Effect of high fiber products on blood lipids and ...

Nutrition Research 24 (2004) 85–93 www.elsevier.com/locate/nutres

Effect of high fiber products on blood lipids and lipoproteins in hamsters Hector E. Martinez-Floresa,*, Yoon Kil Changb, Fernando Martinez-Bustosc, Valdomiro Sgarbierid a

Escuela de QuimicoFarmacobiologia. Universidad Michoacana de San Nicolas de Hidalgo. Tzintzuntzan 173. Col. Matamoros. CP 58240. Morelia, Mich., Mexico b DTA/FEA/UNICAMP. Cidade Universitaria, Baraõ Geraldo, CEP 13080-970. Campinas, SP, Brazil c CINVESTAV-IPN. Libramiento Norponiente 2000. Fracc. Real de Juriquilla. CP 76230. Queretaro, Mexico d Centro de Quimica e Nutriçaõ Aplicada. Instituto de Tecnologia de Alimentos. Avenida Brasil, 2880. CEP 13.073-001. Campinas, SP, Brazil Received 8 April 2003; received in revised form 26 August 2003; accepted 28 August 2003

Abstract Serum and liver lipidemic responses in hamsters fed diets containing 2% cholesterol and different dietary fiber sources were studied. The following diets were made from: a) the control diet made from extruded cassava starch (CSH) contained 9.3% cellulose, b) cassava starch extruded with 9.7% resistant starch (CS-RS), c) cassava starch extruded with 9.9% oat fiber (CS-OF), d) the reference diet contained 9.5% cellulose, and no cholesterol was added. Total cholesterol, LDL⫹VLDL-cholesterol and triglycerides were significantly lower (P ⬍ 0.05) in serum of hamsters fed on the CS-RS (17.87%, 62.92% and 9.17%, respectively) and CS-OF (15.12%, 67.41% and 18.35%, respectively) diets, as compared to hamster fed with the CSH diet. Similar results were found in the livers of hamsters fed on the CS-RS and CS-OF diets, as compared to hamsters fed with the CSH diet. The diets containing these fibers could be used as active ingredients in human diets to improve the human health. © 2004 Elsevier Inc. All rights reserved. Keywords: Resistant starch; Oat fiber; Cholesterol; Lipids; Triglycerides; Hamster

* Corresponding author. Tel.: ⫹52-443-314-2809; fax: ⫹52-443-314-2152. E-mail address: [email protected] (Hector Martinez-Flores). 0271-5317/04/$ – see front matter © 2004 Elsevier Inc. All rights reserved. doi:10.1016/S0271-5317(03)00206-9

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H.E. Martinez-Flores et al. / Nutrition Research 24 (2004) 85–93

1. Introduction The human consumption of products containing dietary fiber has increased due to the health benefits that they offer. Dietary fiber has been associated with cholesterol lowering properties [1,2], which can reduce the danger of arterial blood diseases [3]. Hypocholesterolemic response has been attributed basically to the presence of water-soluble fiber [4,5]. The mechanism by which fiber lowers blood cholesterol and lipid levels has not yet been clearly established. One explanation is that this is due to its ability to form a gel that interferes with the absorption of lipids and cholesterol. Another possible mechanism is that soluble dietary fiber increases sterol excretion. Certain varieties of dietary fiber can bind bile salts and neutral sterols [6], and in this way enhance their removal from the body. Bile salts excreted in the feces are removed from the enterohepatic cycle and smaller amounts of bile salts are available for lipid absorption. Moreover, serum and liver cholesterol are used for bile acid synthesis to compensate depleted pools. Several animal models have been used to evaluate the physiological response to dietary fiber. Hamsters, unlike rats, have a gall bladder, and hamsters are more responsive to dietary factors than rats [7]. Moreover, hamsters are the animals preferred for evaluating cholesterol [8], since their metabolism is similar to that of human beings [9,10]. Resistant starch from cereals and other sources is becoming a potential food ingredient. Resistant starch is defined as dietary starch that does not digest in the small intestine. In the large intestine, resistant starch is fermented by colonic microflora to short-chain fatty acids (SCFA), carbon dioxide, methane and ethane [11]. The conversion of resistant starch to SCFA (mainly acetate, propionate and butyrate), is probably the most important physiological effect preventing health disorders [12]. The presence of these SCFA inhibits the diffusion of cholesterol and bile acids at the microvillous boundary layer, interfering with their absorption into the body [13]. Also, SCFA aid in the prevention of colon cancer [14,15]. The purpose of this study was to determine the effect on the cholesterol, triglycerides and total lipid levels in hamsters of adding extruded oat fiber or resistant starch to Cassava starch in their diets.

2. Materials and methods The Cassava starch, containing 4% dietary fiber, was obtained from B.R. Carvalho and Co., LTD in Brazil. Oat fiber, containing 93.8% dietary fiber, was provided by Canadian Harvest, L.P. in Canada. The resistant starch, containing 29.7% dietary fiber, was supplied by the National Starch and Chemical Company of Brazil. 2.1. Extrusion conditions The Cassava starch was blended with either resistant starch or oat fiber for 10 minutes using a mixer (Kitchen Aid model K45SS, St. Joseph, Michigan, USA). Then water was added to the blends until a moisture content of 18% was reached. Samples were placed in


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Table 1 Diet composition (% dry basis) Diets Diet 1

Extruded product (EP) Starch from EP Fiber from EP Casein cellulose mineral salts vitamins cistine choline tert-butilhidroquinone Soybean oil cholesterol coconut oil

Reference

Control

CS-RS

CS-OF

59.9 58.2 1.5 20.0 8 3.5 1.0 0.3 0.3 0.0014 7.0 na na

47.9 46.6 1.3 20.0 8 3.5 1.0 0.3 0.3 0.0014 7.0 2.0 2.0

55.9 46.2 9.7 20.0 na 3.5 1.0 0.3 0.3 0.0014 7.0 2.0 2.0

55.9 46.0 9.9 20.0 na 3.5 1.0 0.3 0.3 0.0014 7.0 2.0 2.0

1

Extruded product as starch and fiber source. Reference. Diet containing extruded Cassava starch. Control. Diet containing extruded Cassava starch with 8% cellulose, 10% coconut oil and 2% cholesterol added. CS-RS. Diet containing extruded Cassava starch with resistant starch, 10% coconut oil and 2% cholesterol added. CS-OF. Diet containing extruded Cassava starch with oat fiber, 10% coconut oil and 2% cholesterol added. na. not added.

sealed bags and maintained at 4°C for 16 hours. Samples were extruded using a single Brabender extruder (model 20 D/N-GNF 1014/2, Brabender OHG, Duisburg, Alemania). The extruder was operated at a feed speed of 70 g/min, and a screw speed of 150 rpm, with a die exit diameter of 4 mm and a screw ratio of 1:4. The temperature in the three heating zones of the extruder barrel was 75°C, 100°C and 150°C. The extruded products were dried overnight at 45°C in a convection oven (FANEM, model 320-SE) and milled in a hammer mill to obtain flour with particle size smaller than 250␮m. 2.2. Animals and diets Four groups of eight-week-old male golden Syriam hamsters (Departamento de Patologia, Faculdade de Veterinaria, Universidade de Sa˜ o Paulo. Sa˜ o Paulo, SP, Brazil) were fed for 20 days. Animals were housed individually in wire-bottom cages in a temperature-controlled room (22⫾1°C), with 12-hr light and dark cycles. The hamsters received food and water ad libitum for 20 days. Feed consumption was measured twice weekly and the animals were weighed once a week. Experimental diets (Table 1) based on extruded Cassava starch (CSH, control diet), Cassava starch extruded with oat fiber (CS-OF) and Cassava starch extruded with resistant starch (CS-RS) were formulated so as to contain 20% casein, 3.5% minerals, 1% vitamins, 0.3% cystine, 0.3% choline, 0.0014% tert-butilhidroquinone, and 7% soybean oil, in accordance with American Institute of Nutrition-93 [16]. These three diets also

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contained 2% cholesterol and 10% coconut oil. The reference diet based on extruded Cassava starch was formulated as described above, but with 8% cellulose and without cholesterol and coconut oil added. 2.3. Sampling procedures After 20 days the hamsters were killed. They were not fed for 16 hr and were anesthetized with ether. The chests were excised and blood was obtained by cutting the portal vein and drawing it into glass tubes. The blood was allowed to clot and then centrifuged at 1,500 X g for 30 min at 4°C to obtain serum. Serum samples were analyzed by enzimatic colorimetric procedures for total cholesterol (Cat. 60-2/100), and triglycerides (Cat. 59-4/50) using kits 60-2/10 and 59-4/50, respectively, from LabTest Diagnostica (Lagoa Santa, MG, Brazil). HDL-Cholesterol was measured after phosphotungstic acid precipitation of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) using kit 13 from Labtest Diagnostica (Lagoa Santa, MG, Brazil). Cholesterol LDL ⫹ VLDL was obtained with the following equation: Cholesterol LDL ⫹ VLDL ⫽ (total cholesterol)-(cholesterol-HDL). Lipids were analyzed using enzymatic kit 1691 from CELM (Barueri, SP, Brazil). Samples of liver were freezedried and stored at 60°C for the measurement of cholesterol, triglycerides and total lipids. Lipids were extracted using ethanol for 24 hr by the Soxhlet method. Total cholesterol and lipids were measured using the same kit as that used for the serum samples. Feces were collected during three consecutive days (days 7 to 9), dried and stored at -20°C until analysis. Lipids from dry feces were analyzed using the Soxhlet method. 2.4. Chemical analyses Raw and extruded materials were analyzed for moisture, protein and ash using the AACC [17] methods 44-9, 44-19 and 3020, respectively. Insoluble and soluble dietary fiber was measured using the Prosky et al. [18] method. Data was statistically analyzed using Duncan’s test, with the SAS [19] Software.

3. Results 3.1. Weight gain, feed intake and food conversion Initial hamster weights were similar for all treatments (100.9⫾1.57). At the end of the trial, animals fed with CS-RS and CS-OF had higher weight gains than animals fed with the other diets. However, the difference was not significant in all cases (P ⬍ 0.05) (Table 2). Feed consumption was greater from the hamsters on the CS-RS (130.6 g/20 days) and CS-OF (131.2 g/20 days) diets, as compared to hamsters fed with the control diet (121.0 g/20 days). However, food conversion was better for hamsters fed with the CS-RS (6.95) and CS-OF (7.25) diets. Hamsters fed with CS-RS and CS-OF diets required less food to increase one


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Table 2 Final weight, weight gain and feed intake of hamsters Diets

Final weight (g)

Weight gain (g/20 days)

Feed intake (g/20 days)

Food conversion ratio

Reference Control CS-RS CS-OF

114.4 ⫾ 5.13a 114.2 ⫾ 7.59a 120.3 ⫾ 10.09a 117.9 ⫾ 10.75a

11.5 ⫾ 4.15a 13.1 ⫾ 7.64a 18.8 ⫾ 9.78a 18.1 ⫾ 10.22a

124.0 ⫾ 10.58a 121.0 ⫾ 11.15a 130.6 ⫾ 13.72a 131.2 ⫾ 12.30a

10.7a 9.20b 6.95c 7.25c

Means with the same letter in the same column are not significantly different (P ⬍ 0.05). Reference. Diet containing extruded Cassava starch. Control. Diet containing extruded Cassava starch with 8% cellulose, 10% coconut oil and 2% cholesterol added. CS-RS. Diet containing extruded Cassava starch with resistant starch, 10% coconut oil and 2% cholesterol added. CS-OF. Diet containing extruded Cassava starch with oat fiber, 10% coconut oil and 2% cholesterol added.

gram, 6.95 and 7.25, respectively, as compared to the hamsters fed with reference (10.78) and control (9.20) diets. 3.2. Total cholesterol and lipoprotein cholesterol serum values The addition of 2% cholesterol and 10% coconut oil to the diets was efficient in the production of the hypercholesterolemia in hamsters. The group of animals fed the control diet had 83% more serum total cholesterol (TC) than those fed the reference diet. The values obtained for TC, high-density lipoprotein cholesterol (HDL-cholesterol), low-density plus very low-density lipoprotein cholesterol (LDL⫹VLDL-cholesterol) are shown in Table 3. The level of TC in the serum varied between 159 mg/dL (reference group) and 270 mg/dL Table 3 Effect of diets on total cholesterol (TC), high density lipoprotein cholesterol (HDL-C), low density plus very low density lipoproteins (LDl⫹VLDL-C), triglycerides (TG) and total lipids (TL) in hamsters. Diets1

Reference Control CS-RS CS-OF

SERUM

LIVER LDL ⫹ VLDL-C (mg/dl)

TC (mg/dl)

HDL-C (mg/dl)

159 ⫾ 6c 291 ⫾ 17a 239 ⫾ 27b 247 ⫾ 11b

119 ⫾ 5c 37 ⫾ 5c b 171 ⫾ 22 114 ⫾ 30a 158 ⫾ 9b 77 ⫾ 11b a 195 ⫾ 13 55 ⫾ 14bc

HDL-C / TG (LDL⫹ (mg/dl) VLDL-C) 3.22 1.50 2.05 3.54

130 ⫾ 18c 218 ⫾ 35a 198 ⫾ 34ab 178 ⫾ 26b

TL (mg/dl)

TC (mg/g)

TG mg/g

829 ⫾ 29c 1661 ⫾ 86a 916 ⫾ 18bc 964 ⫾ 48c

3.4 ⫾ 0.34b 8.9 ⫾ 0.46a 3.3 ⫾ 0.26b 2.9 ⫾ 0.19bc

14.0 ⫾ 1.55b 18.7 ⫾ 2.17a 11.2 ⫾ 1.03c 9.1 ⫾ 0.74c

Means with the same letter in the same column are not significantly different (P ⬍ 0.05). Reference. Diet containing extruded cassava starch. Control. Diet containing extruded Cassava starch with 10% coconut oil and 2% cholesterol added. CS-RS. Diet containing extruded Cassava starch with resistant starch, 10% coconut oil and 2% cholesterol added. CS-OF. Diet containing extruded Cassava starch with oat fiber, 10% coconut oil and 2% cholesterol added.

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(control group). Hamsters fed the reference diet had lower TC serum than those fed any other diet, since cholesterol was not added to the control diet. Hamsters fed the CS-RS and CS-OF diets had significantly lower TC serum (11.5% and 8.5%, respectively, P ⬎ 0.05) compared to the CSH group. The serum cholesterol-lowering effect of the diet containing CS-OF is in agreement with the studies done by other researchers [8,20]. The reduced levels of total cholesterol could be due to the greater excretion of a higher fecal bile salts and neutral sterols, as noted by Anderson and Chen [21]. HDL-cholesterol was higher in the group fed the CS-OF diet (195 mg/dL) and was significantly different for the groups fed with CS-RS diet (158 mg/dL) and the CSH diet (171 mg/dL). A previous study done by Rieckhoff et al. [22] shows that HDL-cholesterol increased in the serum of hamsters fed diets containing oat. The lowest value corresponds to the reference diet (119 mg/dL). A higher content of HDL-cholesterol is very important because it is correlated with reduced coronary heart disease risk [23]. On the other hand, LDL⫹VLDL-cholesterol serum values were reduced in hamsters fed with CS-RS and CS-OF diets as compared to those obtained from hamsters fed with reference diet. Ranhotra et al. [24] also found reduced values of LDL⫹VLDL-cholesterol serum in hamsters fed diets based on resistant starch and cellulose. Diets containing fiber showed decreases of 32.4% (CS-RS diet) and 51.7% (CS-OF diet), as compared to the CSH diet. Reductions in LDL⫹VLDL-cholesterol are beneficial, reducing coronary heart disease risk. As a result of the increasing HDL-cholesterol and lowering LDL-VLDL-cholesterol values, the ratio HDL-cholesterol/LDL⫹VLDL-cholesterol increased in diets containing fiber (2.05 for the CS-RS diet and 3.04 for the CS-OF diet). The CSH and control diets had ratios of 1.50 and 3.22, respectively. 3.3. Triglycerides and total lipids serum values Hamsters fed the control, CS-RS and CS-OF diets had significantly (P ⬍ 0.05) lower serum triglycerides than the reference hamsters. This is likely due to the lower fat content of the reference diet. The CS-RS and CS-OF diets showed decreases of 9.2% and 18.3%, respectively, in serum triglyceride and total lipid concentrations in the hamsters as compared to the control group. This trend was also observed by Ranhotra et al. [24]. Total lipid values were significantly (P ⬍ 0.05) lower in hamsters fed the CS-RS diet (916 mg/dL) and the CS-OF diet (964.48 mg/dL) than in those fed the CS diet (1661 mg/dL). Dietary fiber (RS and OF) is the chief cause of reduction in lipid serum content in hamsters. The reference diet had the lowest value (829 mg/dL), since coconut oil was not added to this diet. 3.4. TC and triglycerides liver values TC and triglycerides content in the liver show similar tendencies to those obtained from serum content. Significant differences (P ⬍ 0.05) in liver TC occurred among hamsters fed the control diet (8.9 mg/g) and those fed CS-RS (3.3 mg/g) and CS-OF (2.9 mg/g) diets. Liver TC in the reference diet was 3.4 mg/g, and there were no significant differences (P ⬍ 0.05) between the values obtained from the CS-RS and CS-OF diets. Reduction in liver TC


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Fig. 1. Fresh and dry fecal matter from hamsters fed different diets. Control ⫽ diet containing extruded Cassava starch and no cholesterol added. CSH ⫽ extruded Cassava starch, 8% cellulose, and 2% cholesterol. CS-RS ⫽ Cassava starch extruded with resistant starch and 2% cholesterol. CS-OF ⫽ Cassava starch extruded with oat fiber and 2% cholesterol.

in the hamsters that were fed the oat fiber diet is in agreement with results reported by Kahlon et al. [8]. The CS-RS diet (11.2 mg/g) and the CS-OF diet (9.1 mg/g) had lower liver triglyceride values than the control diet (18.7 mg/g). Liver triglyceride concentration in hamsters fed the reference diet had a value of 14.0 mg/g. 3.5. Lipids in fresh and dry fecal matter The total fecal matter was significantly different (P ⬍ 0.05) among all groups of animals. Hamsters fed with CS-OF, CS-RS, control and reference diets excreted fresh fecal matter at a rate of 0.99 g/day, 0.70 g/day, 0.34 g/day and 0.21 g/day, respectively (Figure 1). The increased fecal values could be due to several causes. One of them was the higher lipid content of the fecal matter (Figure 2). The lipid content of the fecal matter obtained from hamsters fed with CS-OF, CS-RS, CSH and control diets was 14.1%, 12.3%, 2.65% and 2.5%, respectively, and was directly related to the weight of the fecal matter. Another possible cause is that the insoluble fiber (not measured) present in the CS-RS and CS-OF diets was not digested nor absorbed in the gastrointestinal tract and was physically present in the organic fecal matter. On the other hand, the fermentation of soluble fiber by the colonic

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Fig. 2. Lipid content in dry fecal matter of hamsters fed with different diets. Control ⫽ diet containing extruded Cassava starch and no cholesterol added. CSH ⫽ extruded Cassava starch, 8% cellulose, and 2% cholesterol. CS-RS ⫽ Cassava starch extruded with resistant starch and 2% cholesterol. CS-OF ⫽ Cassava starch extruded with oat fiber and 2% cholesterol.

microflora produces short chain fatty acids, that may be used by fecal microorganisms. Then, proliferation of fecal microorganisms could increases the fecal weight. 4. Conclusions Diets containing Cassava starch blended with either resistant starch or oat fiber had hypocholesterolemic properties, significantly lowering total cholesterol levels in the serum and liver, as compared to the diet of Cassava starch without added fiber. Diets containing fiber increased the ratio of high-density lipoprotein to low-density plus very low-density lipoproteins. This is very important because this can contribute to reducing the risk of coronary heart disease. We would like to thank the Fundaç a˜ o de Amparo a Pesquisa do Estado de Sa˜ o PauloFAPESP, SP, Brazil, DTA/FEA/UNICAMP, Campinas, SP, Brazil, and CICATA-IPN, Queretaro, Qro., Me´ xico for their financial support. References [1] Kahlon TS, Edwards RH, Chow FI. Effect of extrusion on hypocholesterolemic properties of rice, oat, corn, and wheat bran diets in hamsters. Cereal Chem 1998;75:897–903.


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