Miremadi, Fatemeh (2018) Hypocholesterolemic and Anti-hypertensive Properties of Lactobacilli and Bifidobacteria. PhD thesis, Victoria University.

Abstract

This project involved the development of a synbiotic dairy product and its use in human feeding trials to quantify the hypocholesterolemic and antihypertensive effects of probiotics on hypercholesterolemic men and women. To this end, eleven strains of Lactobacilli and three strains of Bifidobacteria were evaluated for their acid and bile tolerance, cholesterol removal ability and hypotensive properties in vitro. Our results showed that all studied strains were able to tolerate pH 2.0 for two hours. However, strains of L. acidophilus in general were more acid-resistant than Bifidobacteria with an average viable count more than 107 CFU mL-1 after 2h incubation at pH 2.0. All selected strains could tolerate the bile salts, with greater tolerance towards oxgall than to taurocholic acid. The ACE inhibition was found to be significantly different among the strains ranging from 8.2% to 78.5%. In addition to the above, all strains tested possessed varying degrees of cholesterol removal capabilities from the growth, either via medium via incorporation of cholesterol into the cellular structure, or binding of cholesterol onto cellular membrane and/or bile salt deconjugation. The affinity towards cholesterol was found to be greater in growing cells than resting or dead cells; nevertheless, this study confirmed that the latter cells could be useful as cholesterol-reducing agents in gastrointestinal tract (GIT). Fatty acid composition analysis of the harvested cells revealed that cells grown in the presence of cholesterol contained higher level of saturated fatty acids and lower level of unsaturated fatty acids than those grown in the absence of cholesterol, suggesting that cholesterol from the medium may be incorporated into the cellular membrane of probiotics. The membrane of each probiotic cell was broken up by centrifuging at 4000 × g for 20 min and fatty acid composition of harvested cells was analysed using gas chromatography. The selected strains were also screened for their bile salt deconjugation ability and bile salt hydrolase activity (BSH). Strains of Lactobacillus in general showed a higher bile salt deconjugation ability and liberated a more substantial amount of cholic acid than Bifidobacterium strains. Substrate preference for BSH activity was more towards sodium glycocholate than sodium taurocholate, however, most strains exhibited higher total BSH activity on bile salt mixtures than to when bile salts were used individually. Based on the above results L. acidophilus CSCC 2404 and L. rhamnosus ASCC 1520 were selected to develop a synbiotic yogurt to be used in human trials. The selected probiotics were subsequently screened in the presence of four prebiotics, sorbitol, mannitol, fructooligosaccharide, and fructooligosaccharides-enriched inulin (Synergy 1), to determine the best type and concentration of prebiotic in order to achieve maximum cholesterol removal and ACE-I activity inhibition in vitro and in vivo. Screening using factorial design showed that the combination of 1% (v/v) L. acidophilus CSCC 2404 and L. rhamnosus ASCC 1520 in the presence of 2.0% (w/v) Synergy 1 achieved the best results in synbiotic yogurt. In addition to synbiotics, there has been an increased interest in dietary sources of antioxidant polyphenols in red fruit juices such as pomegranate (Punica granatum L.) due to their strong antioxidant activity (AA). Therefore, to further improve the health-promoting properties of the synbiotic yogurt, different levels of pomegranate juice concentrate (PJC) (10, 12.5, 15 and 20 %) were added to yogurt milk before incubation, and the AA, antihypertensive activities and hypocholesterolemic properties of the synbiotic yogurt was evaluated during refrigerated storage for 21 days. Despite the slight adverse effect of PJC supplementation at 20% level on probiotic numbers, the probiotic population at the end of storage period was still within the acceptable range of colony- forming unit (CFU) to deliver health-promoting properties (> 106 CFU mL-1). The resulting synbiotic yogurt showed increased polyphenol levels by 63%, AA by 94%, and angiotensin-converting-enzyme (ACE) inhibition by 75% compared to the control plain yogurt. The sensory evaluation of the PJC-enriched sybniotic yogurt revealed that by increasing the PJC level, the overall acceptability among the panellists increased significantly (P < 0.05). To provide evidence to support the positive effects of this PJC-supplemented synbiotic yogurt on serum lipid profile and blood pressure (BP), an 8-week parallel, doubleblinded, randomised trial was designed and conducted with 48 male and female volunteers. The volunteers were aged between 30-65 years, with serum total cholesterol (TC) and triglyceride (TG) levels less than 6·2 and 2.3 mmol L-1, respectively, a body mass index (BMI) between 25 and 35 kg/m2, and willing to consuming a daily serving of 200g yogurt. Subjects were assigned to three groups; Group 1 (control) consumed the yogurt samples which only contained 0.02% (w/v) freeze-dried YC-380 (Streptococcus thermophilus 1275 and L. bulgaricus 1842), Group 2 consumed the synbiotic yogurt (Syn) containing 0.02% (w/v) freeze-dried YC-380, 0.5% (v/v) L. acidophilus ASCC 2404, L. rhamnosus ASCC 1520, and 2.0% (w/v) Synergy 1, and Group 3 consumed the same yogurt supplemented with 20% (v/v) PJC (Synp). The yogurts were produced and distributed to the participants on weekly intervals. Fasting blood samples, 3-day dietary records, anthropometric measurements and BP were collected at baseline and at the end of four and eight weeks. Comparison of the mean differences of serum TC, TG, and high-density lipoprotein cholesterol (HDL-C) levels among the three groups confirmed a drop of 9.81% and 18.10 % in TG level and TC: HDL-C ratio (P < 0.05) respectively, from baseline to week eight. However, no significant changes from the baseline were observed in systolic blood pressure (SBP) and diastolic blood pressure (DBP) (P > 0.05). The post hoc test showed that the decrease in serum TC and LDL-C was significant for both the Syn and Synp groups (P < 0.001 for both). Consumption of Synp yogurt resulted in 19% and 23% decrease in serum TC and LDL-C levels, respectively, at the end of the study, whereas in the Syn group, these levels were only reduced by 14% and 17% respectively. Overall, the selected probiotics L. acidophilus CSCC 2404 and L. rhamnosus ASCC 1520 proved to have the ability to tolerate acid and bile conditions and were optimised with 2.0% (w/v) Synergy 1 and 20% (v/v) PJC for maximum cholesterol removal in vitro and in vivo. Therefore, this study indicates the combined effectiveness of probiotics, prebiotics and polyphenols in ameliorating cardiovascular disease risk factors in both women and men.

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