Dietary changes are recommended as the first line of intervention for moderate dyslipidemia. This study evaluated the potential cardiovascular protective effects of an isoflavone-supplemented soy yogurt. We also investigated the isolated contributions of the probiotic microrganism and isoflavones to the observed effects.
Feeding excessive amounts of cholesterol to rabbits induces rapid development of hyperlipidemia and atherosclerosis [9, 27, 28]. In this study, the cholesterol added to the diet induced hypercholesterolemia in all experimental groups and none of the treatments applied to these groups reduced the serum lipids to basal levels.
In this study, we observed a decrease in the total (26.6%) and non-HDL cholesterol (27.8%) in rabbits fed the placebo product (unfermented soy product), suggesting that the ingredients of the soy yogurt mixture may contribute to this effect. The ingredients used to obtain the placebo and the soy yogurt included, in each 100 g, 3.39 g of soy protein and 5.24 mg of total isoflavones, components that have potential cholesterol-reducing effects .
Though no significant differences were observed in TC or n-HDL-C levels of animals treated with placebo, soy yogurt and isoflavone-supplemented soy yogurt, the product fermented with E. faecium CRL 183 promoted the greatest reduction (P > 0.05) in TC and non-HDL-C (38.1% and 38.7%, respectively). The supplementation of soy yogurt with isoflavones did not enhance its hypolipidemic effect. In an earlier investigation of our group, using rabbits with diet-induced hypercholesterolemia, the serum TC content was reduced about 18% by ingestion of the unsupplemented soy yogurt . In another study, rats fed fermented soy product enriched with isoflavones had significantly lower (P < 0.05) serum total cholesterol (15.5%) than rats fed on a hypercholesterolemic diet. Non-HDL cholesterol was lower (P < 0.05) in the rats fed fermented soy product, with (27.4%) or without (23.2%) added isoflavones, than in untreated hypercholesterolemic rats .
In a previous study, E. faecium CRL 183 reduced the cholesterol by 54% in an in vitro model . However, the results of the present study did not confirm this effect in rabbits. The E. faecium culture did not promote any improvement in TC or n-HDL-C, indicating that the probiotic microrganism alone was not responsible for the beneficial effects of soy yogurt on these lipid fractions.
Epidemiological studies have demonstrated that HDL-C is a strong, independent, inverse predictor of cardiovascular disease risk [31–33]. The soy yogurt, supplemented or not with isoflavones, placebo and the E. faecium culture prevented the reduction of HDL-C levels, relative to the control group, and raised the HDL-C concentration relative to group H. The probiotic microorganism (E. faecium CRL 183) seems to be decisive for the improvement of the HDL-C level, since in the placebo group the reduction of this fraction in the blood was higher. In a previous experiment with rabbits, the HDL-C level increased 17.8% in the group that consumed the fermented product . Also, the same fermented product, without isoflavone supplementation, was capable of raising the HDL-C level by 10% in normocholesterolemic men . The increase of HDL-C could be an important health benefit of soy yogurt, since each 1-mg increase in HDL-C is associated with an estimated 2-3% reduction in cardiovascular risk .
The triglycerides level varied (P < 0.05) between groups at the beginning of the study, probably because of high biological variability of this lipid. In the current study, rabbits fed the pure culture of E. faecium, soy yogurt supplemented with isoflavones or placebo showed triglycerides concentrations significant lower, after 60 days on these diets. In a previous study with the same fermented product, with or without isoflavones, no effect on triglycerides levels was detected [18, 19, 30].
The differences in the magnitude of the observed effects on blood lipid levels, between the present and earlier studies that evaluated the same soy yogurt, might be explained by differences in experimental design. An important difference between the previous and present studies is the cholesterol concentration added to the diet to induce hypercholesterolemia and atherosclerosis. Rossi et al.  induced hypercholesterolemia by feeding the rabbits a cholesterol-enriched diet (0.15% wt/wt) during the first 15 days of the experiment. In the present study, rabbits were fed a cholesterol-enriched diet for the entire 60 days (1% wt/wt during the first 30 days and 0.7% wt/wt thereafter) to induce atherosclerotic lesions. The TC concentration of group H after this time was about 11 - fold higher than that observed in the earlier study and this could influence the effect of the soy yogurt on lipid parameters.
There are numerous reports showing the hypocholesterolemic effects of probiotics. Xiao et al  showed in rats that milk fermented with Bifidobacterium longum BL1 reduced the total and LDL cholesterol, while no change in HDL-C concentration was observed. Paik et al  found that Bacillus polyfermenticus SCD reduced plasma LDL-C and triglycerides in rats after 6 weeks of treatment. The mechanisms involved in the reduction of serum cholesterol by probiotics, although not completely known, include deconjugation of bile salts, cholesterol assimilation by probiotic cells and fermentation of indigestible carbohydrates from the diet to produce short-chain fatty acids [35, 36]. The lipid profile effects observed here probably involve the ingredients of soy yogurt, the probiotic microorganism and its bioactive metabolites.
The isoflavone mixture reduced the TC and n-HDL-C (19.5%) after 30 days of feeding. However, this effect was not maintained until the end of the protocol. Several studies have assessed isoflavones as a hypocholesterolemic agent and the results are contradictory. Antony et al [37, 38] showed that soy protein without isoflavones was less effective at lowering blood cholesterol levels and preventing the development of atherosclerosis in monkeys than isoflavone-rich soy protein. In contrast, Adams et al  showed that monkeys fed a soy protein diet with low isoflavone content exhibited a lipid profile similar to those fed a similar diet high in isoflavones. Wilson et al  observed that soy protein, with or without isoflavones, lowered TC and n-HDL-C concentrations in hamsters.
The association between oxidized LDL, hypercholesterolemia and atherosclerosis has been demonstrated in numerous studies in animals and humans [15, 41]. Oxidative modification of LDL induces the formation of immunogenic epitopes in the LDL, leading to the generation of antibodies against oxidized LDL (oxLDL Ab) . In this study, the formation of autoantibodies against ox LDL was higher (P < 0.05) in the hypercholesterolemic group (H) by the end of the treatment and the intake of soy yogurt, supplemented with isoflavones or not, prevented this rise in oxLDL Ab during the experiment. The reduction in non-HDL-C concentrations (LDL+VLDL+IDL), with a consequent reduction in LDL particles available for oxidation, observed in the HY and HIY groups could, partially, explain this effect.
The importance of autoantibodies against oxLDL in atherogenesis remains controversial because of the complexity of oxLDL Ab system . An atherogenic role was supported by studies that found elevated concentrations of oxLDL Ab in patients with atherosclerosis [43–45]. Conversely, other studies found an inverse relation between autoantibodies and atherosclerosis development [4, 46]. Analyzing the whole aorta of the rabbits, we observed that only soy yogurt supplemented with isoflavones reduced the atherosclerosis. The variables analyzed in this study are insufficient to define the exact causes or mechanisms involved in the antiatherogenic effect of the soy yogurt. However, given that atherosclerosis is a chronic immune inflammatory disease, it is possible that increase in levels of antibodies against oxidized LDL in the H, HP, HE and HI groups is linked to the development of the atherosclerotic lesion. On the other hand, the results indicate that the soy yogurt supplemented with isoflavones had an atheroprotective effect that may include the anti inflammatory properties of the probiotic product already described  and the antioxidant effect of isoflavones .