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Blue sweet lupins (BSL) are considered as an alternative protein source to soybean meal (SBM) in diets for growing pigs. Some recent studies revealed that grinding intensity and hydrothermal processing might improve the nutritional value of BSL (Kim et al. 2009; Pieper et al. 2015). Altered particle size and viscosity due to processing might also change the bacterial activity in the upper gastrointestinal tract (Kim et al. 2009). It is well established that dietary particle size and digesta viscosity may influence the gastric emptying rate in the pig (Rainbird and Low 1986; Gregory et al. 1990), but little is yet known to which extent this may influence small intestinal microbial ecology patterns during the postprandial phase. It is hypothesised that digesta flow and particle size are important factors driving the substrate availability to the indigenous bacteria. In the current study, we thus determined postprandial kinetics of bacterial counts and activity at the terminal ileum in growing pigs fed differentially processed BSL.
Blue sweet lupins were processed in a hammer mill passing either a 3 mm sieve (coarsely ground blue lupins; CBL), a 1 mm sieve (finely ground blue lupins; FBL), or ground to pass a 1 mm sieve and subsequently expanded in a modified single screw extrusion-cooker TS-45 (ZMCh Metalchem Gliwice, PL) at 110 to 120°C (expanded blue lupins, EBL). Four experimental diets were formulated based on wheat and barley and either soybean meal or lupins as main protein source. A soybean meal-based diet (SBM) served as control and had similar particle size distribution as the EBL diet. Twelve PIC × Danbred crossbred pigs with an initial body weight of 20 kg were surgically fitted with a simple T-cannula at the terminal ileum and offered the experimental diets twice daily in mash form in a 3 × 4 Latin square design. After a 7-day adaptation period, ileal digesta samples were taken every 2 h over the course of 12 h after the morning meal. Microbial metabolites were determined by HPLC (D-/L-lactate), gas chromatography (short chain fatty acids; SCFA) and colorimetrically (ammonia, NH3). Total DNA and RNA were extracted from ileal digesta using commercially available kits. The 16S ribosomal DNA and RNA copy numbers were determined by quantitative polymerase chain reaction using primers specific for Lactobacilli, Enterobacteria, Bacteroides-Porphyromonas-Prevotella, and Clostridial clusters I, IV and XIVa. Data analyses were conducted with sampling time and experimental period considered as repeated observations (IBM SPSS, Version 23.0; USA).
Both time point and dietary treatment had statistically significant effects on bacterial metabolites and bacterial DNA and RNA copy numbers. Concentration of SCFA in ileal digesta increased (P < 0.05) with SBM and EBL and peaked (20 and 23 mmol/L, respectively) after 4 h, whereas CBL and FBL diets showed only minor effects on SCFA concentration (maximum 9 and 7 mmol/L, respectively). Similar patterns were observed for individual SCFA, although concentration of propionate and butyrate were generally low compared to acetate. In contrast, total lactate was highest (90 mmol/L; P < 0.05) after 4 h in CBL-fed pigs, whereas SBM (56 mmol/L) and FBL (54 mmol/L) showed peaks after 6 h, and EBL diets peaked (76 mmol/L) at 8 h after the morning meal. Lactate concentration was correlated to (R2 = 0.40) Lactobacilli 16S rRNA copy numbers and also correlated positively to Enterobacteria counts. Although an antagonistic relationship between Lactobacilli and Enterobacteria would be expected, their 16S rRNA counts showed a positive correlation (R2 = 0.42) revealing that easily accessible substrates are the main driving force for bacterial growth in the pig small intestine. The NH3 concentration was generally low (<5 mmol/L), and showed only minor responses to dietary treatment as well as during the postprandial phase. The results revealed clear differences in postprandial kinetics of bacterial metabolites and 16S rRNA copy numbers at the ileum of pigs fed diets containing differentially processed lupins. These differences seem to be related to particle size and digesta transit. Thus, choice of sampling time point is crucial for interpretation of microbial ecology in digesta contents.