Effect of feeding synbiotic products on the serum cholesterol, HDL cholesterol and non- HDL cholesterol level of albino rats

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1 B-3505 [1-6] Indian J. Anim. Res., Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Effect of feeding synbiotic products on the serum cholesterol, HDL cholesterol and non- HDL cholesterol level of albino rats Rakesh Kumar*, Binita Rani, Sonia Kumari and A.K. Jha Sanjay Gandhi Institute of Dairy Technology, Patna , Bihar, India. Received: Accepted: DOI: /ijar.B-3505 ABSTRCT Considering the excellent bio-therapeutic benefits of Lactobacillus and Bifidobacterium, this study exploited the probiotic characteristics of Lactobacillus acidophilus-015, Lactobacillus casei-297 and Bifidobacterium bifidum-229 strains with natural prebiotic substances viz. banana powder, malto-dextrin and honey to produce synbiotic food formulations and to study the effect of feeding their on the cholesterol level (mg/dl of albino rats. On the basis of the preliminary investigation, synbiotic products with 2% banana powder, with 2% malto-dextrin, with 3% honey and synbiotic product with 2% each of, banana powder and malto-dextrin and 1% of honey were selected for the investigation of the total cholesterol, HDLcholesterol and non-hdl cholesterol. The products showed a significant decrease in total cholesterol level, however the control group receiving the cholesterol rich laboratory diet showed a significant increase (P<0.01 in the total cholesterol after 45 days of feeding. There was to 45.05% reduction of serum cholesterol was observed in case of feeding synbiotic formulations. Increase in the HDL-cholesterol and decrease in the non-hdl cholesterol were also significant. Therefore, synbiotic formulation with all these three natural prebiotic have enhanced ability to decreased down the blood cholesterol level. Key words: HDL-cholesterol, non-hdl cholesterol, Prebiotics, Synbiotics, Total cholesterol INTRODUCTION Increased industrialization, urbanization and mechanization are being associated with the dramatic changes in perception of health beneficial food which in turn has led to a significant decrease in the occurrence of chronic non-communicable diseases viz: obesity, diabetes-mellitus, cardiovascular diseases, stroke and hypertension and some type of hypocholesterolemic problems. In recent years, a number of new food products having magical impact on human health have been introduced in the world food market with the rapid technological advancement in the field of microbial fermentation technology. So many new terms have appeared in the world food market, e.g., Functional food, Foodiceuticals, Nutraceuticals, Bioceuticals, Juiceceuticals, Therapeutic foods, Pharma foods, Designer foods, Medifoods, Super foods, Bio-foods, Bio-active food, Performance foods, Dietetic foods, Probiotic foods, Prebiotic foods, Power foods, Energy foods, Ethnic foods, Convenience foods, Natural foods, Organic foods, Sportsman foods or in general Health foods. The latest in this chain and the most exciting and revolutionary power food in the world today is Synbiotic food. A number of different probiotic fruit and vegetable juices have been gaining popularity in the last few years (Yoon et al., The hypocholesterolemic effect of fermented milk was first discovered more than 45 years ago during studies conducted in Maasai tribesmen in Africa (Mann et al A number of human clinical studies have also assessed the cholesterol-lowering effects of fermented milk products (Sanders, Elevated levels of certain blood lipids are a risk factor for cardiovascular disease. The observation that conventional animals excrete higher levels of cholesterol in feces than germ-free animals suggests that colonizing microbes may influence cholesterol excretion, with potential implications for serum cholesterol levels (Eyssen, In recent years, probiotics are also gaining importance because of their innumerable benefits, especially treating hypercholesterolemia, atopic dermatitis, eczema, gastrointestinal disorders and angioedema by strengthening the immunological functions (Shovna et al., Studies of the effects of culture-containing dairy products or probiotic bacteria on cholesterol levels have yielded equivocal results (Taylor and Williams, Sahin et. al. (2008 reported that adding a synbiotic supplements in the diet had a significant effect on blood parameters, including total serum protein and cholesterol. Schafsma et al. (1985 also observed that total cholesterol and LDL cholesterol levels could be lowered in humans as a result of administering milk fermented by L. acidophilus with the addition of fructooligosaccharides. It is evident that the market for synbiotic products is booming specially with natural prebiotic added *Corresponding author s rakesh.dt27@gmail.com

2 2 INDIAN JOURNAL OF ANIMAL RESEARCH ingredients. Modern consumers are increasingly interested in their personal health, and expect the food that they eat to be healthy or even capable of preventing illness. Keeping in view the excellent therapeutic benefits of Lactobacillus and Bifidobacterium, the present study was proposed to exploit the probiotic characteristics of Lactobacillus acidophilus, Lactobacillus casei and Bifidobacterium bifidum spp. and their combined effect with natural prebiotic substances viz. banana powder, maltodextrin and honey on Total cholesterol, HDL cholesterol and Non HDL cholesterol level of albino rats. MATERIAL AND METHODS Procurement of standard Probiotic cultures: Three probiotic cultures of lactic acid bacteria viz, Lactobacillus acidophilus NCDC-015, Lactobacillus casei NCDC-297 and Bifidobacterium bifidum NCDC-229, respectively, were procured from the National Collection of Dairy Cultures (NCDC, National Dairy Research Institute, Karnal, (India. Propagation and maintenance of cultures: Lactobacillus acidophilus-015 and Lactobacillus casei-297 were propagated and maintained in litmus milk (Davis,1955 and Bifidobacterium bifidum-229 culture was propagated and maintained in supplemented sterile skim milk (Anand et al.,1984 having the following composition: yeast extract, 0.1g; dextrose, 1g and skim milk 100 ml. This milk were filled up to the neck in standard corning screw capped tube ( mm and sterilized by steaming for 30 min on three consecutive days. All the cultures were propagated by incubating at 37±1 0 C for 48 h. After incubation these culture tubes were preserved at C in refrigerator and transferred once in a week in the respective medium for their reactivation. Procurement of prebiotic: Three natural prebiotic substances banana powder, malto dextrin and honey were selected after preliminary trials. Spray dried banana powder obtained from Dry tech processes Pvt Ltd., Andheri (e Mumbai, Malto dextrin from Gujrat Ambuja Exports Ltd., Navrangpura, Ahmedabad and Honey from Dabur india Ltd, New Delhi. Production of synbiotic fermented dairy products: An attempt was made to develop the methodology for the preparation of synbiotic fermented products (Fig.-1. The preliminary trial for selection of the best proportion of selected culture combination with individual prebiotics for preparing three different synbiotic products and a combination of all three prebiotics for preparing fourth synbiotic product was conducted. The selection of best combination of probiotic culture and natural prebiotic substances for synbiotic products were based on the activity (acid production during incubation at different interval of time, 0, 2, 4, 6, 8, 10 and 12 h, changes in probiotic log count (Lactobacillus and Bifidobacterium of selected culture combination with different proportion of prebiotics at different interval of time, 0, 4, 8, 12 h and the sensory evaluation of all the combination after achieving 0.65% of titrable acidity and cooling the product at refrigeration temperature (7±1 0 C at the same interval of time. Finally four synbiotic products were selected for further investigation viz.: a Synbiotic fermented dairy product with 2% banana powder (SFDP b SFDP with 2% malto-dextrin c SFDP with 3% honey and d SFDP with 2% of banana powder, 2% malto-dextrin and 1% honey Clinical trial: Thirty male albino rats (Wistar albino of five to six weeks age with a body weight ranging from 135 to 140 gm were purchased. All rats were initially given a standard laboratory stock feed (Hindustan Lever Ltd. on ad libitum for one week before the treatment. Rat feeding trials: A minimum of 3 albino rats were used to test each product in each trial. The albino rats were housed individually in stainless steel metallic cages at the temperature 25±1 0 C, 50% RH and 12 h of light. The products were available ad libitum and clean feed containers fresh product were exchanged daily for the prior day s residue, so that the feed consumption could be determined. The products were prepared weekly and stored in refrigerator at 7±1 0 C. Before feeding, the products were kept in water bath at 37±1 0 C for about 1 h to obtain normal temperature of products and then provided to the animal to consume. Except for the laboratory diet, the synbiotic products were the only food available to the albino rats. Distilled water was made available to them round the clock. The control albino rats were given the normal laboratory stock feed (Hindustan Lever Ltd.. The feed without protein was formulated containing 40 g starch, 10 g sucrose, 10 g maltose, 20 g dextrose, 10 g ghee, 2 g sodium chloride and 800 mg vitamin B-complex. The feeding trial was carried up to 45 days and its influence on faecal flora and blood cholesterol was studied. Feeding schedule: Four feeding group was prepared (All the rats were Probiotic combination inoculation in reconstituted fatless skim milk. Group I (control : Laboratory stock feed (LSF Group II (treatment : LSF+ Fatless fermented milk (FLFM. Group III (treatment : LSF + 2% banana powder added fatless synbiotic fermented milk (FSFM. Group IV (treatment : LSF+ 2% malto-dexrin added FSFM Group V (treatment Group VI (treatment : LSF + 3% honey added FSFM. : LSF + 2% each of banana powder, malto-dextrin and 1% honey added FSFM.

3 Vol. Issue, ( reconstituted milk filtration warming up to 45 0 C addition of ingredients (banana 2% / malto 2% 3% in three separate synbiotic products and in forth product 2% banana powder, 2% malto dextrin and 1% honey were used for preparing four types of synbiotic products heat treatment (95 0 C / 30 min cooling (37 0 C Inoculation of cultures L. acidophilus-015; L. casei-297 and B. bifidum-229 2% in all synbiotic formulations separately incubation (37±1 0 C until Acidity reaches 0.65% L.A. cooling at 4±1 0 C to solidify the product and then storage at 7±1 0 C for further investigation Fig 1: Chart for preparation of synbiotic dairy products. Determination of total serum Cholesterol, HDL Cholesterol and Non HDL-Cholesterol: The total cholesterol level was estimated according to the method described by Wybenga and Pileggi (1970 by using the diagnostic kits (Span Diagnostics, Surat. Principle: Cholesterol reacts with hot solution of Ferric Perchlorate, Ethyl acetate and Sulphuric acid (Cholesterol reagent and gives a lavender coloured complex which is measured at 560 nm. The concentration of cholesterol is expressed as mg/dl. Reagents supplied in the kit for cholesterol testing Reagent1 : Cholesterol Reagent. Reagent 2 : Working cholesterol standard, 200 mg% Reagent 3 : Precipitating Reagent Stirring storage of stirred synbiotic products (7±1 0 C Collection of blood sample: Blood samples were collected directly from the heart of control and treatment group of rats on 0, 15, 30 and 45 day of feeding experiment under the sedation of Ketamine hydrochloride (35 mg/kg body weight; I.M. Blood samples were collected in dry test tubes and kept in slanting position for 2-3 h. Serum was separated and stored at C till estimation of cholesterol. Calculation and preparation of data: 3 ml of reagent-1 was taken into blank (B, standard (S and test (T tubes ml of reagent 2 was added into standard tube (S ml serum was taken into test (T tube with the help of micropipette. All the tubes were mixed well and kept immediately in the boiling water bath exactly for 90 Secs and immediately cooled to room temperature under running tap water. Required sample was taken in a cuvette, and Absorbance reading was taken on spectrophotometer at 560 nm. Cholesterol level was measured using the formula as

4 4 INDIAN JOURNAL OF ANIMAL RESEARCH Total Cholesterol (mg/dl = O.D Test O.D Std. 200 Where, O. D Test = Optical Density of required sample O. D Std = Optical Density of standard solution, which was given by manufacturer HDL-cholesterol was estimated by taking 0.2 ml of serum sample was into centrifuge tube and mixed with 0.2ml of precipitating reagent, mixed well and kept at room temperature for 10 min and then centrifuged to obtain a clear supernatant. 3 ml of reagent-1 was taken into blank (B, standard (S and test (T tubes ml (15µl of reagent- 2 was added into standard tube (S. 0.12ml (120µl supernatant was taken into test (T tube with the help of micropipette. All the tubes were mixed well and kept immediately in boiling water bath exactly for 90 Secs and immediately cooled to room temperature under running tap water. Absorbance was measured with a yellow green filter at 560 nm using the formula: Serum/ Plasma HDL-Cholesterol (mg/dl = O.D Test 50 O.D Std. Where, O. D Test = Optical Density of required sample O. D Std = Optical Density of standard solution which was given by manufacturer Where 50 = 8 Determination of non HDL-cholesterol: Non HDLcholesterol was determined by substracting the HDLcholesterol level from the total cholesterol level. Statistical analysis: Results obtained during the present investigation for the effect of individual cultures and culture combination on body weight, total cholesterol, HDLcholesterol and non HDL-cholesterol were analysed using one way Analysis of Variance (ANOVA with the general linear model and univariate data from the statistical Analysis System software package version SPSS 10.0 with the significance level set at P<0.05. The experimental data are presented as the means and standard error of the means. Multiple comparisons have been done by Duncan multiple range tests (Montgomery,1991 which was used to detect differences between treatment means. RESULTS AND DISCUSSION Effect of feeding synbiotic products on the serum cholesterol, HDL-cholesterol and non- HDL cholesterol level (mg/dl of albino rats: The effect of feeding synbiotic products on the total cholesterol, HDL-cholesterol and non- HDL cholesterol in mg/dl was recorded on the 0, 15 th, 30 th and 45 th day interval by analyzing the blood sample of albino rat and the results are expressed as mean ± standard values are depicted in Table-1. There was a significant decrease (P<0.01 in total cholesterol observed during the feeding in case of S 2, S 5 and S 16 but in case of S 9 (product with 3% honey showed a non-significant decrease between 0 and 15 th day, 15 th and 30 th day as well as between 30 th and 45 th day. S 16 product showed a maximum decrease in total cholesterol level followed by S 2, S 5 and S 9 ; however the group-i receiving the cholesterol rich laboratory diet showed a significant increase in the total cholesterol after 45 th days of feeding. The rat group-ii fed with fermented food used as control showed a non-significant decrease of total serum cholesterol up to 30 days of feeding and overall effect was found to be non significant (Table-2. There was 45.05% reduction of serum cholesterol observed in case of feeding synbiotic formulation S 16 followed by 36.51% (S 2, 35.00% (S 5 and 25.81% (S 9. Effect on HDL-cholesterol (mg/dl and non-hdl cholesterol (mg/dl of albino rats: There was proportionate significant increase (P<0.01 in HDL-cholesterol in all the synbiotic products on each interval of analysis. It could be concluded that the amount of HDL-cholesterol was significantly increased (P<0.01 during the feeding of synbiotic products. In all the treatment the initial 0 day HDL-cholesterol level was about % of the corresponding serum cholesterol of the same product on 0 day. There was significant increase (P<0.01 in HDL-cholesterol at all interval with significantly decrease in serum cholesterol (mg/dl. In case of S 2 product the result of HDL-cholesterol on 0 day was 36.04±0.87 mg/dl which was 42% of the serum cholesterol reading (85.97±1.55 mg/dl on the same day. After 45 days of feed in S 2 product a highly significant increase (P<0.01 in the HDL-cholesterol was observed i.e ±1.66 mg/dl which was 53.4% of the corresponding serum cholesterol (59.21±1.02 mg/dl on the 45 th day analysis of the blood sample of albino rat. The maximum increase in the HDL-cholesterol level was observed in case of S 16 product (23.69±0.99. It was increase up to 55.21% of the corresponding reduced total serum cholesterol result (42.92±1.70 mg/dl recorded on the 45 th day of experiment. The increase in the HDL-cholesterol was significantly higher in case of S 16 as compared to other products followed by S 2, S 5, S 9 and control group of albino rats. The reduction observed in HDL-cholesterol level in case of control and S 9 product showed a very negligible effect of honey added synbiotic product in the HDL-cholesterol however the result observed in both the cases on 0 day and 45 th day showed a significant increase (P<0.01 in the HDL-cholesterol level. The mechanism underlying the hypocholesterolaemic activity of lactic acid bacteria have been proposed to involve an inhibition of exogenous cholesterol absorption from small intestine, by binding of the cholesterol and bile acids to the bacterial cells and by the assimilation of cholesterol, as well as by suppressing bile acid resorption by deconjugation as a function of bacterial bile salt hydrolase activity (Xiao, 2003.Various other mechanisms; namely assimilation, binding to surface of cells, incorporation into cellular membrane, and co-precipitation with de-conjugated

5 Vol. Issue, ( Table 1: Effect of feeding synbiotic products on the serum cholesterol, hdl cholesterol Non-HDL cholesterol level mg dl albino rats Type of feed Total cholesterol(mg/dl HDL- cholesterol(mg/dl Non HDL- cholesterol(mg/dl Interval for blood analysis of rat (days P value Interval for blood analysis of rat (days P value Interval for blood analysis of rat (days P value 0 Day 15 Day 30 Day 45 Day 0 Day 15Day 30 Day 45 Day 0 Day 15Day 30 Day 45 Day Laboratory 73.31±.66 c 76.55±.42 bc 80.60±2.31 b 85.04±2.53 a 0.00** 30.60±.48 c 32.47±.42 bc 33.65±.74 b 35.75±.73 a 0.00** 42.71±.19 c 44.17±.37 bc 46.97±.61 b 49.29±1.83 a 0.00** stock feed Control 70.82±3.97 a 65.31±3.55 ab 59.62±3.56 ab 54.58±3.31 b 0.06 NS 29.73±1.67 a 28.73±1.50 a 27.54±1.53 a 25.92±1.66 a 0.24 NS 41.08±2.30 a 36.58±2.05 b 32.08±2.03 b 28.66±1.66 b 0.01** Product with 85.97±1.55 a 75.60±1.39 b 66.67±1.29 c 59.21±1.02 d 0.00** 36.04±.87 b 34.24±.80 a 33.06±.56 b 31.62±.55 c 0.00** 49.93±.68 a 41.36±.60 b 33.61±.70 c 27.59±.48 d 0.00** 2% banana powder( S 2 Product with 73.93±1.39 a 64.41±1.39 b 55.80±1.29 c 48.05±1.34 d 0.00** 30.87±.69 b 29.05±.79 a 27.06±.72 b 24.84±.72 c 0.00** 43.06±.70 a 35.36±.60 b 28.74±.60 c 23.21±.59 d 0.00** 2% maltodextrin( S 5 Product with 76.0±2.54 a 69.31±2.50 ab 63.30±2.33 bc 56.38±1.69 c 0.00** 31.78±1.04 b 30.35±1.26 a 29.56±1.14 ab 27.62±.84 b 0.00** 44.24±1.52 a 38.96±1.23 b 33.74±1.19 bc 28.76±.84 c 0.00** 3% honey ( S 9 Product with 78.12±.90 a 63.68±.32 b 54.19±1.49 c 42.92±1.70 d 0.00** 33.06±.33 a 29.67±.14 a 28.12±.86 b 23.69±.99 c 0.00** 45.06±.93 a 34.01±.19 b 26.07±.63 c 19.23±1.70 d 0.00** 2% each of banana powder and malto -dextrin and 1% honey ( S 16 * Products with selected culture combination (C 1 +C 2 +C 3, 2% and selected proportion of prebiotics were used for feeding rats after achieving 0.65% LA; C =Lactobacillus acidophilus-015; 1 C 2 = Lactobacillus casei-297 and C 3 = Bifidobacterium bifidum-229 Cultures were incubated at 37±1 0 C in skim milk (95 0 C/30 min ** Highly significant (P < 0.01; NS- Non-significant Similar superscript marked groups are non-significant and dissimilar superscripts are significant at the level of P < 0.01.

6 6 INDIAN JOURNAL OF ANIMAL RESEARCH bile Liong and Shah, 2005.a,b may also be involved. Other important mechanisms about the above result may be due to enzymatic de-conjugation of bile acids by bile-salt hydrolase of probiotics. Bile, a water-soluble end product of cholesterol in the liver, is stored and concentrated in the gallbladder, and released into the duodenum upon ingestion of food ( Begley, It consists of cholesterol, phospholipids, conjugated bile acids, bile pigments and electrolytes. Once deconjugated, bile acids are less soluble and absorbed by the intestines, leading to their elimination in the feces. Cholesterol is used to synthesize new bile acids in a homeostatic response, resulting in lowering of cholesterol. Similar explanation was given by Gilliland et al., 1985; Klaver and Meer, 1993; Kawase et al., 2000; Oyetayo et al., 2003.The explanation of S 16 having maximum anticholesterilemic activity may be due to presence of dietary fiber which enhances the activity of probiotic to lower down the cholesterol level. Liong and Shah (2006 Showed that the combination of L. casei ASCC 292, FOS, and maltodextrin is beneficial for pathological cholesterol levels. Adebawo et al., (2008 also noted that L.acidophilus was suitable as starter culture for the production of indigenous maize based fermented food products which might serve as medium for the delivery of L. acidophilus as probiotic for hypocholesterolemic effect. CONCLUSION S 16 synbiotic formulation with 2% banana powder, 2% malto-dextrin and 1% honey which showed significantly decrease (P<0.01 in total cholesterol and non-hdl cholesterol can be recommended for improving gastrointestinal environment as well as controlling blood cholesterol level. REFERENCES Adebawo, O.O., Banjoko, Y. and Osilesi,T. (2008. Evaluation of the hypochol-esterolemic effect of Lactobacillus acidophilus fermented maize meal (ogi in rats. The FASEB Journal 22: Anand, S.K., Srinivasan, R.A. and Rao, L.K. (1984. Antimicrobial activity associated with Bifidobacterium bifidum. Cultured Dairy Product Journal 20: Begley,M., Hill, C., Gahan,C.G.M (2006 Bile salt hydrolase activity in probiotics. Appllied. Environmantal. Microbiology. 72: Davis, J.G. (1955 Dictionary of Dairying 2 nd Ed..Leonard Hill Ltd. London pp Eyssen, H. (1973 Role of gut microflora in metabolism of lipids and sterols. Proceding of Nutrition Society 32: Gilliland, S.E., Nelson,C.R..and Maxwell, C. 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