Effect of low inulin doses with different polymerisation degree on lipid metabolism, mineral absorption, and intestinal microbiota in rats with fat-supplemented diet

Food Chemistry 113 (2009) 1058–1065

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Effect of low inulin doses with different polymerisation degree on lipid metabolism, mineral absorption, and intestinal microbiota in rats with fat-supplemented diet María Azorín-Ortuño a, Cristina Urbán a, José J. Cerón b, Fernando Tecles b, Ana Allende a, Francisco A. Tomás-Barberán a, Juan Carlos Espín a,* a b

Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 164, 30100 Campus de Espinardo, Murcia, Spain Department Medicine and Animal Surgery, Faculty of Veterinary, University of Murcia, 30100 Campus de Espinardo, Murcia, Spain

a r t i c l e

i n f o

Article history: Received 15 April 2008 Received in revised form 31 July 2008 Accepted 20 August 2008

Keywords: Prebiotic Artichoke Intestinal microbiota Phosphatase alkaline Iron bioavailability

a b s t r a c t Inulin with different degree of polymerisation from chicory (Orafti BeneoTMGR, GR) and artichoke (ArtBiochem ArtinulinTM, Art) as well as the mixture of these inulins with chicory oligofructose (Orafti BeneoTMSynergy1, Syn; and ArtinulinTM plus Orafti BeneoTMP95 oligofructose, Art+P95) were assayed on developing rats fed with fat-supplemented diet. The effect of these inulins was evaluated on seven haematological and 16 serobiochemical parameters, three minerals, feedstuff intake, growth rate, food utility index and intestinal microbiota upon consumption of a human (60 kg weight) equivalent dose of 0.82 g/day for 75 days. The low inulin dose assayed exerted beneficial effects on rats with fat-supplemented diet upon increase of iron absorption (Art), regulation of ALP (phosphatase alkaline) activity (Art, Syn and Art+P95), increase of growth rate without modification of the final weight (Syn1 and Art+P95), increase of HDL-cholesterol (GR, Syn1 and Art+P95) and reduction of enterobacteria (GR, Syn1 and Art+P95). Ó 2008 Elsevier Ltd. All rights reserved.

1. Introduction Inulin and oligofructose are fructans composed of linear chains of fructosyl monomers linked by b(2?1) bounds. Inulins are widely distributed in some edible plants including asparagus, garlic, chicory, leek, onion and artichoke as storage carbohydrates (Kaur & Gupta, 2002). Inulins from different plant origins differ in their degree of polymerisation (DP), chain distribution and product purity (López-Molina et al., 2005). The average DP (DPav) can range from 2 to 10 in oligofructose to 40 and even more in some inulins. The main industrial source of inulin and oligofructose is chicory fresh root (Cichorium intybus) with a DPav of 12 (Roberfroid, 2007). Artichoke (Cynara scolymus, L.) inulin has an unusual high DPav value (DPav = 46) compared to other inulins. The degree of polymerisation can influence some properties of these products such as digestibility, prebiotic activity, caloric value, sweetening power, water binding capacity, etc. (López-Molina et al., 2005; Van de Wiele, Boon, Possemiers, Jacobs, & Verstraete, 2007). Inulin-type fructans are currently considered ‘functional ingredients’. This involves that their regular consumption within a wellbalanced diet has been correlated with the improvement of life’s quality by means of physiological, psychological and behavioural * Corresponding author. Tel.: +34 968 396344; fax: +34 968 396213. E-mail address: [email protected] (J.C. Espín). 0308-8146/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2008.08.062

performances (Roberfroid, 2007). One of the interesting functions of inulin and oligofructose in human nutrition is related to their prebiotic effect, i.e. the specific stimulation of growth and/or activity of a limited number of colonic bacteria beneficial to the host, as well as the growth inhibition of pathogens and harmful microorganisms (Roberfroid, 2007). The combination of prebiotics and probiotics has given rise to the so-called ‘synbiotics’, with promising healthy properties (Buriti, Cardarelli, Filisetti, & Saad, 2007; PoolZobel & Sauer, 2007). The intake of inulin and oligofructose enhances gastrointestinal mineral absorption such as calcium, magnesium and iron (Kaur & Gupta, 2002; Yeung, Glahn, Welch, & Miller, 2005) which has been related to the protection against mineral deficiencies and the prevention of osteoporosis in the case of calcium (Abrams, Griffin, & Hawthorne, 2007). In addition, experimental data demonstrate that inulin and oligofructose affect processes and parameters involved in lipid metabolism and thus producing a beneficial effect on diseases related with lipid disorders such as atherosclerosis (Kaur & Gupta, 2002). Despite the well-recognised health-benefits of dietary inulins, however, its consumption should be limited for people with fructose malabsorption. It is believed that up to 36% of the European population have fructose malabsorption with different degree of severity and about half of them are symptomatic (Born, Zech, Stark, Classen, & Lorenz, 1994), who should limit the intake of fructans

1059

M. Azorín-Ortuño et al. / Food Chemistry 113 (2009) 1058–1065

ingredients such as inulins (Muir et al., 2007; Shepherd & Gibson, 2006). In addition, high fructose loads trigger irritable bowel syndrome symptoms in patients with this disease (Shepherd & Gibson, 2006). Most studies reporting inulin-derived benefits have assayed relatively high inulin doses, especially in animals. However, the present study was designed to examine whether a low inulin intake (compatible with that permitted for people with fructose malabsorption) could also exert some benefits by exploring differential effects on intestinal microbiota, lipid metabolism and mineral absorption. For this purpose we tested inulins with different DPav, alone or in combination with oligofructose, to examine whether these effects could be more evident in inulins with an extra-long DPav.

Table 1 Inulins assayed in the present study and their polymerisation degree Rat groups

Diet

Inulin source

Average of polymerisation degree (DPav)

Control Fat-control ArtInulinTM (Art) BeneoTMGR (GR) BeneoTMSynergy1 (Syn1)

Standard feedstuff Feedstuff + 20% lard Feedstuff + 20% lard Feedstuff + 20% lard Feedstuff + 20% lard

– – Artichoke Chicory Chicory

– – DPav 46 DPav 25 DP 2–10 and 10–60

Feedstuff + 20% lard

Artichoke + Chicory

DPav 46

(Art+P95) ArtInulinTM (90%) + BeneoTMP95 oligofructose (10%)

DPav 4

2. Materials and methods 2.1. Reagents Reagents for the biochemical analyses were purchased from Spin React (Gerona, Spain). BeneoTMGR inulin, BeneoTMSynergy1 inulin and BeneoTMP95 oligofructose were kindly supplied by ORAFTI Spain S.L. (Barcelona, Spain). Artichoke inulin (ArtInulinTM) was kindly provided by ArtBiochem S.L. (Murcia, Spain).

18 mg inulin per rat (mean of 210 g body weight) at the beginning of the assay to 22 mg inulin per rat (mean of 256 g body weight) at the end of the assay ensured a mean HED of 0.82 g/day in a 60 kg adult person. Body weight, food and water consumption were recorded daily. Growth rate was calculated as the difference between the final and initial weights divided by the 75 days intervention period. Food utility index was calculated as the weekly body weight gain divided by the food consumption.

2.2. Animals and diets Female Sprague–Dawley rats (n = 36) with weights ranging from 183 g to 249 g (mean 210 ± 8.4 g) were provided by the Animal Centre of the University of Murcia (Spain). Handling and killing of rats were in full accordance with national and international law and policies. Animals were randomly assigned to six dietary groups (n = 6/group) and housed in groups of three rats per cage in a temperature-controlled environment (22 ± 2 °C) with 55 ± 10% relative humidity and controlled lighting (12 h light/dark cycle). Rats were fed with standard 2014 Teklad Global 14% protein rodent maintenance diet (Harlan Teklad, Madison, WI, USA) that was grinded to guarantee a homogeneous mixture with inulins. The feedstuff contained 14.5% proteins, 4.7% ash, 51.2% starch, 4.3% sugar and 4% fat (3.2 kcal/g). Regarding micronutrients, the feedstuff contained 0.72% calcium, 0.6% phosphorus, 0.23% magnesium and 0.25% chloride among others. The detailed feedstuff composition is available at . The six groups were distributed as follows: one group was fed with standard feedstuff (control) and the other five groups were fed with a ‘high-fat diet’ provided by the intake of 20% lard-enriched feedstuff. One group fed with high-fat diet without inulin was the fat-control group. The other four groups with high-fat diet were supplemented with different types of inulin. Inulins differed in both DPav and plant origin (Table 1). Groups were termed according to their diets, i.e., control, fat-control, GR (Orafti BeneoTMGR inulin), Art (ArtBiochem ArtinulinTM, Artichoke inulin), Syn1 (Orafti BeneoTMSynergy1), Art+P95 (90% ArtBiochem ArtinulinTM plus 10% Orafti BeneoTMP95 oligofructose). The composition of the high-fat diet was 11.6% proteins, 51.12% carbohydrates and 23.2% fat (4.36 kcal/g). The inclusion of inulins did not modify significantly this composition. Both solid diet and water were consumed ad libitum. The inulin content in the rat diet was adapted during the experiment according to the increasing weight of the rats. Instead of the direct animal weight–human weight extrapolation, the dose translation from animal to human studies is better calculated using a body surface area normalisation method. For this purpose, the human equivalent dose (HED) was calculated following the equation: HED = animal dose in mg/kg  (animal weight in kg/human weight in kg)0.33 (Reagan-Shaw, Nihal, & Ahmad, 2008). The addition of

2.3. Sampling procedure Rats were anaesthetized with a mixture (1:1 v/v; 1 mL/kg body weight) of xylazine (Xilagesic 2%, Calier Laboratories, Barcelona, Spain) and ketamine (Imalgene 1000, Merial laboratories, Barcelona, Spain). Blood samples (approximately 2 mL) were obtained every 2 weeks by cardiac punction and collected in heparinised tubes. Heparinised blood was used to obtain the haematological profile. For the determination of sero biochemical parameters, blood was immediately separated in plasma by centrifugation at 14,000g for 15 min at 4 °C in a Sigma 1–13 microcentrifuge (Braun Biotech. International, Germany). The plasma was immediately frozen at 80 °C for further analyses. Fresh fecal samples (about 1 g) were collected in a sterile tube with buffer solution (5 mL). At the end of the intervention study (75 days), animals, previously anaesthetized as described above, were sacrificed by exsanguination. Liver and kidneys were macroscopically examined and weighted. Fat accumulation and disposition was also determined. 2.4. Haematology and clinical chemistry Haematological parameters were determined using an automated haematological analyser (Abacus Junior Vet, CVM S.L., Navarra, Spain), with specific software for rat blood samples. The parameters analysed were red blood cell number (RBC); haemoglobin concentration (Hb); haematocrit (HCT); mean corpuscular volume (MCV); mean corpuscular haemoglobin (MCH); mean corpus cular haemoglobin concentration (MCHC); and white blood cell count (WBC). Biochemical parameters were determined in heparinised plasma using a Cobas Mira Plus analyser (HORIBA ABX Diagnostic, Montpellier, France). The parameters analysed were glucose, urea, creatinine, cholesterol, HDL-cholesterol, triglycerides, total protein, albumin, globulins, alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), gamma glutamyl transferase (GGT), creatine kinase (CK), amylase, calcium, phosphorus and iron.

1060

M. Azorín-Ortuño et al. / Food Chemistry 113 (2009) 1058–1065

The normal values range (NVR) was calculated for each parameter as the mean of control group value ±2  SD. GGT, bilirubin and CK were not parametrically distributed and were expressed as mean and percentiles. NVR are influenced by reagents, methods, analysers or even blood extraction systems. For this reason, the establishment of reference values by each laboratory is highly recommended.

A

2.5. Isolation and enumeration of bacteria Fresh fecal samples of about 1 g were collected immediately prior to the experiment and homogenized in maximum recovery diluent (Oxoid CM 733, Hampshire, UK). Homogenised samples were serially diluted with the same diluent. Dilutions of the samples were then inoculated in duplicate onto selective agars:

0.20

b

Control Control-Fat Art GR Syn1 Art+P95

b

Food utility index

0.15

ab

ab a

ab

0.10

a

a a

a

ab

a

b

b

ab* ab

a

0.05

a* 0.00 24

47

75

Days

B

25

20 Feed-stuff intake (g)

a 15

b

b

b

b

c

10

5

0

b

C

b

Growth rate

0.90

ab a

a a

0.45

0.00 Control

Control-Fat

Art

GR

S yn1

Art+P95

Fig. 1. Food utility index (A), feedstuff intake (B), and growth rate in rat groups over the study (C). Bars (treatments) with different letters are significantly different (p < 0.05). In the case of Fig. 1A, asterisks designate differences (p < 0.05) among sampling periods.

1061

M. Azorín-Ortuño et al. / Food Chemistry 113 (2009) 1058–1065

BHI-Blood agar (Oxoid, CM 225 with 1.6% agar and 50 mL sheep blood/L) for total anaerobic; Lactobacillus anaerobic MRS with Vancomycin and Bromocresol Green (LAMVAB) according to the instructions by the authors for lactobacilli; and MacConkey agar (Oxoid, CM 007) for enterobacteria. Inoculated agar plates were incubated in an anaerobic environment at 37 °C for 48 h, except for MacConkey agar plates, which were incubated aerobically at 37 °C for 24 h. Anaerobic conditions were generated in airtight bags or jars using AnaeroGenTM compacts (Oxoid). Microbial colonies were counted with an automated plate counter (ProtoCOL, Synoptics, Cambridge, UK). Microbial counts were expressed as log10 cfu/g. 2.6. Statistical analysis Results were analysed using the SPSS software 14.0 (SPSS Inc, Chicago, IL, USA) Haematological and serobiochemical data were analysed using the Levene’s test to check homogeneity of variances (homocedasticity). Those data with homocedasticity were analysed using analysis of variance (ANOVA) followed by Tukey’s test. Data with no homocedasticity were treated using the non-parametric Mann–Whitney U-test. Data were deemed significant at p < 0.05 (*). For microbial evaluation ANOVA followed by the Tukey or T3 of Dunnett methods with a significant level of p < 0.005 (*) were performed. A significant level of p < 0.005 was selected to determine the microbial variations to consider the inherent variability of microbial testing methods. 3. Results and discussion 3.1. Growth rate and food utility index Body weight of rats and feedstuff intake were daily monitored. The mean rat weight, taking into account all groups, increased from 210 ± 8.4 g at the beginning of the study to 256 ± 15.2 g after 75 days. Some logical fluctuations due to animal handling and cardiac punctures for blood collection were observed over the study (results not shown). The food utility index decreased after the 24 first days in these growing rats due to the progressive decrease in weight gain during the following weeks except in the case of the GR group which remained constant (Fig. 1A) probably due to the slower growth rate of this group (Fig. 1C). It is of note that

the feedstuff intake was significantly higher (p < 0.05) in control rats than in the rest of the groups fed with the high-fat diet (Fig. 1B). This could be due to an increase in the energetic value of the high-fat diet (4.36 kcal/g) respect to the standard diet (3.2 kcal/g). Previous studies have reported that rats modify the intake by means of an energetic regulation process (Corwin et al., 1998). A significant increase (p < 0.05) in the Art+P95 group intake respect to the rest of high-fat diet groups was observed (Fig. 1B). The possible explanation could be based on the inhibition of fat absorption by artichoke inulin + oligofructose P95. In this context, as the energetic value of the feedstuff decreased, the feedstuff intake was increased due to energetic regulation. In addition, although all the groups reached approximately the same weight at the end of the intervention, it is noticeable that the growth rate in the Art+P95 and Syn1 groups (p < 0.05) was significantly faster than that of the other groups (Fig. 1C). 3.2. Haematological and serobiochemical parameters Different haematological and serobiochemical parameters were routinely assayed to assess potential harmful effects due to the dietary fat-supplementation and animal handling. No significant changes were observed in the haematological parameters during the study (results not shown) which discarded any potential haemostatic disorder, inflammatory conditions, anaemia, etc. (Azorín et al., 2008). The majority of serobiochemical parameters were not affected by the dietary fat-supplementation and animal handling (Table 2). Creatinine concentrations were significantly increased in all groups, including control group, with respect to the beginning of the assay (p < 0.001) (Table 2). These values were within our laboratory reference values and thus no pathological status was associated to the mild increases observed (Meyer & Harvey, 1998). Liver parameters (ALT, AST and bilirubin) were normal and CK activity did not show significant differences with respect to the control at the end of the study (Table 2). However, serum ALP activity was significantly increased in all groups fed with the high-fat diet at day 14 (p < 0.001) with respect to day 0 (Table 3). Fat control group maintained significantly high ALP activity throughout the study whereas rats receiving inulin, with the exception of inulin GR group, showed a decrease on serum ALP activity although without achieving the initial values at the end of the experiment (Table 3). The increase of ALP activity in intesti-

Table 2 Serobiochemical parameters in controls and inulin-fed ratsa Parameter

Control

Fat-control

Art

GR

Syn1

Art+P95

NVR

Glucose (mg/dL) Urea (mg/dL) Creatinine (mg/dL) Cholesterol (mg/dL) HDL-Cholesterol (mg/dL) Triglycerides (mg/dL) Total proteins (g/dL) Albumins (g/dL) Globulins (g/dL) Bilirubin (mg/dL) ALP (units/L) AST (units/L) ALT (units/L) GGT (units/L) CK (units/L) Amylase (units/L) Calcium (mg/dL) Phosphorus (mg/dL) Iron (lg/dL)

288 ± 31.3 27.6 ± 2.6 0.58 ± 0.06*** 113.0 ± 11.4 48.9 ± 5.2 64.1 ± 14.6 6.75 ± 0.31**

273.6 ± 28.9 21.5 ± 4.0 0.62 ± 0.08*** 101.9 ± 15.7 45.6 ± 3.9 57.7 ± 12.7 6.54 ± 0.35 3.38 ± 0.22 3.16 ± 0.24 0.27 (0.21–0.35) 379.8 ± 94.9** 111.2 ± 48.8 44.0 ± 13.6 0.0 (0.0–0.23) 674 (437–816) 729.8 ± 68.9 9.29 ± 2.36 5.25 ± 0.81 247.7 ± 53.7

267.4 ± 42.4 24.0 ± 1.7 0.60 ± 0.08*** 109.7 ± 8.0 44.9 ± 2.5 52.6 ± 19.6 6.40 ± 0.23 3.43 ± 0.14 2.97 ± 0.10 0.12 (0.10–0.36) 338.5 ± 83.3 171.1 ± 49.1*** 49.5 ± 32.8 0.4 (0.0–0.9) 1072 (1005–1139) 729.2 ± 57.7 10.59 ± 2.05 7.17 ± 0.86 321.5 ± 61.6*

245.4 ± 21.9 19.1 ± 2.4 0.52 ± 0.01*** 108.5 ± 11.9 60.0 ± 10.5** 43.8 ± 4.1 6.12 ± 0.14 3.53 ± 0.13 2.59 ± 0.22** 0.13 (0.12–0.13)* 474.8 ± 101.5*** 84.1 ± 18.5 40.9 ± 16.6 0.0 (0.0–0.0) 272 (234–309)*

257.2 ± 36.9 19.7 ± 6.8 0.63 ± 0.04*** 104.2 ± 10.0 43.9 ± 5.6** 43.9 ± 10.4 6.58 ± 0.66 3.30 ± 0.15 3.28 ± 0.56 0.22 (0.19–0.23) 337.6 ± 97.6 145.6 ± 72.4 65.7 ± 19.8 0.0 (0.0–0.0) 712 (454–879) 726.2 ± 99.5 9.71 ± 0.58 5.66 ± 1.36 269.4 ± 33.3

307.4 ± 18.2 19.9 ± 4.4 0.59 ± 0.08*** 121.7 ± 6.4*** 42.9 ± 9.2* 58.1 ± 21.3 6.43 ± 0.46 3.28 ± 0.22 3.15 ± 0.30 0.22 (0.15–0.32) 293.9 ± 27.1 135.9 ± 24.6 49.7 ± 14.4 0.0 (0.0–0.0) 905 (763–1040) 732.2 ± 92.4 8.98 ± 0.75 7.02 ± 0.92 297.9 ± 18.6

150.6–413.8 20.3–32.3 0.3–0.7 80.1–128.1 24.8–58.4 23.3–98.9 5.5–7.1 3.1–3.5 1.7–4.1 0.2 (0.1–0.3) 83.1–306.7 20.9–168.5 22.2–63 0.0 (0.0–0.1) 441 (374–563) 565.7–808.1 7.1–12.3 3.7–7.3 134.6–300.6

3.32 ± 0.16 3.43 ± 0.22* 0.16 (0.15–0.19)* 163.9 ± 28.6 125.1 ± 49.2 50.3 ± 13.4 0.0 (0.0–0.14) 725 (483–944) 728.6 ± 75.1 8.47 ± 0.80 5.18 ± 0.44 216.9 ± 29.2

685.5 ± 37.1 9.43 ± 1.86 4.07 ± 0.66 258.7 ± 62.2

a Results are expressed as mean ± SD except in bilirubin, GGT and CK in which mean and percentiles (25–75) are shown. Normal values range (NVR) was calculated as the mean of reference values ±2  SD. Results shown correspond to the end of the experiment (day 75). * (p < 0.05); ** (p < 0.01); *** (p < 0.001).

1062

M. Azorín-Ortuño et al. / Food Chemistry 113 (2009) 1058–1065

nal mucosa upon consumption of a high-fat diet has been previously reported in rats (Kaur, Madan, Hamid, Singla, & Mahmood, 2007). This increase is further reflected in high serum ALP activity because the origin of the serum ALP is usually intestinal (Kaur et al., 2007). The results obtained by DeSchryver-kecskemeti, Eliakim, Green, and Alpers (1991) claimed the important function of intestinal ALP in the trans-epithelial transport of triglycerides. Moreover, the use of inhibitors for intestinal transport of lipids have proved to inhibit the secretion of this enzyme in the rat intestine, and thus blocking the increase of the levels of serum ALP (Kaur et al., 2007). Taking into account the above, our results suggest that inulins, despite the low amount assayed in this study, could hamper fat absorption resulting in lower serum ALP activity. This effect was strongly observed in the animals receiving the inulin with the longest chain (ArtInulinTM) as well as in the groups that received the combinations that included oligofructose mixed with long-chain inulins (Syn1 and Art+P95) (Table 3). The fat disposition in rat organs after sacrifice was also explored. The rats fed with the high-fat diet showed more perivisceral fat surrounding both the digestive tract and uteri than rats from the control group. No differences were observed between rats from fat-control group and inulin groups (results not shown). No differences were observed in the kidneys of any rat. On the contrary, the livers belonging to rats fed with the high-fat diet were more yellowish in colour than livers from control rats, although no differences in weight were observed. No differences were observed between rats from fat-control group and rats from inulin groups (results not shown).

HDL cholesterol is high compared to humans. Total cholesterol concentrations only increased significantly in rats receiving Art+P95 from 28th day to the end of the experiment (Table 2). This increase could be related with an elevation in the cholesterol fraction in HDL (HDL-cholesterol) (Table 4). Significant increases in this parameter were observed in GR group at the end of the assay as well as in Syn1 and Art+P95 groups at 28, 42 and 71th day (Table 4). The increase of serum HDL-cholesterol concentrations in these groups ranged from 42% to 49% at the end of the experimental procedure compared with the initial values. Our results are in agreement with those of Kim and Shin (1998) who found elevated levels of HDL-cholesterol respect to control groups when rats were fed with diets containing 1% or 5% chicory extract or 5% inulin for 4 weeks. On the other hand, Trautwein, Rieckhoff, and Erbersdobler (1998) did not find any significant changes in this parameter when different concentrations of inulin (8%, 12% and 16%) were added to hamster diet. It is well established that the decrease in serum HDLcholesterol is a potential risk factor involved in atherosclerosis development. This lipoprotein has an important role in reverse cholesterol transport, from peripheral tissues to liver where is metabolize into bile acids (Stein & Stein, 1999). Therefore, despite the low inulin dose assay compared with most of studies reported in the literature, our results suggest that BeneoTMGR inulin, BeneoTMSynergy1 inulin and ArtInulinTM+BeneoTMP95 could potentially exert beneficial effects against atherosclerosis development.

3.3. Serum lipids

Several studies have previously suggested that the intake of inulin-enriched diets increase calcium absorption in both rats and humans (Abrams et al., 2007; Kaur & Gupta, 2002). In the present study, no significant differences in calcium level were observed among the rat groups at the end of the experiment (Table 2). However, in contrast to the low dose assayed in our study (0.82 g/day HED), the amount of inulin used in the rat experiments mentioned above (Delzenne, Aertssens, Verplaetse, Roccaro, & Roberfroid, 1995) was 10% of the diet (a dose that cannot be achieved in human trials) and 8 g inulin in the human trial (Abrams et al., 2007). Iron concentration tended to increase in all groups except in controls and rats receiving GR-inulin. This increase was higher in the two groups whose diets contained artichoke inulin although this increment was only significant in the artichoke inulin group (p < 0.05) at the end of the experiment (increase of 53.60%) (Fig. 2). In this case, the increase could be related to the extra-long DPav of this inulin which could be also related with a more pronounced prebiotic effect (Van de Wiele et al., 2007). The increase of iron absorption upon inulin consumption has been previously reported (Yap, Mohamed, Yazid, & Maznah, 2005). In addition, Yeung et al. (2005) launched the hypothesis that prebiotics, such as inulin, enhance iron absorption through the fermentation of prebiotics by colonic microbiota which decrease the pH of the luminal content and improve iron solubility, promote reduction of Fe(III) to

Previous studies in rats fed with a supplement of these fructans have demonstrated a decrease in serum triglycerides, cholesterol, VLDL- and LDL-cholesterol lipoproteins (Fiordaliso et al., 1995) and an increase of HDL-cholesterol (Kim & Shin, 1998). However, when these studies are carried out in humans, controversial results have been reported. Some studies did not report any changes in these parameters in patients treated with inulin and oligofructose (Pedersen, Sandström, & Van Amelsvoort, 1997). On the other hand, other authors have reported an improved serum lipid profile in patients (Kaur & Gupta, 2002). These conflicting results could be due to the lower doses used in human trials respect to animal experiments. Although the inulin doses are rather high in all the studies, the inulin doses usually assayed in the animal assays are much higher (10% of the diet; Fiordaliso et al., 1995), than those assayed in humans (from 8 g/day to 10 g/day; Williams & Jackson, 2002). No differences in triglycerides were observed in the serum of the high-fat fed rats compared to controls over the study (Table 2). Diet containing 23.2% fat did not significantly increased triglyceride concentration in the rats of this study which could be due to the resistance of the rat to the induction of hyperlipidemia (Loeb, 1997). The level of serum cholesterol in rats is rather low compared to humans (only one-third than that expected in humans) whereas

3.4. Minerals

Table 3 Evolution of serum ALP activity over the studya Group

Day 0

Day 14

Day 28

Day 42

Day 56

Day 75

Control Fat-control Art GR Syn1 Art + P95

228.9 ± 47.7 239.5 ± 34.2 245.1 ± 51.6 277.7 ± 47.1 222.4 ± 17.9 198.9 ± 18.5

213.7 ± 46.4 380.4 ± 59.1** 407.8 ± 48.5** 472.1 ± 73.2*** 429.1 ± 96.2** 363.2 ± 32.7***

211.2 ± 57.7 428.7 ± 51.6** 328.4 ± 33.7 429.0 ± 78.5*** 364.8 ± 87.9 346.8 ± 76.0***

183.3 ± 44.3 417.1 ± 73.8** 262.5 ± 24.9 336.3 ± 63.4 372.3 ± 99.8** 248.0 ± 84.4

149.4 ± 85.4 360.1 ± 79.4** 260.3 ± 71.6 393.0 ± 56.6 312.8 ± 56.1 276.4 ± 33.2

163.9 ± 28.6 379.8 ± 94.9** 338.5 ± 83.3 474.8 ± 101.5*** 337.6 ± 97.6 293.9 ± 27.1

a

Results are expressed as mean ± SD. Normal values range (NVR) for ALP is shown in Table 2. ** (p < 0.01); *** (p < 0.001).

1063

M. Azorín-Ortuño et al. / Food Chemistry 113 (2009) 1058–1065 Table 4 Changes in HDL-cholesterol concentration over the studya HDL-cholesterol (mg/dL)

Day 0

Day 14

Day 28

Day 42

Day 56

Day 75

Incrementb %

Control Fat-control Art GR Syn1 Art+P95

42.3 ± 3.5 38.6 ± 3.8 42.1 ± 2.9 40.1 ± 8.4 30.7 ± 6.1 28.8 ± 7.7

30.3 ± 2.4 33.5 ± 9.0 31.8 ± 6.7 40.3 ± 3.9 38.6 ± 5.1 37.8 ± 2.6

40.6 ± 4.7 38.3 ± 4.8 42.5 ± 3.1 44.8 ± 5.2 41.4 ± 3.6** 45.9 ± 12.7*

42.9 ± 15.2 44.8 ± 17.9 41.9 ± 6.2 39.2 ± 4.6 47.2 ± 7.2** 39.7 ± 4.9*

45.6 ± 4.5 45.7 ± 6.3 45.4 ± 8.1 42.5 ± 4.2 40.1 ± 6.4 43.5 ± 3.4**

48.9 ± 5.2 45.6 ± 3.9 44.9 ± 2.5 55.2 ± 5.1** 43.9 ± 5.6** 42.9 ± 9.2*

15.6 18.1 6.7 37.6 42.9 48.9

a b

Results are expressed as   mean ± SD. Normal values range (NVR) for HDL-cholesterol is shown in Table 2. 75100 100; * (p < 0.05); ** (p < 0.01). Increment = dayday 0

Fe(II), stimulate proliferation of epithelial cells to expand the absorptive surface area by the production of short-chain fatty acids (SCFA), and potentially stimulate expression of mineral-transport proteins in epithelial cells. 3.5. Microbiota analysis Previous studies have reported that important groups of the gastrointestinal community in the rat can be affected not only by the diet but also by the age. In fact, the outcome of the dietary modification with synbiotics has been reported to depend on the ages of the animals (Lesniewska et al., 2006). Total anaerobic microbiota of the rats was evaluated up to 75 days without significant changes among the dietary treatments (Fig. 3A). This agrees with Kleessen, Hartmann, and Blaut (2001) who found that total bacterial counts of fecal samples were very similar in rats fed with or without inulin for eight days. In addition, total bacterial levels remained unaffected after the ingestion of inulin in previously reported human trials (Kleessen, Sykura, Zunft, & Blaut, 1997). On the other hand, several studies showed significant differences in the microbiota of rats fed with inulin, although this has been reported when other specific microorganisms were studied such as bifidobacteria (Kleessen et al., 2001). In the present study, probably due to the low inulin intake, the

lactobacilli load of faeces samples was not affected by the inulin treatments (Fig. 3B) in accordance to Campbell, Fahey, and Wolf (1997). Many potentially pathogenic/harmful bacteria belong to the Enterobacteriacea family. Thus, the reduction or the control of proliferation of this potentially harmful bacteria play a role in maintaining health and well-being and in reducing the risk of some diseases (Roberfroid, 2007). In this study, enterobacteria of both control and control-fat groups increased over the study (p < 0.005), while their number slightly decreased in inulin-fed rats (Fig. 3C). According to this, Kleessen et al. (1997) reported that inulin decreased enterococci in number and enterobacteria in frequency. Therefore, our results suggest that the ingestion of low doses of inulins could positively affect the gut microbiota by a reduction of enterobacteria. 4. Conclusion Most studies reporting inulin-derived benefits have assayed relatively high inulin doses, i.e., 5–10% animal diet, 8–10 g/day in humans. However, our results suggest that the intake of low doses of inulin (HED = 0.82 g/day), compatible with the intake for subjects with fructose malabsorption, exerts beneficial effects in developing rats with fat-supplemented diet. These effects might take place

500

Control Control-Fat Art GR Syn1 Art+P95

Iron concentration (μg/dL)

400

a 300

a

a a

a

* b a

a a

a a a

200

100

0 Day 0

Day 75

Fig. 2. Iron concentration in rat groups. Bars (treatments) with different letters are significantly different (p < 0.05). Asterisk designates differences (p < 0.05) between the two sampling periods.

1064

M. Azorín-Ortuño et al. / Food Chemistry 113 (2009) 1058–1065

A Total anaerobic growth (log cfu/g)

10.4

B

9.6

a,b a,b a,b

Art GR

Control Control-Fat

a

Syn1 Art+P95 a

a a,b b,c a,b

a,b b

a

b,c c

a,b b,c

8.8

a

a

a,b a,b,c

a,b a

a,b,c

aa a

b,c,d

b a,b

a

c,d d

b

c b,c

a

8.0

0.0 9.6

a a,b a,b a,b

C

8.8

b

a

a

a,b a,b b a,b a

b a,b

b b

* a,ba

a

a

a b,c

a a,b

b

c

b

b,c b,c cb,c

8.0

b

7.2 0.0 8.0 a

Enterobacteria growth (log cfu/g)

a

a,b

a

a

a,b

7.2

a,b a,b

a

a a,b b a,b

c

6.4

*a

a

a

a,b b,c

b

a

a a aa a

a,b,c

Lactobacilli growth (log cfu/g)

a a,b

a,b

b

b

c

b bb

b

*b *

b

* c

5.6 0.0 0

15

35

45

60

75

Days Fig. 3. Total anaerobic (A), lactobacilli (B) and enterobacteria (C) growth in rat groups over the study. Bars (treatments) with different letters are significantly different (p < 0.005). Asterisk designates differences (p < 0.005) between the initial and final sampling periods.

mainly upon regulation of ALP activity; increase of growth rate, iron absorption and HDL-cholesterol as well as by reducing enterobacteria. In general, these effects were more pronounced in rats fed with diets containing inulins with the longest DPav such as artichoke inulin.

Acknowledgements This work has been partially supported by the Project Consolider Ingenio 2010, CSD2007–00063 (Fun-C-Food). M.A.O. and A.A. are holder of a JAE-Predoc fellowship and a JAE-Doc-07 contract,

M. Azorín-Ortuño et al. / Food Chemistry 113 (2009) 1058–1065

respectively, from the Spanish ‘‘Consejo Superior de Investigaciones Científicas” (CSIC). Authors are grateful to Orafti (Barcelona, Spain) for providing chicory-derived inulins and oligofructose and to ArtBiochem (Archena, Murcia, Spain) for providing artichoke inulin. References Abrams, S. A., Griffin, I. J., & Hawthorne, K. M. (2007). Young adolescents who respond to an inulin-type fructan substantially increase total absorbed calcium and daily calcium accretion to the skeleton. Journal of Nutrition, 137, 2524S–2526S. Azorín, M., Urbán, C., Cerón, J. J., Tecles, F., Gil-Izquierdo, A., Pallarés, F. J., TomásBarberán, F. A., & Espín, J. C. (2008). Safety evaluation of an oak-flavored milk powder containing ellagitannins upon oral administration in the rat. Journal of Agricultural and Food Chemistry, 56, 2857–2865. Born, P., Zech, J., Stark, M., Classen, M., & Lorenz, R. (1994). Carbohydrate substitutes: comparative study of intestinal absorption of fructose, sorbitol and xylitol. Medizinische Klinik, 89, 575–578. Buriti, F. C. A., Cardarelli, H. R., Filisetti, T. M. C. C., & Saad, S. M. I. (2007). Synbiotic potential of fresh cream cheese supplemented with inulin and Lactobacillus paracasei in co-culture with Streptococcus thermophilus. Food Chemistry, 104, 1605–1610. Campbell, J. M., Fahey, G. C. Jr., & Wolf, B. W. (1997). Selected indigestible oligosaccharides affect large bowel mass, cecal and fecal short-chain fatty acids, pH and microflora in rats. Journal of Nutrition, 127, 130–136. Corwin, R. L., Wojnicki, F. H., Fisher, J. O., Dimitriou, S. G., Rice, H. B., & Young, M. A. (1998). Limited access to a dietary fat option affects ingestive behavior but not body composition in male rats. Physiology and Behavior, 65, 545–553. Delzenne, N., Aertssens, J., Verplaetse, H., Roccaro, M., & Roberfroid, M. (1995). Effect of fermentable fructo-oligosaccharides on mineral, nitrogen and energy digestive balance in the rat. Life Sciences, 57, 1579–1587. DeSchryver-kecskemeti, K., Eliakim, R., Green, K., & Alpers, D. H. (1991). A novel intracellular pathway for rat intestinal digestive enzymes (alkaline phosphatase and sucrase) via a lamellar particle. Laboratory Investigation, 65, 365–373. Fiordaliso, M. F., Kok, N., Desager, J. P., Goethals, F., Deboyser, D., Roberfroid, M., & Delzenne, N. (1995). Dietary oligofructose lowers triglycerides, phospholipids and cholesterol in serum and very low density lipoproteins of rats. Lipids, 30, 163–167. Kaur, J., & Gupta, A. K. (2002). Applications of inulin and oligofructose in health and nutrition. Journal of Biosciences, 27, 703–714. Kaur, J., Madan, S., Hamid, A., Singla, A., & Mahmood, A. (2007). Intestinal alkaline phosphatase secretion in oil-fed rats. Digestive Diseases and Sciences, 52, 665–670. Kim, M., & Shin, K. (1998). The water-soluble extract of chicory influences serum and liver lipid concentrations, cecal short-chain fatty acid concentrations and fecal lipid excretion in rats. Journal of Nutrition, 128, 1731–1736.

1065

Kleessen, B., Hartmann, L., & Blaut, M. (2001). Oligofructose and long-chain inulin influence the gut microbial ecology of rats associated with a human faecal flora. British Journal of Nutrition, 86, 291–300. Kleessen, B., Sykura, B., Zunft, H. J., & Blaut, M. (1997). Effects of inulin and lactose on fecal microflora, microbial activity, and bowel habit in elderly constipated persons. American Journal of Clinical Nutrition, 65, 1397–1402. Lesniewska, V., Rowland, I., Cani, P. D., Neyrinck, A. M., Delzenne, N. M., & Naughton, P. J. (2006). Effect on components of the intestinal microflora and plasma neuropeptide levels of feeding Lactobacillus delbrueckii, Bifidobacterium lactis, and inulin to adult and elderly rats. Applied and Environmental Microbiology, 72, 6533–6538. Loeb, W. F. (1997). Clinical biochemistry of laboratory rodents and rabbits. In J. J. Kaneko, J. W. Harvey, & M. L. Bruss (Eds.), Clinical biochemistry of domestic animals (5th ed., pp. 845–856). New York: Academic Press. López-Molina, D., Navarro-Martínez, M. D., Rojas Melgarejo, F., Hiner, A. N., Chazarra, S., & Rodríguez-López, J. N. (2005). Molecular properties and prebiotic effect of inulin obtained from artichoke (Cynara scolymus L.). Phytochemistry, 66, 1476–1484. Meyer, D. J., & Harvey, J. W. (1998). Veterinary laboratory medicine interpretation and diagnosis. Philadelphia: WB Saunders Company. Muir, J. G., Shepherd, S. J., Rosella, O., Rose, R., Barrett, J. S., & Gibson, P. R. (2007). Fructan and free fructose content of common Australian vegetables and fruit. Journal of Agricultural and Food Chemistry, 55, 6619–6627. Pedersen, A., Sandström, B., & Van Amelsvoort, J. M. M. (1997). The effect of ingestion of inulin on blood lipids and gastrointestinal symptoms in healthy females. British Journal of Nutrition, 78, 215–222. Pool-Zobel, B. L., & Sauer, J. (2007). Overview of experimental data on reduction of colorectal cancer risk by inulin-type fructans. Journal of Nutrition, 137, 2580S–2584S. Reagan-Shaw, S., Nihal, M., & Ahmad, N. (2008). Dose translation from animal to human studies revisited. Faseb Journal, 22, 659–661. Roberfroid, M. B. (2007). Inulin-type fructans: Functional food ingredients. Journal of Nutrition, 137, 2493S–2502S. Shepherd, S. J., & Gibson, P. R. (2006). Fructose malabsorption and symptoms of irritable bowel syndrome: Guidelines for effective dietary management. Journal of the American Dietetic Association, 106, 1631–1639. Stein, O., & Stein, Y. (1999). Atheroprotective mechanism of HDL. Atherosclerosis, 144, 285–301. Trautwein, E. A., Rieckhoff, D., & Erbersdobler, H. F. (1998). Dietary inulin lowers plasma cholesterol and triacylglycerol and alters biliary bile acid profile in hamsters. Journal of Nutrition, 128, 1937–1943. Van de Wiele, T., Boon, N., Possemiers, S., Jacobs, H., & Verstraete, W. (2007). Inulintype fructans of longer degree of polymerization exert more pronounced in vitro prebiotic effects. Journal of Applied Microbiology, 102, 452–460. Williams, C. M., & Jackson, K. G. (2002). Inulin and oligofructose: effects on lipid metabolism from human studies. British Journal of Nutrition, 87, S261–S264. Yap, K. W., Mohamed, S., Yazid, A. M., & Maznah, I. (2005). Doses–response effects of inulin on the faecal short-chain fatty acids content and mineral absorption of formula-fed infants. Nutrition and Food Sciences, 35, 208–219. Yeung, C. K., Glahn, R. P., Welch, R. M., & Miller, D. D. (2005). Prebiotics and iron bioavailability-Is there a connection? Journal of Food Science, 70, R88–R92.