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Effects on Cholesterol Balance and LDL Cholesterol in the Rat of a Soft-Ripened Cheese Containing Vegetable Oils

Laboratoire de Lipochimie Alimentaire, Département de Biologie, Université de Bordeaux I, Talence Cedex, FRANCE

Address reprint requests to: Alexandrine During, PhD, USDA-ARS, Beltsville Human Nutrition Research Center, DHPL, Bldg. 308, Beltsville, MD, 20705. E-mail: during{at}bhnrc.arsusda.gov

	ABSTRACT

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Objective: The purpose of this study was to examine effects of a modified soft-ripened cheese containing vegetable oils on cholesterol status, using the rat as the experimental model and the traditional soft-ripened cheese as the control.

Methods: Adult male Wistar rats (370 g) were divided into two dietary groups (20 rats/group) and fed either the standard diet (STD, containing traditional cheeses made from whole milk) or the experimental diet (EXP, containing modified cheeses made from the combination of skim milk with the following fat mixture: milk fat/oleic acid-enriched sunflower oil/soybean oil mixture). Lipids of the diets came solely from cheeses (14 g/100 g diet); the EXP diet contained (3-fold) less saturated fat, (2-fold) less cholesterol, and (15-fold) more phytosterols than the STD diet.

Results: Although serum triglyceride and total cholesterol concentrations were not affected by the type of diet, the EXP diet resulted in a significant reduction of LDL-cholesterol (31%, p<0.001) and a significant increase of HDL-cholesterol (11%, p<0.05), compared to the STD diet. Thus, a marked reduction (39%) of serum LDL/HDL cholesterol ratio was observed in the EXP group (p<0.001). In addition, the two quantitative balances (excreted/ingested) of cholesterol and total neutral sterols (for which phytosterols were excluded) were significantly higher by 183% and 174%, respectively for the EXP group, compared to the STD group (p<0.05). On another hand, rats fed the EXP diet excreted more cholesterol than they ingested dietary cholesterol (cholesterol balance 1), indicating that those animals eliminated some endogenous cholesterol in their feces, while the opposite was true for rats fed the STD diet (cholesterol balance < 1). Finally, fecal bile salt concentration was not significantly different between the two dietary groups.

Conclusions: The partial substitution of milk fat by vegetable oils in soft-ripened cheese resulted in a decreased blood LDL/HDL cholesterol ratio and an increased fecal excretion of endogenous cholesterol and neutral sterols and, thus, markedly improved its nutritional qualities. Therefore, the consumption of the described modified cheese may meet the demand of subjects who wish to lower their risk for atherosclerosis and cardiovascular disease.

Key words: soft-ripened cheese, PUFA, phytosterols, cholesterol, rats

Abbreviations: STD=standard (diet or group) • EXP=experimental (diet or group) • FA=fatty acids • SFA=saturated fatty acids • MUFA=monounsaturated fatty acids • PUFA=polyunsaturated fatty acids • CHL=cholesterol • LDL-C=cholesterol in low density lipoproteins • HDL-C=cholesterol in high density lipoproteins

	INTRODUCTION

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All sources of milk fat combined (including cheeses) were estimated to contribute 30% to 40% and 15% to 20% of total fat intake per day and per person, respectively in France [1] or the United States [2] in 1988. Thus, dairy products are an important source of fat in the human diet for the inhabitants of these countries; however, a decline in consumption of dairy-derived products has occurred during the last decade because of their negative health image. The association of dairy food intake to risk of coronary heart disease (CHD) has been a long-standing topic of discussion [3]. In general, this negative effect has been attributed to the high content in cholesterol (CHL) and saturated fatty acids (SFA) of dairy products. Indeed, these nutrients (CHL and SFA) were clearly associated with high blood CHL concentration, atherosclerosis and CHD in humans. As a consequence, nutritionists have emphasized a reduction of animal fats (rich in CHL and SFA), particularly butter fat, as well as an increase of vegetable fats (rich in phytosterols and unsaturated fatty acids) in the daily food intake.

In response to these dietary recommendations, the dairy industry has developed new products with modified lipid content increasing unsaturated fats and/or phytosterols levels to satisfy the demand of more health conscious consumers. Several studies have reported the nutritional properties of different types of modified dairy products, e.g. margarine and mozzarella cheese. For instance, both polyunsaturated dairy products produced by modifying the bovine diet [4] and a linoleate-enriched mozzarella cheese product [5] reduced serum CHL concentrations in hyperlipidemic or hypercholesterolemic adults. In addition, in 1995, Lai et al. [6] demonstrated that a change in fatty acid (FA) profile of butterfat by fractionation process may improve its nutritional qualities. Furthermore, recent studies indicated that plant sterol-fortified margarines reduced plasma CHL levels in normocholesterolemic and mildly hypercholesterolemic subjects [7,8]. However, to our knowledge, no study has reported modification of the fat composition of soft-ripened cheeses, which are widely consumed. For example, in France, the daily consumption of soft cheeses is 16 g per adult (32% of the total amount of cheeses consumed per day) [9].

Thus, the aim of this study was to examine effects of a modified soft-ripened cheese made from a mixture of skim milk with butter fat and vegetable oils on lipid metabolism in the rat, compared with the traditional soft-ripened cheese. Effects of cheeses included blood lipids (triglycerides (TG), total CHL and CHL in lipoproteins), intestinal FA absorption and liver sterol. Fecal CHL, bile salts and neutral sterols were also determined.

	MATERIALS AND METHODS

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Preparation of Cheeses and Cheese-Containing Diets
Milk from Poitou-Charentes cows was used either as whole milk to make control cheeses or as skim milk combined with the following fat mixture: low melting point milk fat fraction/oleic acid-enriched sunflower oil/soybean oil (25:50:25, by wt) to make experimental cheeses. The two milks were processed to soft-ripened cheeses according to the traditional techniques used in manufacturing and described previously [10]. For both types of cheeses, two cheesemaking runs of 40 cheeses were made at two different periods of the year (February and December). At the end of ripening (or packaging), cheeses were sampled, lyophilized and sent to our laboratory. At their arrival, lyophilized cheeses were blended into powders (Waring Blendor) stored in airtight jars at 4°C and in the dark.

The basal purified diet "UAR" (Usine d’Alimentation Rationnelle, Epinay sur Orge, 91, France) contained all nutrients necessary for rat growth, except lipids and proteins (Table 1). Powder of either control cheeses or experimental cheeses was incorporated into the basal diet in order to yield a final lipid concentration of 14 g per 100 g of standard (STD) or experimental (EXP) diet, respectively (Table 1). The food given ad libitum was renewed each day to prevent lipid oxidation in diets.

Rats were first stabilized by feeding the STD diet for three weeks. At d0 (zero day of the experiment), rats (370 ± 30 g) were divided into two dietary groups (20 rats/group) and fed either the STD diet or the EXP diet for four weeks. During the feeding period, at d17 and d18, animals (16 rats/group) were housed individually in metabolic cages to determine their individual food intake and to collect their feces (separated from urine). Feces removed from food particles were weighed and immediately placed at -20°C until analyzed. At the end of the experiment, at d28 and d29 after an overnight fast (10 hr), rats were killed by decapitation and their blood and livers harvested. The experimental procedures used in this study met the Guide for the care and use of laboratory animals of the University.

Analyses of Lipids in Serum
Serum TG and total CHL concentrations were determined using the reactional Kits of TG: GPO-PAP method and CHL: CHOD-PAP method, respectively (Boehringer, France).

Two sera of rats from the same dietary group were chosen randomly and combined to obtain a sufficient volume for the lipoprotein fractionation. The sample (4.5 mL) placed in the ultracentrifuge tube was overlaid with 0.5 mL of d = 1.0063 g/mL density solution and then ultracentrifuged (L5-50B model Beckman Ultracentrifuge, Beckman Instruments, Inc., California, USA, SW 50 rotor, 20 hr, 250000 g, 16°C) [11]. The VLDL fraction was obtained from 1.5 mL of the top. The pellet was resuspended with the remaining supernatant, and 250 µL of the suspension was mixed with 25 µL of the Boehringer reagent (0.55 mmol/L phosphotungstic acid and 25 mmol/L MgCl₂) to precipitate LDL [12]. The HDL fraction was obtained from the supernatant of the centrifugation at 4000 rpm for 30 minutes. The LDL fraction corresponded to the pellet resuspended in 0.5 mL of sodium bicarbonate at 100 g/L. CHL in the different lipoprotein fractions was measured by the same colorimetric method used for total CHL determination (see above). Extinction coefficients of CHL in VLDL, HDL and LDL were 6.05, 5.67 and 1.62, respectively.

Preparation of Liver Microsomes
Microsomes were prepared from an aliquot (4 g liver) according to the procedure of Maisterrena et al. [13]. Proteins in microsomes were determined by the method of Lowry et al. [14] using bovine serum albumin as the standard.

Lipid Extraction from Diets and Liver Microsomes
Total lipids of the diets and liver microsomes were extracted using the method of Folch et al. [15]. The resultant lipid extract ("Folch extract") in chloroform/methanol (2:1, v/v) was stored at -20°C until use.

Saponification of Lipids in Diets, Liver Microsomes, and Feces
For diets and liver microsomes, an aliquot of the "Folch extract" (250 µg sterols) was saponified (100°C, 1 min) in the presence of 2N KOH in ethanol (0.5 mL). Then, water (1 mL) and hexane (2 mL) were added (at room temperature), the mixture vigorously mixed, and the hexane phase ("unsaponified (NS) fraction") stored at -20°C for sterol analyses.

For feces, both "NS fraction" and "saponified (S) fraction" were obtained for analyses of sterols, bile salts and FA [16,17]. Briefly, feces (d17 + d18) were first hydrated in water (20 mL) overnight and then blended. An aliquot of the homogenate (4 g) was saponified at 100°C for 1 hr in presence of water (10 mL) and 1N NaOH in ethanol (20 mL) under reflux. The mixture was then extracted with hexane (4 x 35 mL), extracts combined, solvent evaporated and the "NS fraction" redissolved in hexane and stored at -20°C for sterol analyses. The remaining hydroalcohol phase was acidified with HCl (pH 2) and extracted once with chloroform/methanol (2:1, v/v) and twice with chloroform. The three extracts were combined, solvents evaporated and the "S fraction" redissolved in chloroform/methanol (2:1, v/v) and kept at -20°C for bile salt and FA analyses.

Sterol Trimethylsilyl Ether Analyses by Gas Chromatography
Sterols of the "NS fraction" were converted to trimethylsilyl ethers according to the method of Mordret et al. [18]. After evaporation of hexane, the dried extract was silylated at 65°C for 30 min in presence of 0.1 mL of bis-(trimethylsilyl)-trifluoroacetamide/trimethylsilyl chloride (80:20, v/v) (Interchim, France). The mixture was analyzed directly using a gas chromatograph (GC) Carlo Erba HRGC, model 5300 (20090 Rodano, Milan, Italy) equipped with a Ross injector and an OV-17 column (30 m x 0.32 mm i.d., A.M.L.-Chromato, France). Sterols were quantified by use of 5-cholestane as internal standard, which was added before the saponification. Phytosterols were determined using the French norm [19] and neutral fecal sterols using standards commercially available (Sigma, USA). When reference standards were unavailable, fecal sterols were tentatively "identified" in relation to their chromatographic behavior [20, 21].

Fatty Acid Methyl Ester and Bile Salt Trimethylsilyl Ether Analyses by Gas Chromatography
Lipids from the diets and the "S fraction" were transmethylated following the procedure of Lepage and Roy [22]. For feces samples, prior to GC analyses, FA methyl esters were separated from bile salt methyl esters by TLC using a 60H silicagel (0.5 mm thick, Merck, Germany). After a first migration with benzene, FA methyl esters were recovered at the solvent front and extracted with hexane (3 x 2 mL) in presence of 1 mL of methanol/water/acetic acid (10:10:1, v/v/v). After a second migration (the same direction as benzene) with a mixture of isooctane/2-propanol/acetic acid (120:40:1, v/v/v), bile salt methyl esters were extracted from the gel with methanol (3x) and chloroform/methanol (2:1, v/v, 1x). The extracts were then combined, solvents evaporated and the dried extract silylated for 1 hr at room temperature in presence 0.2 mL of N-trimethylsilyl-imidazol.

Bile salt trimethylsilyl ethers were analyzed directly by GC using similar analytical conditions described for sterol analyses (see above). Bile salts in feces were quantified by use of cholestanol as internal standard (added before the step of silylation) and were identified with standards commercially available (Sigma). When reference standards were not available, fecal bile salts were tentatively "identified" by their chromatographic properties compared with a previous report using similar analytical conditions [23].

Fatty acid methyl esters were analyzed using a GC Carlo Erba, model 4160 equipped with a DBWAX capillary column (30 m x 0.32 mm i.d., J&W Scientific, Folsom, CA 95630, USA) and quantified using 19:0 as internal standard added before the procedure of transmethylation.

Statistical Analyses
All data were expressed as mean ± standard deviation (SD). The comparison of the two dietary group effects on each parameter analyzed were performed using the Student’s t test. Differences were considered statistically significant at p < 0.05.

	RESULTS

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Rats were fed either the STD diet (containing 31.5 g of traditional cheese/100 g diet) or the EXP diet (containing 29 g of fat-modified cheese/100 g diet) for 4 wk and, in the two diets, the lipid and protein content was derived solely from cheeses (Table 1). Lipid concentration in both STD and EXP diets was 14.2 g/100 g of diet (Table 1). Both daily food intake (23 ± 2 g either 7 g cheese/d per rat) and body weight gain (3.5 ± 0.3 g/d) were similar in the two dietary groups (data not shown).

Fatty acid and sterol profiles of the diets are indicated in Tables 2 and 3, respectively. As expected, the STD diet exhibited a FA profile common to dairy products: 71% SFA, 23% MUFA, and 3% PUFA with 50% of CHL-raising SFA [sum of lauric acid (12:0), myristic acid (14:0) and palmitic acid (16:0)] (% of total FA) (Table 2). In contrast, FA profile of the EXP diet was 26% SFA, 49% MUFA and 23% PUFA [mainly linoleic acid (18:2 n-6) (21%) and linolenic acid (18:3 n-3) (2%)]. In the EXP diet, CHL-raising SFA represented only 18% of total FA (Table 2). CHL was the major sterol in the STD diet (93% of total sterols), while phytosterols (mainly ß-sitosterol and campesterol) were predominant in the EXP diet (69%) (Table 3). Finally, CHL concentration was 37 vs. 17 mg/100 g and phytosterol concentration was 2.5 vs. 35 mg/100 g, respectively in the STD diet vs. the EXP diet.

View larger version (22K): [in this window] [in a new window]   Fig. 1. Representations of cholesterol balance (R1) and total neutral sterol balance (R2) of rats fed either the STD diet containing traditional cheeses made from whole milk or the EXP diet containing experimental cheeses made from the recombination of skim milk with milk fat and vegetable oils for 4 weeks (see Materials & Methods for details). R1 = ratio of the quantity of cholesterol excreted via the feces divided by the quantity of cholesterol ingested, R2 = ratio of the quantity of total neutral sterols excreted in feces (minus dietary phytosterols) divided by the quantity of cholesterol ingested for each rat. *Values of the EXP group were significantly different from those of the STD group (p < 0.05).

	DISCUSSION

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The purpose of this study was to examine nutritional responses of the fat-modified cheese in comparison with those of the traditional cheese, in the rat. The data show that rats fed experimental cheeses exhibited higher intestinal absorption rates for all classes of FA, particularly for PUFA, compared to rats fed traditional cheeses. We indicated earlier [10] that, in the experimental cheese, the fat mixture was not structurally arranged as the typical fat globule characteristic of whole milk, resulting in higher lipolysis rates during cheese ripening, compared with the traditional cheese. These previous observations may explain the increased "bioavailability" of total FA from experimental cheeses, which contained a fat organization more accessible to pancreatic lipases in the lumen. Moreover, SFA were predominantly eliminated in feces (54% of total FA excreted) (data not shown), even when rats fed the experimental diet containing 75% of unsaturated fats. This preferential excretion of SFA could be related to formation of insoluble calcium-SFA soaps in the gut of animals, since both diets were rich in calcium (1000 mg Ca/100 g diet). Indeed, Fakambi [28] reported that calcium bound preferentially long chain SFA in the lumen to form insoluble calcium soaps, which can not be absorbed and thus are excreted, dragging SFA in feces. As a result, in our two dietary groups, long-chain unsaturated FA (18:1, 18:2 and 18:3) showed higher intestinal absorption rates than long-chain SFA (12:0, 14:0, 16:0 and 18:0). Quantitatively, rats fed experimental cheeses absorbed 60% less SFA, 120% more MUFA and 770% more PUFA than rats fed standard cheeses.

Both n-3 and n-6 PUFA series are recognized as essential constituents of cell membranes as well as precursors of eicosanoids involved in numerous biological functions such as those in the developing of the nervous system [29]. Due to their competitive effects on functional and metabolic functions, an appropriate balance of n-6 and n-3 PUFA in the diet is required. In the present study, the experimental cheese exhibited a n-6/n-3 PUFA ratio of 10 (20% of 18:2 n-6 to 2% of 18:3 n-3), which is somewhat higher than the recommended n-6/n-3 PUFA ratio value of 5 [30], but lower than that provided by the typical North America "Western" diet (up to 25) [31]. Finally, rats fed modified cheeses exhibited an 1.5-fold increase of n-6/n-3 PUFA ratio (defined as 18:2 n-6 + arachidonic acid (20:4 n-6)/docosahexaenoic acid (22:6 n-3) ratio) in both serum and liver microsomes, compared with rats fed control cheeses (data not shown).

In contrast to SFA, unsaturated fats (MUFA and PUFA) have been clearly associated with a reduction of both blood lipids and risk of atherosclerosis [32]. In the present study, ingestion of the unsaturated fat-enriched cheese induced a slight decrease (16%), but not significant, of serum TG concentration compared with the traditional cheese. Similar to our data, most human studies reported no effect of unsaturated dairy products on blood TG concentration, compared with traditional dairy products [4, 5, 33]. However, in spite of no significant change in serum TG level, serum fatty acid profile was significantly enriched in PUFA and diminished in cholesterol-raising SFA when rats fed experimental cheeses (data not shown). In addition, the unsaturated fat-enriched cheese did not produce the expected decrease of serum total CHL level, compared with the traditional saturated cheese. This fact may be due to the low CHL status of our rats, which fed low dietary CHL levels (37 mg CHL/100 g of STD or EXP diet). Similarly, Lai et al. [6] reported no effect of both the liquid butterfat fraction and corn oil on plasma CHL level in rats fed 35 mg CHL/100 g diet, while they reduced plasma CHL level in hypercholesterolemic rats fed 1000 mg CHL/100 g diet, when compared with a solid butterfat fraction. In human studies, the effect of unsaturated dairy products on blood CHL concentration varied; no effect [33] or a lowering effect were observed in healthy or hypercholesterolemic adults [4,5].

In rats fed experimental cheeses, serum LDL-C level was reduced and serum HDL-C level increased, resulting in a significant decrease of the LDL-C/HDL-C ratio considered as an indice of CHD risk. Davis et al. [5] also reported a decrease of plasma LDL-C, but no change in HDL-C, with ingestion of dairy products enriched in 18:2 n-6 (65% of total FA). Moreover, O’Callaghan et al. [33] found that patients fed polyunsaturated dairy products showed lower plasma HDL-C level than patients fed monounsaturated dairy products. Indeed, it has been established that MUFA are as effective as PUFA to reduce LDL-C, while MUFA do not reduce HDL-C as reported with PUFA (such as 18:2 n-6 and 18:3 n-3) [32]. Thus, our modified cheese may contain the "best" balance between MUFA (49%) and PUFA (21%) to produce a reduction of LDL-C associated with an increase of HDL-C, since serum total CHL was not affected.

Unsaturated TG are not the only component in vegetable oils which could result in decreased LDL-C/HDL-C ratio; indeed, dietary phytosterols can also reduce plasma LDL-C level [34]. Vegetable oils used here to make experimental cheeses (25 g of oleic acid-enriched sunflower and 12 g of soybean oil/100 g dried cheese) contained a significant and similar amount of phytosterols (200 mg/100 g oil either 70 mg phytosterols/100 g dried cheese), resulting in a final phytosterol concentration of 35 mg/100 g in the EXP diet. Thus, the daily intake of 6.5 mg phytosterols/d per rat (either 13 mg/d · kg body wt) was somewhat lower than the usual dose of 2–3 g phytosterols/d (either 30–50 mg/d · kg body wt) used in human studies to see a beneficial decrease of plasma LDL-C levels [7,8,34].

The CHL balance (excreted/ingested) was approximately twice higher in animals fed experimental cheeses than in animals fed traditional cheeses. On another hand, rats fed fat-modified cheeses excreted same quantity (or more) of CHL as they ingested (CHL balance 1), while rats fed control cheeses increased their body CHL pool (CHL balance <1). In our experimental conditions, it is not possible to distinguish between the two origins of CHL (dietary vs. endogenous) excreted in feces. However, the data suggest that, in the experimental group, some endogenous CHL could be eliminated, since it is known that dietary CHL is at least partly absorbed (range of 20% to 50%), even in presence of phytosterols. We found that intestinal absorption of ³H-cholesterol was 30%, when a single dose of fats containing 5.2 mg CHL and 12.7 mg phytosterols (quantities close to daily intakes of CHL and phytosterols in the experimental group) was given to mesenteric duct-cannulated rats and using ¹⁴C-triolein as reference (data not shown). This fact indicated that, even with a low CHL diet containing phytosterols such as the experimental cheese, a significant part of dietary CHL was absorbed. In terms of total neutral sterol balance (excreted/ingested), we hypothesized that only dietary CHL participated in the endogenous neutral sterol pool because dietary phytosterols are poorly absorbed (see below). Similar to the CHL balance, the neutral sterol balance (with or without fecal CHL participation) was significantly (170%) higher in rats fed experimental cheeses, compared with rats fed control cheeses. Finally, daily excretion of bile acids (direct products of endogenous CHL metabolism) was similar in the two dietary groups.

Those observations in favor of increased CHL and neutral sterol excretion could be attributed to the presence of phytosterols in the experimental cheese. Indeed, similar to our data, dietary phytosterols were reported to increase fecal CHL elimination as CHL itself, usually not as bile salts, and to enhance excretion of endogenous neutral sterols in humans [34]. In the rat, ingestion of sitosterol or stigmasterol inhibited CHL absorption from 42% to 23% [35]. The exact mechanism of phytosterol inhibition on CHL absorption is still unclear. It is suggested that phytosterols, by their structure close to CHL, may compete with CHL for incorporation into mixed micelles and uptake by the mucosal cell membrane (two essential steps in the intestinal absorption process), while those phytosterols show a limited absorption (2% to 5%) [35]. Similarly, we also found that, after administration of a single dose of a phytosterol mixture (mainly ß-sitosterol (60%) and campesterol (20%)) to cannulated rats, lymphatic recoveries were low: 0% fucosterol, 2% stigmastenol, 4% ß-sitosterol, 5% stigmasterol and 13% campesterol of the initial dose (data not shown). As a result, two phytosterols (campesterol and ß-sitosterol, the two major phytosterols present in the EXP diet) were detected in rat liver microsomes, probably in proportion to their respective small amount absorbed.

By reducing intestinal CHL absorption, phytosterols were expected to decrease CHL pools in body. However, no significant reduction of both total serum and hepatic CHL concentrations were found in rats fed fat-modified cheeses, compared with rats fed control cheeses. Although phytosterol doses of at least 1 g/d (16 mg/d · kg body wt) were recommended to observe a decrease of blood CHL level (as well as LDL-C level), smaller doses (<16 mg/d · kg body wt) were shown to reduce intestinal absorption of CHL and other sterols in humans [36]. Thus, in the present study, phytosterol intake (13 mg/d · kg body wt) could be sufficient to reduce CHL absorption, without affecting serum and hepatic CHL levels. In addition to the dose, the composition of phytosterol mixture in the diet may also affect plasma CHL-lowering efficacy of phytosterols; for instance, 1.5 g of sitostanol was shown to reduce plasma CHL level as effectively as ß-sitosterol given in amounts fourfold higher [37]. The experimental cheese contained mainly ß-sitosterol (48% either 6 mg/d · kg body wt), followed by campesterol (24% either 3 mg/d · kg) (% of total phytosterols). Finally, the lack of response to phytosterols in tissues could be also attributed to the low CHL status of our rats (<17 mg CHL ingested/d · kg body wt). Indeed, Denke [38] suggested that a low dietary CHL intake (<200 mg/d for a human adult either 3 mg/d · kg body wt) may have an indirect effect on apparent phytosterol efficacy via the serum CHL modifying effect of the control low-CHL diet. Therefore, the data suggest that phytosterol efficacy to decrease tissue CHL pools, independently to their reducing effect on CHL absorption, may require an appropriate phytosterol/CHL ratio in the diet.

In conclusion, the experimental soft-ripened cheese containing vegetable oils exhibited more desirable nutritional properties than the traditional cheese. Indeed, due to its high content in unsaturated fatty acids and phytosterols, the modified cheese resulted in an enrichment of PUFA in membranes, a significant reduction of LDL-C/HDL-C ratio related to both decreasing LDL-C and increasing HDL-C levels and a fecal excretion of CHL and neutral sterols. If applicable to humans, the data indicate that the experimental cheese may be beneficial for maintaining health, particularly for subjects who enjoy consuming cheeses and are at risk for atherosclerosis and CHD.

	FOOTNOTES

 
Dr. Alexandrine During is now at USDA-ARS, BHNRC, Diet and Human Performance Laboratory, Bldg. 308, Beltsville, MD, 20705.

Dr. Stephane Mazette is now at Institut des Corps Gras (ITERG), rue Monge, Parc Industriel, 33600 Pessac, FRANCE.

Received February 1, 2000. Revised May 24, 2000. Accepted May 24, 2000.

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