Heart disease is a major cause of death in humans( Reference Atkins, Whincup and Morris 1 ). Nutritional studies have indicated that high concentrations of total cholesterol (TC) and LDL-cholesterol correlate highly with the incidence of CHD. Thus, considerable research has been conducted to determine factors that are effective in lowering concentrations of serum cholesterol, including dietary modifications( Reference Kannel, Doyle and Osteld 2 ). β-Cyclodextrin (BCD) is a cyclic oligosaccharide consisting of seven glucose units and is produced from starch using the enzyme cyclodextrin glycotransferase – to break the polysaccharide chain – giving the molecule its affinity for molecules such as cholesterol( Reference Alonso, Cuesta and Fontecha 3 – Reference Szente and Szejtli 5 ). Several reports( Reference DiRienzo 6,7 ) have indicated that consumption of certain cultured dairy products supplemented with Lactobacillus acidophilus reduced concentrations of serum cholesterol during growth in laboratory media. The presence of bile and anaerobic conditions during growth enable L. acidophilus to assimilate cholesterol( Reference Gilliland, Nelson and Maxwell 8 – Reference Corzo and Gilliland 10 ). Such assimilation of cholesterol in the small intestine may be important for reducing the absorption of dietary cholesterol from the digestive system into the blood. Pigs were selected as the animal model for this study as their digestive system, distribution of coronary arteries and atherosclerotic tendencies resemble those of humans( Reference Ratcliffe 11 ). As L. acidophilus exhibits host specificity in the intestinal tract( Reference Gilliland, Bruce and Bush 12 ), we had to use strains of L. acidophilus of pig origin.
The objective of the present study was to investigate the effects of a BCD diet rich in cholesterol supplemented with a culture of L. acidophilus on TC and LDL-cholesterol levels in blood serum in twenty-four 6–7-week-old Yorkshire gilts randomly assigned to individual pens, divided into four treatments, with six pigs per treatment.
Methods
Chemicals
BCD was obtained from Cavamax w7 (Wacker) and cholesterol (Sigma Co.). All the other reagents were of the highest commercially available quality.
Source of culture and bile tolerance
L. acidophilus EP32 used in this experiment was from our laboratory stock culture collection and was originally isolated from the intestinal contents of pigs( Reference Gilliland, Nelson and Maxwell 8 ). The organisms were cultured in the presence of bile by individual inoculation (1 %) into sterile MRS-THIO broth (lactobacilli de Man, Rogosa and Sharpe broth supplemented with 0·2 % sodium thioglycolate) with and without 0·3 % oxgall (bovine bile), and the culture was incubated for 3 h at 37°C. Increases in absorbance at 620 nm during the 2-h incubation period were used to compare growth of the culture.
Deconjugation of sodium taurocholate and β-cyclodextrin
About 30 ml of MRS-THIO broth containing 0·2 % sodium taurocholate was inoculated (1 %) with L. acidophilus EP32 and incubated at 37°C in the presence of 1 % BCD. One tube of each culture was removed at 3-h intervals throughout the 18 h of incubation. A 1:10 dilution was made from each tube using sterile peptone diluent (1 %), and the absorbance at 620 nm was measured to determine the relative amount of growth. Free cholic acid liberated by each culture was measured( Reference Walker and Gilliland 13 ). Absorbance was read at 660 nm against a reagent blank and compared with a standard curve to determine the concentration of free cholic acid. The results are expressed as micromoles of cholic acid per millilitre.
Animals
A total of twenty-four 6–7-week-old Yorkshire gilt female pigs weighing approximately 9–10 kg obtained from the pig research unit, Oklahoma State University, were used for the study. All the procedures involving animals in this study were conducted with the approval of the Oklahoma State University, Animal Ethics Committee.
Experimental design
Pigs were randomly assigned to individual pens and subsequently divided into four treatment groups with six pigs per treatment. The location of each pig was random with respect to the treatments (Fig. 1). The experimental basal diet (902 kg) was prepared by mixing using a Marion mixer (Rapids Machinery Co.); 228·0 kg of the diet was removed for adjustment period feeding, and 2·1 kg of cholesterol, with purity at least equivalent to the United State Pharmacopeia recommendations (Sigma Co.), was added and mixed to the remaining 674·0 kg. Including cholesterol contents from butter, the experimental diet contained 1775 mg of cholesterol/kg. After mixing, 10·0 kg BCD was added to 337·0 kg to provide the BCD experimental diet. All the pigs were fed a maize basal diet for the adjustment period for 1 week without added crystalline cholesterol twice daily (morning and afternoon); the experimental period began at the 2nd week. All the pigs were then fed the maize experimental diet (Table 1) at the start of the trial according to the four treatment groups: 1M, control high-cholesterol diet plus 50 ml of sterile non-fat milk; 1C, high-cholesterol diet plus 50 ml of sterile non-fat milk containing 5×1010 cells of L. acidophilus EP32; 2M, 3 % BCD high-cholesterol diet plus 50 ml of sterile non-fat milk; and 2C, 3 % BCD high-cholesterol diet plus 50 ml of sterile non-fat milk containing 5×1010 cells of L. acidophilus EP32. Pigs were fed twice daily and water was available at all times. During the experimental period, the pigs were fed for 21 d at 10 % of the metabolic body weight (kg0·70). If food was refused following the morning meal, it was offered again with the afternoon meal. The quantity of food remaining at the end of the day was recorded.
Fig. 1 Flow chart detailing the experimental design of each treatment. SNFM, sterile non-fat milk; BCD, β-cyclodextrin; L. acidophilus, Lactobacillus acidophilus.
Table 1 Ingredients and nutritional values of the experimental basal diets
1M, control high-cholesterol diet plus 50 ml of sterile non-fat milk; 1C, high-cholesterol diet plus 50 ml of sterile non-fat milk containing 5×1010 cells of Lactobacillus acidophilus EP32; 2M, 3 % β-cyclodextrin (BCD) high-cholesterol diet plus 50 ml of sterile non-fat milk; 2C, 3 % BCD high-cholesterol diet plus 50 ml of sterile non-fat milk containing 5×1010 cells of L. acidophilus EP32.
* The vitamin trace mineral premix supplied 1760 mg of riboflavin, 8800 mg of pantothenic acid, 8800 mg of niacin, 8·8 mg of vitamin B12, 176 000 mg of choline chloride, 528·28 mg of vitamin A, 4·42 mg of vitamin D3, 3235·29 mg of vitamin E, 44 mg of menadiane dimethyl-primidionol bisulfite, 39·6 mg of Se, 299·2 mg of I, 19·8 g of Fe, 11 g of Mn, 2·2 g of Cu and 39·6 g of Zn/kg of premix.
Blood collection
Blood samples were collected weekly from the indwelling jugular catheter just before the 1st day of the training period and continued for additional 28 d after initiation of the trial. Immediately following collection, the tubes were placed in ice-cold water bags and then centrifuged for 20 min at 3000 g , and the serum samples were transferred into screw-cap vials and stored at −20°C until the samples were analysed. Duplicate samples of serum were analysed for TC. Concentrations of LDL-cholesterol were calculated by the difference between TC and HDL-cholesterol. All the analyses were performed using enzymatic kits (Sigma Chemical Co.).
Statistical analysis
The statistical design was a 2×2 factorial design, and time was included as the factor for repeated-measures analysis to determine the significance of meal effect (treatment 1 (cyclodextrin: yes, no), treatment 2 (probiotic: yes, no), time) and interactions (treatment 1×treatment 2, treatment 1×time, treatment 2×time, treatment 1×treatment 2×time). For each time point ANOVA (two-way full factorial) was performed using SAS software( 14 ). Statistical significance was accepted at P<0·05.
Results
All the animals remained in good health throughout the experiment. There were no significant differences (P<0·05) in feed intake and weight gain as well as the growth and development between treatments.
The effects of Lactobacillus and BCD on serum TC and LDL-cholesterol during the 4 weeks of treatment are presented in Fig. 2, according to the statistical model presented in experimental methods. Pigs fed L. acidophilus and BCD (2C) had a significant interaction (P=0·021) of plasma TC than those without BCD and L. acidophilus (1M) (2·63 (SD 0·21) v. 3.20 (SD 0·26) mmol/l) (17 % lower) at the end of the treatment period. Similarly, pigs fed BCD and milk (2M) had a significant interaction (P=0·028) of TC than those without BCD and L. acidophilus (2C) (2·82 (SD 0·27) v. 3·20 (SD 0·26) mmol/l) with a decrease of 12 %. No significant interaction (P=0·253) was observed between basal diet with L. acidophilus and BCD (1C, 2M) in the two experimental groups at 3 weeks (2·93 (SD 0·24) v. 2·82 (SD 0·27) mmol/l). As expected, total serum cholesterol concentrations decreased for all the treatment groups containing L. acidophilus, BCD or both in the diet after 3 weeks of treatment. Similar results were observed for LDL-cholesterol for the four treatments. No significant interactions (P=0·089; 0·095; 0·091) were observed for the separate treatments with L. acidophilus and BCD diets (1C, 2C and 2M) (1·42 (SD 0·18), 1·28 (SD 0·18) and 1·25 (SD 0·22) mmol/l), but a significant interaction (P=0·022; 0·029; 0·023) of those treatments with the control group was found (basal diet and milk, 1M) (1·83 (SD 0·18) mmol/l) with a decrease of 23 %. The main significant interaction (P=0·017) was for the group fed L. acidophilus and BCD (2C) compared with the control (1M) (1·28 (SD 0·18) v. 1·83 (SD 0·18) mmol/l) with a decrease of over 27 % after 3 weeks of treatment.
Fig. 2 Serum concentrations (mmol/l) of total cholesterol (TC) and LDL-cholesterol of pigs previously fed a high-cholesterol diet with β-cyclodextrin (BCD) and Lactobacillus acidophilus during the three experimental weeks. Treatments sharing the same letter are not significantly different at 95 % CI. 
, Milk; 
, milk+L. acidophilus; 
, milk+BCD; 
, L. acidophilus+BCD.
Discussion
Serum TC concentrations increased, as expected, by a diet supplemented with crystalline cholesterol and butter. This ability to increase serum cholesterol levels of pigs by increasing dietary cholesterol has been reported in other studies( Reference Jensen, Mazur and Pettigew 15 , Reference Sun, Monagas and Jang 16 ). High concentrations of serum TC and LDL-cholesterol are strongly associated with an increased risk for CHD( 17 ). Reduction in TC and LDL-cholesterol in hypercholesterolomic men has been reported to reduce the incidence of CVD( 14 ). Thus, to assay different ways to reduce serum cholesterol levels are important. This study shows that dietary supplementation with L. acidophilus EP32 and BCD diet reduced total serum cholesterol in pigs previously fed a high-cholesterol diet more significantly compared with pigs that did not receive L. acidophilus and BCD. These findings are in accordance with those reported for L. acidophilus in humans( Reference Harrison and Peat 18 ), swine( Reference Gilliland, Nelson and Maxwell 8 ) and rats( Reference Grunewald 19 ) as well as for BCD in pigs( Reference Juste, Domingo and Lafont 20 ). At present, there are no studies that have combined L. acidophilus and BCD in the same diet. Some authors, using other strains of Lactobacillus, have found no effect of L. acidophilus on serum cholesterol concentration( Reference Lin, Ayres and Winkler 21 ), and a possible explanation for these results is that the strain of L. acidophilus used in the study was not selected for its ability to take up cholesterol during growth or its ability to deconjugate bile acids. In fact, the L. acidophilus assayed by those authors was only moderately active in assimilating cholesterol and bile resistance( Reference Gilliland and Walker 22 ), and no data have been reported on its ability to deconjugate bile acids. These factors may have limited the ability of this strain to survive and grow in the intestinal tract and to exert a beneficial effect by regulating serum cholesterol levels. The L. acidophilus EP32 used in our study is very active in taking up cholesterol and in deconjugating bile acids( Reference Walker and Gilliland 13 ). L. acidophilus EP32 tested in this study in the presence of BCD exhibited a greater degree of bile tolerance and the ability to deconjugate sodium taurocholate releasing more cholic acid and growing slightly more in the broth media with BCD. Deconjugation of sodium taurocholate in the in vitro assay in this study was higher (P<0·05) when L. acidophilus was grown in presence of 1 % BCD (4·4±0·4 mm) compared with controls (3·1±0·3 mm) as BCD enhances deconjugation of bile salts.
Deconjugated bile acids are less well absorbed from the small intestine than are the conjugated bile acids. Thus, deconjugation of bile acids in the small intestine could result in greater excretion of bile acid from the intestinal tract, especially because free bile acids are excreted more rapidly than conjugated bile acids( Reference Noh, Kim and Gilliland 9 , Reference Corzo and Gilliland 10 ). An excretion of bile acids should result in lower serum bile acids, which in turn would decrease the amount of bile acids reaching the liver for secretion back into the intestine via the enterohepatic circulation( Reference Pereira and Gibson 23 ).
Taking into account the high capacity of BCD to bind to cholesterol( Reference Riottot, Oliver and Caboche 24 ) it may be assumed that BCD acts as a cholesterol trap in the lumen, and thus inhibits the absorption of both dietary and endogenous cholesterol in the small intestine. However, endogenous cholesterol entering the lumen from the bile can act as a competitor for the binding process( Reference van Munster, Tangerman and Nagengast 25 ). The inhibition of cholesterol absorption is not sufficient to explain the overall action of BCD with respect to bile acid metabolism. As BCD binds bile acids with a relatively high affinity in vitro ( Reference Juste, Domingo and Lafont 20 ) and enhances the deconjugation of bile salts by L. acidophilus EP32 in bile acids in the combined diet of this study, it is likely that this carbohydrate also plays a role analogous to that described for bile acid chelators such as cholestyramine( Reference Boucarel, Ceryak and Robins 26 ). These compounds, which are not metabolised in the digestive tract, may act as hypocholesterolaemic agents in pigs by these mechanisms. In conclusion, addition of BCD to the cholesterol-rich diet plus L. acidophilus prevented the elevation of plasma LDL-cholesterol and lowered TC levels.
Acknowledgements
L. A. is grateful to Stan Gilliland for his valuable help and assistance at Food and Agricultural Products Centre (Oklahoma State University).
The present study was supported by the Ministry of Economy and Competitiveness of Spain (grant number AGL-2011-26713).
The authors’ contributions are as follows: L. A. and P. C. contributed to the study design, data collection, performed the data analysis and writing the manuscript. J. F. performed the statistical analysis. All the authors contributed to the discussion and interpretation of the results. All the authors read and approved the final version of the manuscript.
The authors declare that there are no conflicts of interest.
