The concept of ‘functional foods’ was pioneered in Japan during the 1980s (Arai, Reference Arai1996). Functional foods ‘should either have a relevant effect on well-being and health or result in a reduced risk of disease’ (Roberfroid, Reference Roberfroid1999). Due to changes in attitudes towards nutrition together with advances in technology in the last two decades, functional foods are generating considerable interest in consumers and researchers alike.
Prebiotics fulfil the criteria of being functional food ingredients due to their effects in both animals and human consumers. Research on inulin and oligofructose has revealed a beneficial influence of these food ingredients on faecal weight and faecal transit time (Alles et al. Reference Alles, Hautvast, Nagengast, Harteming, Van Laere and Jansen1996; Gibson et al. Reference Gibson, Rastall, Roberfroid, Gibson and Roberfroid1999), the mineral absorption (Coudray et al. Reference Coudray, Bellanger, Castiglia-Delavaud, Rémésy, Vermorel and Rayssignuier1997; van den Heuvel et al. Reference van den Heuvel, Muys, Van Dokkum and Schaafsma1998, Reference van den Heuvel, Schaafsma, Muys and Van Dokkum1999; van Loo et al. Reference van Loo, Cummings and Delzenne1999; Griffin et al. Reference Griffin, Davilla and Abrams2002), the risk of colon cancer (Reddy, Reference Reddy1999; Buddington et al. Reference Buddington, Danahoo and Buddington2002; Pool-Zobel et al. Reference Pool-Zobel, Van Loo and Roberfroid2002), the immune system (Schley & Field, Reference Schley and Field2002; Kelly-Quagliana et al. Reference Kelly-Quagliana, Nelson and Buddington2003) and lastly on concentration of blood lipids (Anderson & Hanna, Reference Anderson and Hanna1999; Brighenti et al. Reference Brighenti, Casiraghi, Canzi and Ferrari1999; Causey et al. Reference Causey, Feirtag, Gallaher, Tungland and Slavin2000; Letexier et al. Reference Letexier, Diraison and Beylot2003).
Health benefits have also been attributed to antioxidants, including a reduced risk of CVD (Weisburger, Reference Weisburger1998; Connor et al. Reference Connor, Ojeda, Sexton, Weidner and Connor2004; Rajasekhar et al. Reference Rajasekhar, Srinivasa, Latheef, Saibaba and Subramanyam2004; Sano et al. Reference Sano, Inami, Seimiya, Ohba, Sakai, Takano and Mizuno2004), and some forms of cancer (Weisburger, Reference Weisburger1998; Cooper et al. Reference Cooper, Eldridge and Peters1999; Lee et al. Reference Lee, Lee and Lee2004; Jian et al. Reference Jian, Du, Lee and Binns2005; Weinstein et al. Reference Weinstein, Wright, Pietinen, King, Tan, Taylor, Virtamo and Albanes2005). In their aetiology, these chronic diseases seem to result from cellular oxidative damage, caused by pro-oxidative agents, such as free radicals affecting lipids, proteins and DNA (Nikoleit, Reference Nikoleit1997). Dietary antioxidants may protect against these oxidative events in the body in cases where insufficient levels of endogenous antioxidants cannot counteract the reactive species (Rietveld & Wiseman, Reference Rietveld and Wiseman2003). Moreover, since the antioxidative activity of flavonoids and carotenoids is assumed to be dose-dependent (Upritchard et al. Reference Upritchard, Schuurman, Wiersma, Tijburg, Coolen, Rijken and Wiseman2003), an enhanced uptake of these nutritional components within functional foods is considered to be of added value.
Tea (Camellia sinensis) and its catechin ingredients have strong antioxidant properties (Trevisanato & Kim, Reference Trevisanato and Kim2000; Peters et al. Reference Peters, Poole and Arab2001; McKay & Blumberg, Reference McKay and Blumberg2002; Rietveld & Wiseman, Reference Rietveld and Wiseman2003), protecting human cells from DNA damage (Glei et al. Reference Glei, Habermann, Osswald, Seidel, Persin, Jahreis and Pool-Zobel2005). Carotenoids, also presumed to have antioxidant capacity, may promote cell–cell communication and influence the immune response (Nikoleit, Reference Nikoleit1997; Murr et al. Reference Murr, Schroecksnadel, Winkler, Ledochowski and Fuchs2005). Although the association between carotenoids and CVD or cancer is to date controversial, many studies suggest a health benefit due to these types of phytochemicals, in particular, carotenoids (Hertog et al. Reference Hertog, Feskens, Hollman, Katan and Kromhout1993; Weisburger, Reference Weisburger1998; Cooper et al. Reference Cooper, Eldridge and Peters1999; Riley & Stouffer, Reference Riley and Stouffer2003; Schabath et al. Reference Schabath, Grossman, Delclos, Hernandez, Day, Davis, Lerner, Spitz and Wu2004; Nkondjock et al. Reference Nkondjock, Ghadirian, Johnson and Krewski2005).
The aim of the present study was to assess the effects of prebiotics and antioxidants incorporated into a food consumed on a daily basis. We determined whether dietary intervention with supplemented breads affects the antioxidative capacity in plasma and immunological parameters in blood.
Subjects and methods
Subjects
Volunteers were recruited via newspaper advertisements. Criteria of exclusion were gastrointestinal disease and diabetes. All volunteers gave their informed written consent. The study was approved by the ethical committee of the Friedrich Schiller University (Jena, Germany).
Twenty current male smokers (smoking five or more cigarettes per d) and eighteen male non-smokers with a BMI of 23·2 kg/m2 were enrolled in the study (Table 1). The thirty-eight men were equally distributed into four groups regarding their smoking status and type of intervention bread (prebiotic bread or prebiotic antioxidant bread (pre-aox-bread)).
Table 1 Characteristics, dietary intake (7 d dietary record) and lipid status of all volunteers and the subgroups (non-smokers and smokers) at the start of the study (Mean values and standard deviations)
All parameters listed in Table 1 (body composition, dietary intakes, blood lipids) did not differ within the subgroups smokers v. non-smokers and within subjects consuming the prebiotic bread v. subjects consuming the pre-aox-bread.
Experimental design
The randomised, parallel, double-blind study consisted of two experimental periods. The trial was conducted for 12 weeks, starting with a baseline period of 2 weeks in which the volunteers were asked to follow their normal diet and to complete a 7 d dietary record. This period was followed by the control period (placebo) and, subsequently, the intervention period, both lasting 5 weeks. All thirty-eight volunteers were asked to eat the placebo bread during the control period. During the intervention period, nineteen subjects consumed the prebiotic bread and the rest ate the pre-aox-bread. Participants were instructed to consume at least 200 g bread per day during both periods. In the last 7 d of both parts of the study, the volunteers were given a standardised diet. The subjects collected 24 h urine samples for 3 d and faeces for 5 d at the end of all periods. On the last day of all phases, blood was collected after overnight fasting and the body composition of the subjects was analysed using bioelectric impedance analysis.
Test breads and standardised diets
The bread provided in the control period was a common German wheat–rye bread (Table 2). The prebiotic bread contained inulin, soya and linseed; the pre-aox-bread was prepared by adding tomato paste, green tea powder and oregano as functional components (which together thus replaced some of the wheat). All breads were baked twice per week in a local bakery and delivered immediately to the study participants. The weight of each bread was recorded before delivery. In order to determine the amount of bread actually consumed, the subjects were asked to hand in or to document the weight of leftover bread. A sensory panel assessed the test breads for taste, consistence and shelf life.
Table 2 Composition and characteristics of the control bread, prebiotic bread and the prebiotic antioxidant bread (pre-aox-bread) during the intervention study
TEAC, trolox equivalent antioxidant capacity.
The standardised diets were provided for 7 d during the control and intervention phases in the last week of each period. Meals consisting of 1 d menus were weighed, documented and packed twice per week for all volunteers. The portions included all the food for the duration of the standardised diet (7 d), mineral water and herbal tea bags. The subjects were instructed to collect and return the leftover food to the supervisor. Volunteers were asked to refrain from eating any fermented or prebiotic and probiotic dairy products, any other bread, soya products, tomato products, green tea or supplements of vitamins and minerals.
Analytical methods
Blood samples were centrifuged for 20 min at 1600 g and the serum was frozen immediately at − 80°C. α-Tocopherol in serum was measured according to Rettenmaier & Schüep (Reference Rettenmaier and Schüep1992) using HPLC (HPLC-LC 10, Shimadzu, Tokyo, Japan; fluorescence detector RF 10AXL, Shimadzu; column, nucleosil 100 NH2; flow rate, 0·8 ml/min; eluent, 96 % n-hexan, 4 % 2-propanol; 292 nm/330 nm; injection volume, 20 μl). Uric acid was determined photometrically via a test kit on a Synchron LX® (Beckman Coulter, Inc., Fullerton, CA, USA).
Blood for immunological assays was collected in EDTA-coated tubes and immediately analysed. The phagocytic activity was measured with the test kit Phagotest® (Orpegen, Heidelberg, Germany) that analyses the percentage of granulocytes which phagocytise fluoresceinisothiocyanat-marked Escherichia coli. The determination was carried out using fluorescence microscopy. The ‘oxidative burst’ (intracellular destruction of oxygen-dependent mechanisms) was measured by flow cytometry using the testkit Phagoburst® (Orpegen). To analyse surface-marked lymphocytes, 100 μl heparinised blood were incubated with 20 μl fluoresceinisothiocyanat-marked antibodies. Lysis of erythrocytes occurred after adding 2 ml fluorescence-activated cell sorter solution (Becton Dickinson, Franklin Lakes, NJ, USA). Cells were then measured by flow cytometer. Two thousand cells per field of lymphocytes were detected and the percentage of the surface-marked lymphocytes was determined.
Samples were immediately placed in dark containers to avoid any unnecessary exposure to light, and antioxidant parameters were measured in plasma and in urine with the ferric reducing ability of plasma (FRAP) assay (Benzie & Strain, Reference Benzie and Strain1996). To measure the antioxidant activity of the test breads, the trolox equivalent antioxidant capacity assay for hydrophilic antioxidants (Miller et al. Reference Miller, Rice-Evans, Davies, Gopinathan and Millner1993) and the trolox equivalent antioxidant capacity assay for lipophilic antioxidants (Boehm et al. Reference Boehm, Puspitasari-Nienaber, Ferruzzi and Schwartz2002) were performed. Total phenols in urine were photometrically detected using the Folin–Ciocalteau method (Singleton & Rossi, Reference Singleton and Rossi1965). Results are given as total polyphenol excretion per 24 h in gallic acid equivalents (GAE). The same method was used to analyse GAE in plasma after incubation with HCl, NaOH and meta-phosphoric acid. After adding an acetone–water mixture the resulting residue was used for performing the determinations. Total β-carotene, isomer (E)-β-carotene, total lycopene and isomer (E)-lycopene in plasma were analysed by HPLC (column C30 Vertex YMC carotenoid S-5 μm; YMC, Schermbeck, Germany) and diode array detector (L4500; Merck, Darmstadt, Germany) using the method of Boehm (Reference Boehm2001). The total radical-trapping antioxidant parameter (TRAP) was measured with a fluorimeter according to the procedure described by Ghiselli et al. (Reference Ghiselli, Serafini, Maiani, Azzini and Ferro-Luzzi1995). The photosensitive chemoluminescence (PCL) method, using trolox as standard, was performed according to the method of Popov & Lewin (Reference Popov, Lewin and Packer1999) using the Photochem® (Analytik Jena AG, Jena, Germany).
Statistics
The data were tested for significant differences between periods with no adjustment for general linear model-repeated measurements. To compare differences between the two types of bread as well as between the smokers and non-smokers, the Mann–Whitney U test was used as a non-parametric test for two independent values. For each comparison, a value of P < 0·05 was considered to be statistically significant. All statistical calculations were performed with the Statistical Package for the Social Sciences (SPSS/PC+, version 11.5; SPSS Inc., Chicago, IL, USA).
Results
Analysis of the breads
The content of total dietary fibre was highest in the prebiotic bread, followed by the pre-aox-bread (Table 2). The trolox equivalent antioxidant capacity value (hydrophilic version of the assay) of the pre-aox-bread was approximately twice as high as that of the prebiotic bread and the trolox equivalent antioxidant capacity value (lipophilic version of the assay) of the pre-aox-bread was approximately thirty times higher than that of the prebiotic bread. The content of α-tocopherol in the pre-aox-bread was about ten times that of the prebiotic bread. The carotenoids (total lycopene, (E )-lycopene, (E )-β-carotene, (9Z )-β-carotene, (15Z )-β-carotene and (E )-lutein), detected in the pre-aox-bread, were not present either in the control bread or in the prebiotic bread.
Intake
The average intake of all breads during the control period was 202 (sd 57) g/d, which was significantly higher than before the start of the study (166 (sd 61) g). In the intervention period the bread intake was 229 (sd 64) g for prebiotic bread and 189 (sd 70) g for pre-aox-bread. None of the volunteers complained of any difficulties while consuming the test breads. In fact, the subjects graded the taste, the consistence and the shelf life of all three breads as ‘good’ (scale of rating given was 1 for ‘excellent’; 2 for ‘good’ and 3 for ‘sufficient’). The placebo bread was graded to be better in taste and shelf life than the two intervention breads. Similar grading results were obtained by a professional sensory panel.
Body composition of the subjects and diets
BMI, body lean mass and body fat mass remained stable during the control and intervention periods (Table 3). Also the average daily intake of energy, fat, protein and carbohydrates revealed no changes during the control and intervention periods. But a significant increase of the average daily intake was observed for total dietary fibre and α-tocopherol. Compared with the start, BMI significantly decreased after the intervention (23·2 to 23·0 kg/m2), whereas the energy intake remained stable.
Table 3 Parameters of the body composition and dietary intake of the subjects during the study (Mean values and standard deviations for thirty-eight male subjects)
EI, energy intake.
* Mean value was significantly different from that for the control period (P = 0·000).
† Data calculated using PRODI 4.4 software (Nutri-Science GmbH, Freiburg, Germany).
‡ Data from analysed food of the standardised diet (see p. 350).
Contents of antioxidants and antioxidant capacity
There was a significant increase (P = 0·018) of the FRAP of participants consuming the pre-aox-bread, but no increase when consuming the prebiotic bread (Table 4). This increase in FRAP value was significant for the non-smokers who consumed the pre-aox-bread, whereas there were no changes in FRAP value for the smokers who ate the pre-aox-bread.
Table 4 Plasma concentrations of β-carotene, lycopene, α-tocopherol, uric acid and ferric-reducing ability of plasma (FRAP) values* (Mean values and standard deviations)
Pre-aox-bread, prebiotic antioxidant bread.
* Subjects were divided into groups consuming the prebiotic bread or pre-aox-bread during the intervention and in non-smokers and smokers consuming the pre-aox-bread.
The concentrations of total phenols (measured in GAE) remained stable (792 to 797 mg/l after consuming the prebiotic bread and 813 to 806 mg/l after intervention with the pre-aox-bread).
Total β-carotene, (E )-β-carotene, total lycopene and (E )-lycopene significantly increased in the pre-aox group during intervention (Table 4), whereas no effects were seen in the subjects who consumed prebiotic bread. The analysed carotenoids increased for smokers and non-smokers after consumption of the pre-aox-bread, but showed a higher statistical significance in the non-smoking group. The increase of the (E )-lycopene in the plasma of non-smokers was significantly higher (P = 0·045) compared with the group of smokers who ate the pre-aox-bread. The concentrations of α-tocopherol and uric acid significantly rose after consumption of both types of bread. This increase of α-tocopherol and uric acid was more significant for the pre-aox-bread group (P = 0·000 and P = 0·001, respectively) than for the prebiotic bread group (P = 0·027 and P = 0·006, respectively; Table 4).
Total phenols and antioxidant capacity in urine
TRAP and PCL increased significantly after consumption of the pre-aox-bread (10·6 to 14·9 mmol/d, P = 0·010; and 2·5 to 3·1 mmol/d, P = 0·043, respectively) as well as after consuming the prebiotic bread (13·2 to 19·1 mmol/d, P = 0·007; and 3·2 to 3·9 mmol/d. P = 0·019, respectively). No significant effects on the mean values of FRAP and GAE in urine were seen after intervention. When comparing both types of breads, and the response parameters of smokers v. non-smokers, there were no significant differences for GAE, FRAP, TRAP and PCL.
Immunological parameters
The numbers of leucocytes (n/μl) in the blood did not change after the intervention period compared with the control period. However, the numbers of lymphocytes and granulocytes significantly increased from the start to the intervention period (data not shown). The relative distribution of the leucocytes (lymphocytes, monocytes and granulocytes) was 37·4 (SD 6·7), 7·69 (SD 1·9) and 55·1 (SD 6·5) %, respectively, at the start and remained stable during the trial. The fraction of CD19 in the lymphocytes increased significantly after intervention for smokers and those on prebiotic bread (Table 5). There was a similar significant increase after intervention in smokers irregardless of the type of bread consumed (n 20; data not shown). Intercellular adhesion molecule-1 (ICAM-1) and CD3+NK+ decreased significantly after intervention. This decrease of ICAM-1 was also found in the subjects testing the prebiotic bread as well as in the non-smokers consuming both breads (n 18). The decrease of ICAM-1, however, was significant only in the group of non-smokers who consumed the pre-aox-bread (n 8). No differences were found for ICAM-1 and CD19 after intervention when comparing the test breads (19:19) and when comparing all smokers to non-smokers (20:18). The decrease of CD3+NK+ was observed in all tested groups (smokers, non-smokers, consumers of the prebiotic bread and the pre-aox-bread). CD3+HLA-DR+ (activated T-cells) increased after consuming the prebiotic bread.
Table 5 Selected immunological parameters of blood dependent on the type of bread (% of lymphocytes) (Mean values and pooled standard deviations for nineteen male subjects)
Pre-aox-bread; prebiotic antioxidant bread; C, control period; I, intervention period; CD, cluster of differentiation; ICAM-1, intercellular adhesion molecule-1; NK, natural killer; HLA, human leucocyte antigen; DR, related to HLA-D locus.
* Statistical significance of difference.
Phagotest® and Phagoburst® values did not change after the intervention. Phagoburst® decreased after the control period and remained at the lower level until the end of the study. There was a tendency of increased values of the Phagotest® (P = 0·076) from the start to the intervention. Other immunological parameters analysed (CD3, CD4, CD8, CD4:CD8, NK, CD57, CD8+CD57+, CD25, CD4+CD25+, CD122 and CD4+CD54+) did not change after the intervention period.
Discussion
Antioxidant capacity
The FRAP value increased in the plasma of subjects testing the pre-aox-bread, particularly in the group of non-smokers (n 8). In contrast, the concentrations of GAE in plasma remained unchanged after consumption of both pre-aox-bread and prebiotic bread. In general, the antioxidant capacity (in this case the FRAP assay) has been shown to correlate with the concentrations of total phenols (Serafini et al. Reference Serafini, Maiani and Ferro-Luzzi1998). Thus, an increase of antioxidant activity in plasma without a change in GAE could be due to a modification of the phenolic pattern. Moreover, it has been shown that polyphenols may be capable of modifying the antioxidant capacity (Pedersen et al. Reference Pedersen, Kyle, Jenkinson, Gardner, McPhail and Duthie2000). It has previously been shown that after intervention with a beverage rich in antioxidants GAE did not result in a change of total phenolics in plasma and in serum whereas the antioxidant capacity increased significantly (Boehm et al. Reference Boehm, Netzel, Kler, Marx, Weiss and Geiss2004). Most of the studies on the potential antioxidative effects of tea in human subjects have reported an increase of the antioxidant capacity in plasma. Benzie et al. (Reference Benzie, Szeto, Strain and Tomlinson1999) observed a rise of the FRAP activity in plasma 20–40 min after ingestion of green tea. Similar increases of the FRAP activity have also been reported for green and black tea (Leenen et al. Reference Leenen, Roodenburg, Tijburg and Wiseman2000; Langley-Evans, Reference Langley-Evans2000). In a study investigating sixty patients with coronary artery disease, however, there was no significant increase of FRAP subsequent to black tea consumption (Duffy et al. Reference Duffy, Keaney, Holbrook, Gokce, Swerdloff, Frei and Vita2001).
Unexpectedly, the correlations between uric acid and FRAP were not significant (r 0·29 after consumption of the prebiotic bread and r 0·44 after consuming the pre-aox-bread). Nevertheless, uric acid seems to be a strong contributor to the FRAP value. Fernandez-Pachon et al. (Reference Fernandez-Pachon, Villano, Troncoso and Garcia-Parrilla2005) also found a correlation between uric acid and the antioxidant capacity determined with FRAP.
The carotenoids in plasma were also significantly increased after the consumption of the pre-aox-bread, whereas no changes were observed in the prebiotic bread group. The most obvious effect was seen in the group of non-smokers who consumed the pre-aox-bread. Levels of all four carotenoids investigated rose significantly.
Three parameters (total lycopene, total β-carotene and (E )-β-carotene) were significantly enhanced and (E )-lycopene increased (P = 0·05) in the group of smokers who consumed the pre-aox-bread (n 11). In addition, the levels of significance for increased carotenoid concentrations were higher for non-smokers than for smokers. The difference of the (E )-lycopene content in plasma between smokers and non-smokers after intervention with pre-aox-bread was statistically significant. It may be assumed that the differences between smokers and non-smokers for total lycopene, total β-carotene, (E )-β-carotene, and FRAP might have been significant with more participants. The low increase of antioxidants in plasma of smokers is supported by the findings of Handelman et al. (Reference Handelman, Packer and Cross1996), who observed a destruction of carotenoids and tocopherols in human plasma by cigarette smoke.
There was a more pronounced increase of α-tocopherol in plasma after pre-aox-bread (P = 0·000) than after the prebiotic bread. This was reflected by higher concentrations of α-tocopherol in this bread in comparison with the control period and the prebiotic bread. A high intake of α-tocopherol (more than 40 mg/d) was associated with a lower risk of CHD in both men (Rimm et al. Reference Rimm, Stampfer, Ascherio, Giovannucci, Colditz and Willett1993) and women (Stampfer et al. Reference Stampfer, Hennekens, Manson, Colditz, Rosner and Willett1993).
TRAP and PCL measured in urine increased significantly after consumption of both intervention breads. However, FRAP and GAE in urine remained unchanged. As with the effects observed in plasma, this might indicate a rise of the antioxidant capacity in urine whereas the total amount of phenolic components remained stable. In a previous study, an increase of FRAP in urine was found 60 to 90 min after consumption of green tea (Benzie et al. Reference Benzie, Szeto, Strain and Tomlinson1999), which was a time frame considerably below the one we used for our analyses (weeks). Thus, these differences at which the measurements were performed may also explain the different effects observed. Similarly, the differences in the antioxidative activity may also depend on the assay used (Day et al. Reference Day, Kemp, Bolton, Hartog and Stansbie1997; Serafini et al. Reference Serafini, Maiani and Ferro-Luzzi1998; Prior & Cao, Reference Prior and Cao1999).
These findings demonstrate an immediate effect of a food, enriched in antioxidants, in human subjects. The notable effect of antioxidants might be explained by the results of a study in which natural antioxidants had an up to sixteen times higher antioxidant potential than synthetic ones (Kranl et al. Reference Kranl, Schlesier, Bitsch, Hermann, Rohe and Boehm2005).
Immunological parameters
The majority of the immunological parameters from peripheral blood showed no fluctuation, whereas four markers changed after consuming the prebiotic bread. ICAM-1 is responsible for co-operation between cells and is, for instance, up regulated in response to virus infection and cellular stress (Roebuck & Finnegan, Reference Roebuck and Finnegan1999). It has been shown that patients with ischaemic stroke also had elevated concentrations of ICAM-1 compared with the control group (Sanchez-Moreno et al. Reference Sanchez-Moreno, Dashe, Scott, Thaler, Folstein and Martin2004). In the smokers and the non-smokers who ate the pre-aox-bread, ICAM-1 decreased after the intervention period. ICAM-1 also decreased in a human study after consumption of a symbiotic yoghurt (Lactobacillus acidophilus and Bifidobacterium sp. and the oligofructose Fibrilose® F90; Klein et al. Reference Klein, Vogelsang, Kuhnt and Jahreis2003). The concentration of ICAM-1 furthermore changed after the intake of Lactobacillus paracasei incorporated into sausages (Jahreis et al. Reference Jahreis, Vogelsang, Kiessling, Schubert, Bunte and Hammes2002). These observations indicate that ICAM-1 is influenced by the intestinal microflora. CD19, which increased for smokers and after the consumption of the prebiotic bread, was also enhanced in participants in a study subjected to stress for 5 d (sleep deprivation, energy restriction and psychological stress) during military training (Gomez-Merino et al. Reference Gomez-Merino, Drogou, Chennaoui, Tiollier, Mathieu and Guezennec2005). A decline of CD19 cells was observed in a survey of 100 healthy volunteers only in participants aged > 50 years (Jentsch-Ullrich et al. Reference Jentsch-Ullrich, Koenigsmann, Mohren and Franke2005). CD3+NK+ was the only immunological parameter which changed after intake of the pre-aox-bread. This is probably due to functional effects.
In vitro studies showed a decrease of oxidative burst activity after incubating macrophages (isolated from fish) with high concentrations of β-glucans (Robertson et al. Reference Robertson, Engstad, Jøensen, Stolen and Fletcher1994; Schmitz, Reference Schmitz2004). This could explain the decrease of Phagoburst® from the beginning to the control period. In fact, it looks as if the modulation was more due to consuming higher quantities of bread (during periods of control and intervention) than to consuming the prebiotic or pre-aox-bread. Kelly-Quagliana et al. (Reference Kelly-Quagliana, Nelson and Buddington2003) found a higher phagocytic activity of peritoneal macrophages and a greater activity of natural killer cells of splenocytes after feeding mice with inulin. The immune functions were primarily affected via gut-associated lymphoid tissues in a study using rats fed a prebiotic supplement (inulin enriched with oligofructose) for 4 weeks (Roller et al. Reference Roller, Rechkemmer and Watzl2004). This can explain the minor effects of prebiotic bread in the present trial on the immunological parameters of peripheral blood as opposed to parameters of the gut-associated lymphoid tissues.
In conclusion, moderate amounts of prebiotics and natural antioxidants incorporated into a main food had an influence on several parameters of the immune system. The added antioxidants enhanced the levels of carotenoids and antioxidative capacity in the plasma (measured as FRAP). There were also changes more related to consuming higher quantities of bread than due to the functional ingredients. These include an effect on the oxidative burst of granulocytes. The antioxidants and the antioxidant capacity, in particular, responded to the functional components. There seem to be indications for differences on the antioxidant status between smokers and non-smokers, but there were marked differences related to the pharmacology (absorption, bioavailability, excretion) of antioxidants. We assume that consuming antioxidative components incorporated into basic food, as opposed to taking supplements, lowers the risk of antioxidants over consumption and its unpredictable consequences (as observed in the CARET and ATBC study and its follow-ups: Rapola et al. Reference Rapola, Virtamo, Ripatti, Huttunen, Albanes, Taylor and Heinonen1997; Virtamo et al. Reference Virtamo, Rapola, Ripatti, Heinonen, Taylor, Albanes and Huttunen1998; Markareetta et al. Reference Markareetta, Törnwall, Virtamo, Korhonen, Virtanen, Taylor, Albanes and Huttunen2004) might be minimised. Long-term research in the field of moderately enriched food with antioxidants and its risks for CHD is necessary, especially with regard to the smoking status of the participants.
Acknowledgements
We thank the volunteers of the study as well as Ute Helms, Peter Moeckel and Irmtraut Schmuck for their excellent technical assistance. We are also indebted to Rainer Schubert, PhD, for his help with the statistical analysis and finally we extend our appreciation to the employees at the local Feid bakery for their outstanding co-operation.
