EFFECT OF SUPPLEMENTATION OF PROBIOTIC BACILLUS COAGULANS UNIQUE IS-2 (ATCC PAT-11748) ON HYPERCHOLESTEROLEMIC SUBJECTS: A CLINICAL STUDY
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1 International Journal of Probiotics and Prebiotics Vol. 6, No. 2, pp , 2011 ISSN print, Copyright 2011 by New Century Health Publishers, LLC All rights of reproduction in any form reserved EFFECT OF SUPPLEMENTATION OF PROBIOTIC BACILLUS COAGULANS UNIQUE IS-2 (ATCC PAT-11748) ON HYPERCHOLESTEROLEMIC SUBJECTS: A CLINICAL STUDY 1 M. Ratna Sudha, 2 Neelam Radkar and 2 Asin Maurya 1 Centre for Research & Development, Unique Biotech Limited, SP Biotech Park, Phase-II, Plot-2, Shameerpet, Hyderabad , AP, India; and 2 Department of Medicince and Nephrology, Government, L.T.M.G Hospital, Sion, Mumbai, Maharastra, India [Received March 9, 2011; Accepted May 19, 2011] ABSTRACT: The objective of this study was to evaluate the effect due to consumption of capsule containing Bacillus coagulans Unique IS-2 (ATCC PAT-11748), on serum lipids for 60 days. Thirty hyperlipidemic (Having serum cholesterol levels of more than 200mg/dl) subjects were divided into 3 groups (n=10). Two group subjects were allotted to receive a daily dose of two capsules of probiotic Bacillus coagulans Unique IS2 (10X10 9 CFU/capsule (Group A) and 20X10 9 CFU/capsule (Group B)) and third group subjects were received standard medication. Serum lipid profile was done at 0, 30 and 60 th day of the study period. At the end of study, group B subjects exhibited slight reduction in total cholesterol (11%) and LDL (0.8%), whereas an increase in HDL cholesterol levels (3.6%). This data suggest that the strain Bacillus coagulans IS-2 has the potential of control in the serum cholesterol in humans suffering with hyperlipidemia. KEY WORDS: Acute toxicity, Bacillus coagulans Unique IS2, Cholesterol, Clinical studies Corresponding Author: Dr. Ratna M. Sudha, Centre for Research & Development, Unique Biotech Limited, SP Biotech Park, Phase-II, Plot-2, Shamirpet, Hyderabad , A.P. India; Tel: ; E mail: sudha.ratna@gmail.com INTRODUCTION Coronary heart disease (CHD) is one of the major causes of death and disability in industrialized countries. Results from epidemiological studies reveal that there is a positive correlation between elevated total serum cholesterol (hyperlipidemia) levels mainly LDL-cholesterol fraction and risk of emergence of CHD (Periera and Gibson 2002). It has been shown that reduction of total plasma cholesterol in population also reduces the risk of CHD (Levine et al. 1995). Currently there is an increased demand for dietary strategies to reduce the serum cholesterol and triglyceride levels. This is mainly due to expense of drug therapy and large numbers of individuals are affected with unwanted side effects (Periera and Gibson 2002). Dietary strategies for prevention of CHD includes, low fat/low saturated diet (Taylor and Williams 1998), diet containing soluble fibers, plant sterols, prebiotic compounds and probiotic bacteria (Periera and Gibson 2002). Probiotics are live microorganisms, play important role in the development of human health. De Vuyst et al. (2004) describes the probiotic as a preparation of or a product containing viable, defined microorganisms in a sufficient numbers, which alter the microbiota (by implantation or colonization) in a compartment of the host and exert beneficial health effects of the host. Few clinical trials provided information on cholesterol lowering effect of probiotics such as Lactobacillus acidophilus (Harrison and Peat 1975) and Enterococcus faecium and two strains of Streptococcus thermophilus (CAUSIO, Agerbaek et al and Richelsen et al. (1996). Similarly Lactobacillus sporogenes (Bacillus coagulans) has been reported to exert hypocholesterolemic effect (Mohan et al., 1990), prevent antibiotic associated diarrhea in children (La Rosa et al., 2003) and facilitate treatment of non-specific vaginitis (Shirodkar et al. 1980). The aim of present investigation was therefore to determine whether probiotic capsule containing Bacillus coagulans Unique IS-2 would alter the serum cholesterol levels in hypercholesterolemic subjects. MATERIAL AND METHODS Study design This was a 3 month, open-label, single centre, noncomparative phase II clinical study (IEC/13/09) conducted at LTMG hospital, Sion, Mumbai, India. A total of 30 patients (15 male and 15 female patients) of either sex aged 30 yrs and above diagnosed with hyperlipidemia (i.e having a serum cholesterol levels of more than 200mg/dl (between 200 to
2 90 Hypocholesterolemic activity of Bacillus coagulans Unique IS2 240 mg/dl)) were included in the study after obtaining written informed consent and ethical committee approval. Subjects diagnosed with diabetes or hypothyroidism as well as those on medications such as statins, anticoagulants, beta-blockers, diuretics, and steroids were excluded from the study. by enzymatic method of Liberman (1958). Triglycerides were estimated using glycerol phosphate oxidase (GPO) method of Fossati and Lorenzo (1982) and HDL was estimated by enzymatic method of Demacker and Hifman (1980). TABLE 1. Demographics and Health Habits of the study population Characterstics Female (n=15) Male (n=15) Total % n % n % Age yr yr Per capita income / month (Rs.) > Habitual diet Vegetarian Non-vegetarian Ovo-vegetarian Other habits Non alcoholic, Non Smoking Non alcoholic, Smoking Non smoking, Alcoholic Alcoholic, Smoking BMI (kg/m 2 ) Statistical analysis Data were analyzed using SPSS/PC+10.0 statistical package. Descriptive statistics were given as Mean ± SD with n as the number of observations. Student s paired t tests were applied to compare basal & final values. Descriptive statistics were calculated on to per protocol population (completed patients). RESULTS A total of 30 patients were recruited and all the patients completed their 60 days treatment (Table1). The physical examination and initial blood sample reports showed that all the subjects were healthy without signs of liver disease, renal disease and diabetes. Serum Cholesterol. Table 2 describes about serum cholesterol level in daily consumption of Bacillus coagulans Unique IS-2 by group A& B subjects and of standard medication by group C subjects. Serum cholesterol concentrations in the Group B &C decreased significantly by approximately 14% after 60 days of treatment (P, 0.05). After 60 days treatment, in Group A there was up to 11% reduction in serum cholesterol level was observed. The diet Thirty participants fulfilling entry criteria were randomized with respect to age and sex, were divided equally into 3 groups, Subjects belonging to Group A & B were given capsules containing Bacillus coagulans Unique IS-2 (10X10 9 CFU/capsule (Group A), 20X10 9 CFU/ capsule (Group B)) twice a day before meals for 60 days. Group C subjects were allotted to receive standard medication. Study assessments The main assessment of clinical efficacy was for lipid profile analysis at initial, 30, and 60 days after treatment. Total cholesterol was estimated TABLE 2. Total cholesterol levels of hypercholesterolemia subjects before and after Bacillus coagulans Unique IS-2 supplementation for different groups at different time periods. Difference in the superscript indicate statistical significance at different time points at P< Category Dosage of Bacilllus Serum cholesterol levels coagulans Unique (mg/dl)mean values ± SD(n=10) IS-2 supplemented 0 day After After Change F value Level Group A 10 x 10 9 CFU of Bacilllus coagulans b c P < Unique IS-2 / a ±6.4 ±6.0 ±9.6 Group B 20 x 10 9 CFU of Bacilllus coagulans 229 a b c P < Unique IS-2 / ±7.7 ±8.9 ±5.6 Group C Nil (on medication) a b c P < ±12.8 ±14.5 ±12.7
3 Hypocholesterolemic activity of Bacillus coagulans Unique IS2 91 Triglycerides. Significant decrease in concentrations of serum triglyceride (~4%) was observed in all groups (Table 3). TABLE 3. Time course of changes in triglyceride levels in hypercholesterolemia subjects after treatment with increasing dosage of Bacillus coagulans Unique IS-2. Difference in the superscript indicate statistical significance at different time points at P< Category Dosage of Bacilllus coagulans Triglyceride levels Unique IS-2 supplemented Group A Group B (mg/dl)mean values ± SD(n=10) 0 day After After Change F value Level 10 x 10 9 CFU of Bacilllus coagulans a b c P < Unique IS-2 / ±36.1 ±36.6 ± x 10 9 CFU of Bacilllus coagulans a b c P < Unique IS-2 / ±43.6 ±43.4 ±43.6 Group C Nil (on medication) a 271 a c P < ±59.2 ±82.4 ±80.0 HDL Cholesterol. Table 4 presents the serum HDL-cholesterol values during initial, 30, 60 days of treatment. The result found to be slight increase (3%) in serum HDL cholesterol level in all the groups. TABLE 4. Time course of changes in HDL cholesterol levels in hypercholesterolemia subjects after treatment with increasing dosage of Bacillus coagulans Unique IS-2. Difference in the superscript indicate statistical significance at different time points at P< Category Dosage of Bacilllus coagulans HDL cholesterol levels Group A Group B Unique IS-2 supplemented (mg/dl)mean values ± SD(n=10) 0 day After After Change F value Level 10 x 10 9 CFU of B acilllus coagulans 41.3 a 42.2 ab 42.6 cb P < 0.01 Unique IS-2 / ±2.2 ±1.9 ± x 10 9 CFU of Bacilllus coagulans 41.6 a 42.3 b 43.1 c P < Unique IS-2 / ±2.2 ±2.5 ±1.9 Group C Nil (on medication) 40.8 a 41.8 b 42 b P < ±1.8 ±2.16 ±1.7 LDL Cholesterol. Table 5 depicts serum LDL-cholesterol values during initial, 30, 60 days after treatment. It was found that about 0.7% decrease in B & C group subjects, but was not significant. Whereas in group A there was no reduction in LDL cholesterol was observed. TABLE 4. Time course of changes in HDL cholesterol levels in hypercholesterolemia subjects after treatment with increasing dosage of Bacillus coagulans Unique IS-2. Difference in the superscript indicate statistical significance at different time points at P< Category Dosage of Bacilllus coagulans LDL cholesterol levels Unique IS-2 supplemented (mg/dl)mean values ± SD(n=10) 0 day After After Change F value Level Group A 10 x 10 9 CFU of Bacilllus coagulans Unique IS-2 / P < 0.01 Group B ±4.64 ±4.2 ± x 10 9 CFU of Bacilllus coagulans Unique IS-2 / P < ±5.41 ±2.34 ±6.57 Group C Nil (on medication) P < ±4.64 ±3.4 ±4.64
4 92 Hypocholesterolemic activity of Bacillus coagulans Unique IS2 DISCUSSION High concentrations of total cholesterol and LDL are highly associated with an increased risk of CHD. Reduction in total and LDL cholesterol in hyperlipidemic subjects reduces the incidence of CHD (Lipid Research Clinics Program. 1984). Dietary managements, such as supplementation of diet with fermented dairy products or probiotic lactic acid bacteria containing product is one way that serum cholesterol may be reduced (Periera and Gibson 2002). Mohan et al., (1990) observed that administration of Lactobacillus sporogenes in hypercholesterolemic patients resulted in a significant reduction in serum cholesterol and LDL levels. Similarly Lactobacillus sporogenes was reported to be potential in probiotic applications due to its stability and also found to reduce the symptoms caused by diarrhea (Gandhi 1988). In the present investigation strain, Bacillus coagulans Unique IS-2 was well characterized for its probiotic properties in vitro (Sudha et al., 2011) and toxicity studies proved to be safe in oral administration (Sudha et al 2011). Hence Bacillus coagulans Unique IS-2 was evaluated for its hypocholesterolemic properties. The results of present study support the use of probiotic capsule containing Bacillus coagulans Unique IS-2 (group B) found to similar with the group C subjects who were on standard medication. These finding were similar with work reported earlier (Danielson et al 1989; Usman and Hosono 2000). However, Grunewald and Michell and Thompson et al (1982) who observed no significant hypocholesterolemic effect from acidophilus milk consumed by mice and humans respectively. Many researchers (Periera and Gibson 2002) suggest that these conflicting results may be due to difference in strains or unsuitable cultures used in the experimental study. In conclusion, 20 x 10 9 CFU of Bacillus coagulans Unique IS-2 /day/individual for a period of 60 days is more effective and beneficial in bringing out remarkable changes in serum lipid profile than 10 x 10 9 CFU of Bacillus coagulans Unique IS-2 / day/ individual for the same functional parameters. Further studies are necessary before use of Bacillus coagulans Unique IS-2 (ATCC PAT-11748) as a food supplement for managementof abnormal lipid levels in the hypercholesterolemic subjects. CONFLICT OF INTEREST The author of this manuscript declares that there is no conflict of interest. REFERENCES Agerbaek, M., Gerdes, L.U., Richelsen, B. (1995). Hypocholesterolemic effect of a new fermented milk product in healthy middleaged men. European Journal of Clinical Nutrition 49: Colditz, G.A., Willett, W.C., Stampfer, M.J., Manson, J.E., Hennekens, C.H., Arky, R.A., Speizer, F.E. (1990). Weight as a risk factor for clinical diabetes in women. American Journal of Epidemiology 132: Danielson, A.D., Peo, E.R., Shahani, K.M. Lewis, A.J., Whalen, P.J., Amer, M. A. (1989). Anticholesterolemic property of Lactobacillus acidophilus yoghurt fed to mature boars. Journal of Animal Science 67: De Vuyst, L., Avonts, L., Makras, E. (2004). Probotics, prebiotics and gut health (Chap. 17). Remacle, C., Reusens, B. (Eds.), Functional Foods, Ageing and Degenerative Disease. Woodhead Publishing Ltd., Cambridge, United Kingdom, Pereira, D.I.A., Gibson, G.R. (2002). Effects of Consumption of Probiotics and Prebiotics on Serum Lipid Levels in Humans. Critical Reviews in Biochemistry and Molecular Biology 37: Demacker, P. N. M., Hifmans, A. G. M. (1980). Estimation of HDL cholesterol by enzymatic method.clinical Chemistry 26: Fossati, P., Lorenzo, P. (1982). Serum triglycerides determined calorimetrically with an enzyme that produces hydrogen peroxide. Clinical Chemistry 28: Friedewald, W.T., Levy, R.I., Fredericson, D.S. (1979). Estimation of the concentration of low-density lipoprotein cholesterol in plasma without diffuse use of preparative ultracentrifugation. Clinical Chemistry 18: Gandhi, A.B. (1988). Lactobacillus sporogenes: An advancement in lactobacillus therapy. The Eastern Pharmacist, Grunewald, K. K., Mitchell. K. (1983). Serum cholesterol levels in mice fed fermented and unfermented acidophilus milk. Journal of Food Protection 46: Harrison, V.C., Peat, G. (1975). Serum cholesterol and bowel flora in the newborn. American Journal of Clinical Nutrition 28: Levine, G.N., Keaney, J.F., Vita, J.A. (1995). Cholesterol reduction in cardiovascular dis280 ease. Clinical benefits and possible mechanisms. New England Journal of Medicine 332: Lieberman, L. L. (1958). A method for the estimation of serum cholesterol. Clinical Chemistry 2: Lipid Research Clinics Program. (1984). The lipid research clinics coronary prevention trial results. 1. Reduction in incidence of coronary heart disease. Journal of the American Medical Association 251:
5 Hypocholesterolemic activity of Bacillus coagulans Unique IS2 93 Manson, J. E., Tosteson, H., Ridker, P.M., Satterfield, S., Herbert, P., Conner, G.T.O. (1992). The primary prevention of myocardial infarction. New England Journal of Medicine 326: Mohan C., Arora, R., Khalilullah, M. (1990). Preliminary observation on effect of Lactobacillus sporogenes on serum lipid levels in hypercholesterolemic patients. Indian Journal of Medical Research 92: Richelsen, B., Kristensen, K., Pedersen, S.B. (1996). Longterm (6 months) effect of a new fermented milk product on the level of plasma lipoproteins a placebo-controlled and double blind study. European Journal of Clinical Nutrition 50: Sudha, R. M., Sunita, M. and Sekhar, B. M. (2011). Safety studies of Bacillus coagulans Unique IS-2 in rats : Morphological,Biochemical and clinical evaluations. International Journal of Probiotics and Prebiotics 6: Sudha, R. M., Chauhan, P., Dixit, K., Babu, S. and Jamil, K. (2011). Molecular typing and probiotic attributes of a new strain of Bacillus coagulans Unique IS-2: a potent biotherapeutic agent. Genetic engineering and Biotechnology Journal GEBJ-7. Taylor, G.R.J., Williams, C.M. (1998). Effects of probiotics and prebiotics on blood lipids. British Journal of Nutrition 80: S225 S230. Thompson, L. U., Jenkins, D.J.A., Amer, M. A., Reichert, R., Jenkins, A., Kamulsky, J. (1982). Effect of fermented and unfermented milks on serum cholesterol. American Journal of Clinical Nutrition 36:
6 94 Hypocholesterolemic activity of Bacillus coagulans Unique IS2
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