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The hypocholesterolaemic effect of milk yoghurt and soy-yoghurt containing bifidobacteria in rats fed on a cholesterol-enriched diet
ARTICLE IN PRESS
International Dairy Journal 15 (2005) 37–44 www.elsevier.com/locate/idairyj
The hypocholesterolaemic effect of milk yoghurt and soy-yoghurt containing bifidobacteria in rats fed on a cholesterol-enriched diet Ibrahim A. Abd El-Gawada,, E.M. El-Sayeda, S.A. Hafezb, H.M. El-Zeinia, F.A. Salehb a
Faculty of Agriculture, Dairy Science and Technology Department, Cairo University, P.O. Box 12613, Giza, Egypt Special Food & Nutrition Department, Food Technology Research Institute, Agriculture Research Centre, Giza, Egypt
b
Received 4 February 2004; accepted 3 June 2004
Abstract The effect of buffalo milk yoghurt and soy-yoghurt supplemented with Bifidobacterium lactis Bb-12 or Bifidobacterium longum Bb-46 on plasma and liver lipids and the faecal excretion of bile acids was determined in rats fed on a cholesterol-enriched diet. The groups fed on a cholesterol-enriched diet supplemented with yoghurt and soy-yoghurt containing Bb-12 or Bb-46 had significantly lower levels of plasma total cholesterol and very low-density lipoprotein (VLDL)+low-density lipoprotein (LDL) cholesterol than the positive control group (without supplementation). Yoghurt or soy-yoghurt containing Bb-46 was more effective in the lowering of plasma and liver cholesterol levels than yoghurt or soy-yoghurt containing Bb-12. Furthermore, the faecal excretions of bile acids were markedly promoted in yoghurt and soy-yoghurt containing Bb-12 and Bb-46 groups compared with the positive control group. The results showed also an inverse relationship between the faecal excretions of bile acids and the levels of total cholesterol in blood plasma from rats fed on a cholesterol-enriched diet with probiotic supplementation. r 2004 Elsevier Ltd. All rights reserved. Keywords: Yoghurt; Soy-yoghurt; Bifidobacteria; Cholesterol; Bile acids
1. Introduction Hypercholesterolaemic is a major risk factor associated with coronary heart diseases, and it is considered that keeping blood cholesterol at a desirable level is one of the major preventive strategies for these diseases. Thus, much attention has been given to the relationship between diet and blood cholesterol levels. Fermented milk products have been recommended as dietary supplements because of their hypocholesterolaemic effect in humans (Mann, 1977) and rats (Suzuki, Kaizu, & Yamauchi, 1991). Since 1986, traditional yoghurt cultures have been supplemented or replaced by Bifidobacterium species to enhance the therapeutic value of yoghurt and to establish its market as a Corresponding author. Tel./fax: +20-27743824.
E-mail address: [email protected] (I.A. Abd El-Gawad). 0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.06.001
functional therapeutic food. Therefore, incorporation of bifidobacteria in various dairy products has become a recent trend (Kailasapathy & Rybka, 1997). On the other hand, although most probiotic foods are derived from milk, the possibility of using other proteinrich substrates such as soymilk to make such foods has not been adequately considered. The potential role of dietary soy in the prevention and treatment of chronic diseases, in particular heart disease, has been recognized for a long time (Nagata, Ishiwaki, & Sugano, 1982). Recently, soy-yoghurt has been prepared from soymilk fermented with lactic acid bacteria or bifidobacteria (Abd El-Gawad, Hefny, El-Sayed, & Saleh, 1998; Kikuchi-Hayakawa et al., 1998b; Chou & Hou, 2000). Also, it was reported that soymilk fermented with Bifidobacterium significantly decreased the levels of total plasma cholesterol and very low-density lipoprotein (VLDL)+low-density lipoprotein (LDL)-cholesterol in rats (Kikuchi-Hayakawa et al., 1998a). Though,
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numerous studies have focused on the health effect of yoghurt, relatively little is yet known regarding the potential role of the health-promoting effects of yoghurt and soy-yoghurt containing bifidobacteria by reducing cholesterol. Therefore, the objective of this investigation was to study the effects of yoghurt and soy-yoghurt containing Bifidobacterium Bb-12 or Bb-46 on the levels of plasma and liver lipids in hypercholesterolaemic rats and the levels of bile acid in their faeces.
2. Materials and methods 2.1. Preparation of yoghurts from buffalo milk Low-fat buffalo milk (1.5%, w/w) was inoculated with 3% (v/v) of Lactobacillius dulbrueekii sub sp. bulgaricus and Streptococcus salivarius sub sp. thermophilus (Chr. Hansen Laboratories, Copenhagen, Denmark), and divided into three portions. One portion (without added bifidobacteria) served as a control yoghurt and was denoted as YBM; a second portion was inoculated at a level of 0.07% (w/v) with a freezedried Bifidobacterium lactis Bb-12 and served as an experimented yoghurt and was denoted as YBMBb-12 yoghurt. The third portion was inoculated at a level of 0.07% (w/v) with a freeze-dried Bifidobacterium longum Bb-46 and served as an experimented yoghurt and was denoted as YBMBb-46 yoghurt. The two strains of bifidobacteria were obtained from Chr. Hansen Laboratories (Copenhagen, Denmark). Following incubation YBM, YBMBb-12 and YBMBb-46 yoghurts were stored at 471 1C. 2.2. Preparation of non-fermented soymilk and soyyoghurts Fresh and non-beany flavoured soymilk was prepared according to the method of Tanteeratarm, Nelson, and
Wei (1993), and divided into three portions. One portion served as a non-fermented soymilk and was denoted NFSM; the other two portions were used for preparation of soymilk yoghurts Bb-12 and Bb-46 according to the method described by El-Sayed, Hefny, Saleh, and Abd El-Gawad (1998). The manufacture of soymilk yoghurts Bb-12 and Bb-46 involved the addition of gelatin at a level of 1% (w/w) to the milk, heating at 95 1C for 5 min, and cooling to 37 1C. One portion was inoculated at a level of 0.07% (w/v) with a freeze-dried B. lactis Bb-12 and was denoted as YSMBb-12 yoghurt; the other portion was inoculated at a level of 0.07% (w/ v) with a freeze-dried B. longum Bb-46 and was denoted as YSMBb-46 yoghurt. After inoculation at 37 1C for 4–5 h, both portions were stored at 471 1C. 2.3. Animal feeding experiments Forty-eight male albino rats with an average weight of between 80 and 100 g were housed in cages with wiremesh floors in a room at 2371 1C and 6075% relative humidity. All animals were fed on a basal diet for one week. After this adaptation period, the rats were divided randomly into 8 experimental groups of 6. One group received a basal diet (cholesterol-free diet) throughout the experimental period of six weeks and served as a negative control group. The other seven groups were fed on the basal diet with cholesterol added at a level of 0.5% (w/w) (cholesterol-enriched diet) for 1–2 weeks to create hypercholesterolaemic rats. One of the 7 groups, which served as a positive control group, was fed only on a cholesterol-enriched diet for the six-week period. The other six groups were fed for five weeks on a cholesterol-enriched diet supplemented with one of the following products: YBM, YBMBb-12, YBM Bb-46, NFSM, YSMBb-12 or YSMBb-46. The experimental diets given to the eight groups are described in Table 1. The rats were allowed free access to experimental diet and water, and their body weights were monitored. At
Table 1 Experimental groups of rats and diets used in the triala Diet treatment
Diet code
Diet formulae
Cholesterol-free diet Cholesterol-enriched diet (CED) CED+yoghurt from buffalo milk (YBM) CED+YBM with added B. lactis Bb-12 (YBMBb-12) CED+YBM with added B. longum Bb-46 (YBMBb-46) CED+non-fermented soymilk (NFSM) CED+yoghurt from soymilk with added B. lactis Bb-12 (YSMBb-12) CED+yoghurt from soymilk with added B. longum Bb-46 (YSMBb-46)
Negative control Positive control YBM YBMBb-12 YBMBb-46 NFSM YSMBb-12
100 g basal diet+50 mL water 99.5 g basal diet+0.5 g cholesterol+50 mL water 99.5 g basal diet+0.5 g cholesterol+50 g YPM yoghurt 99.5 g basal diet+0.5 g cholesterol+50 g yoghurt YPM Bb-12 99.5 g basal diet+0.5 g cholesterol+50 g yoghurt YPM Bb-46 99.5 g basal diet+0.5 g cholesterol+50 g NFSM 99.5 g basal diet+0.5 g cholesterol+50 g soy-yoghurt YSM Bb-12
YSMBb-46
99.5 g basal diet+0.5 g cholesterol+50 g soy-yoghurt YSM Bb-46
a
The basal diet consisted of : 15%, (w/w), casein; 10%, (w/w), maize oil; 10%, (w/w), cellulose; 4%, (w/w), mineral mix; 1%, (w/w), vitamin mix and 60%, (w/w), starch.
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the end of the 6-week experimental period, blood samples were collected from the eye vein under diethyl ether anesthesia. The samples were collected in tubes, with EDTA as an anti-coagulating agent. The tubes were centrifuged at 3000 rpm for 15 min to obtain the plasma, which was kept frozen ( 2371 1C) until analysis. Faeces were collected on the last 2 days of the feeding period and kept frozen (2371 1C) until analysed for bile acids. The rats were sacrificed and the liver, heart, kidney and spleen were excised immediately and weighed. The liver was washed with ice-cold saline solution (0.9%, w/v, NaCl) and stored at 2371 1C until analysed.
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S. salivarius sub sp. thermophilus) were enumerated according to the method of Lee, Vedamuthu, Washam, and Reinbold (1973) in which the yoghurt sample was added to Lee’s agar and incubated at 43 1C for 3 days. Table 2 shows the viable count of bifidobacteria and yoghurt culture in the experimental products. 2.4.2. Determination of plasma lipid Total plasma cholesterol, high-density lipoprotein (HDL) and triglycerides were determined using the enzymatic colourimetric method according to Trinder (1969). The VLDL+LDL was calculated as follows: VLDL+LDL cholesterol=total cholesterolHDL cholesterol.
2.4. Analytical methods 2.4.1. Counts of bifidobacteria and yoghurt cultures The count of bifidobacteria in soy-yoghurts YSMBb12 and YSMBb-46, which did not contain yoghurt culture, was enumerated by poured plate method on Lactobacilli MRS-agar medium containing 0.05%, w/v, L-cycteine-HCl according to the method described by Samona and Robison (1991). The count of bifidobacteria in buffalo milk yoghurts (YBMBb-12 and YBMBb46), which contain yoghurt culture as well as bifidobacteria, was enumerated according to the method of Dinakar and Mistry (1994), in which a mixture of antibiotics, including 2 g paromomycin sulphate, 0.3 g nalidixic acid, and 60 g lithium chloride, was dissolved in 1 L distilled water, filter-sterilised (0.2 mm) and stored at 4 1C until use. The antibiotic mixture (5 mL) was added to 100 mL MRS-agar medium. L-Cysteine-HCl 0.5% (w/ v) (Sigma Chemical Co., St Louis, MO, USA) was also added to decrease the redox potential of the medium. Plates were incubated at 37 1C for 48 h anaerobically. Yoghurt cultures (L. dulbrueekii sub sp. bulgaricus and
Table 2 The viable count of yoghurt culture and bifidobacteria in experimental treatmentsa,b,c Diet treatmentd
YBM YBMBb-12 YBMBb-46 NFSM YSMBb-12 YSMBb-46 a
Microbial count ( 108 cfu mL1) Yoghurt culture
Bifidobacteria
88.7 57.4 43.7 ND ND ND
NDe 1.9 1.7 ND 4.1 5.8
The data presented are the means of three replicate trials. Yoghurt culture: L. dulbrueekii sub sp. bulgaricus and S. salivarius sub sp. thermophilus. c B. lactis Bb-12 or B. longum Bb-46. d See Table 1 and text for details of diet treatments. e ND: not determined. b
2.4.3. Determination of cholesterol and triglycerides in liver Cholesterol and triglycerides were extracted from the liver by the method of Fernandez et al. (1997), and were measured by the method of Trinder (1969). 2.4.4. Determination of bile acids in faeces Extraction of bile acids from faeces was carried out according to the method of Kikuchi-Hayakawa et al. (1998b). The level of extracted bile acids was determined according to the method of Roda, Piazza, and Baraldini (1998), using a Hewlett Packard HPLC (Series 1050, HP, USA) equipped with an autosampling injector, solvent degasser, ultra-violet (UV) detector, and quarter HP pump (Series 1050, HP, USA). Thirty five microlitres of sample extracted was injected into the Sphrosorb BDS C18 column (250 mm 4.6 mm, ID 5 mm particle size). The operating parameters were set as follows: column temperature, 35 1C; flow rate 1.0 mL/ min; UV-detector, 210 nm. The isocratic elution was with methanol/acetonitryl/0.01% M phosphate buffer pH 5.3 (50/25/25, v/v/v) as a mobile phase. The standard bile acids used were cholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid (Sigma Chemical Co., St Louis, MO, USA). Each sample was injected twice. The peak area of bile acids standard solution was used to prepare a standard curve. From these standard curves, the concentration of bile acids in the test samples were calculated. 2.5. Statistical analysis Data are presented as means and standard deviation. The significance differences between groups/treatments were evaluated using the general linear model procedure of Statistical Analysis Systems (SAS) (1990) (SAS Institute, Inc., Cary, NC, USA) to analyse the biological examination data by least significant difference (LSD) at Po0:05.
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3. Results and discussion 3.1. Plasma and liver lipids The effect of the experimental diets on the levels of plasma and liver lipids in rats are presented in Table 3. 3.1.1. Plasma lipids 3.1.1.1. Total cholesterol. The total concentration of plasma cholesterol was significantly reduced from 301 mg 100 mL1 in the positive control to a mean value of 155 mg 100 mL1 in both YBMBb-12 and YBMBb-46 groups and to a mean value of 189 mg 100 mL1 in both YSMBb-12 and YSMBb-46 groups. This decrease corresponds to a 48.7% and 37.3% reduction, respectively. In contrast, there was no significant difference in the total level of plasma cholesterol detected between YBMBb-12, YBMBb-46, YSMBb-12, YSMBb-46, and negative control groups (Table 3). It can also be seen that the total levels of plasma cholesterol in the NFSM and YBM groups were 23% and 33% lower than that in the positive control group, respectively. While the cholesterol-enriched diet supplemented with YBMBb-12, YBMBb-46, YSMBb-12 or YSMBb-46 was more efficient than the corresponding diet supplemented with YBM or NFSM for decreasing total plasma cholesterol, none of these differences were significant (Table 3). The diet supplemented with YBMBb-46 or YSMBb-46 was more effective in reducing plasma cholesterol than the corresponding diet supplemented with YBMBb-12 or YSMBb-12. The YBMBb-12 and YBMBb-46 diets were more effective at lowering total cholesterol than the diets of YSMBb12 and YSMBb-46. The YBM diet was more effective in reducing the total cholesterol level than the NFSM diet.
Beena and Prasad (1997) found lower serum cholesterol in rats fed on yoghurt containing Bifidobacterium bifidum (120 mg 100 mL1) compared to a positive control (172 mg 100 mL1) after 30 days. However, the administration of fermented milk containing bifidobacteria (109 cfu g1) to hypercholesterolaemic human subjects resulted in a decrease in the total cholesterol level from 300 to 150 mg 100 mL1 (Homma, 1988). Schaarmann, Schneider, Zorn, Vilser, and Jahreis (2001) reported that the consumption of the probiotic yoghurt made by B. longum and Lactobacillus acidophilus decreased total cholesterol in hypercholesterolaemic women from 293 mg 100 mL1 at the beginning of the experiment to 255 mg 100 mL1 after 153 days. KikuchiHayakawa et al. (1998b) reported that consumption of bifidobacterium fermented soymilk significantly reduced plasma cholesterol, whereas consumption of soymilk resulted in a non-significant decrease in the bloodcholesterol level in hamsters. These results were confirmed also by Sirtori et al. (1999) and Onning, Akesson, Oste, and Lundquist (1998).
3.1.1.2. HDL-cholesterol. As shown in Table 3, there was no significant difference in the plasma HDLcholesterol level between the negative control group and other experimental groups at the end of the 6-week experimental period. Endo, Nakano, Shimizu, Fukushima, and Miyoshi (1999) reported that the addition of the probiotic to the diet had no effect on the HDLcholesterol level. Our data agree well with this finding. Beena and Prasad (1997) found that the bifidus yoghurt fortified with lactose-hydrolysed condensed whey had little effect on HDL-cholesterol, whereas KikuchiHayakawa et al. (1998b) reported that consumption of both soymilk and Bifidobacterium-fermented soymilk
Table 3 Levels of plasma and liver lipids in rats fed on experimental diets for six weeksa Diet treatment
Negative control Positive control YBM YBMBb-12 YBMBb-46 NFSM YSMBb-12 YSMBb-46 a
Plasma
Liver
Total cholesterol (mg 100 mL1)
Triglycerides (mg 100 mL1)
HDLcholesterol (mg 100 mL1)
VLDL+LDLcholesterolb (mg 100 mL1)
Atherosclerotic indexc (mg 100 mL1)
Cholesterol (mg g1)
Triglycerides (mg g1)
127.4720.1d 301.4716.1f 201.5720.9d,i 159.6726.3d,i 149.9714.9d 231.7754.9f,i 1 95.1717.4d,i 183.074.3d,i
40.975.5e 105.7712.5f 86.572.7f,i 65.170.6d,e,i 51.678.9d,e 69.7712.4d,i 64.6712.0d,e,i 40.377.8e
38.172.3f 24.874.3f 16.672.1f 20.671.9f 29.171.8f 22.4710.2f 31.4710.2f 33.6712.2f
89.371.1g 276.670.6f 184.970.8d 139.071.2j 120.970.1k 209.470.5i 163.771.3e 149.470.9h
2.470.1h 11.170.1f 11.270.2f 6.970.2d 4.270.3e 9.470.1e 5.270.4e 4.470.6e
2.8870.0d 7.3470.1f 2.3570.0e 2.2470.2e 2.2270.1e 4.5370.1i 1.7770.1h 2.3370.1e
17.7870.5i 25.1871.1f 17.6370.1i 17.8971.5i 21.0070.1fi 18.4371.0i 18.5073.4i 16.4570.4i
See Table 1 and text for details of diet treatment. VLDL: Very low-density lipoprotein-cholesterol; LDL: low-density lipoprotein-cholesterol; VLDL+LDL-cholesterol=Total cholesterolHDLcholesterol. c Atherosclerotic index=(VLDL+LDL-cholesterol)/(HDL-cholesterol). d–k Mean values (7SD; n ¼ 6) within the same column bearing different superscripts are significantly different (P40.05). b
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increased the HDL-cholesterol level in hamsters fed on a cholesterol-enriched diet. 3.1.1.3. VLDL+LDL-cholesterol. Rats fed on YBM, YBMBb-12, YBMBb-46, NFSM, YSMBb-12 or YSMBb-46 diet had significantly lower plasma VLDL+LDL-cholesterol than the positive control group of rats. The YBMBb-12, YBMBb-46, YSMBb12 and YSMBb-46 diets were more effective in lowering plasma VLDL+LDL-cholesterol levels than the NFSM and YBM diets (Table 3). Also, it is noteworthy from the same table that the plasma VLDL+LDL-cholesterol levels in the rats fed the YBMBb-46 and YSMBb-46 diets (120.86 and 149.39 mg 100 mL1) were markedly lower than those in the rats fed on the YBMBb-12 and YSMBb-12 diets (163.67 and 138.99 mg 100 mL1, respectively). Beena and Prasad (1997), found that yoghurt containing B. bifidum markedly lowered the levels of LDLcholesterol in rats fed on a cholesterol-enriched diet (positive control) from 97 mg 100 mL1 to 22, 16 and 15 mg 100 mL1, respectively, in rats fed on yoghurt containing B. bifidum fortified with skimmed milk, condensed whey or lactose-hydrolysed condensed whey at the end of the 30-day experimental period. Schaarmann et al. (2001) reported that the consumption of probiotic yoghurt made using B. longum and L. acidophilus reduced the LDL-cholesterol level in normocholesterolaemic and cholesterolaemic women. Kikuchi-Hayakawa et al. (2000) studied the effect of soymilk and fermented soymilk on lipid metabolism in ovariectomized Syrian hamsters. They found that soymilk fermented by Bifidobacterium breve was more effective than soymilk in reducing the level of VLDL+LDL-cholesterol (77.5 and 86.2 mg 100 mL1, respectively) when compared with the control group fed on a basal diet (89.2 mg 100 mL1). 3.1.1.4. Atherosclerotic index. The data in Table 3 indicate that feeding YBMBb-12, YBMBb-46, YSMBb12 or YSMBb-46 significantly decreased the values of atherosclerotic index of rats by 37.8%, 62.7%, 53.2% and 60.1%, respectively, below that in rats fed the cholesterol-enriched diet (positive control group). For rats fed on cholesterol-enriched diets (Table 3), the YBMBb-12, YBMBb-46, YSMBb-12 and YSMBb46 diets were more effective in reducing the atherosclerotic index values than the YBM and NFSM, which did not contain bifidobacteria. These findings are in agreement with those reported by Kikuchi-Hayakawa et al. (1998b, 2000). 3.1.1.5. Triglycerides. The levels of plasma triglycerides in rats fed on YBMBb-12, YBMBb-46, YSMBb-12, YSMBb-46 or NFSM diets were significantly lower than in those fed the cholesterol-enriched diet (positive
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control). The reduction in triglyceride levels with the above-mentioned groups was 38.4%, 51.2%, 38.9%, 61.8% and 34.0%, respectively (Table 3). The level of plasma triglycerides in the YBM group was also lower than that in the positive control group, but not significantly so. It is noteworthy from the data in Table 3 that the YBMBb-46 and YSMBb-46 diets were more effective in lowering plasma triglycerides than the YBMBb-12 and YSMBb-12 diets. Schaarmann et al. (2001) reported that the consumption of probiotic yoghurt made with B. longum and L. acidophilus lowered triglycerides in hypercholesterolaemic women from 114 mg 100 mL1 to 91 mg 100 mL1 after 153 days. However, KikuchiHayakawa et al. (2000) reported that feeding ovariectomised hamsters on fermented soymilk decreased the triglycerides from 232 mg 100 mL1 in the control to 180 mg 100 mL1 after four weeks. The results in Table 3 also suggest that the NFSM diet significantly decreased the level of plasma triglycerides, but the YSMBb-12 and YSMBb-46 diets were more effective in reducing plasma triglycerides. While the YBM diet resulted in a non-significant reduction in plasma triglycerides, the YBMBb-12 and YBMBb-46 diets significantly reduced the level of plasma triglycerides. 3.1.2. Liver lipids 3.1.2.1. Liver cholesterol. The data in Table 3 show that the content of liver cholesterol in the positive control group (7.34 mg g1) was significantly higher than that in the negative control group (2.88 mg g1). There was a significant difference between rats fed on the positive control diet and those fed on YBM, YBMBb12, YBMBb-46, YSMBb-12, YSMBb-46 or NFSM diets (Table 3). It can be observed from the data in Table 3 that the YBMBb-12, YBMBb-46, YSMBb-12 and YSMBb-46 diets were more effective in lowering the levels of liver cholesterol than YBM and NFSM diets. These results are in agreement with those reported by Kheadr, Abd El-Rahman, Abd El-Soukkary, and Abd El-Rahman (2000) who suggested that the yoghurt diets supplemented with B. bifidum reduced cholesterol in rat liver tissues. Kikuchi-Hayakawa et al. (1998b) found that the total level of liver cholesterol in hamsters fed on a cholesterol-free diet, soymilk or soymilk fermented with B. breve, were lower than that in the control hamsters fed on basal diet. 3.1.2.2. Liver triglycerides. The content of liver triglycerides was significantly higher in rats fed on the positive control diet than those fed the negative control diet (Table 3). In contrast, the levels of liver triglycerides in rats fed the experimental diet groups were significantly lower than in rats fed on the positive control diet, except for rats fed the YSMBb-46 diet for which the decrease
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was not significant. Kikuchi-Hayakawa et al. (1998a) reported that the contents of liver triglycerides in ovariectomized rats fed on both soymilk and Bifidobacterium-fermented soymilk were lower than that of the control group of rats fed on a basal diet. Kikuchi-Hayakawa et al. (1998b) concluded that the content of liver triglycerides was not affected by soymilk or Bifidobacterium-fermented soymilk in hamsters fed either cholesterol-free diet or a cholesterol-enriched diet. On the contrary KikuchiHayakawa, et al. (2000) found that soymilk fermented with Bifidobacterium had a hypolipidaemic effect in ovariectomised hamsters.
3.2. Faecal bile acid excretion As shown in Table 4, there was no significant difference in the levels of total bile acids between rats fed the negative control and positive control diets. Rats fed on YBM, YBMBb-12, YBMBb-46, NFSM, YSMBb-12 or YSMBb-46 diets excreted significantly higher level of bile acids than those fed either the positive or negative diets. The excretion of bile acids in rats fed on the YBMBb12, YBMBb-46, YSMBb-12 or YSMBb-46 diets was significantly higher than those fed on YBM or NFSM diets. The ratio of cholic acid to chenodeoxycholic acid in rats fed on the YBMBb-12, YBMBb-46, YSMBb-12 or YSMBb-46 diets (6.7:1–9.7:1) was higher than that in rats fed on the negative (5.8:1) or positive (6.1:1) control diets (Table 4). In contrast, the corresponding ratios in rats fed on the YBM or NFSM groups were 6.1:1 and 6.5:1, respectively (Table 4). Metabolism of bile acids, especially the change in the ratio of cholic to chenodeoxycholic acids in bile or faeces, has been associated with cholesterol 7a-hydroxylase deficiency (Schwarz et al., 1996). Uchida, Nomura, and Takeuchi (1980) suggested that cholic acid, but not chenodeoxycholic acid, promotes absorption of cholesterol. Therefore, an
increase in the ratio of cholic acid to chenodeoxycholic acid may promote cholesterol absorption. The data in Tables 3 and 4 showed an inverse relationship between the level of bile acids excreted in the faeces and the total levels of plasma cholesterol or VLDL+LDL-cholesterol in rats fed on the YBMBb-12, YBMBb-46, YSMBb-12 and YSMBb-46 diets. These findings are in accordance with those found by KikuchiHayakawa et al. (1998b). However, the study of Nagata et al. (1982) showed that feeding soybean protein increased the excretion of faecal bile acids in rats. This finding and the results of our study, suggest that hepatic cholesterol metabolism could change in order to provide more cholesterol for synthesis of bile acids. Imaizumi, Hirata, Zommara, Sugano, and Suzuki (1992) and Suzuki (1995) found that Bifidobacterium species stimulated both the secretion of bile acids and the activity of cholesterol 7ahydroxylase, a rate-limiting enzyme in the synthesis of bile acids. Furthermore, the fermentation of soymilk by Bifidobacterium spp. resulted in compositional changes in isoflavones, phytate and saponins (Anderson & Wolf, 1995). Bifidobacteria cells can remove cholesterol from the growth culture medium through both assimilation and coprecipitation with deconjugated bile salts, as found in an in vitro study by Tahri, Crociani, Ballongue, and Schneider (1995). Consequently, bifidobacteria caused an increase in the excretion of free bile salts in the faeces (Chikai, Nakao, & Uchida, 1987; Dambekodi & Gilliland, 1998). 3.3. Body weight gain and organs rats weight The value of body weight gain was significantly higher in rats fed YBMBb-12, YBMBb-46 or YSMBb-12 than that in rats fed the negative control diet. The body weight gain in rats fed the YSMBb-46 diet was nonsignificantly higher than that in rats fed the negative control diet (Table 5). These results are in agreement with those reported by Kikuchi-Hayakawa et al.
Table 4 Excretion of bile acids in faeces in rats fed on experimental diets for six weeksa,b Diet treatment
Cholic acid (mg 100 g1)
Chenodeoxycholic acid (mg 100 g1)
Deoxycholic acid (mg 100 g1)
Lithocholic acid (mg 100 g1)
Total bile acids (mg 100 g1)
Negative control Positive control YBM YBMBb-12 YBMBb-46 NFSM YSMBb-12 YSMBb-46
205.970.7c,d 201.770.6d 240.271.6c,e 272.472.3e,h 285.271.9g,h 243.273.8e 268.270.8e,h 283.670.4g
35.471.0c,e 33.372.5c 39.471.2g,h 28.971.2d 29.370.5d 37.371.3e,h 38.171.2e,h 42.671.2g
18.970.8e 18.271.1c,e 16.471.0c 21.370.4h 21.171.1h 21.570.8h 20.170.7e,h 26.371.6g
9.970.6d 16.070.5c 17.170.5c 19.771.2e 30.971.2g 22.070.8h 18.171.4c,e 24.170.8h
270.171.2f 269.171.2f 313.174.1d 342.372.4e 366.573.5h 324.071.6c 344.571.5e 376.573.1g
a
Values are the means for three replicate experiments. See Table 1 and text for details of diet treatments. c–h Mean values (7SD; n ¼ 6) within the same column bearing different superscripts are significantly different (P40:05). b
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Table 5 Body weight gain and organ weight of rats fed on experimental diets for six weeksa Diet treatment
Negative control Positive control YBM YBMBb-12 YBMBb-46 NFSM YSMBb-12 YSMBb-46
Body weight gain (%)b
29.975.3c 48.8715.0c,f 50.2723.1c,e,f 51.672.5e,f 71.376.3e 37.173.7f,c 52.3715.0e,f 47.7710.4c,f
Organ weight (g) Liver
Heart
Kidney
Spleen
3.970.2d 7.970.3e 5.570.6c 5.670.4c 5.870.7c,f 7.370.8e,f 5.570.5c,d 5.870.5c,f
0.770.0c,e,f 0.870.1c,e,f 0.870.1e,f 0.970.1e 0.670.0c 0.970.1e 0.670.1c,f 0.970.0e
0.970.0c 1.370.1e 0.970.0c,f 1.070.1c,f 1.070.1c,f 1.170.0f 1.070.1c,f 1.070.1c,f
0.370.1e 0.470.0e 0.370.1e 0.470.1e 0.570.1e 0.470.1e 0.370.0e 0.470.0e
a
See Table 1 and text for details of diet treatments. % Body weight gain =[(final weightinitial weight)/initial weight] 100. c–f Mean values (7SD; n ¼ 6) within the same column bearing different superscripts are significantly different (P40:05). b
(1998b). Suzuki et al. (1991) found no difference in body weight gain between rats fed skim milk and rats fed skim milk fermented with L. acidophilus or bifidobacteria. No significant differences in the weights of the heart or spleen were detected between rats fed on the YBMBb-12, YBMBb-46, YSMBb-12 or YSMBb-46 diets and rats fed the two control diets. There was a significant difference in kidney weight between rats fed the positive control diet and rats fed the other diets. The liver weight in rats fed the positive control diet was significantly higher than that in rats fed the negative control diet. There was a significant difference in the liver weight between rats fed the positive control diet and rats fed the other experimental diets. However, the difference between rats fed on the positive control diet and rats fed on NFSM diet was not significant. Similar results were reported by Mohan, Kadirvel, Natarajan, and Bhaskaran (1996).
4. Conclusions This study has demonstrated that the inclusion of buffalo milk yoghurt, soymilk yoghurt (with or without bifidobacteria) or non-fermented soymilk in the diet of rats fed a cholesterol-enriched diet had a marked affect on the levels of plasma and liver lipids. Buffalo milk yoghurt and soymilk yoghurt containing Bifidobacterium Bb-12 or Bb-46 were very effective in lowering the levels of plasma and liver lipids in rats. Buffalo milk yoghurt or soymilk yoghurt containing Bifidobacterium Bb-46 was more affective in reducing the level of plasma and liver cholesterol than buffalo milk yoghurt or soymilk yoghurt containing Bifidobacterium Bb-12. Buffalo milk yoghurt and soymilk yoghurt containing Bifidobacterium Bb-12 or Bb-46 markedly enhanced the excretion of bile acids in the faeces. The results also showed an inverse relationship between the excretion of faecal bile acids and the levels of total plasma
cholesterol or VLDL+LDL-cholesterol in rats fed on a cholesterol-enriched diet supplemented with buffalo milk yoghurt or soymilk yoghurt containing Bifidobacterium Bb-12 or Bb-46. These hypocholesterolaemic effects of buffalo milk yoghurt and soymilk yoghurt containing Bifidobacterium Bb-12 or Bb-46, which have been demonstrated in the rats in the present study, could make an effective and economic contribution in treating hypercholesterolaemia, if these effects could be confirmed in human volunteers.
References Abd El-Gawad, I.A., Hefny, A.A., El-Sayed, E.M., Saleh, F.A. (1998). Reduction of flatulence-causing soymilk oligosaccharides by different starter cultures. In Proceeding of the Seventh Egyptian Conference for Dairy Science & Technology (pp. 125–144). Egyptian society of Dairy Science: Cairo, Egypt. Anderson, R. L., & Wolf, W. J. (1995). Compositional changes in trypsin inhibitors, phytic acid, saponins and isoflavones releted to soybean processing. Journal of Nutrition, 125, 581S–588S. Beena, A., & Prasad, V. (1997). Effect of yoghurt and bifidus yoghurt fortified with skim milk powder, condensed whey and lactosehydrolysed condensed whey on serum cholesterol and triacylglycerol levels in rats. Journal of Dairy Research, 64, 453–457. Chikai, T., Nakao, H., & Uchida, K. (1987). Deconjugation of bile acids by human intestinal bacteria implanted in germ free rats. Lipids, 22, 669–672. Chou, C. C., & Hou, J. W. (2000). Growth of bifidobacteria in soymilk and their survival in the fermented soymilk drink during storage. International Journal of Food Microbiology, 56(2/3), 113–121. Dambekodi, P. C., & Gilliland, S. E. (1998). Incorporation of cholesterol into the cellular membrane of Bifidobacterium longum. Journal of Dairy Science, 81, 1818–1824. Dinakar, P., & Mistry, V. V. (1994). Growth and viability of Bifidobactrium bifidum in chedder cheese. Journal of Dairy Science, 77, 2854–2864. El-Sayed, E. M., Hefny, A. A., Saleh, F. A., & Abd El-Gawad, I.A. (1998). Bifidobacteria as a starter for the manufacture of soyyoghurt products. In Proceeding of the Seventh Egyptian Conference for Dairy Science & Technology (pp. 259–294). Cairo, Egypt.
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Endo, T., Nakano, M., Shimizu, S., Fukushima, M., & Miyoshi, S. (1999). Effects of a probiotic on the lipid metabolism of cocks fed on a cholesterol-enriched diet. Bioscience Biotechnology and Biochemistry, 63, 1569–1575. Fernandez, M. L., Vega, S., Ayala, M. T., Shen, H., Conde, K., Vergara-Jimenez, M., & Robbins, A. (1997). Vitamin C level and dietary fat saturation alter hepatic cholesterol homeostasis and plasma LDL metabolism in Guinea pigs. Nutritional Biochemistry, 8, 414–424. Homma, N. (1988). Bifidobacteria as a resistance factor in human beings. Bifidobacteria Microflora, 7, 35–43. Imaizumi, K., Hirata, K., Zommara, M., Sugano, M., & Suzuki, Y. (1992). Effects of cultured milk products by Lactobacillus and Bifidobactrium species on the secretion of bile acids in hepatocytes and in rats. Journal of Nutrition Science and Vitaminology, 38(5), 343–351. Kailasapathy, K., & Rybka, S. (1997). Lactobacillus acidophilus and Bifidobacterium spp. their therapeutic potential and survival in yoghurt. Australian Journal of Dairy Technology, 52, 28–35. Kheadr, E. E., Abd El-Rahman, A. M., Abd El-Soukkary, F. A. H., & Abd El-Rahman, A. M. (2000). Impact of yoghurt and probiotic strains on serum cholesterol and lipoprotein profile in rats. Alexandria Journal of Agricultural Research, 45(3), 81–100. Kikuchi-Hayakawa, H., Onodera, N., Masuoka, S., Kano, M., Matsubara, S., Yasuda, E., & Ishikawa, F. (2000). Effect of soy milk and Bifidobacterium-fermented soy milk on plasma and liver lipids in ovariectomized Syrian hamsters. Journal of Nutritional Science and Vitaminology, 46, 105–108. Kikuchi-Hayakawa, H., Onodera, N., Matsubara, S., Yasuda, E., Chonan, O., Takahashi, R., & Ishikawa, F. (1998a). Effects of soy milk and bifidobacterium fermented soy milk on lipid metabolism in aged ovariectomized rats. Bioscience, Biotechnology and Biochemistry, 62(9), 1688–1692. Kikuchi-Hayakawa, H., Onodera, N., Matsubara, S., Yasuda, E., Shimakawa, Y., & Ishikawa, F. (1998b). Effects of soya milk and Bifidobacterium-fermented soya milk on plasma and liver lipids, and faecal steroids in hamsters fed on a cholesterol-free or cholesterol-enriched diet. British Journal of Nutrition, 79, 97–105. Lee, S. Y., Vedamuthu, E. R., Washam, C. J., & Reinbold, B. W. (1973). An agar medium for the differential enumeration of yoghurt starter bacteria. Journal of Milk Food Technology, 37, 272–276. Mann, G. V. (1977). A factor in yoghurt which lowers cholesterolaemia in man. Atherosclerosis, 26, 335–340. Mohan, B., Kadirvel, Natarajan, A., & Bhaskaran, M. (1996). Effect of probiotic supplementation on growth, nitrogen utilization and serum cholesterol on broilers. British Poultry Science, 37, 394–401.
Nagata, Y., Ishiwaki, N., & Sugano, M. (1982). Studies on the mechanism of the antihypercholesterolemic action of soy protein and soy protein type amino acid mixtures in relation to their casein counterparts in rats. Journal of Nutrition, 112, 1614–1625. Onning, G., Akesson, B., Oste, R., & Lundquist, I. (1998). Effects of consumption of oat milk, soyamilk or cows milk on plasma lipids and antioxidative capacity in healthy subjects. Annals of Nutrition and Metabolism, 42, 211–220. Roda, A., Piazza, F., & Baraldini, M. (1998). Separation techniques for bile salts analysis. Journal of Chromatography B, 717, 263–278. Samona, A., & Robison, R. K. (1991). Enumeration of bifidobacteria in dairy products. Journal of the Society of Dairy Technology, 44, 64–64. Schaarmann, G., Schneider, J., Zorn, A., Vilser, C., & Jahreis, G. (2001). Influence of probiotic yoghurt on serum lipids in women. American Journal of Clinical Nutrition, 73(suppl), 496S. Schwarz, M., Lund, E. G., Setchell, K. D. R., Kayden, H. J., Zerwekh, J. E., Bjorkhem, I., Herz, J., & Russell, D. W. (1996). Disruption of cholesterol 7a-hydroxylase gene in mice. Journal of Biological Chemistry, 271, 18024–18031. Sirtori, C. R., Pazzucconi, F., Colombo, P., Battisin, P., Bondioli, A., & Descheemaeker, K. (1999). Double-blind study of the addition of high-protein soya milk v. cows milk to the diet of patients with severe hypercholesterolaemia and resistance to or intolerance of statins. British Journal of Nutrition, 82, 91–96. Statistical Analysis Systems (1990). User Guide Statistics, Version 6.0. SAS Inst., Inc., Cary, NC. Suzuki, Y. (1995). Effects of fermented milk on lipid metabolism. Snow Brand Reports, 105, 67–108. Suzuki, Y., Kaizu, H., & Yamauchi, Y. (1991). Effect of cultured milk on serum cholesterol concentrations in rats fed high-cholesterol diets. Animal Science and Technology, 62, 565–571. Tahri, K., Crociani, J., Ballongue, J., & Schneider, F. (1995). Effect of three strains of bifidobacteria on cholesterol. Letters in Applied Microbiology, 21, 151–449. Tanteeratarm, K., Nelson, A. I., & Wei, L. S. (1993). Manufacturing of bland soymilk. In Soybean Extrusion and Soymilk technology, Soy Food Products and Home Utilization. Urbana-Champaign, IL: University of Illinois, Soybean Program (INTSOY). Trinder, P. (1969). Determination of total cholesterol, high density lipoprotein cholesterol and triglycerids by enzymatic colourimetric method. Journal of Clinical Pathology, 22, 158. Uchida, K., Nomura, Y., & Takeuchi, N. (1980). Effect of cholic acid, chenodeoxycholic acid, and their related bikle acids on cholesterol, phospholipid and bile acids level in serum, liver, bile and feces of rats. Journal of Biochemistry, 87, 187–194.
International Dairy Journal 15 (2005) 37–44 www.elsevier.com/locate/idairyj
The hypocholesterolaemic effect of milk yoghurt and soy-yoghurt containing bifidobacteria in rats fed on a cholesterol-enriched diet Ibrahim A. Abd El-Gawada,, E.M. El-Sayeda, S.A. Hafezb, H.M. El-Zeinia, F.A. Salehb a
Faculty of Agriculture, Dairy Science and Technology Department, Cairo University, P.O. Box 12613, Giza, Egypt Special Food & Nutrition Department, Food Technology Research Institute, Agriculture Research Centre, Giza, Egypt
b
Received 4 February 2004; accepted 3 June 2004
Abstract The effect of buffalo milk yoghurt and soy-yoghurt supplemented with Bifidobacterium lactis Bb-12 or Bifidobacterium longum Bb-46 on plasma and liver lipids and the faecal excretion of bile acids was determined in rats fed on a cholesterol-enriched diet. The groups fed on a cholesterol-enriched diet supplemented with yoghurt and soy-yoghurt containing Bb-12 or Bb-46 had significantly lower levels of plasma total cholesterol and very low-density lipoprotein (VLDL)+low-density lipoprotein (LDL) cholesterol than the positive control group (without supplementation). Yoghurt or soy-yoghurt containing Bb-46 was more effective in the lowering of plasma and liver cholesterol levels than yoghurt or soy-yoghurt containing Bb-12. Furthermore, the faecal excretions of bile acids were markedly promoted in yoghurt and soy-yoghurt containing Bb-12 and Bb-46 groups compared with the positive control group. The results showed also an inverse relationship between the faecal excretions of bile acids and the levels of total cholesterol in blood plasma from rats fed on a cholesterol-enriched diet with probiotic supplementation. r 2004 Elsevier Ltd. All rights reserved. Keywords: Yoghurt; Soy-yoghurt; Bifidobacteria; Cholesterol; Bile acids
1. Introduction Hypercholesterolaemic is a major risk factor associated with coronary heart diseases, and it is considered that keeping blood cholesterol at a desirable level is one of the major preventive strategies for these diseases. Thus, much attention has been given to the relationship between diet and blood cholesterol levels. Fermented milk products have been recommended as dietary supplements because of their hypocholesterolaemic effect in humans (Mann, 1977) and rats (Suzuki, Kaizu, & Yamauchi, 1991). Since 1986, traditional yoghurt cultures have been supplemented or replaced by Bifidobacterium species to enhance the therapeutic value of yoghurt and to establish its market as a Corresponding author. Tel./fax: +20-27743824.
E-mail address: [email protected] (I.A. Abd El-Gawad). 0958-6946/$ - see front matter r 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2004.06.001
functional therapeutic food. Therefore, incorporation of bifidobacteria in various dairy products has become a recent trend (Kailasapathy & Rybka, 1997). On the other hand, although most probiotic foods are derived from milk, the possibility of using other proteinrich substrates such as soymilk to make such foods has not been adequately considered. The potential role of dietary soy in the prevention and treatment of chronic diseases, in particular heart disease, has been recognized for a long time (Nagata, Ishiwaki, & Sugano, 1982). Recently, soy-yoghurt has been prepared from soymilk fermented with lactic acid bacteria or bifidobacteria (Abd El-Gawad, Hefny, El-Sayed, & Saleh, 1998; Kikuchi-Hayakawa et al., 1998b; Chou & Hou, 2000). Also, it was reported that soymilk fermented with Bifidobacterium significantly decreased the levels of total plasma cholesterol and very low-density lipoprotein (VLDL)+low-density lipoprotein (LDL)-cholesterol in rats (Kikuchi-Hayakawa et al., 1998a). Though,
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numerous studies have focused on the health effect of yoghurt, relatively little is yet known regarding the potential role of the health-promoting effects of yoghurt and soy-yoghurt containing bifidobacteria by reducing cholesterol. Therefore, the objective of this investigation was to study the effects of yoghurt and soy-yoghurt containing Bifidobacterium Bb-12 or Bb-46 on the levels of plasma and liver lipids in hypercholesterolaemic rats and the levels of bile acid in their faeces.
2. Materials and methods 2.1. Preparation of yoghurts from buffalo milk Low-fat buffalo milk (1.5%, w/w) was inoculated with 3% (v/v) of Lactobacillius dulbrueekii sub sp. bulgaricus and Streptococcus salivarius sub sp. thermophilus (Chr. Hansen Laboratories, Copenhagen, Denmark), and divided into three portions. One portion (without added bifidobacteria) served as a control yoghurt and was denoted as YBM; a second portion was inoculated at a level of 0.07% (w/v) with a freezedried Bifidobacterium lactis Bb-12 and served as an experimented yoghurt and was denoted as YBMBb-12 yoghurt. The third portion was inoculated at a level of 0.07% (w/v) with a freeze-dried Bifidobacterium longum Bb-46 and served as an experimented yoghurt and was denoted as YBMBb-46 yoghurt. The two strains of bifidobacteria were obtained from Chr. Hansen Laboratories (Copenhagen, Denmark). Following incubation YBM, YBMBb-12 and YBMBb-46 yoghurts were stored at 471 1C. 2.2. Preparation of non-fermented soymilk and soyyoghurts Fresh and non-beany flavoured soymilk was prepared according to the method of Tanteeratarm, Nelson, and
Wei (1993), and divided into three portions. One portion served as a non-fermented soymilk and was denoted NFSM; the other two portions were used for preparation of soymilk yoghurts Bb-12 and Bb-46 according to the method described by El-Sayed, Hefny, Saleh, and Abd El-Gawad (1998). The manufacture of soymilk yoghurts Bb-12 and Bb-46 involved the addition of gelatin at a level of 1% (w/w) to the milk, heating at 95 1C for 5 min, and cooling to 37 1C. One portion was inoculated at a level of 0.07% (w/v) with a freeze-dried B. lactis Bb-12 and was denoted as YSMBb-12 yoghurt; the other portion was inoculated at a level of 0.07% (w/ v) with a freeze-dried B. longum Bb-46 and was denoted as YSMBb-46 yoghurt. After inoculation at 37 1C for 4–5 h, both portions were stored at 471 1C. 2.3. Animal feeding experiments Forty-eight male albino rats with an average weight of between 80 and 100 g were housed in cages with wiremesh floors in a room at 2371 1C and 6075% relative humidity. All animals were fed on a basal diet for one week. After this adaptation period, the rats were divided randomly into 8 experimental groups of 6. One group received a basal diet (cholesterol-free diet) throughout the experimental period of six weeks and served as a negative control group. The other seven groups were fed on the basal diet with cholesterol added at a level of 0.5% (w/w) (cholesterol-enriched diet) for 1–2 weeks to create hypercholesterolaemic rats. One of the 7 groups, which served as a positive control group, was fed only on a cholesterol-enriched diet for the six-week period. The other six groups were fed for five weeks on a cholesterol-enriched diet supplemented with one of the following products: YBM, YBMBb-12, YBM Bb-46, NFSM, YSMBb-12 or YSMBb-46. The experimental diets given to the eight groups are described in Table 1. The rats were allowed free access to experimental diet and water, and their body weights were monitored. At
Table 1 Experimental groups of rats and diets used in the triala Diet treatment
Diet code
Diet formulae
Cholesterol-free diet Cholesterol-enriched diet (CED) CED+yoghurt from buffalo milk (YBM) CED+YBM with added B. lactis Bb-12 (YBMBb-12) CED+YBM with added B. longum Bb-46 (YBMBb-46) CED+non-fermented soymilk (NFSM) CED+yoghurt from soymilk with added B. lactis Bb-12 (YSMBb-12) CED+yoghurt from soymilk with added B. longum Bb-46 (YSMBb-46)
Negative control Positive control YBM YBMBb-12 YBMBb-46 NFSM YSMBb-12
100 g basal diet+50 mL water 99.5 g basal diet+0.5 g cholesterol+50 mL water 99.5 g basal diet+0.5 g cholesterol+50 g YPM yoghurt 99.5 g basal diet+0.5 g cholesterol+50 g yoghurt YPM Bb-12 99.5 g basal diet+0.5 g cholesterol+50 g yoghurt YPM Bb-46 99.5 g basal diet+0.5 g cholesterol+50 g NFSM 99.5 g basal diet+0.5 g cholesterol+50 g soy-yoghurt YSM Bb-12
YSMBb-46
99.5 g basal diet+0.5 g cholesterol+50 g soy-yoghurt YSM Bb-46
a
The basal diet consisted of : 15%, (w/w), casein; 10%, (w/w), maize oil; 10%, (w/w), cellulose; 4%, (w/w), mineral mix; 1%, (w/w), vitamin mix and 60%, (w/w), starch.
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the end of the 6-week experimental period, blood samples were collected from the eye vein under diethyl ether anesthesia. The samples were collected in tubes, with EDTA as an anti-coagulating agent. The tubes were centrifuged at 3000 rpm for 15 min to obtain the plasma, which was kept frozen ( 2371 1C) until analysis. Faeces were collected on the last 2 days of the feeding period and kept frozen (2371 1C) until analysed for bile acids. The rats were sacrificed and the liver, heart, kidney and spleen were excised immediately and weighed. The liver was washed with ice-cold saline solution (0.9%, w/v, NaCl) and stored at 2371 1C until analysed.
39
S. salivarius sub sp. thermophilus) were enumerated according to the method of Lee, Vedamuthu, Washam, and Reinbold (1973) in which the yoghurt sample was added to Lee’s agar and incubated at 43 1C for 3 days. Table 2 shows the viable count of bifidobacteria and yoghurt culture in the experimental products. 2.4.2. Determination of plasma lipid Total plasma cholesterol, high-density lipoprotein (HDL) and triglycerides were determined using the enzymatic colourimetric method according to Trinder (1969). The VLDL+LDL was calculated as follows: VLDL+LDL cholesterol=total cholesterolHDL cholesterol.
2.4. Analytical methods 2.4.1. Counts of bifidobacteria and yoghurt cultures The count of bifidobacteria in soy-yoghurts YSMBb12 and YSMBb-46, which did not contain yoghurt culture, was enumerated by poured plate method on Lactobacilli MRS-agar medium containing 0.05%, w/v, L-cycteine-HCl according to the method described by Samona and Robison (1991). The count of bifidobacteria in buffalo milk yoghurts (YBMBb-12 and YBMBb46), which contain yoghurt culture as well as bifidobacteria, was enumerated according to the method of Dinakar and Mistry (1994), in which a mixture of antibiotics, including 2 g paromomycin sulphate, 0.3 g nalidixic acid, and 60 g lithium chloride, was dissolved in 1 L distilled water, filter-sterilised (0.2 mm) and stored at 4 1C until use. The antibiotic mixture (5 mL) was added to 100 mL MRS-agar medium. L-Cysteine-HCl 0.5% (w/ v) (Sigma Chemical Co., St Louis, MO, USA) was also added to decrease the redox potential of the medium. Plates were incubated at 37 1C for 48 h anaerobically. Yoghurt cultures (L. dulbrueekii sub sp. bulgaricus and
Table 2 The viable count of yoghurt culture and bifidobacteria in experimental treatmentsa,b,c Diet treatmentd
YBM YBMBb-12 YBMBb-46 NFSM YSMBb-12 YSMBb-46 a
Microbial count ( 108 cfu mL1) Yoghurt culture
Bifidobacteria
88.7 57.4 43.7 ND ND ND
NDe 1.9 1.7 ND 4.1 5.8
The data presented are the means of three replicate trials. Yoghurt culture: L. dulbrueekii sub sp. bulgaricus and S. salivarius sub sp. thermophilus. c B. lactis Bb-12 or B. longum Bb-46. d See Table 1 and text for details of diet treatments. e ND: not determined. b
2.4.3. Determination of cholesterol and triglycerides in liver Cholesterol and triglycerides were extracted from the liver by the method of Fernandez et al. (1997), and were measured by the method of Trinder (1969). 2.4.4. Determination of bile acids in faeces Extraction of bile acids from faeces was carried out according to the method of Kikuchi-Hayakawa et al. (1998b). The level of extracted bile acids was determined according to the method of Roda, Piazza, and Baraldini (1998), using a Hewlett Packard HPLC (Series 1050, HP, USA) equipped with an autosampling injector, solvent degasser, ultra-violet (UV) detector, and quarter HP pump (Series 1050, HP, USA). Thirty five microlitres of sample extracted was injected into the Sphrosorb BDS C18 column (250 mm 4.6 mm, ID 5 mm particle size). The operating parameters were set as follows: column temperature, 35 1C; flow rate 1.0 mL/ min; UV-detector, 210 nm. The isocratic elution was with methanol/acetonitryl/0.01% M phosphate buffer pH 5.3 (50/25/25, v/v/v) as a mobile phase. The standard bile acids used were cholic acid, chenodeoxycholic acid, deoxycholic acid and lithocholic acid (Sigma Chemical Co., St Louis, MO, USA). Each sample was injected twice. The peak area of bile acids standard solution was used to prepare a standard curve. From these standard curves, the concentration of bile acids in the test samples were calculated. 2.5. Statistical analysis Data are presented as means and standard deviation. The significance differences between groups/treatments were evaluated using the general linear model procedure of Statistical Analysis Systems (SAS) (1990) (SAS Institute, Inc., Cary, NC, USA) to analyse the biological examination data by least significant difference (LSD) at Po0:05.
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3. Results and discussion 3.1. Plasma and liver lipids The effect of the experimental diets on the levels of plasma and liver lipids in rats are presented in Table 3. 3.1.1. Plasma lipids 3.1.1.1. Total cholesterol. The total concentration of plasma cholesterol was significantly reduced from 301 mg 100 mL1 in the positive control to a mean value of 155 mg 100 mL1 in both YBMBb-12 and YBMBb-46 groups and to a mean value of 189 mg 100 mL1 in both YSMBb-12 and YSMBb-46 groups. This decrease corresponds to a 48.7% and 37.3% reduction, respectively. In contrast, there was no significant difference in the total level of plasma cholesterol detected between YBMBb-12, YBMBb-46, YSMBb-12, YSMBb-46, and negative control groups (Table 3). It can also be seen that the total levels of plasma cholesterol in the NFSM and YBM groups were 23% and 33% lower than that in the positive control group, respectively. While the cholesterol-enriched diet supplemented with YBMBb-12, YBMBb-46, YSMBb-12 or YSMBb-46 was more efficient than the corresponding diet supplemented with YBM or NFSM for decreasing total plasma cholesterol, none of these differences were significant (Table 3). The diet supplemented with YBMBb-46 or YSMBb-46 was more effective in reducing plasma cholesterol than the corresponding diet supplemented with YBMBb-12 or YSMBb-12. The YBMBb-12 and YBMBb-46 diets were more effective at lowering total cholesterol than the diets of YSMBb12 and YSMBb-46. The YBM diet was more effective in reducing the total cholesterol level than the NFSM diet.
Beena and Prasad (1997) found lower serum cholesterol in rats fed on yoghurt containing Bifidobacterium bifidum (120 mg 100 mL1) compared to a positive control (172 mg 100 mL1) after 30 days. However, the administration of fermented milk containing bifidobacteria (109 cfu g1) to hypercholesterolaemic human subjects resulted in a decrease in the total cholesterol level from 300 to 150 mg 100 mL1 (Homma, 1988). Schaarmann, Schneider, Zorn, Vilser, and Jahreis (2001) reported that the consumption of the probiotic yoghurt made by B. longum and Lactobacillus acidophilus decreased total cholesterol in hypercholesterolaemic women from 293 mg 100 mL1 at the beginning of the experiment to 255 mg 100 mL1 after 153 days. KikuchiHayakawa et al. (1998b) reported that consumption of bifidobacterium fermented soymilk significantly reduced plasma cholesterol, whereas consumption of soymilk resulted in a non-significant decrease in the bloodcholesterol level in hamsters. These results were confirmed also by Sirtori et al. (1999) and Onning, Akesson, Oste, and Lundquist (1998).
3.1.1.2. HDL-cholesterol. As shown in Table 3, there was no significant difference in the plasma HDLcholesterol level between the negative control group and other experimental groups at the end of the 6-week experimental period. Endo, Nakano, Shimizu, Fukushima, and Miyoshi (1999) reported that the addition of the probiotic to the diet had no effect on the HDLcholesterol level. Our data agree well with this finding. Beena and Prasad (1997) found that the bifidus yoghurt fortified with lactose-hydrolysed condensed whey had little effect on HDL-cholesterol, whereas KikuchiHayakawa et al. (1998b) reported that consumption of both soymilk and Bifidobacterium-fermented soymilk
Table 3 Levels of plasma and liver lipids in rats fed on experimental diets for six weeksa Diet treatment
Negative control Positive control YBM YBMBb-12 YBMBb-46 NFSM YSMBb-12 YSMBb-46 a
Plasma
Liver
Total cholesterol (mg 100 mL1)
Triglycerides (mg 100 mL1)
HDLcholesterol (mg 100 mL1)
VLDL+LDLcholesterolb (mg 100 mL1)
Atherosclerotic indexc (mg 100 mL1)
Cholesterol (mg g1)
Triglycerides (mg g1)
127.4720.1d 301.4716.1f 201.5720.9d,i 159.6726.3d,i 149.9714.9d 231.7754.9f,i 1 95.1717.4d,i 183.074.3d,i
40.975.5e 105.7712.5f 86.572.7f,i 65.170.6d,e,i 51.678.9d,e 69.7712.4d,i 64.6712.0d,e,i 40.377.8e
38.172.3f 24.874.3f 16.672.1f 20.671.9f 29.171.8f 22.4710.2f 31.4710.2f 33.6712.2f
89.371.1g 276.670.6f 184.970.8d 139.071.2j 120.970.1k 209.470.5i 163.771.3e 149.470.9h
2.470.1h 11.170.1f 11.270.2f 6.970.2d 4.270.3e 9.470.1e 5.270.4e 4.470.6e
2.8870.0d 7.3470.1f 2.3570.0e 2.2470.2e 2.2270.1e 4.5370.1i 1.7770.1h 2.3370.1e
17.7870.5i 25.1871.1f 17.6370.1i 17.8971.5i 21.0070.1fi 18.4371.0i 18.5073.4i 16.4570.4i
See Table 1 and text for details of diet treatment. VLDL: Very low-density lipoprotein-cholesterol; LDL: low-density lipoprotein-cholesterol; VLDL+LDL-cholesterol=Total cholesterolHDLcholesterol. c Atherosclerotic index=(VLDL+LDL-cholesterol)/(HDL-cholesterol). d–k Mean values (7SD; n ¼ 6) within the same column bearing different superscripts are significantly different (P40.05). b
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increased the HDL-cholesterol level in hamsters fed on a cholesterol-enriched diet. 3.1.1.3. VLDL+LDL-cholesterol. Rats fed on YBM, YBMBb-12, YBMBb-46, NFSM, YSMBb-12 or YSMBb-46 diet had significantly lower plasma VLDL+LDL-cholesterol than the positive control group of rats. The YBMBb-12, YBMBb-46, YSMBb12 and YSMBb-46 diets were more effective in lowering plasma VLDL+LDL-cholesterol levels than the NFSM and YBM diets (Table 3). Also, it is noteworthy from the same table that the plasma VLDL+LDL-cholesterol levels in the rats fed the YBMBb-46 and YSMBb-46 diets (120.86 and 149.39 mg 100 mL1) were markedly lower than those in the rats fed on the YBMBb-12 and YSMBb-12 diets (163.67 and 138.99 mg 100 mL1, respectively). Beena and Prasad (1997), found that yoghurt containing B. bifidum markedly lowered the levels of LDLcholesterol in rats fed on a cholesterol-enriched diet (positive control) from 97 mg 100 mL1 to 22, 16 and 15 mg 100 mL1, respectively, in rats fed on yoghurt containing B. bifidum fortified with skimmed milk, condensed whey or lactose-hydrolysed condensed whey at the end of the 30-day experimental period. Schaarmann et al. (2001) reported that the consumption of probiotic yoghurt made using B. longum and L. acidophilus reduced the LDL-cholesterol level in normocholesterolaemic and cholesterolaemic women. Kikuchi-Hayakawa et al. (2000) studied the effect of soymilk and fermented soymilk on lipid metabolism in ovariectomized Syrian hamsters. They found that soymilk fermented by Bifidobacterium breve was more effective than soymilk in reducing the level of VLDL+LDL-cholesterol (77.5 and 86.2 mg 100 mL1, respectively) when compared with the control group fed on a basal diet (89.2 mg 100 mL1). 3.1.1.4. Atherosclerotic index. The data in Table 3 indicate that feeding YBMBb-12, YBMBb-46, YSMBb12 or YSMBb-46 significantly decreased the values of atherosclerotic index of rats by 37.8%, 62.7%, 53.2% and 60.1%, respectively, below that in rats fed the cholesterol-enriched diet (positive control group). For rats fed on cholesterol-enriched diets (Table 3), the YBMBb-12, YBMBb-46, YSMBb-12 and YSMBb46 diets were more effective in reducing the atherosclerotic index values than the YBM and NFSM, which did not contain bifidobacteria. These findings are in agreement with those reported by Kikuchi-Hayakawa et al. (1998b, 2000). 3.1.1.5. Triglycerides. The levels of plasma triglycerides in rats fed on YBMBb-12, YBMBb-46, YSMBb-12, YSMBb-46 or NFSM diets were significantly lower than in those fed the cholesterol-enriched diet (positive
41
control). The reduction in triglyceride levels with the above-mentioned groups was 38.4%, 51.2%, 38.9%, 61.8% and 34.0%, respectively (Table 3). The level of plasma triglycerides in the YBM group was also lower than that in the positive control group, but not significantly so. It is noteworthy from the data in Table 3 that the YBMBb-46 and YSMBb-46 diets were more effective in lowering plasma triglycerides than the YBMBb-12 and YSMBb-12 diets. Schaarmann et al. (2001) reported that the consumption of probiotic yoghurt made with B. longum and L. acidophilus lowered triglycerides in hypercholesterolaemic women from 114 mg 100 mL1 to 91 mg 100 mL1 after 153 days. However, KikuchiHayakawa et al. (2000) reported that feeding ovariectomised hamsters on fermented soymilk decreased the triglycerides from 232 mg 100 mL1 in the control to 180 mg 100 mL1 after four weeks. The results in Table 3 also suggest that the NFSM diet significantly decreased the level of plasma triglycerides, but the YSMBb-12 and YSMBb-46 diets were more effective in reducing plasma triglycerides. While the YBM diet resulted in a non-significant reduction in plasma triglycerides, the YBMBb-12 and YBMBb-46 diets significantly reduced the level of plasma triglycerides. 3.1.2. Liver lipids 3.1.2.1. Liver cholesterol. The data in Table 3 show that the content of liver cholesterol in the positive control group (7.34 mg g1) was significantly higher than that in the negative control group (2.88 mg g1). There was a significant difference between rats fed on the positive control diet and those fed on YBM, YBMBb12, YBMBb-46, YSMBb-12, YSMBb-46 or NFSM diets (Table 3). It can be observed from the data in Table 3 that the YBMBb-12, YBMBb-46, YSMBb-12 and YSMBb-46 diets were more effective in lowering the levels of liver cholesterol than YBM and NFSM diets. These results are in agreement with those reported by Kheadr, Abd El-Rahman, Abd El-Soukkary, and Abd El-Rahman (2000) who suggested that the yoghurt diets supplemented with B. bifidum reduced cholesterol in rat liver tissues. Kikuchi-Hayakawa et al. (1998b) found that the total level of liver cholesterol in hamsters fed on a cholesterol-free diet, soymilk or soymilk fermented with B. breve, were lower than that in the control hamsters fed on basal diet. 3.1.2.2. Liver triglycerides. The content of liver triglycerides was significantly higher in rats fed on the positive control diet than those fed the negative control diet (Table 3). In contrast, the levels of liver triglycerides in rats fed the experimental diet groups were significantly lower than in rats fed on the positive control diet, except for rats fed the YSMBb-46 diet for which the decrease
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was not significant. Kikuchi-Hayakawa et al. (1998a) reported that the contents of liver triglycerides in ovariectomized rats fed on both soymilk and Bifidobacterium-fermented soymilk were lower than that of the control group of rats fed on a basal diet. Kikuchi-Hayakawa et al. (1998b) concluded that the content of liver triglycerides was not affected by soymilk or Bifidobacterium-fermented soymilk in hamsters fed either cholesterol-free diet or a cholesterol-enriched diet. On the contrary KikuchiHayakawa, et al. (2000) found that soymilk fermented with Bifidobacterium had a hypolipidaemic effect in ovariectomised hamsters.
3.2. Faecal bile acid excretion As shown in Table 4, there was no significant difference in the levels of total bile acids between rats fed the negative control and positive control diets. Rats fed on YBM, YBMBb-12, YBMBb-46, NFSM, YSMBb-12 or YSMBb-46 diets excreted significantly higher level of bile acids than those fed either the positive or negative diets. The excretion of bile acids in rats fed on the YBMBb12, YBMBb-46, YSMBb-12 or YSMBb-46 diets was significantly higher than those fed on YBM or NFSM diets. The ratio of cholic acid to chenodeoxycholic acid in rats fed on the YBMBb-12, YBMBb-46, YSMBb-12 or YSMBb-46 diets (6.7:1–9.7:1) was higher than that in rats fed on the negative (5.8:1) or positive (6.1:1) control diets (Table 4). In contrast, the corresponding ratios in rats fed on the YBM or NFSM groups were 6.1:1 and 6.5:1, respectively (Table 4). Metabolism of bile acids, especially the change in the ratio of cholic to chenodeoxycholic acids in bile or faeces, has been associated with cholesterol 7a-hydroxylase deficiency (Schwarz et al., 1996). Uchida, Nomura, and Takeuchi (1980) suggested that cholic acid, but not chenodeoxycholic acid, promotes absorption of cholesterol. Therefore, an
increase in the ratio of cholic acid to chenodeoxycholic acid may promote cholesterol absorption. The data in Tables 3 and 4 showed an inverse relationship between the level of bile acids excreted in the faeces and the total levels of plasma cholesterol or VLDL+LDL-cholesterol in rats fed on the YBMBb-12, YBMBb-46, YSMBb-12 and YSMBb-46 diets. These findings are in accordance with those found by KikuchiHayakawa et al. (1998b). However, the study of Nagata et al. (1982) showed that feeding soybean protein increased the excretion of faecal bile acids in rats. This finding and the results of our study, suggest that hepatic cholesterol metabolism could change in order to provide more cholesterol for synthesis of bile acids. Imaizumi, Hirata, Zommara, Sugano, and Suzuki (1992) and Suzuki (1995) found that Bifidobacterium species stimulated both the secretion of bile acids and the activity of cholesterol 7ahydroxylase, a rate-limiting enzyme in the synthesis of bile acids. Furthermore, the fermentation of soymilk by Bifidobacterium spp. resulted in compositional changes in isoflavones, phytate and saponins (Anderson & Wolf, 1995). Bifidobacteria cells can remove cholesterol from the growth culture medium through both assimilation and coprecipitation with deconjugated bile salts, as found in an in vitro study by Tahri, Crociani, Ballongue, and Schneider (1995). Consequently, bifidobacteria caused an increase in the excretion of free bile salts in the faeces (Chikai, Nakao, & Uchida, 1987; Dambekodi & Gilliland, 1998). 3.3. Body weight gain and organs rats weight The value of body weight gain was significantly higher in rats fed YBMBb-12, YBMBb-46 or YSMBb-12 than that in rats fed the negative control diet. The body weight gain in rats fed the YSMBb-46 diet was nonsignificantly higher than that in rats fed the negative control diet (Table 5). These results are in agreement with those reported by Kikuchi-Hayakawa et al.
Table 4 Excretion of bile acids in faeces in rats fed on experimental diets for six weeksa,b Diet treatment
Cholic acid (mg 100 g1)
Chenodeoxycholic acid (mg 100 g1)
Deoxycholic acid (mg 100 g1)
Lithocholic acid (mg 100 g1)
Total bile acids (mg 100 g1)
Negative control Positive control YBM YBMBb-12 YBMBb-46 NFSM YSMBb-12 YSMBb-46
205.970.7c,d 201.770.6d 240.271.6c,e 272.472.3e,h 285.271.9g,h 243.273.8e 268.270.8e,h 283.670.4g
35.471.0c,e 33.372.5c 39.471.2g,h 28.971.2d 29.370.5d 37.371.3e,h 38.171.2e,h 42.671.2g
18.970.8e 18.271.1c,e 16.471.0c 21.370.4h 21.171.1h 21.570.8h 20.170.7e,h 26.371.6g
9.970.6d 16.070.5c 17.170.5c 19.771.2e 30.971.2g 22.070.8h 18.171.4c,e 24.170.8h
270.171.2f 269.171.2f 313.174.1d 342.372.4e 366.573.5h 324.071.6c 344.571.5e 376.573.1g
a
Values are the means for three replicate experiments. See Table 1 and text for details of diet treatments. c–h Mean values (7SD; n ¼ 6) within the same column bearing different superscripts are significantly different (P40:05). b
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Table 5 Body weight gain and organ weight of rats fed on experimental diets for six weeksa Diet treatment
Negative control Positive control YBM YBMBb-12 YBMBb-46 NFSM YSMBb-12 YSMBb-46
Body weight gain (%)b
29.975.3c 48.8715.0c,f 50.2723.1c,e,f 51.672.5e,f 71.376.3e 37.173.7f,c 52.3715.0e,f 47.7710.4c,f
Organ weight (g) Liver
Heart
Kidney
Spleen
3.970.2d 7.970.3e 5.570.6c 5.670.4c 5.870.7c,f 7.370.8e,f 5.570.5c,d 5.870.5c,f
0.770.0c,e,f 0.870.1c,e,f 0.870.1e,f 0.970.1e 0.670.0c 0.970.1e 0.670.1c,f 0.970.0e
0.970.0c 1.370.1e 0.970.0c,f 1.070.1c,f 1.070.1c,f 1.170.0f 1.070.1c,f 1.070.1c,f
0.370.1e 0.470.0e 0.370.1e 0.470.1e 0.570.1e 0.470.1e 0.370.0e 0.470.0e
a
See Table 1 and text for details of diet treatments. % Body weight gain =[(final weightinitial weight)/initial weight] 100. c–f Mean values (7SD; n ¼ 6) within the same column bearing different superscripts are significantly different (P40:05). b
(1998b). Suzuki et al. (1991) found no difference in body weight gain between rats fed skim milk and rats fed skim milk fermented with L. acidophilus or bifidobacteria. No significant differences in the weights of the heart or spleen were detected between rats fed on the YBMBb-12, YBMBb-46, YSMBb-12 or YSMBb-46 diets and rats fed the two control diets. There was a significant difference in kidney weight between rats fed the positive control diet and rats fed the other diets. The liver weight in rats fed the positive control diet was significantly higher than that in rats fed the negative control diet. There was a significant difference in the liver weight between rats fed the positive control diet and rats fed the other experimental diets. However, the difference between rats fed on the positive control diet and rats fed on NFSM diet was not significant. Similar results were reported by Mohan, Kadirvel, Natarajan, and Bhaskaran (1996).
4. Conclusions This study has demonstrated that the inclusion of buffalo milk yoghurt, soymilk yoghurt (with or without bifidobacteria) or non-fermented soymilk in the diet of rats fed a cholesterol-enriched diet had a marked affect on the levels of plasma and liver lipids. Buffalo milk yoghurt and soymilk yoghurt containing Bifidobacterium Bb-12 or Bb-46 were very effective in lowering the levels of plasma and liver lipids in rats. Buffalo milk yoghurt or soymilk yoghurt containing Bifidobacterium Bb-46 was more affective in reducing the level of plasma and liver cholesterol than buffalo milk yoghurt or soymilk yoghurt containing Bifidobacterium Bb-12. Buffalo milk yoghurt and soymilk yoghurt containing Bifidobacterium Bb-12 or Bb-46 markedly enhanced the excretion of bile acids in the faeces. The results also showed an inverse relationship between the excretion of faecal bile acids and the levels of total plasma
cholesterol or VLDL+LDL-cholesterol in rats fed on a cholesterol-enriched diet supplemented with buffalo milk yoghurt or soymilk yoghurt containing Bifidobacterium Bb-12 or Bb-46. These hypocholesterolaemic effects of buffalo milk yoghurt and soymilk yoghurt containing Bifidobacterium Bb-12 or Bb-46, which have been demonstrated in the rats in the present study, could make an effective and economic contribution in treating hypercholesterolaemia, if these effects could be confirmed in human volunteers.
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