in human. Further research is needed to determine whether or not ingestion of cells of strain F0822 could effectively decrease serum cholesterol levels in adult humans with primary hypercholesterolaemia after an appropriate risk assessment.
References Al-Sheraji, S. H., Ismail, A., Manap, M. Y., Mustafa, S., Yusof, R. M., & Hassan, F. A. (2012). Hypocholesterolaemic effect of yoghurt containing Bifidobacterium pseudocatenulatum G4 or Bifidobacterium longum BB536. Food Chemistry, 135, 356e361. Amresh, G., Singh, P. N., & Rao, C. V. (2008). Toxicological screening of traditional medicine Laghupatha (Cissampelos pareira) in experimental animals. Journal of Ethnopharmacology, 116, 454e460. Anderson, J. W., & Gilliland, S. E. (1999). Effect of fermented milk (yogurt) containing Lactobacillus acidophilus L1 on serum cholesterol in hypercholesterolemic humans. Journal of the American College of Nutrition, 18, 43e50. Berg, R. D. (1995). Bacterial translocation from the gastrointestinal tract. Trends in Microbiology, 3, 149e154. Berg, R. D., & Garlington, A. W. (1979). Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse model. Infection and Immunity, 23, 403e411. Bhupathiraju, S. N., & Tucker, K. L. (2011). Coronary heart disease prevention: nutrients, foods, and dietary patterns. Clinica Chimica Acta, 412, 1493e1514. Charach, G., Rabinovich, A., Argov, O., Weintraub, M., & Rabinovich, P. (2012). The role of bile acid excretion in atherosclerotic coronary artery disease. International Journal of Vascular Medicine, 2012, 949672. Crowell, M. J., & Macdonald, I. A. (1980). Enzymic determination of 3a-, 7a- and 12a-hydroxyl groups of fecal bile salts. Clinical Chemistry, 26, 1298e1300. Czubayko, F., Beumers, B., Lammsfuss, S., Lütjohann, D., & von Bergmann, K. (1991). A simplified micro-method for quantification of fecal excretion of neutral and acidic sterols for outpatient studies in humans. Journal of Lipid Research, 32, 1861e1867. DeRodas, B. Z., Gilliland, S. E., & Maxwell, C. V. (1996). Hypocholesterolaemic action of Lactobacillus acidophilus ATCC 43121 and calcium in swine with hypercholesterolemia induced by diet. Journal of Dairy Science, 79, 2121e2128. Fletouris, D. J., Botsoglou, N. A., Psomas, I. E., & Mantis, A. I. (1998). Rapid determination of cholesterol in milk and milk products by direct saponification and capillary gas chromatography. Journal of Dairy Science, 81, 2833e2840. Guo, C. F., Zhang, L. W., Han, X., Li, J. Y., Du, M., Yi, H. X., et al. (2011). Short communication: a sensitive method for qualitative screening of bile salt hydrolase-active lactobacilli based on thin-layer chromatography. Journal of Dairy Science, 94, 1732e1737. Guo, C.-F., Zhang, L.-W., Han, X., Yi, H.-X., Li, J.-Y., Tuo, Y.-F., et al. (2012). Screening for cholesterol-lowering probiotic based on deoxycholic acid removal pathway and studying its functional mechanisms in vitro. Anaerobe, 18, 516e522. Gupta, A. K., Savopoulos, C. G., Ahuja, J., & Hatzitolios, A. I. (2011). Role of phytosterols in lipid-lowering: current perspectives. QJM-An International Journal of Medicine, 104, 301e308. Haarman, M., & Knol, J. (2006). Quantitative real-time PCR analysis of fecal Lactobacillus species in infants receiving a prebiotic infant formula. Applied and Environmental Microbiology, 72, 2359e2365. Hagio, M., Matsumoto, M., Fukushima, M., Hara, H., & Ishizuka, S. (2009). Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS. Journal of Lipid Research, 50, 173e180. Hamilton, J. P., Xie, G., Raufman, J.-P., Hogan, S., Griffin, T. L., Packard, C. A., et al. (2007). Human cecal bile acids: concentration and spectrum. American Journal of Physiology-Gastrointestinal and Liver Physiology, 293, G256eG263. Hofmann, A., & Mysels, K. (1992). Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2þ ions. Journal of Lipid Research, 33, 617e626.
149
Huang, Y., & Zheng, Y. (2010). The probiotic Lactobacillus acidophilus reduces cholesterol absorption through the down-regulation of Niemann-Pick C1-like 1 in Caco-2 cells. British Journal of Nutrition, 103, 473e478. Jenkins, D. J. A., Mirrahimi, A., Srichaikul, K., Berryman, C. E., Wang, L., Carleton, A., et al. (2010). Soy protein reduces serum cholesterol by both intrinsic and food displacement mechanisms. Journal of Nutrition, 140, 2302Se2311S. Kumar, R., Grover, S., & Batish, V. K. (2011). Hypocholesterolaemic effect of dietary inclusion of two putative probiotic bile salt hydrolase-producing Lactobacillus plantarum strains in Sprague-Dawley rats. British Journal of Nutrition, 105, 561e573. Kumar, M., Nagpal, R., Kumar, R., Hemalatha, R., Verma, V., Kumar, A., et al. (2012). Cholesterol-lowering probiotics as potential biotherapeutics for metabolic diseases. Experimental Diabetes Research, 2012, 902917. Li, T., & Chiang, J. Y. L. (2009). Regulation of bile acid and cholesterol metabolism by PPARs. PPAR Research, 2009, 501739. Lipid Research Clinics Program. (1984). The lipid research clinics coronary primary prevention trial results. I. Reduction in incidence of coronary heart disease. Journal of the American Medical Association, 251, 351e364. Lye, H.-S., Rahmat-Ali, G. R., & Liong, M.-T. (2010). Mechanisms of cholesterol removal by lactobacilli under conditions that mimic the human gastrointestinal tract. International Dairy Journal, 20, 169e175. Ma, L., Deitch, E., Specian, R., Steffen, E., & Berg, R. (1990). Translocation of Lactobacillus murinus from the gastrointestinal tract. Current Microbiology, 20, 177e184. Martinez-Augustin, O., & de Medina, F. S. (2008). Intestinal bile acid physiology and pathophysiology. World Journal of Gastroenterology, 14, 5630e5640. Matsubara, M., Maruoka, S., & Katayose, S. (2002). Decreased plasma adiponectin concentrations in women with dyslipidemia. Journal of Clinical Endocrinology and Metabolism, 87, 2764e2769. Matsumoto, K., Takada, T., Shimizu, K., Moriyama, K., Kawakami, K., Hirano, K., et al. (2010). Effects of a probiotic fermented milk beverage containing Lactobacillus casei strain Shirota on defecation frequency, intestinal microbiota, and the intestinal environment of healthy individuals with soft stools. Journal of Bioscience and Bioengineering, 110, 547e552. McNamara, D. J., Proia, A., & Miettinen, T. A. (1981). Thin-layer and gas-liquid chromatographic identification of neutral steroids in human and rat feces. Journal of Lipid Research, 22, 474e484. Nijjar, P. S., Burke, F. M., Bloesch, A., & Rader, D. J. (2010). Role of dietary supplements in lowering low-density lipoprotein cholesterol: a review. Journal of Clinical Lipidology, 4, 248e258. Ooi, L. G., Ahmad, R., Yuen, K. H., & Liong, M. T. (2011). Lactobacillus acidophilus CHO220 and inulin reduced plasma total cholesterol and low-density lipoprotein cholesterol via alteration of lipid transporters. Journal of Dairy Science, 94, 2658. Ramchandran, L., & Shah, N. P. (2011). Yogurt can beneficially affect blood contributors of cardiovascular health status in hypertensive rats. Journal of Food Science, 76, H131eH136. Reeves, P. G. (1997). Components of the AIN-93 diets as improvements in the AIN76A diet. Journal of Nutrition, 127, 838Se841S. Ridlon, J. M., Kang, D.-J., & Hylemon, P. B. (2006). Bile salt biotransformations by human intestinal bacteria. Journal of Lipid Research, 47, 241e259. Rosengren, A., Hagman, M., Wedel, H., & Wilhelmsen, L. (1997). Serum cholesterol and long-term prognosis in middle-aged men with myocardial infarction and angina pectoris: a 16-year follow-up of the primary prevention study in Goteborg, Sweden. European Heart Journal, 18, 754e761. Samuel, P., Saypoi, G. M., Meilman, E., Mosbach, E. H., & Chafizadeh, M. (1968). Absorption of bile acids from the large bowel in man. Journal of Clinical Investigation, 47, 2070e2078. Shu, Q., Zhou, J. S., Rutherfurd, K. J., Birtles, M. J., Prasad, J., Gopal, P. K., et al. (1999). Probiotic lactic acid bacteria (Lactobacillus acidophilus HN017, Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019) have no adverse effects on the health of mice. International Dairy Journal, 9, 831e836. Siigur, U., Tamm, E., Torm, S., Lutsar, I., Salminen, S., & Midtvedt, T. (1996). Effect of bacterial infection and administration of a probiotic on faecal short-chain fatty acids. Microbial Ecology in Health and Disease, 9, 271e277. Thornton, J. R. (1981). High colonic pH promotes colorectal cancer. Lancet, 317, 1081e1083. Usman, & Hosono, A. (2000). Effect of administration of Lactobacillus gasseri on serum lipids and fecal steroids in hypercholesterolemic rats. Journal of Dairy Science, 83, 1705e1711. Usman, & Hosono, A. (2001). Hypocholesterolaemic effect of Lactobacillus gasseri SBT0270 in rats fed a cholesterol-enriched diet. Journal of Dairy Research, 68, 617e624. Wang, J., Zhang, H., Chen, X., Chen, Y., Menghebilige, & Bao, Q. (2012). Selection of potential probiotic lactobacilli for cholesterol-lowering properties and their effect on cholesterol metabolism in rats fed a high-lipid diet. Journal of Dairy Science, 95, 1645e1654. Wolever, T. M., Spadafora, P., & Eshuis, H. (1991). Interaction between colonic acetate and propionate in humans. American Journal of Clinical Nutrition, 53, 681e687. Wong, J. M. W., & Jenkins, D. J. A. (2007). Carbohydrate digestibility and metabolic effects. Journal of Nutrition, 137, 2539Se2546S.
Contents lists available at SciVerse ScienceDirect
International Dairy Journal journal homepage: www.elsevier.com/locate/idairyj
Hypocholesterolaemic action of Lactobacillus casei F0822 in rats fed a cholesterol-enriched diet C.F. Guo*, J.Y. Li School of Food Science and Engineering, Northwest A & F University, Yangling 712100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 December 2012 Received in revised form 22 March 2013 Accepted 2 April 2013
Elevated serum cholesterol is a major risk factor for coronary artery disease. Probiotics may manage elevated cholesterol. The hypocholesterolaemic effects of Lactobacillus casei F0822 and its functional mechanisms were investigated in rats. The serum total cholesterol, low-density lipoprotein cholesterol and liver total cholesterol levels significantly decreased in rats fed a high-cholesterol diet plus drinking water supplemented with viable cells of strain F0822; however, there was no significant difference in the serum high-density lipoprotein cholesterol levels among the treatment groups. The hypocholesterolaemic mechanisms of strain F0822 were attributed to its ability to suppress the reabsorption of bile acids into the enterohepatic circulation though hydrolysis of conjugated bile acids in the small intestine, binding of deoxycholic acid and hyodeoxycholic acid in the large intestine, and increase in propionate:acetate ratio in the large intestine in rats. Strain F0822 may be a promising probiotic culture with potential hypocholesterolaemic action in human. Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved.
1. Introduction Results of several studies have shown that elevated total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) levels in serum are associated with an increased risk of developing coronary heart disease (CHD) (Lipid Research Clinics Program, 1984; Rosengren, Hagman, Wedel, & Wilhelmsen, 1997). Efforts to reduce TC and LDL-C levels by diet play an important role in the prevention of CHD (Bhupathiraju & Tucker, 2011). Thus, much attention has thus been drawn to different dietary ways of reducing the serum TC and LDL-C levels. The reduction of serum cholesterol levels could be effected by consumption of appropriate food containing low cholesterol, dietary fibre (Nijjar, Burke, Bloesch, & Rader, 2010), soy protein (Jenkins et al., 2010), plant sterols (Gupta, Savopoulos, Ahuja, & Hatzitolios, 2011), or lactic acid bacteria (LAB) (Ooi, Ahmad, Yuen, & Liong, 2011). LAB, especially lactobacilli, have recently attracted more focus as potential cholesterol lowering agents. It has been shown that fermented milk containing certain strains of lactobacilli could reduce serum cholesterol levels in humans (Anderson & Gilliland, 1999) and animals (Ramchandran & Shah, 2011). However, the cholesterol-lowering mechanisms of lactobacilli are not yet completely understood. The mechanisms that have been proposed involve inhibition of exogenous cholesterol absorption from small intestine by the binding or assimilation of cholesterol (Lye, * Corresponding author. Tel.: þ86 29 87091917. E-mail address: [email protected] (C.F. Guo).
Rahmat-Ali, & Liong, 2010), inhibition of deoxycholic acid (DCA) resorption from large intestine by the binding of DCA, downregulation of NiemannePick C1-Like 1(NPC1L1) gene expression (Huang & Zheng, 2010), as well as suppressing bile acids resorption by deconjugation of bile acids as a function of the bacterial bile salt hydrolase (BSH) activity (Kumar, Grover, & Batish, 2011). Lactobacillus casei is an indigenous and dominant Lactobacillus species that is present in the gastrointestinal tract of most healthy adults (Haarman & Knol, 2006). It has been recognised as a potentially beneficial microorganism for health in the human gastrointestinal tract. In our previous study it was reported that L. casei F0822 exhibited greater tolerance to artificial gastric juice and bile, better adhesion to epithelial cells, greater BSH activity and increased DCA removal ability than others of the 150 LAB strains of human origin tested (Guo et al., 2011, 2012). Although strain F0822 expressed better probiotic properties and stronger cholesterollowering potential in vitro, it is not yet understood if it can exert cholesterol-lowering activity in vivo. The objective of this study was to evaluate hypocholesterolaemic activity of strain F0822 and study its functional mechanisms in rats. 2. Materials and methods 2.1. Source and maintenance of cultures L. casei strain F0822 used in this study was isolated from a human faecal sample by spreading the faecal material onto LAMVAB
0958-6946/$ e see front matter Crown Copyright Ó 2013 Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.idairyj.2013.04.001
C.F. Guo, J.Y. Li / International Dairy Journal 32 (2013) 144e149
agar plates, and identified to the species level by partially sequencing the 16S rDNA gene. The culture was maintained by subculture in MRS broth (Oxoid Ltd., Basingstoke, Hampshire, UK) supplemented with 0.05% (w/v) L-cysteine using a 1% inoculum and 18 h of anaerobic incubation at 37 C. The culture was serially transferred three times in the MRSC broth before experimental use. Unless otherwise indicated, all reagents and materials used in this work were obtained from SigmaeAldrich (St. Louis, MO, USA). 2.2. Animal feeding and grouping Twenty four male Wistar rats were purchased from Laboratory Animal Center, Harbin Medical University, China, at the body weight (BW) of 180e200 g. The rats were individually housed in metal cages in a temperature-controlled room (22 2 C) with a 12 h light/dark cycle and humidity 55 5%. After a 5-day adaptation period, the rats were divided into four groups of six each. Group 1 (negative control) received cholesterol-free diet plus drinking water, group 2 (high cholesterol control) received cholesterol-enriched diet plus drinking water, group 3 (low-dose F0822) received cholesterol-enriched diet plus drinking water supplemented with 1 108 cfu mL1 viable cells of strain F0822, and group 4 (high-dose F0822) received cholesterol-enriched diet plus drinking water supplemented with 1 109 cfu mL1 viable cells of strain F0822. During the experimental period lasting 3 weeks, food and water were available ad libitum for all rats. Food and water consumption was monitored daily, and BW was recorded at the beginning and end of the study. The composition of the cholesterol-free and cholesterol-enriched diets was designed based on AIN 93M recommendation (Reeves, 1997), with protein content of 14% (Table 1). Casein, sucrose, soybean oil, cellulose, cornstarch and dextrinised cornstarch were obtained from Aladdin Reagents (Shanghai, China). Choline bitartrate, Lcystine, t-butylhydroquinone, cholesterol and sodium cholate were purchased from SigmaeAldrich (St. Louis, MO, USA). Mineral mixture (AIN-93M mineral mixture) and vitamin mixture (AIN-93M vitamin mixture) were obtained from ICN Biochemicals (Aurora, OH, USA).
145
2.4. Assay for liver lipids, relative organ weight and bacterial translocation After an animal had been killed, the viscera was opened, and the liver, spleen and kidneys were removed, rinsed with sterile physiological saline solution, blotted dry with sterile filter paper, and weighed quickly. Relative organ weight was calculated as the ratio of the absolute organ weight to body weight. Bacterial translocation to the above organs was determined by the method described by Shu et al. (1999), and liver TC contents were analysed by the direct saponification-gas chromatographic method (Fletouris, Botsoglou, Psomas, & Mantis, 1998). 2.5. Assay for faecal steroids Faecal samples were collected for the last 3 d of the experimental period, freeze-dried, weighed, and then stored at 80 C until analysis. Faecal neutral sterols (cholesterol, coprostanol and coprostanone) were analysed by a simplified micro-method based on gas chromatography using 5a-cholestane as internal standard (Czubayko, Beumers, Lammsfuss, Lütjohann, & von Bergmann, 1991). Faecal bile acids were extracted using the method described by Crowell and Macdonald (1980), then individual bile acids were analysed by high performance liquid chromatographye electrospray tandem mass spectrometry (HPLCeESI-MS/MS, model 4000 Q-Trap, Applied Biosystems, Foster City, CA, USA) (Hagio, Matsumoto, Fukushima, Hara, & Ishizuka, 2009), and total bile acids (TBA) were quantified enzymatically with a commercial kit (BioSino Bio-technology and Science Inc., Beijing, China). 2.6. Assay for caecal pH and short-chain fatty acid content After an animal had been killed, caecal contents (about 2 g) were removed rapidly, centrifuged at 20,000 g for 10 min, and supernatants were used to determine pH using a compact pH meter (Model C-1, Horiba, Tokyo, Japan) and short chain fatty acids (SCFAs; acetate, butyrate and propionate) using an Agilent 1100 HPLC system (Agilent Technologies, Wilmington, DE, USA) (Matsumoto et al., 2010).
2.3. Assay for serum lipids 2.7. Statistical analyses At the end of experiment, the rats were deprived of food overnight (16 h), and then anesthetised with an intraperitoneal injection of sodium pentobarbital at 50 mg kg1 BW. Blood samples were collected from the femoral artery, and serum was separated from the blood by centrifugation at 3000 g for 10 min. Serum TC, LDL-C and high-density lipoprotein cholesterol (HDL-C) were measured enzymatically with commercial kits (BioSino Bio-technology and Science Inc., Beijing, China). Atherogenic index was calculated by the formula: (TC HDL-C)/HDL-C (Matsubara, Maruoka, & Katayose, 2002).
The difference in the numbers of animals with bacterial translocation was tested by a contingency table using StatView 5.0 software (SAS Institute Inc., Cary, NC, USA). Other data are expressed as mean standard deviation (SD), and statistical analysis was performed by one-way analysis of variance (ANVOA) followed by Tukey’s multiple comparison tests using SPSS 15.0 software (SPSS Inc., Chicago, IL, USA). A difference was considered statistically significant when P < 0.05.
Table 1 Composition of the experimental diets (g kg1).
3. Results
Ingredient
Cholesterol-free diet
Cholesterol-enriched diet
Cornstarch Dextrinised cornstarch Casein Sucrose Soybean oil Cellulose Choline bitartrate L-Cystine t-Butylhydroquinone Mineral Vitamin Cholesterol Sodium cholate
465.692 155 140 100 40 50 2.5 1.8 0.008 35 10 e e
459.442 155 140 100 40 50 2.5 1.8 0.008 35 10 5 1.25
3.1. BW gain, food intake, feeding efficiency, relative organ weight and bacterial translocation The administered strain F0822 did not result in any significant (P > 0.05) change in food intake, body gain, feeding efficiency of rats, or the relative organ weight or bacterial translocation for liver, kidneys and spleen (data not shown). 3.2. Serum lipids Serum lipid levels of rats fed different diets are shown in Table 2. At the end of the experimental period of 3 weeks, high
146
C.F. Guo, J.Y. Li / International Dairy Journal 32 (2013) 144e149
Table 2 Changes in serum cholesterol levels of rats fed different diets.a
80.2 245.4 180.0 149.2
)
6.3c 26.4a 16.5b 20.8b
HDL-C (mg dL 48.0 52.7 48.0 54.5
4.0a 5.7a 5.6a 4.5a
)
a
1400 1
LDL-C (mg dL 15.1 125.4 89.1 58.7
)
2.1d 11.4a 5.3b 14.2c
Means in the same column with different superscript letters are significantly different (P < 0.05). Abbreviations are: TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol. a Results are expressed as means standard deviation of means (n ¼ 6).
cholesterol control rats showed significantly (P < 0.05) higher serum cholesterol levels when compared with negative control rats. This indicated that the high cholesterol diet-induced experimental hypercholesterolaemic rat model had been established. There was no significant difference (P > 0.05) in serum HDL-C levels between low or high-dose F0822 fed rats and high cholesterol control rats; however, both low and high-dose F0822 fed rats had significantly (P < 0.05) lower levels of serum TC and LDL-C when compared with high cholesterol control rats. The serum TC and LDL-C levels were 25.7% and 28.9% lower in the low-dose F0822 fed rats than in the high cholesterol control rats. The serum TC and LDL-C levels were 39.2% and 63.2% lower in the high-dose F0822 fed rats than in the high cholesterol control rats. There were no significant differences (P > 0.05) in serum TC and HDL-C levels between high-dose F0822 fed rats and low-dose F0822 fed rats. However, the high-dose F0822 fed rats showed significantly (P < 0.05) levels of serum LDL-C when compared with the low-dose F0822 fed rats. The serum LDL-C levels were 34.1% lower in the high-dose F0822 fed rats than in the low-dose F0822 fed rats. 3.3. Arteriosclerotic index Arteriosclerotic index of rats fed different diets is shown in Fig. 1. There was no significant difference (P > 0.05) in arteriosclerotic index between the low-dose F0822 fed rats and high cholesterol control rats. However, the high-dose F0822 fed rats showed significantly (P < 0.05) lower levels in arteriosclerotic index when compared with the high cholesterol control rats. The
1200 -1
TC (mg dL
1
Liver TC (mg 100 g )
Group Negative control High cholesterol control Low-dose F0822 High-dose F0822
1
b
1000
b
800 600 400
c
200 0
NC
HCC
LD
HD
Fig. 2. Changes in liver total cholesterol (TC) level of rats fed different diets: NC, negative control; HCC, high cholesterol control; LD, low dose F0822; HD, high dose F0822. Results are expressed as means standard deviation of means (n ¼ 6). Different letters indicate significantly different means (P < 0.05).
arteriosclerotic index was 47.8% lower in the high-dose F0822 fed rats than in the high cholesterol control rats. Moreover, there was no significant difference (P > 0.05) in arteriosclerotic index between the low-dose F0822 fed rats and high-dose F0822 fed rats. 3.4. Liver lipids Liver TC levels of rats fed different diets are shown in Fig. 2. High cholesterol control rats showed significantly (P < 0.05) higher liver TC levels than did negative control rats. This resulted from oral intake of cholesterol of high cholesterol control rats. Both low and high-dose F0822 fed rats showed significantly (P < 0.05) lower levels in liver TC than the high cholesterol control rats. The liver TC levels were 25.0% and 41.0% respectively lower in the low-dose F0822 fed rats and high-dose F0822 fed rats than in the high cholesterol control rats. However, there was no significant difference (P > 0.05) in liver TC levels between the high-dose F0822 fed rats and low-dose F0822 fed rats. 3.5. Faecal neutral steroid excretion
4.5
a
Arteriosclerotic index
4.0 3.5 ab
3.0 b
2.5 2.0 1.5 1.0
Daily faecal excretion levels of neutral steroids of rats fed different diets are shown in Table 3. The high cholesterol control rats showed significantly (P < 0.05) more increased levels of daily faecal excretion of cholesterol, coprostanol, cholestanol, and total neutral steroids (TNS) when compared with the negative control rats. This also resulted from administration of cholesterol in the high cholesterol control rats. However, there were no significant differences (P > 0.05) in daily faecal excretion levels of cholesterol, Table 3 Changes in daily faecal excretion levels of neutral steroids of rats fed different diets.a
c
Group
0.5 0.0
NC
HCC
LD
HD
Fig. 1. Changes in arteriosclerotic index of rats fed different diets: NC, negative control; HCC, high cholesterol control; LD, low dose F0822; HD, high dose F0822. The atherogenic index was calculated as (total cholesterol high-density lipoprotein cholesterol)/high-density lipoprotein cholesterol. Results are expressed as means standard deviation of means (n ¼ 6). Different letters indicate significantly different means (P < 0.05).
Negative control High cholesterol control Low-dose F0822 High-dose F0822
Cholesterol a
Coprostanol a
Cholestanol a
TNS
5.2 0.9 42.9 7.9b
4.6 1.0 30.8 6.6b
1.4 0.3 8.6 1.7b
11.2 2.1a 82.3 11.4b
42.3 10.8b 49.3 9.3b
39.1 11.8b 42.1 6.3b
11.6 2.2b 9.5 3.3b
93.0 10.5b 100.9 10.9b
Means in the same column with different superscript letters are significantly different (P < 0.05). TNS (total neutral steroids) ¼ cholesterol þ coprostanol þ cholestanol. a Results, in milligrams, are expressed as means standard deviation of means (n ¼ 6).
C.F. Guo, J.Y. Li / International Dairy Journal 32 (2013) 144e149
147
Table 4 Changes in daily faecal excretion levels of bile acids of rats fed different diets.a Group
Total bile acids
Individual bile acids CA
Negative control High cholesterol control Low-dose F0822 High-dose F0822
7.1 13.6 18.2 26.9
d
0.3 1.1 1.2 1.4
0.5 1.3c 2.4b 3.2a
DCA
b
0.1 0.3a 0.3a 0.4a
1.5 4.7 7.5 9.4
LCA d
0.3 0.6c 0.4b 1.0a
0.7 0.8 1.6 2.0
b-MCA
HDCA
b
0.3 0.2b 0.4a 0.3a
2.2 2.7 5.6 8.2
b
0.4 0.5b 0.7a 1.1a
1.0 1.5 1.3 1.7
u-MCA a
0.3 0.4a 0.5a 0.7a
1.5 1.7 1.2 1.7
0.4a 0.3a 0.4a 0.5a
Means in the same column with different superscript letters are significantly different (P < 0.05). Total bile acids were determined enzymatically; individual bile acids were determined by high performance liquid chromatographyeelectrospray tandem mass spectrometry. Abbreviations are: CA, cholic acid; DCA, deoxycholic acid; LCA, lithocholic acid; HDCA, hyodeoxycholic acid; b-MCA, b-muricholic acid; u-MCA, u-muricholic acid. a Results (in mmol) are expressed as means standard deviation of means (n ¼ 6).
Table 5 Changes in caecal pH and short-chain fatty acid (SCFA) concentrations of rats fed different diets.a Group Negative control High cholesterol control Low-dose F0822 High-dose F0822
Acetate (mmol L1)
pH 7.0 7.1 6.9 6.8
a
0.2 0.2a 0.1a 0.2a
60.6 55.2 61.3 65.3
a
6.2 6.7a 8.0a 7.0a
Propionate (mmol L1) 21.4 20.8 32.5 37.2
b
2.5 3.8b 4.5a 4.1a
Butyrate (mmol L1) 20.8 20.5 27.0 24.0
a
4.8 2.8a 5.3a 4.2a
Propionate:acetate 0.36 0.38 0.53 0.57
b
0.04 0.03b 0.04a 0.07a
Total SCFA (mmol L1) 102.9 96.5 120.8 126.5
10.1bc 12.9c 17.7ab 12.0a
Means in the same column with different superscript letters are significantly different (P < 0.05). Total SCFA is acetate þ propionate þ butyrate. a Results are expressed as means standard deviation of means (n ¼ 6).
coprostanol, cholestanol and TNS between the low or high-dose F0822 fed rats and high cholesterol control rats. 3.6. Faecal bile acid excretion Daily faecal bile acid excretion levels of rats fed different diets are shown in Table 4. The high cholesterol control rats showed significantly (P < 0.05) more increased daily faecal TBA excretion levels when compared with the negative control rats. This resulted from oral administration of bile acid (sodium cholate) in the high cholesterol control rats. Both the low and high-dose F0822 fed rats showed significantly (P < 0.05) more increased levels of daily faecal TBA excretion when compared with the high cholesterol control rats. Daily faecal TBA excretion levels were 33.8% and 97.8% higher, respectively, in the low-dose F0822 fed rats and high-dose F0822 fed rats than in the high cholesterol control rats. Moreover, the high-dose F0822 fed rats showed significantly (P < 0.05) more increased levels of daily faecal TBA excretion than did the lowdose F0822 fed rats. Daily faecal TBA excretion levels were 47.8% higher in the high-dose F0822 fed rats than in the low-dose F0822 fed rats. There was a significantly (P < 0.05) negative correlation between the daily faecal TBA excretion levels and serum or liver TC levels among the 18 rats fed the cholesterol-enriched diets. The Pearson’s correlation coefficients (r) were 0.83 and 0.85 respectively between serum TC levels and daily faecal TBA excretion levels and between liver TC levels and daily faecal TBA excretion levels. The results from the HPLCeESI-MS/MS analysis showed that the major faecal bile acids in rats consisted of cholic acid (CA), DCA, lithocholic acid (LCA), hyodeoxycholic acid (HDCA), b-muricholic acid (b-MCA), and u-muricholic acid (u-MCA). These bile acids accounted for more than 90% of faecal TBA. Both the low and highdose F0822 fed rats showed significantly (P < 0.05) more increased levels of daily faecal excretion of DCA, LCA and HDCA when compared with the high cholesterol control rats. Daily faecal excretion levels of DCA, LCA and HDCA were 59.6%, 100.0% and 107.4% higher respectively in the low-dose F0822 fed rats than in the high cholesterol control rats. Daily faecal excretion levels of DCA, LCA and HDCA were 100.0%, 150.0% and 203.7% higher respectively in the high-dose F0822 fed rats than in the high
cholesterol control rats. However, there were no significant differences (P > 0.05) in daily faecal excretion levels of CA, b-MCA and u-MCA between the low or high-dose F0822 fed rats and high cholesterol control rats. 3.7. Caecal pH and short chain fatty acid concentrations Caecal pH, SCFA concentrations and propionate:acetate ratio of rats fed different diets are shown in Table 5. There were no significant differences (P > 0.05) in caecal pH between rats fed different diets. However, both the low and high-dose F0822 fed rats showed significantly (P < 0.05) higher levels of caecal propionate and total SCFAs, and propionate:acetate ratio when compared with the high cholesterol control rats. Caecal propionate and total SCFA concentrations and propionate:acetate ratio were 25.2%, 56.3% and 28.3% higher, respectively, in the low-dose F0822 fed rats than in the high cholesterol control rats. Caecal propionate and total SCFA concentrations and propionate:acetate ratio were 50.0%, 78.8% and 31.1% higher respectively in the high-dose F0822 fed rats than in the high cholesterol control rats. However, no significant differences (P > 0.05) were found in caecal acetate and butyrate concentrations between the low or high-dose F0822 fed rats and high cholesterol control rats. 4. Discussion The present study showed that strain F0822 significantly reduced serum TC and LDL-C levels of rats fed high cholesterol diets, but had no significant effect on serum HDL-C levels of rats. This is in agreement with earlier observations on the hypocholesterolaemic activity of lactobacilli (Ooi et al., 2011) and bifidobacteria (Al-Sheraji et al., 2012) in rats. The strain F0822 resulted in a reduction in serum TC and LDL-C levels of rats by 39.2% and 63.2% respectively at a dose of 1 109 cfu mL1 drinking water. It was well-matched in the hypocholesterolaemic effects to Lactobacillus gasseri SBT0270 from Snow Brand Milk Products Co. Ltd. that resulted a reduction in serum TC and LDL-C levels of rats by 37.4% and 65.1% respectively at a dose of 2 109 cfu mL1 drinking water (Usman & Hosono, 2001).
148
C.F. Guo, J.Y. Li / International Dairy Journal 32 (2013) 144e149
The primary hypothesis of the mechanism responsible for the cholesterol-lowering effect of lactobacilli and bifidobacteria is an increased excretion of cholesterol and/or bile acids. The digestive balance of cholesterol may be altered by an inhibition of its absorption in the small intestine, since sterols reaching the large intestine are considered as non reabsorbable. Cholesterol can be converted by intestinal bacteria to corresponding coprostane and ketonic derivatives during intestinal transit in both human and rats. Cholesterol and these derivatives are called faecal neutral steroids. Although faecal neutral steroid pattern is highly complex, about 95% of the faecal neutral steroids consist of cholesterol, coprostanol and coprostanone in human and rats (McNamara, Proia, & Miettinen, 1981). No significant differences were found in the daily faecal excretion of the individual neutral steroids and TNS between the low or high-dose F0822 fed rats and high cholesterol control rats. This demonstrated that hypocholesterolaemic mechanisms of strain F0822 were unrelated with increase in daily faecal excretion of TNS. Bile acids are amphipathic molecules synthesised from cholesterol in the liver. Bile acid synthesis is a major pathway for endogenous cholesterol metabolism in human and other animals (Li & Chiang, 2009). Enhancement of faecal bile acid excretion is an important manner for reducing serum cholesterol levels (Charach, Rabinovich, Argov, Weintraub, & Rabinovich, 2012). In the present study, it was found that strain F0822 significantly increased dairy faecal TBA excretion levels. This is agreement with previous observations on effect of BSH-active lactobacilli on faecal TBA excretion of experimental animals (Kumar et al., 2011; Usman & Hosono, 2000; Wang et al., 2012). The presence of the significantly negative correlation between the daily faecal TBA excretion levels and serum TC levels demonstrated that the hypocholesterolaemic activity of strain F0822 in rats resulted from enhancement of daily faecal TBA excretion levels of rats. Bile acids are efficiently conserved under normal physiological conditions by enterohepatic recirculation process. Conjugated and free bile acids are absorbed by active transport in the terminal ileum and by passive diffusion along the entire intestine (Ridlon, Kang, & Hylemon, 2006). In human small intestine, bile acids are mainly presented in conjugated form. Ileal bile salt transport is highly efficient, but approximately 5% (300e600 mg) of bile acid escapes the enterohepatic circulation daily and enters large intestine (Kumar et al., 2012). The strain F0822 showed stronger BSH activity against human bile acids in our previous study (Guo et al., 2011). This suggested that it has potential to hydrolyse the conjugated bile acids in human ileum and produce free bile acid. Thus, administration of strain F0822 is likely to increase amounts of free bile acids that enter the human large intestine. The free bile acids that enter the large intestine are rapidly modified by indigenous bacteria, and the most abundant converted products consist of DCA and LCA in human (Hamilton et al., 2007). DCA can be absorbed from the large intestine in significant amounts (Samuel, Saypoi, Meilman, Mosbach, & Chafizadeh, 1968), whereas LCA is poorly absorbed from the large intestine duo to its lower solubility under physiological conditions of the large intestine (Martinez-Augustin & de Medina, 2008). It was found in our previous study that strain F0822 was able to remove DCA from a laboratory medium simulated conditions in the human large intestine through binding of S-layer protein of strain F0822 (Guo et al., 2012). DCA removal from the large intestine by cells of strain F0822 can further increase faecal TBA excretion. This could reduce the quantities of bile acids recycled to the liver via the portal vein, and thus to maintain the necessary levels of bile acids for the enterohepatic circulation, the excreted bile salts have to be replaced by synthesis of new ones in the body from cholesterol, thus providing the potential to reduce the pool of cholesterol in the body (DeRodas, Gilliland, & Maxwell, 1996).
Free bile acid patterns in the large intestine in rats are different from that in human. In the large intestine of rats, the abundant bile acids consists of CA, DCA, HDCA, b-MCA and u-MCA and LCA (Hagio et al., 2009); however, in the large intestine of human, it consists of only DCA and LCA (Hamilton et al., 2007). The three free bile acids (HDCA, b-MCA and u-MCA) are not present in the human body. Strain F0822 had no significant effect on daily faecal excretion levels of the trihydroxy bile acids (CA and b-MCA and u-MCA), but significantly increased daily faecal excretion levels of the monohydroxy bile acid (LCA) and dihydroxy bile acids (DCA and HDCA). Enhancement of daily faecal excretion levels of LCA resulted from its lower solubility in the large intestine, whereas enhancement of daily faecal excretion levels of DCA and HDCA could at least partially be attributed to their binding by cells of strains F0822 based our previous in vitro observation (Guo et al., 2012). Strain F0822 is impossible to remove HDCA from the large intestine in human. However, DCA concentration in the human large intestine is higher than in the large intestine of rats (Hagio et al., 2009; Hamilton et al., 2007), thus strain F0822 is likely to remove more DCA from the large intestine in human than in rats. Specific SCFAs in the large intestine may reduce the risk of developing CHD. Butyrate has been shown to decrease the transformation of primary to secondary bile acids as a result of colonic acidification (Thornton, 1981). Acetate is the principal SCFA in the large intestine, and after absorption it has been shown to increase cholesterol synthesis, however, propionate has been shown to inhibit cholesterol synthesis (Wolever, Spadafora, & Eshuis, 1991). Thus, substrates that can increase the propionate:acetate ratio may reduce serum lipid levels and possibly CHD risk (Wong & Jenkins, 2007). In this study, oral administered strain F0822 significantly increased the propionate:acetate ratio. This should be one of hypocholesterolaemic mechanisms of strain F0822 in rats. Lactobacilli do not produce SCFAs with more than two carbon atoms (Siigur et al., 1996), thus this elevated propionate concentration is most likely not the direct effect of the oral administrated strain F0822. The major product of fermentation in strain F0822 is lactic acid, which, in turn, is a substrate for many other intestinal bacteria that ferment it to propionate. The drop in large intestinal pH can decrease the solubility of free bile acids, which may suppress their reabsorption from the intestinal lumen and thereby increase their faecal excretion levels (Hofmann & Mysels, 1992). However, although strain F0822 significantly increased caecal SCFA concentrations, it did not significantly affect caecal pH of rats since the large intestinal juice was a buffer system. The development of any therapy, even those using biological products, requires the methodological research of the potential side effects. Organ:BW ratio is an indication of organ swelling, atrophy or hypertrophy (Amresh, Singh, & Rao, 2008). The lack of significant effect on the liver-, spleen- and kidney:BW ratios suggested that the strain F0822 at the two tested dose levels did not cause any swelling, atrophy and hypertrophy of the organs investigated in this study. Bacterial translocation is defined as the passage of viable bacteria from the gastrointestinal tract through the mucosal epithelium to other sites, such as the mesenteric lymph nodes, liver, spleen, kidney, and blood (Berg, 1995). There were several cases of translocation of lactobacilli (Ma, Deitch, Specian, Steffen, & Berg, 1990) and indigenous intestinal bacteria (Berg & Garlington, 1979) from the gut lumen into the extra-intestinal tissues. For this reason, bacterial translocation to the liver, spleen or kidneys was studied after the 21st day of feeding with strain F0822. No significant differences were observed in the number of animals with bacterial translocation to the liver, spleen or kidneys between rats fed different diets. This indicated that the strain F0822 neither translocated nor resulted in translocation of other intestinal bacteria from the intestinal lumen into the extra-intestinal tissues.
C.F. Guo, J.Y. Li / International Dairy Journal 32 (2013) 144e149
5. Conclusions The present study showed that L. casei F0822 exerted hypocholesterolaemic effect in rats fed a cholesterol-enriched diet through hydrolysis of conjugated bile acids in the small intestine, binding of free bile acids (DCA and HDCA) in the large intestine, and increase in propionate:acetate ratio in the large intestine. Because strains for most effective use as dietary adjuncts or probiotic for human should likely originate from human intestines, strain F0822 may be a promising candidate for use as an adjunct culture in fermented dairy products with hypocholesterolaemic potential on consumption in human. Further research is needed to determine whether or not ingestion of cells of strain F0822 could effectively decrease serum cholesterol levels in adult humans with primary hypercholesterolaemia after an appropriate risk assessment.
References Al-Sheraji, S. H., Ismail, A., Manap, M. Y., Mustafa, S., Yusof, R. M., & Hassan, F. A. (2012). Hypocholesterolaemic effect of yoghurt containing Bifidobacterium pseudocatenulatum G4 or Bifidobacterium longum BB536. Food Chemistry, 135, 356e361. Amresh, G., Singh, P. N., & Rao, C. V. (2008). Toxicological screening of traditional medicine Laghupatha (Cissampelos pareira) in experimental animals. Journal of Ethnopharmacology, 116, 454e460. Anderson, J. W., & Gilliland, S. E. (1999). Effect of fermented milk (yogurt) containing Lactobacillus acidophilus L1 on serum cholesterol in hypercholesterolemic humans. Journal of the American College of Nutrition, 18, 43e50. Berg, R. D. (1995). Bacterial translocation from the gastrointestinal tract. Trends in Microbiology, 3, 149e154. Berg, R. D., & Garlington, A. W. (1979). Translocation of certain indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes and other organs in a gnotobiotic mouse model. Infection and Immunity, 23, 403e411. Bhupathiraju, S. N., & Tucker, K. L. (2011). Coronary heart disease prevention: nutrients, foods, and dietary patterns. Clinica Chimica Acta, 412, 1493e1514. Charach, G., Rabinovich, A., Argov, O., Weintraub, M., & Rabinovich, P. (2012). The role of bile acid excretion in atherosclerotic coronary artery disease. International Journal of Vascular Medicine, 2012, 949672. Crowell, M. J., & Macdonald, I. A. (1980). Enzymic determination of 3a-, 7a- and 12a-hydroxyl groups of fecal bile salts. Clinical Chemistry, 26, 1298e1300. Czubayko, F., Beumers, B., Lammsfuss, S., Lütjohann, D., & von Bergmann, K. (1991). A simplified micro-method for quantification of fecal excretion of neutral and acidic sterols for outpatient studies in humans. Journal of Lipid Research, 32, 1861e1867. DeRodas, B. Z., Gilliland, S. E., & Maxwell, C. V. (1996). Hypocholesterolaemic action of Lactobacillus acidophilus ATCC 43121 and calcium in swine with hypercholesterolemia induced by diet. Journal of Dairy Science, 79, 2121e2128. Fletouris, D. J., Botsoglou, N. A., Psomas, I. E., & Mantis, A. I. (1998). Rapid determination of cholesterol in milk and milk products by direct saponification and capillary gas chromatography. Journal of Dairy Science, 81, 2833e2840. Guo, C. F., Zhang, L. W., Han, X., Li, J. Y., Du, M., Yi, H. X., et al. (2011). Short communication: a sensitive method for qualitative screening of bile salt hydrolase-active lactobacilli based on thin-layer chromatography. Journal of Dairy Science, 94, 1732e1737. Guo, C.-F., Zhang, L.-W., Han, X., Yi, H.-X., Li, J.-Y., Tuo, Y.-F., et al. (2012). Screening for cholesterol-lowering probiotic based on deoxycholic acid removal pathway and studying its functional mechanisms in vitro. Anaerobe, 18, 516e522. Gupta, A. K., Savopoulos, C. G., Ahuja, J., & Hatzitolios, A. I. (2011). Role of phytosterols in lipid-lowering: current perspectives. QJM-An International Journal of Medicine, 104, 301e308. Haarman, M., & Knol, J. (2006). Quantitative real-time PCR analysis of fecal Lactobacillus species in infants receiving a prebiotic infant formula. Applied and Environmental Microbiology, 72, 2359e2365. Hagio, M., Matsumoto, M., Fukushima, M., Hara, H., & Ishizuka, S. (2009). Improved analysis of bile acids in tissues and intestinal contents of rats using LC/ESI-MS. Journal of Lipid Research, 50, 173e180. Hamilton, J. P., Xie, G., Raufman, J.-P., Hogan, S., Griffin, T. L., Packard, C. A., et al. (2007). Human cecal bile acids: concentration and spectrum. American Journal of Physiology-Gastrointestinal and Liver Physiology, 293, G256eG263. Hofmann, A., & Mysels, K. (1992). Bile acid solubility and precipitation in vitro and in vivo: the role of conjugation, pH, and Ca2þ ions. Journal of Lipid Research, 33, 617e626.
149
Huang, Y., & Zheng, Y. (2010). The probiotic Lactobacillus acidophilus reduces cholesterol absorption through the down-regulation of Niemann-Pick C1-like 1 in Caco-2 cells. British Journal of Nutrition, 103, 473e478. Jenkins, D. J. A., Mirrahimi, A., Srichaikul, K., Berryman, C. E., Wang, L., Carleton, A., et al. (2010). Soy protein reduces serum cholesterol by both intrinsic and food displacement mechanisms. Journal of Nutrition, 140, 2302Se2311S. Kumar, R., Grover, S., & Batish, V. K. (2011). Hypocholesterolaemic effect of dietary inclusion of two putative probiotic bile salt hydrolase-producing Lactobacillus plantarum strains in Sprague-Dawley rats. British Journal of Nutrition, 105, 561e573. Kumar, M., Nagpal, R., Kumar, R., Hemalatha, R., Verma, V., Kumar, A., et al. (2012). Cholesterol-lowering probiotics as potential biotherapeutics for metabolic diseases. Experimental Diabetes Research, 2012, 902917. Li, T., & Chiang, J. Y. L. (2009). Regulation of bile acid and cholesterol metabolism by PPARs. PPAR Research, 2009, 501739. Lipid Research Clinics Program. (1984). The lipid research clinics coronary primary prevention trial results. I. Reduction in incidence of coronary heart disease. Journal of the American Medical Association, 251, 351e364. Lye, H.-S., Rahmat-Ali, G. R., & Liong, M.-T. (2010). Mechanisms of cholesterol removal by lactobacilli under conditions that mimic the human gastrointestinal tract. International Dairy Journal, 20, 169e175. Ma, L., Deitch, E., Specian, R., Steffen, E., & Berg, R. (1990). Translocation of Lactobacillus murinus from the gastrointestinal tract. Current Microbiology, 20, 177e184. Martinez-Augustin, O., & de Medina, F. S. (2008). Intestinal bile acid physiology and pathophysiology. World Journal of Gastroenterology, 14, 5630e5640. Matsubara, M., Maruoka, S., & Katayose, S. (2002). Decreased plasma adiponectin concentrations in women with dyslipidemia. Journal of Clinical Endocrinology and Metabolism, 87, 2764e2769. Matsumoto, K., Takada, T., Shimizu, K., Moriyama, K., Kawakami, K., Hirano, K., et al. (2010). Effects of a probiotic fermented milk beverage containing Lactobacillus casei strain Shirota on defecation frequency, intestinal microbiota, and the intestinal environment of healthy individuals with soft stools. Journal of Bioscience and Bioengineering, 110, 547e552. McNamara, D. J., Proia, A., & Miettinen, T. A. (1981). Thin-layer and gas-liquid chromatographic identification of neutral steroids in human and rat feces. Journal of Lipid Research, 22, 474e484. Nijjar, P. S., Burke, F. M., Bloesch, A., & Rader, D. J. (2010). Role of dietary supplements in lowering low-density lipoprotein cholesterol: a review. Journal of Clinical Lipidology, 4, 248e258. Ooi, L. G., Ahmad, R., Yuen, K. H., & Liong, M. T. (2011). Lactobacillus acidophilus CHO220 and inulin reduced plasma total cholesterol and low-density lipoprotein cholesterol via alteration of lipid transporters. Journal of Dairy Science, 94, 2658. Ramchandran, L., & Shah, N. P. (2011). Yogurt can beneficially affect blood contributors of cardiovascular health status in hypertensive rats. Journal of Food Science, 76, H131eH136. Reeves, P. G. (1997). Components of the AIN-93 diets as improvements in the AIN76A diet. Journal of Nutrition, 127, 838Se841S. Ridlon, J. M., Kang, D.-J., & Hylemon, P. B. (2006). Bile salt biotransformations by human intestinal bacteria. Journal of Lipid Research, 47, 241e259. Rosengren, A., Hagman, M., Wedel, H., & Wilhelmsen, L. (1997). Serum cholesterol and long-term prognosis in middle-aged men with myocardial infarction and angina pectoris: a 16-year follow-up of the primary prevention study in Goteborg, Sweden. European Heart Journal, 18, 754e761. Samuel, P., Saypoi, G. M., Meilman, E., Mosbach, E. H., & Chafizadeh, M. (1968). Absorption of bile acids from the large bowel in man. Journal of Clinical Investigation, 47, 2070e2078. Shu, Q., Zhou, J. S., Rutherfurd, K. J., Birtles, M. J., Prasad, J., Gopal, P. K., et al. (1999). Probiotic lactic acid bacteria (Lactobacillus acidophilus HN017, Lactobacillus rhamnosus HN001 and Bifidobacterium lactis HN019) have no adverse effects on the health of mice. International Dairy Journal, 9, 831e836. Siigur, U., Tamm, E., Torm, S., Lutsar, I., Salminen, S., & Midtvedt, T. (1996). Effect of bacterial infection and administration of a probiotic on faecal short-chain fatty acids. Microbial Ecology in Health and Disease, 9, 271e277. Thornton, J. R. (1981). High colonic pH promotes colorectal cancer. Lancet, 317, 1081e1083. Usman, & Hosono, A. (2000). Effect of administration of Lactobacillus gasseri on serum lipids and fecal steroids in hypercholesterolemic rats. Journal of Dairy Science, 83, 1705e1711. Usman, & Hosono, A. (2001). Hypocholesterolaemic effect of Lactobacillus gasseri SBT0270 in rats fed a cholesterol-enriched diet. Journal of Dairy Research, 68, 617e624. Wang, J., Zhang, H., Chen, X., Chen, Y., Menghebilige, & Bao, Q. (2012). Selection of potential probiotic lactobacilli for cholesterol-lowering properties and their effect on cholesterol metabolism in rats fed a high-lipid diet. Journal of Dairy Science, 95, 1645e1654. Wolever, T. M., Spadafora, P., & Eshuis, H. (1991). Interaction between colonic acetate and propionate in humans. American Journal of Clinical Nutrition, 53, 681e687. Wong, J. M. W., & Jenkins, D. J. A. (2007). Carbohydrate digestibility and metabolic effects. Journal of Nutrition, 137, 2539Se2546S.