Three totally different bread merchandise have been used: a industrial entire grain rye bread, a industrial entire grain oat bread, and a refined wheat bread containing oat endosperm flour (25% of flour). Ingredient lists and different particulars are offered in Supplementary Desk S3 on-line. The breads have been freeze-dried for five h at 30 °C and 0.01 mbar, adopted by roughly 20 h at 0 °C and 1.5 mbar. Dried samples have been milled in a laboratory mill to go a 0.5 mm display. After milling, the bread samples have been saved at −20 °C.
Elimination of lipids, accessible starch, and savinase-degradable protein from bread
Freeze-dried and milled bread samples (35 g) have been weighed into centrifuge bottles. To take away lipids, every pattern was combined with 50 mL of n-heptane, vortexed twice for two min, and centrifuged (10 min, 2000×g), after which the heptane layer was discarded. For rye and wheat bread samples, this heptane washing was repeated as soon as, whereas for the oat bread samples it was repeated twice, after which the residues have been air-dried.
To extract fructan, the dried residue was combined with 250 mL of ethanol (80% v/v) and incubated at 80 °C for 45 min with magnetic stirring (500 rpm). After incubation, the pattern was centrifuged (10 min, 1000×g) and the supernatant was collected. Thereafter, the pattern was washed 3 times by including 30 mL of ethanol (80% v/v), mixing, and centrifuging (10 min, 1000×g), with the supernatant collected after every centrifugation. Ethanol was faraway from the pooled supernatants by vacuum rotor evaporation, and the unevaporated residue containing fructan was combined with 50–100 mL of deionized water and frozen at −20 °C.
To take away starch, an amylolytic remedy was carried out. The strong residue from ethanol washing was dried in a single day at 40 °C and dispersed in 175 mL of acetate buffer (0.1 M, pH 5.0 and 5 mM CaCl2) in a bottle. Then 1.75 mL thermostable α-amylase (3000 U/mL) was added and the pattern was incubated at 100 °C for 60 min, with mixing 3 times throughout incubation. The answer was cooled to 40 °C, adopted by addition of 10.5 mL of amyloglucosidase answer (140 U/mL), and in a single day incubation at 60 °C in a shaking water tub. For oat bread samples, 25 mL of acetate buffer have been added earlier than amyloglucosidase remedy, to make sure homogeneous mixing.
To take away proteins, the dispersion was cooled to room temperature, and 1.85 mL Savinase (≥ 16 U/g, Sigma-Aldrich) was added, adopted by incubation for 3 h at 50 °C in a shaking water tub. Thereafter, the pattern was cooled to room temperature and ethanol (99.5% v/v) was added to make 80% (v/v) ethanol answer. The answer was shaken vigorously for two min, centrifuged (15 min, 1000×g), and the supernatant liquid was discarded. The pellet was washed three extra instances with 60 mL of ethanol (80% v/v). The strong residue was dried in a single day at 40 °C, and combined with the extract containing fructan. The combination was frozen, freeze-dried, and milled as described above, and saved at −20 °C.
Chemical evaluation of bread and substrate samples
Chemical composition of bread samples and of substrates derived from the bread samples was analyzed in duplicate, with the outcomes offered on a dry weight foundation after drying at 105 °C for 16 h. Dietary fiber content material and composition have been analyzed based on the AOAC Methodology 994.133. 4with beforehand described modifications35 to investigate the extractable and non-extractable dietary fiber individually. For the evaluation of substrates, pattern quantity of 75 mg was used. The β-glucan content material was analyzed with Okay-BGLU equipment (Megazyme) as beforehand described36. The fructan content material was decided with a Okay-FRUC equipment (Megazyme) as beforehand described37, with modifications described in Supplementary strategies on-line. Starch content material was analyzed enzymatically based on a beforehand printed technique38. Protein content material was analyzed based on the Kjeldahl technique39 as Kjeldahl-N × 6.25. Fats content material was analyzed as beforehand described40. The focus of glucose, fructose, sucrose, maltose, and raffinose was analyzed as beforehand described (modified)41.
Research topics and fecal pattern assortment
Wholesome examine topics (n = 10) have been recruited and screened based on exclusion and inclusion standards (Supplementary Desk S4 on-line) to search out two fecal donors with contrasting intestine microbiota composition. The Swedish Moral Assessment Authority accredited the examine protocol (utility quantity 2019-04229) and the examine was carried out following the related pointers and laws. All examine topics signed an knowledgeable consent earlier than being enrolled.
All examine topics collected a screening fecal pattern utilizing EasySampler for stool assortment (GP Medical Units) and a small pattern tube. The screening fecal samples have been saved at −80 °C. For speedy screening of donor microbial profile, the molecular fingerprinting technique terminal restriction fragment size polymorphism (T-RFLP) was used, based on a beforehand described protocol42. The T-RFLP information generated by screening samples from all examine topics have been evaluated to be able to determine two donors with totally different microbial group composition, with regard to terminal restriction fragments related to Bacteroides and Prevotella in earlier research. Primarily based on the T-RFLP information, two donors with contrasting microbiota composition have been chosen to offer fecal samples for the in vitro experiments.
Fecal samples for the fermentation experiments have been collected inside two hours earlier than every experiment (together with pattern processing described under). The donors collected pattern at residence utilizing an EasySampler and a plastic beaker (500 mL) with a sealed cap for amassing minimal 30 g of feces, and the samples have been saved at room temperature till the experiment. Roughly 1 g of every fermentation fecal pattern was frozen and saved at −80 °C for microbiota composition evaluation.
In vitro fermentation experiments
4 batch fermentation experiments have been carried out with fecal samples from every donor at two separate events, leading to 4 replicates of every substrate and donor mixture. The quantity of fermentation substrate was energy-standardized between the breads. As well as, inulin (Merck KGaA) was utilized in management samples to watch the fermentation course of. Substrate (1.65 g of oats, 2.35 g of rye, 1.03 g of wheat substrate, or 1.00 g of inulin) was added to fermentation bottles. Thereafter, 50 mL of buffer (8.5 g NaHCO35.8gK2HPO30.5 g (NH4)2HPO41.0 g NaCl, 0.5 g MgSO47 Hrs20.01g FeSO47 Hrs20.0.1g CaCl2 to 1 L of deionized water, pH 7.0)43 have been added to every bottle and to 2 bottles with out substrate (clean controls). All bottles have been handled with CO2 fuel till addition of inoculate. Inoculate was produced by mixing fecal pattern (20 g) with buffer (1500 mL) in a bottle with CO2 fuel remedy, to acquire 1% (w/v) answer for the fermentation. The fecal slurry was filtered via a kitchen sieve and one layer of polyester filter material, and 50 mL have been instantly added to the bottles containing buffer and substrate or clean controls. The bottles have been closed and incubated at 37 °C for twenty-four h. Bottle contents have been combined with a motor stirrer all through the experiment (60 s stirring, 60 s break). Gasoline manufacturing was measured all through the experiment utilizing the Gasoline Endeavor system (Bioprocess Management) to comply with the fermentation course of.
At time factors 8 h and 24 h, liquid (5 mL) was collected from every bottle with a syringe and divided into three 1 mL-aliquots, and pH was measured. Aliquots have been saved at −20 °C for later evaluation of microbiota composition and unstable compounds. After 24 h of fermentation and pattern assortment, the fermentation residue materials was centrifuged (5 min, 5000×g), and the supernatant liquid was separated from the pellet. The supernatant and pellet have been autoclaved at 125 °C for 15 min, frozen to − 20 °C, freeze-dried as described above, and saved at − 20 °C.
Evaluation of fermentation samples
Fecal samples, inoculates, and fermentation samples at time factors 8 h and 24 h have been analyzed for microbiota composition with 16S rRNA gene sequencing as described in Supplementary strategies on-line. Acetate, propionate, butyrate, valerate, BCFA, and lactate concentrations have been analyzed as beforehand described44.
Dietary fiber quantity and composition after fermentation was analyzed utilizing the fermentation residue materials. Pellet composition was analyzed to estimate insoluble fiber degradation, and supernatant composition to estimate soluble fiber degradation. Dietary fiber was analyzed based on the AOAC Methodology 994.133. 4 with printed modifications35, and extra modifications described in Supplementary Strategies on-line. All samples have been analyzed in duplicate, and outcomes are offered on a dry weight foundation, after drying at 105 °C for 16 h.
Information processing and statistical evaluation
To estimate fiber degradation, the quantity of every insoluble and soluble sugar residue in fermentation samples was calculated as a share of whole sugar residues (insoluble plus soluble) within the fermentation substrate. Complete SCFA content material at 8 h and at 24 h was calculated because the sum of acetate, propionate, butyrate, and valerate, whereas whole BCFA content material was calculated because the sum of isobutyrate and isovalerate. The microbiota composition information have been analyzed to find out relative abundance on genus stage. The cut-off worth for information was set at 0.9% of common relative abundance, which represented 85% of whole genera abundance. These comprised the 20 most plentiful genera within the dataset and have been utilized in additional information evaluation.
PCA was used for exploratory information evaluation of microbiota information (Simca v. 16, Umetrics). For PCA modelling, the information have been scaled (univariate scaling) and log-transformed. Evaluation of similarities (ANOSIM) was used to statistically check for multivariate variations in microbiota and SCFA information between categorical variables (donor, substrate, time level and experiment event) (PAST v. 4.114. 5). The ANOSIM was primarily based on Bray Curtis metrics the place the impact of substrate, time and experiment was evaluated for every donor individually. SCFA and BCFA ranges, fiber degradation, and pH have been statistically in contrast between the fermentation samples with fecal microbiota from the 2 donors and between the totally different fermentation substrates, utilizing a generalized linear fixed-effects mannequin and two-way ANOVA with interplay (RStudio v. 1.2.501946). The generalized linear mannequin included the next fixed-effects variables: donor, substrate, the interplay between donor and substrate, and experiment event. Homoscedasticity and normality of residuals in every linear mannequin have been checked and, if both was detected, the response variables have been log-transformed. This was the case for 8 h butyrate, 8 h valerate, and soluble glucose residues. Statistically vital interactions between donor and substrate variables have been examined with an interactions plot, and put up hoc pairwise comparability of estimated marginal means was carried out (R package deal emmeans47). All analyzes have been adjusted for a number of comparisons (Tukey’s HSD). Inulin controls and clean samples weren’t included within the statistical analyses. Descriptive statistical evaluation was carried out in Microsoft Excel.