AI: Atherogenic Index; BM: Buffalo Milk; ANOVA: Analysis of Variance; AOAC: Association of Official Analytical Chemist; CVD: Cardiovascular Disease; HDL: High Density Lipoprotein; LAB: Lactic acid Bacteria; LDL: Low Density Lipoprotein; NCDC: National Collection of Dairy Cultures; NDRI: National Dairy Research Institute; SCFA: Short Chain Fatty Acids; TAGs: Triacylglycerols; VLDL: Very Low Density Lipoprotein

Cardiovascular disease (CVD) is a major cause of death in adults across the world and about 17.5 million people died from CVDs in 2005, representing 30% of all global deaths [1]. Increased levels of certain blood lipids have been reported to be the predominant risk factor for cardiovascular disease [2-4]. More recently it has been reported that probiotics have innumerable therapeutics benefits, such as lowering blood cholesterol [5,6], anticarcinogenesis [7-9], prevention of diarrhoea [10], improving lactose tolerance [11], immunomodulation [12], as an antidiabetic agent [13] and enhancement of resistance against various pathogens [14]. The possible mechanisms involved in the hypocholesterolemic effect include an assimilation of cholesterol by growing cells, the binding of cholesterol to the microbial cellular surface, the deconjugation of bile by bile salt hydrolase (Bsh), the coprecipitation of cholesterol with deconjugate bile, the binding action of bile by fiber, and the microbial production of short-chain fatty acids (SCFAs) in the colon [6-9,14,15-18]. Several recent studies from our laboratory have shown multifactorial health benefits of probiotics in animal models such as anti-aging [19,20], anticarcinogenic [21], detoxification [22] and hypocholesterolemic [23] effects. An indigenous isolate LaVK2 identified as Lactobacillus acidophilus by moleculartyping methods was studied extensively for its functional and probiotic attributes viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. LaVK2 isolate could survive 2 h incubation at pH 1.5-2.0 and toxicity of 1.5-2.0% oxgall bile. LaVK2 could deconjugate major bile salts like taurocholic acid (TCA), glycocholic acid (GCA) and tauro-deoxycholic acid (TDCA), indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of approximately 41.7% and possesses a functional bile salt hydrolase (BSH) enzyme activity that could help in the depletion of cholesterol in the host. Presence of these characteristics suggested the possibility of specific interaction and colonization potential of LaVK2 isolate in the gut. The isolate LaVK2 demonstrated higher free radical scavenging activity and also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus (Unpublished data). Dahi is an Indian fermented milk product containing mixed mesophillic cultures of lactococci, which are not probiotic in nature. In the present study, we have developed the technology to incorporate the LaVK2 strain into Dahi that could be used as a good medium for delivery of probiotic strains for the protection against cardiovascular disease. In this context, the present study was undertaken to explore the therapeutic potential of probiotic LaVK2 Dahi, with a particular fous on the cholesterol-lowering potential for lowering the risks of CVD.

Bacterial strains

Lactococcus lactis ssp. cremoris NCDC-86 and L. lactis ssp. lactis biovar diacetylactis NCDC-60 were obtained from National Collection of Dairy Cultures (NCDC), National Dairy Research Institute (NDRI), Karnal, India. L. acidophilus LaVK2 is our laboratory isolate with probiotic attributes tested through a battery of tests as per FAO/WHO guidelines. This isolate was identified by molecular-typing methods and studied extensively for their functional and probiotic attributes viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities (Unpublished data). Lactobacilli and lactococci were propagated and maintained in MRS-broth and M17 broth (Himedia Laboratories Pvt. Ltd., Mumbai, India) at 37 and 30°C, respectively, and were stored at 4-8°C between transfers.

Preparation of dahi and probiotic LaVK2 dahi

Bacterial cultures were revitalized three times in reconstituted and autoclaved skim milk prior to use for preparation of fermented milk. Buffalo milk obtained from the cattle yard of the Institute and standardized to 3.0% fat, was heated to 90°C for 15 min and was then cooled to 37°C. Dahi was prepared by culturing standardized buffalo milk with Dahi starter culture (Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis biovar diacetylactis, 1% each) for 8 h at 30°C. Probiotic LaVK2 Dahi was prepared by inoculating buffalo milk with L. acidophilus LaVK2 (1%) and mesophillic Dahi culture (1%). The final product contained 2×10⁹ cfu/g of lactococci, and 2×10⁹ cfu/g of L. acidophilus.

Animals, diet and experimental design

Twenty-one male Wistar rats (each 110 day old) were allocated to one of three groups (n=7/group). The animals were maintained in small animal house of Institute at a controlled temperature of 25 ± 2°C and relative humidity of 55 ± 5% at a 12-h light/12-h dark cycle. After the supplements (LaVK2 Dahi, Dahi and BM) were consumed, the animals were fed a hypercholesterolemic diet ad libitum (Table 1). The animals were used and cared for in accordance with the principles and guidelines for human use, and the protocol was approved by the Institutional Ethics Committee.

Table 1: Composition of hypercholesterolemic basal diet

Blood sampling and analytical procedures

Blood samples were collected from the orbital venous plexus of 12- hour fasted and anesthetized rats using heparinized tubes containing 10 μl of heparin solution (20 IU), and spun at 1000 x g for 10 minutes at 4°C for plasma separation. Plasma was analyzed for total cholesterol, HDL-cholesterol and triacylglycerols (TAGs) using Liquid Gold kits (Span Diagnostics Ltd, Surat, India) based on enzymatic oxidation of these molecules. Low density lipoprotein (LDL) concentration was calculated using the equations of Friedewald [24]. At the end of the experimental period, the animals were sacrificed. Fat was extracted from the aorta and liver with chloroform-methanol (2:1) mixture [25] evaporated to dryness under the stream of nitrogen, reconstituted in ethanol, and analyzed for plasma total cholesterol and TAGs.

Statistical analysis

The results were expressed as the means ± standard deviation for each group (n=7) and analyzed by 1-way analysis of variance followed by the Tukey post hoc test (SYSTAT version 6.0.1, SPSS Inc, Chicago, Illinois). Differences were considered significant at P<0.05.

Average feed intake and body weight gained among LaVK2 Dahi, Dahi and BM were not significantly different (data not shown). This observation suggests that BM, Dahi or LaVK2 Dahi administration did not affect the overall feed intake and body weight gain in the entire experiment. To investigate the protective effect of probiotic Dahi in lowering cholesterol in rats fed with hypercholesterolemic diet, plasma cholesterol was measured every 30 day interval upto 120 days. The effects of three dietary treatments on the plasma total cholesterol are presented in Table 2. Plasma total cholesterol was elevated throughout experimental period and increased above 73% when compared to baseline level in rats fed with BM. In rats fed with LaVK2 Dahi, plasma total cholesterol level was decreased by 22.6% when compared to rats fed with BM. The overall mean plasma total cholesterol was significantly lower in rat fed with LaVK2 Dahi and Dahi as compared to rats fed with BM. Plasma HDL-cholesterol levels were increased in all three dietary groups: BM, Dahi and LaVK2 Dahi (Table 3). However, increase in plasma HDL-cholesterol was non-significant among the 3 dietary groups. Plasma LDL- and very-low-density-lipoprotein (VLDL)-cholesterol in BM- and Dahi fed groups were elevated by 84.6% and 73%, respectively, on day 120, as compared to baseline level in the respective groups (Table 4). The decrease in level of LDL and VLDL-cholesterol was 89% on day 120 as compared to baseline level in LaVK2 Dahi group.

Table 2: The effect of feeding buffalo milk, Dahi or probiotic LaVK2 Dahi on plasma total cholesterol concentration in rats

Table 3: The effect of feeding buffalo milk, Dahi or probiotic LaVK2 Dahi dahi on plasma HDL-cholesterol concentration in rats

Table 4: The effect of feeding buffalo milk, Dahi or probiotic LaVK2 Dahi on plasma LDL + VLDL cholesterol concentration in rats

The statistical analysis showed that plasma LDL cholesterol levels differed significantly among the exprimental groups (P<0.05). As shown in Table 5, plasma levels of LDL were remarkably increased in Dahi and BM fed groups and remained elevated throughout the experimental period. In the LaVK2 Dahi group, level of LDLcholesterol transiently increased on day 30 and then subsequently decreased below the baseline levels by 390.0% on days 120 as compared to baseline levels. In addition, the decreased levels in plasma LDLcholesterol was significantly lower (P<0.001) in LaVK2 Dahi group as compared to Dahi and BM fed groups. As shown in Table 6, plasma levels of TAGs remained increased in Dahi- and BM-fed groups and remained elevated throughout the experimental period. In the LaVK2 Dahi group, the levels of TAGs transiently increased on day 30 and then subsequently decreased and reached below baseline level by 64.2%. The levels of plasma TAGs were significantly (P<0.05) lower in LaVK2 Dahi-fed group when compared to Dahi- and BM-fed groups. The atherogenic index (AI), a ratio of VLDL + LDL cholesterol to HDL cholesterol, remained unaffected throughout the experimental period in rats fed with BM. In contrast, the AI decreased in rats fed with LaVK2 Dahi (Table 7). On day 120, the AI was significantly (P<0.05) lower in LaVK2 Dahi-fed rats when compared to BM- and Dahi-fed groups.

Table 5: The Effect of feeding buffalo milk, Dahi or probiotic LaVK2 Dahi on plasma LDL cholesterol concentration in rats

Table 6: The effect of feeding buffalo milk, Dahi or probiotic LaVK2 Dahi on plasma triacylglycerols concentration in rats

Table 7: The Effect of feeding buffalo milk, Dahi or probiotic LaVK2 Dahi on atherogenic index† in rats

The contents of cholesterol and TAGs in aortic tissue were significantly lower in rats fed with Dahi and LaVK2 Dahi (Table 8). As anticipated, the LaVK2 Dahi was more efficacious than Dahi in reducing the levels of cholesterol and TAGs in aortic tissue. The contents of cholesterol in aortic tissue decreased by 7.2 and 26.4% on Dahi and LaVK2 Dahi groups, respectively, relatively to BM group and the corresponding declines in TAG contents in the aortic tissue were 14.4 and 29.1%, respectively. In addition, LaVK2 Dahi was also more efficacious than dahi in reducing the deposition of cholesterol and TAGs in liver. Deposition of cholesterol in liver reduced by 14.0% and 29.8% in Dahi and LaVK2 Dahi-fed animals, respectively, compared to BM fed group (Table 9), the corresponding decline in TAG in the liver was 9.5% and 35.7%, respectively.

Table 8: The effect of feeding buffalo milk, Dahi or probiotic LaVK2 Dahi on cholesterol and triacylglycerols contents in aortic tissue in rats

Table 9: The effect of feeding buffalo milk, Dahi or probiotic LaVK2 Dahi on contents of cholesterol and triacylglycerols in liver in rats

It is well known that increased cholesterol levels constitute the predisposing factor associated with an increased risk of CVD. Hence, lowering plasma cholesterol levels in hypercholesterolemic patients lowers the incidence of CVD. A probiotic fermented dietary intervention could be a promising and cost-effective approach in improving the lipid profile and, hence, may play a crucial role in the management of CVD. Several studies showed that regular supplementation of functional foods such as probiotics may reduce plasma cholesterol levels [5,6,15,23]. Hence, the present study was designed specifically to explore the antihypercholesterolemic potential of probiotic LaVK2 Dahi in rat model. The present study clearly showed that rats fed with LaVK2 Dahi exhibit significantly (P<0.05) lower levels of plasma total cholesterol, TAGs, LDL-cholesterol, LDL+VLDL-cholesterol, AI and accumulations of tissue lipids in aorta and liver than Dahi- and BMfed animals. The overall positive effects showed by LaVK2 Dahi group on the lipid profile in experimental animals were better than those rendered by Dahi and BM groups. These observations are consistent with preveiously published studies from author’s laboratory on the hypocholesterolemic effect of probiotic fermented milk products [5]. These observations suggest that the major lipids in plasma and hepatic or aortic tissues of animals fed with buffalo milk along with hypercholesterolemic diets went beyond the level of the GI tract at each interval resulting in enhanced tissue uptake and/or de novo synthesis of these lipids in plasma during the 4 months. These observations of up-regulation of plasma and hepatic or aortic cholesterol and TAGs are in agreement with the results of other workers [26]. Though, the effects of consumption of LaVK2 Dahi on plasma lipids as well as on the major lipids of hepatic/aortic tissues have not previously been reported, the results of present study indicate that feeding rats with LaVK2 Dahi resulted in significant decrease (P<0.05) in plasma total cholesterol at day 60 and 120. The results were consistence with other studies [27,28]. Mechanistically, enhanced cholesterogenesis observed in the liver of animals may be attributed to activation of HMG CoA reductase [29]; while the accumulation of cholesterol by the aorta during this feeding period may have been mediated by an enhanced cholesterol efflux from the liver, since most extra-hepatic tissues depend on the liver for their cholesterol needs [30]. The immediate substrates for triacylglycerols and lipid synthesis are the free fatty acids (FFA). They are also the major source of energy in many tissues. In the present study, the excessive accumulation of TAGs in the tissues upon consumption of the BM and Dahi along with hypercholesterolemic diets indicates that most of the absorbed FFA may be directed towards the synthesis of TAGs. This may suggests an impairment of mitochondrial β-oxidation of FFA, hence, compromising energy production in these tissues.

In the present study, animals fed with LaVK2 Dahi reversed the hypercholesterolemia induced by the consumption of the hypercholesterolemic diet. Several interpretations of this observation could be considered. At the outset, the probiotic LaVK2 Dahi reduced plasma cholesterol and TAGs suggesting that the effects of the probiotic Dahi might have commenced at the level of the GI tract, inhibiting absorption of these lipids. Cholesterol reduction by lactic acid bacteria has already been attributed to the ability of these bacteria to bind cholesterol in the small intestine [31-35]. Our statistics indicate that in addition to cholesterol and TAGs could also be bound by these micro-organisms resulting in their decreased absorption and homeostatic response, resulting in lowering of these plasma lipids. Secondly, with reference to lipid metabolism in the tissues, ingestion of probiotic dahi could have inhibited the activity of HMG CoA reductase, reducing the amount of cholesterol synthesized in the liver and aorta. This reduced cholesterol content of the hepatic tissues is consistent with the fact that their cholesterol is obtained from the liver. In addition to reducing hepatic cholesterol and hepatic TAGs, aortic tissue lipids were also decreased by ingestion of the probiotic Dahi. Furthermore, the indigenous L. acidophilus LaVK2 strain has also shown to possess bile salt hydrolase (BSH) activity in vitro and this activity remains in the product up to seven days (data not shown), thereby suggesting that this BSH activity could be responsible for lowering the plasma total cholesterol concentration.

Several workers have attributed cholesterol-lowering property of lactobacilli to their ability to deconjugate bile salts, resulting in excretion of free bile acids in faeces, which in turn enhances the requirement of cholesterol to produce more bile acids hepatic tissue [36,37].

Recently, a novel protein having a molecular weight of 12KDa has been characterized from L. acidophilus ATCC 43121 responsible for reduction of cholesterol in vitro [38]. It has also been suggested that lactic acid bacteria (LAB) may incorporate cholesterol from the surrounding environment into cellular membranes during growth, thus exerting beneficial effects on serum cholesterol levels [36,37]. Cholesterol incorporated into or attached to LAB cells in the intestine cannot be absorbed into the blood [34]. As observed in the present study, deconjugation of major bile salts by the probiotic LaVK2 strain could be responsible for lowering the cholesterol level in vivo. The deconjugation of bile salts such as cholate, chenodeoxycholate and deoxycholate may lead to decreased solubility, and hence, lowering the reabsorption in enterohepatic system, thereby, resulting in an increased demand for cholesterol as a precursor of bile salts [39]. The BSH activity present in LaVK2 strain could be responsible for depletion of cholesterol, hence may be involved in ameliorating the hypercholesterolemia.

In the present study, the levels of plasma LDL+VLDL cholesterol was significantly lower (P<0.05) on rats fed with LaVK2 Dahi when compared to Dahi and BM. These observations are in agreement with studies of Fukushima and Nakano [40] who have reported that the probiotic preparation reduced plasma lipids including the VLDL+LDL cholesterol and a decrease in hydroxymethylglutaryl coenzyme A reductase (NADPH) activity in liver and increased neutral and acidic sterol excretion in faeces of rats fed with probiotic preparation. The results recently obtained by Rajpal and Kansal [23] from the author’s laboratory also support the significant decrease in blood and tissue lipids observed in present study. HDL-cholesterol is considered as good cholesterol and its level in plasma correlated inversely to CVD [5]. This is an important factor since HDL-cholesterol can prevent arteriosclerosis. In present study, levels of HDL-cholesterol in plasma statistically non-significant in groups fed with Dahi, BM and LaVK2 Dahi. This is inconsistent with the reports of other workers who reported augmentation in HDL-cholesterol levels in rats fed with probiotic Dahi containing L. casei, L. acidophilus and Lactococcus lactis [41].

Administration of LaVK2 Dahi significantly lowered the plasma LDL-cholesterol at the conclusion of the experiment. These results are in agreement with the reports of several studies [42,43]. In the present study, the level of plasma TAGs was significantly (P<0.05) reduced in rats fed with LaVK2 Dahi when compared to Dahi or BM. These results are in agreement with the reports of Usman and Hosono [44,45] who studied the effect of L. gasseri on lipid profile in rats fed cholesterolenriched diet, and observed reduction in serum total cholesterol, LDLcholesterol, HDL-cholesterol and triglycerides levels and diminution in AI. It has also been proposed that various milk components such as orotic acid, retentate, pyrimidine-like nucleotide, calcium or hydroxyl methyl glutaric acid reduces the de-novo synthesis of cholesterol through inhibition of NADPH formation (a reducing power required for biosynthesis of cholesterol) by HMG-CoA synthase (a rate limiting step in cholesterol biosynthesis) in liver and increases the cholesterol clearance from blood stream by enhancing the excretion of bile acids [46]. In addition, fermentation of milk with LAB increases the bioavailability of these components, and their absorption as well as function of such components in gastrointestinal tract [47]. Probably, this may be the reason why LaVK2 Dahi is more effective than BM.

Recently, the author’s laboratory has shown that probiotics downregulate carcinogen activating cytochrome P450 enzymes CYP1A1, CYP1A2 and CYP1B1 in liver, and up-regulate carcinogen detoxifying γ-glutamyltranspeptidase, UDP-glucuronosyl transferase and quinone reductase activities in liver as well as in colon [22]. The potential of this product to improve macrophage and lymphocyte functions [20], and antioxidative status [19] has also been established. Findings of this study indicate that consumption of probiotic Dahi may be more efficacious in reducing diet-induced hypercholesterolemia and decreasing the plasma, hepatic, and aortic lipids than dahi. However, future research may be needed to explore the molecular mechanism underlying the cholesterol-lowering effect of probiotic Dahi and production of bacterial metabolites that may modulate the hepatic cholesterol biosynthesis or degradation.

In conclusion, the present study has presented the antihypercholesterolemic effect of the probiotic LaVK2 Dahi in rats with diet-induced hypercholesterolemia, where plasma total cholesterol, LDL, VLDL, and TAG levels were found to be decreased significantly, and HDL cholesterol levels were observed to be increased but not significantly. Our study reveals that probiotic LaVK2 Dahi was more efficacious than Dahi in reducing plasma cholesterol and TAGs in diet-induced hypercholesterolemic rats. These findings suggest that probiotic LaVK2 Dahi could be exploited as potential alternate biotherapeutic agent to decrease cholesterol levels and lower the risk of CVD, although the area is open for further studies.

The authors wish to acknowledge the research fellowships and necessary facilities provided by University Grant Commission (UGC), Department of Biotechnology (DBT) and Indian Council of Agricultural Research (ICAR), New Delhi, and National Dairy Research Institute (NDRI), Karnal, India. The authors declare that there is no conflict of interest.