Ten healthy non-smoking volunteers (5 men and 5 women) aged 26.0 ± 1.1 y with normal body mass indices (22.6 ± 0.4 kg/m2) and without drug therapy participated in the study. All subjects had normal fasting plasma glucose concentrations (5.5 ± 0.1 mmol/L). The subjects were recruited in March 2007 and the study was performed from April to June 2007. All subjects gave their informed consent and were aware of the possibility of withdrawing from the study at any time they desired. Approval of the study was obtained from the regional ethical review board in Lund, Sweden (reference number 109/2007)
Four rye breads, boiled rye and wheat kernels and a white wheat bread reference were included in the study. Whole grain rye flour, kernels and endosperm rye flour (commercial blends) were provided by Lantmännen R&D (Järna, Sweden). Commercial white wheat flour was obtained from Kungsörnen AB (Järna, Sweden). Whole wheat kernels (Tiger) were provided by BFEL (Germany). Dry yeast was obtained from Jästbolaget AB (Sollentuna, Sweden) and lactic acid (88-92% extra pur) from Riedel-de Haën (Morris Township, NJ, USA).
The white wheat bread (WWB) was made from 540 g of white wheat flour, 360 g water, 4.8 g dry yeast, 4.8 g NaCl and baked in a bread baking machine (BM 3983, Severin, Sundern, Germany) using a program for white bread.
Endosperm rye bread (ERB) was made from 5000 g endosperm rye flour, 3413 g water, 84 g dry yeast and 43 g NaCl (containing 5 mg KI/100 g). The dough was mixed for 8 minutes and proofed at room temperature for 30 minutes. It was divided into pieces of 1000 g each and placed in baking tins. The dough was subjected to a second proofing (38°C, 85 % humidity) during 30 minutes for the endosperm rye breads and 45 minutes for the whole grain rye breads. Baking was performed initially at 250°C with 3 sec of steam. The temperature was then immediately lowered to 200°C and the breads were baked for 40 min.
Endosperm rye bread with lactic acid (ERB-lac) was made from 5000 g endosperm rye flour, 3322 g water, 90 g lactic acid, 84 g dry yeast and 43 g NaCl (containing 5 mg KI/100 g). The bread was made using the same method as for ERB.
Whole grain rye bread (WGRB) was made from 5000 g coarse whole grain rye flour, 3661 g water, 84 g dry yeast and 43 g NaCl (containing 5 mg KI/100 g). The bread was made using the same method as for ERB but was baked for 45 min.
Whole grain rye bread with lactic acid (WGRB-lac) was made from 5000 g coarse whole grain rye flour rye flour, 3574 g water, 90 g lactic acid, 84 g dry yeast and 43 g NaCl (containing 5 mg KI/100 g). The bread was made using the same method as for WGRB.
The WWB was left to cool for 1 hour and the rye breads for 21 hours under cover. Thereafter, the crust was removed and the breads were sliced and wrapped in aluminum foil in portions sizes, put into plastic bags and stored in a freezer (-20°C) until use. The day before the experiment, the breads were taken from the freezer and were thawed over night at ambient temperature, still wrapped in aluminum foil and in the plastic bag.
RK and WK
The wholegrain wheat kernels (WK) and rye kernels (RK) were prepared on the day of the experiment. 97.1 g of whole wheat kernels and 0.5 g NaCl were boiled in 156.4 g water for 40 minutes. 106.6 g whole rye kernels and 0.5 g NaCl were boiled in 189.5 g water for 35 minutes. All water was absorbed by the kernels.
Composition of the lunch buffet
An ad libitum lunch buffet was served at 270 min after breakfast to measure voluntary food intake. The buffet meal was a common Swedish lunch meal and was composed of meatballs (ICA Handlarnas AB, Solna, Sweden), pasta (Kungsörnen AB, Järna, Sweden), ketchup (Procordia Food AB, Eslöv, Sweden) and cucumbers. The cucumbers were fresh, and were peeled and sliced prior to serving to ensure homogeneity. Meatballs (240 g) were heated in a microwave oven (MS 2334B, LG, LG Electronics Inc., Seoul, Korea) for 4 min at 850 W. The pasta (325 g dry weight) was boiled for 8 min in 3 liters of water with 2 teaspoons (13 g) NaCl. One tablespoon of rapeseed oil (Di Luca & Di Luca AB, Stockholm, Sweden) was added to the pasta after boiling.
Chemical analysis of the breakfast products
Prior to analysis of the total starch, fiber and protein content, the breakfast products were air dried and milled (1.0 mm screen, Cyclotec, Tecator, Höganäs, Sweden). Measurements of resistant starch (RS) was performed on products as is. Total and resistant starch was analyzed according to Björck et al. [27
] and Åkerberg et al. [28
]. The available starch was calculated by subtracting RS from total starch. Insoluble and soluble dietary fiber were determined with a gravimetric, enzymatic method described by Asp et al. [29
]. Protein content was determined by Kjeldahl analysis (Kjeltec Auto 1030 Analyser, Tecator, Höganäs, Sweden). Fat content in the products was calculated using data from endosperm and wholegrain rye and wheat flours from Lantmännen. Energy content of the test meals were calculated using fat, carbohydrate and protein contents of the meals (17 kJ per gram of protein and available carbohydrate and 37 kJ per gram fat). The rate of starch hydrolysis (HI) was determined using an in vitro procedure based on chewing [30
], with WWB as a reference. The nutritional composition and HI of the products are shown in Table .
Composition of the breakfast products
The test and reference products were provided as breakfasts, on 7 different occasions, in random order separated by approximately 1 wk. The subjects were instructed to eat a standardized evening meal (9:00-10:00 P.M) prior to the test, consisting of a few slices of white wheat bread. No eating or drinking except for small amounts of water was then allowed until the start of the test. The subjects were also told to avoid alcohol and excessive physical exercise the day before each test, and otherwise as far possible maintain their regular life style throughout the entire study. The subjects arrived at the laboratory at 07.45 a.m. on the test day. A peripheral venous catheter (BD Venflon, Becton Dickinson, Helsingborg, Sweden) was inserted into an antecubital vein.
Fasting blood samples were taken prior to the breakfast meal at time 0. Thereafter the test meals, contributing with 50 g of available starch, were served with 250 ml of tap water. The test subjects finished the breads within 14 minutes and the kernels within 25 minutes. At 120 min after the breakfast, the test subjects were served 250 ml of tea, coffee or water without any sweeteners or milk products. The chosen beverage remained consistent for each individual at all 7 visits.
At 270 min after commencing the breakfast meals, the subjects were provided the lunch buffet and were instructed to eat the amount needed to reach comfortable satiation. At the following visits they should eat until they reached the same degree of satiation as on their first occasion. On their first visit, the subjects could drink as much water as they desired, and the same amount of water was then served at the following 6 visits. The subjects had to finish their lunch within 30 min, before the next blood sampling occasion at 300 min after commencing breakfast. The weight of the different food items ingested was registered individually to allow calculation of the energy intake at the buffet lunch meal. The energy content of the foods in the lunch buffet was obtained from the manufacturer of the products, and that of the cucumber from food tables (Swedish National Food Administration).
Capillary blood samples were taken for analysis of plasma glucose (p-glucose). Venous blood samples were drawn for the analysis of serum insulin, serum free fatty acids (s-FFA), s-adiponectin and p-ghrelin. Breath hydrogen excretion (H2) was measured as a marker of colonic fermentation, using an EC 60 gastrolyzer (Bedfont EC60 Gatrolyzer, Rochester, England). In addition, the subjects were asked to fill in their subjective feeling of fullness, hunger and desire to eat, respectively, using a 100 mm Visual Analogue Scale (VAS).
Glucose and insulin were measured at 0, 15, 30, 45, 60, 90, 120, 180, 240 and 270 min. FFA and adiponectin were measured at 0, 180 and 270 min. Ghrelin was measured at 0, 60, 90, 120, 270, 330, 360 and 390 min. Subjective appetite ratings were performed every 30 min throughout the experimental day and also at 15 and 315 min. H2 was measured every 30 min.
After sampling, serum and plasma (EDTA) tubes were left in ice bath for approximately 1 h before being centrifuged for 11 min (1800 * g, 4°C,). Serum and plasma were thereafter immediately separated and the samples were frozen at -20°C (serum) or -40°C (plasma) until analysis. Plasma for ghrelin analysis was sampled into tubes containing 500 KIU aprotinin (Bayer HealthCare AG, Leverkusen, Germany) per ml of whole blood.
Glucose was analyzed using a p-glucose analyzer (Glucose 201+, Hemocue, Ängelholm). The s-insulin analysis was performed on an integrated immunoassay analyzer (CODA Open Microplate System; Bio-rad Laboratories, Hercules, CA, USA) using an enzyme immunoassay kit (Mercodia AB, Uppsala, Sweden). S-FFA were analyzed using an enzymatic colometric method (NEFA C, ACS-ACOD method, WAKO CHEMICALS gmBH, Germany). S-adiponectin was analyzed using an enzyme immunoassay kit (Mercodia AB, Uppsala, Sweden), and p-ghrelin with a radioimmunoassay kit (Linco research Inc., St. Charles, MO, USA).
Calculations and statistical methods
One subject was excluded from the analysis of data from the WGRB breakfast due to having a cold at that particular test day. The data for WGRB is therefore analyzed with n = 9. Data are expressed as means ± SEM.
The total area under curve (AUC) was calculated for each subject and test meal, using the trapezoid model. The glycemic index (GI) is defined as the incremental positive area under the blood glucose curve after a test product, expressed as a percentage of the corresponding area after an equi-carbohydrate reference product taken by the same subject [31
]. The insulinemic index (II) is calculated from the corresponding insulin areas. Thus, GI and II were calculated using the net incremental AUC (0-120 min), with WWB as a reference. Incremental peaks for glucose and insulin were calculated as maximum postprandial increase from baseline.
The glycemic profile (GP) defined as the duration of the glucose curve divided with the incremental glucose peak was calculated [18
], with the modification that in cases where the glucose remained above fasting for the entire 270 min before lunch, the duration value was set to 270 min.
Hydrolysis index were calculated from the 180 min AUC for in vitro starch hydrolysis, in a similar way of calculating GI and II values, using WWB as a reference [30
Time x treatment interactions for glucose, insulin, ghrelin, satiety, breath hydrogen, FFA and adiponectin responses were analyzed using a mixed model (PROC MIXED in SAS release 8.01, SAS Institute Inc., Cary, NC) with repeated measures and an autoregressive covariance structure. Subjects were modeled as a random variable and corresponding baseline (fasting values) value were modeled as covariate. The data were analyzed using a mixed model analysis of covariance (ANCOVA) with subject as a random variable and corresponding baseline (fasting values) as a covariate. For voluntary energy intake at lunch and HI, a mixed model analysis of variance (ANOVA) was used with subject as a random variable. Differences between groups were identified using Tukey's multiple comparison tests. (MINITAB, release 16, Minitab Inc., State College, PA). In the cases of unevenly distributed residuals (tested with Anderson-Darling test), Box Cox transformation were performed on the data prior to the ANCOVA and ANOVA. Correlation analysis was conducted to evaluate the relation among dependent measures with the use of Spearman's partial correlation coefficients controlling for subjects and corresponding baseline values (two-tailed test), (SPSS software, version 19; SPSS Inc., Chicago, IL, USA). p < 0.05 was considered statistically significant.