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Journal of Animal Science - Animal Nutrition

Effects of feeding fermented wheat with Lactobacillus reuteri on gut morphology, intestinal fermentation, nutrient digestibility, and growth performance in weaned pigs1

 

This article in JAS

  1. Vol. 94 No. 11, p. 4677-4687
     
    Received: June 02, 2016
    Accepted: Aug 23, 2016
    Published: October 27, 2016


    2 Corresponding author(s): ruurd.zijlstra@ualberta.ca
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doi:10.2527/jas.2016-0693
  1. M. H. A. Le*,
  2. S. Galle*,
  3. Y. Yang*,
  4. J. L. Landero*,
  5. E. Beltranena*†,
  6. M. G. Gänzle* and
  7. R. T. Zijlstra 2*
  1. * Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, Canada T6G 2P5
     Alberta Agriculture and Forestry, Edmonton, AB, Canada T6H 5T6

Abstract

Feeding fermented feed to weaned pigs may improve nutrient digestibility and gut health and thereby reduce diarrhea incidence. Effects of feeding wheat grain fermented for 24 h with Lactobacillus reuteri were evaluated with 36 weaned pigs (7.3 kg BW). Fermented wheat grain contained (DM basis) 14.2% CP, 0.45% chemically available Lys, and 7.8% NDF, whereas unfermented wheat grain contained 16.4% CP, 0.45% chemically available Lys, and 9.9% NDF. Pigs were fed 6 mash wheat-based diets balanced for water content during 2 phases: Phase 1 diets for 1 wk (d 0–7) with 20% unfermented or fermented wheat and, subsequently, Phase 2 diets for 2 wk (d 8–21) with 50% unfermented or fermented wheat. The 6 diets were unfermented wheat (CTRL), unfermented and chemically acidified wheat (ACD), fermented wheat with L. reuteri TMW1.656 and 10% sucrose, fermented wheat with L. reuteri TMW1.656 and 5% glucose + 5% fructose, fermented wheat with L. reuteri LTH5794 and 10% sucrose, and fermented wheat with L. reuteri LTH5794 and 5% glucose + 5% fructose. Diets were formulated to provide 2.5 and 2.4 Mcal NE/kg and 5.3 and 5.0 g standardized ileal digestible Lys/Mcal NE for Phase 1 and 2 diets, respectively. Feeding fermented wheat reduced (P < 0.05) apparent total tract digestibility (ATTD) of diet DM (84.7 vs. 85.4%), GE (84.4 vs. 85.3%), and CP (81.8 vs. 83.6%) for d 15 through 21 compared with the CTRL and ACD diets. Weaned pigs fed fermented wheat diets had lower (P < 0.05) ADFI than pigs fed the CTRL and ACD diets for d 0 through 7. The ADFI, ADG, and G:F did not differ between pigs fed fermented and unfermented diets. Concentrations of acetic, propionic, and branched-chain fatty acids and total VFA in feces increased (P < 0.05) for pigs fed fermented wheat diets containing exopolysaccharides (EPS). However, VFA did not differ in ileal digesta. Villus height in the duodenum and jejunum increased in pigs fed fermented wheat without EPS (P < 0.05) compared with pigs fed fermented wheat with EPS. However, pigs fed the CTRL and ACD diets had longer (P < 0.05) villi and deeper crypts in the ileum than pigs fed fermented wheat. The ratio of villus height to crypt depth did not differ in the 3 segments of small intestine of weaned pigs. In conclusion, feeding fermented wheat grain diets to weaned pigs did not affect gut morphology, intestinal fermentation, growth performance, and ATTD of nutrients; however, EPS stimulated hindgut fermentation and may promote health benefits.



INTRODUCTION

Weaning imposes various stresses to piglets including consuming dry feed, change of environment, and challenges with pathogens. Combined with an immature digestive tract and immune system, these sudden changes increase the susceptibility of piglets to postweaning diarrhea (Lallès et al., 2007). Removal of antibiotics as growth promoters from swine feed may further increase diarrhea incidence (Stein, 2002; Laine et al., 2008). Although various antibiotic alternatives exist, combined approaches that promote health benefits, prevent enteric diseases, and manage growth of weaned pigs are more effective than single approaches (Stein and Kil, 2006). Fermentation of cereal grains may provide combined benefits to young pigs.

Lactobacillus reuteri TMW1.656 and LTH5794 produce the exopolysaccharides (EPS) reuteran and levan in the presence of sucrose as a substrate, respectively (Gänzle and Vogel, 2003; Gänzle et al., 2007). Levan and reuteran inhibit adhesion of enterotoxigenic Escherichia coli to porcine cells (Wang et al., 2010) and decrease net fluid loss induced by enterotoxigenic Escherichia coli, indicating their potential to reduce postweaning diarrhea losses (Chen et al., 2014). Fermentation of wheat supports production of EPS by L. reuteri (Tieking and Gänzle, 2005); therefore, feed fermentation may deliver EPS to weaned pigs. However, effects of feeding fermented wheat grain require evaluation in weaned pigs in terms of individual effects of fermentation, organic acids, and the EPS reuteran and levan.

Our null hypothesis was that pigs fed fermented wheat grain with L. reuteri TMW1.656 or LTH5794 with and without EPS would have similar gut morphology, intestinal fermentation, nutrient digestibility, and growth performance. The objectives were to determine effects of feeding diets containing unfermented or fermented wheat grain with or without EPS on apparent total tract digestibility of GE, CP, and DM of diets, growth performance, and intestinal morphology and fermentation products in weaned pigs.


MATERIALS AND METHODS

Animal Housing and Experimental Design

Animal care use and procedures were reviewed by the University of Alberta Animal Care and Use Committee for Livestock and followed principles established by the Canadian Council on Animal Care (2009). The study was performed at the Swine Research and Technology Centre (SRTC), University of Alberta (Edmonton, AB, Canada).

In total, 36 crossbred pigs (Duroc × Large White/Landrace F1; Hypor Inc., Regina, SK, Canada) were weaned at 19 ± 1 d of age. Pigs were selected based on BW (7.3 ± 1.7 kg) at weaning. Pigs were individually housed in metabolism pens.

Six dietary treatments were randomly allotted to pigs housed in adjacent pens by area of the room in a randomized block design for 6 replicate pens per treatment. Individual metabolism pens (0.5 m wide by 1.22 m long by 0.76 m high) were made of solid plastic planking, had a window allowing nose-to-nose contact with the neighbor piglet, and were raised 0.8 m from the concrete floor. Pen floors were fully slatted with extruded plastic flooring. Each pen was equipped with a stainless steel, wet/dry self-feeder (0.17 m wide by 0.15 m high trough) attached to the front of the pen, and a single cup drinker attached to the side wall (0.09 m above pen floor). Room temperature was maintained within the thermoneutral zone of pigs, using a negative pressure ventilation system. Lighting was provided for a 12-h light (0600–1800 h) and 12-h dark cycle. Pigs had free access to feed and water during the trial.

Experimental Diets

Pigs were fed 6 mash wheat-based diets balanced for water content during growth phases (Table 1): Phase 1 diets for 1 wk (d 0–7) with 20% unfermented or fermented wheat grain and, subsequently, Phase 2 diets for 2 wk (d 8–21) with 50% unfermented or fermented wheat grain. Six diets were prepared as described by Yang et al. (2015a): 1) unfermented wheat (CTRL), 2) unfermented and chemically acidified wheat (ACD), 3) fermented wheat with L. reuteri TMW1.656 and 10% sucrose, 4) fermented wheat with L. reuteri TMW1.656 and 5% glucose + 5% fructose, 5) fermented wheat with L. reuteri LTH5794 and 10% sucrose, and 6) fermented wheat with L. reuteri LTH5794 and 5% glucose + 5% fructose. Lactobacillus reuteri TMW1.656 is a sourdough isolate with the gene coding for reuteransucrase, gtfA, whereas L. reuteri LTH5794 originates from the human intestine with gene ftfA coding for levansucrase (Wang et al., 2010; Yang et al., 2015a). Diets without antimicrobials or growth promoters were formulated to provide 2.5 and 2.4 Mcal NE/kg and 5.3 and 5.0 g standardized ileal digestible Lys/Mcal NE for Phase 1 and 2 diets, respectively. Titanium oxide was added as an indigestible marker to diets.


View Full Table | Close Full ViewTable 1.

Ingredient composition (as-fed basis) and analyzed nutrient content (DM basis) of Phase 1 and Phase 2 diets

 
Phase 1 diets1
Phase 2 diets
Lactobacillus reuteri R2
L. reuteri L3
L. reuteri R
L. reuteri L
Ingredient, % CTRL ACD REU+ REU– LEV+ LEV– CTRL ACD REU+ REU– LEV+ LEV–
Wheat, ground 20 20 50 50
Wheat, ground, fermented 20 20 20 20 50 50 50 50
Corn, ground 31.54 31.54 31.54 31.54 31.54 31.54 1.76 1.76 1.76 1.76 1.76 1.76
Soybean meal 15 15 15 15 15 15 15 15 15 15 15 15
Lactose 15 15 15 15 15 15 10 10 10 10 10 10
Canola meal 5 5 5 5 5 5
Wheat DDGS4 5 5 5 5 5 5
Soy protein concentrate 3 3 3 3 3 3 2.5 2.5 2.5 2.5 2.5 2.5
Herring fish meal 6 6 6 6 6 6 2.5 2.5 2.5 2.5 2.5 2.5
Canola oil 4 4 4 4 4 4 3.4 3.4 3.4 3.4 3.4 3.4
Limestone 1.15 1.15 1.15 1.15 1.15 1.15 1.1 1.1 1.1 1.1 1.1 1.1
Mono-/dicalcium phosphate 1.3 1.3 1.3 1.3 1.3 1.3 1.0 1.0 1.0 1.0 1.0 1.0
Vitamin premix5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Mineral premix6 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Salt 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
TiO27 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
l-Lys HCl 0.45 0.45 0.45 0.45 0.45 0.45 0.4 0.4 0.4 0.4 0.4 0.4
l-Thr 0.29 0.29 0.29 0.29 0.29 0.29 0.18 0.18 0.18 0.18 0.18 0.18
dl-Met 0.14 0.14 0.14 0.14 0.14 0.14 0.11 0.11 0.11 0.11 0.11 0.11
l-Trp 0.08 0.08 0.08 0.08 0.08 0.08 0.02 0.02 0.02 0.02 0.02 0.02
Choline chloride 0.05 0.05 0.05 0.05 0.05 0.05 0.03 0.03 0.03 0.03 0.03 0.03
Analyzed nuterients8
    GE, Mcal/kg 4.5 4.5 4.5 4.5 4.5 4.5 4.6 4.6 4.6 4.6 4.6 4.6
    CP, % 21.6 21.3 21.5 21.7 21.2 21.8 24.4 24.0 25.1 24.9 25.0 25.2
    Crude fat, % 5.79 5.95 5.80 5.92 5.97 6.26 5.84 5.69 5.18 5.30 5.16 4.98
    NDF, % 7.74 8.16 7.93 8.04 7.71 8.14 10.8 9.54 12.6 10.5 10.5 11.8
    ADF, % 3.61 3.08 3.43 3.23 3.55 3.56 4.95 5.04 4.70 5.04 5.21 5.22
    Crude fiber, % 2.17 2.02 2.11 2.20 2.23 2.32 2.89 2.91 3.15 3.12 3.10 3.31
    Ash, % 7.17 7.23 7.20 7.17 7.35 7.15 6.56 6.67 6.68 6.87 6.65 6.59
1CTRL = unfermented wheat; ACD = unfermented and chemically acidified wheat; REU+ = fermented wheat with L. reuteri TMW1.656 and 10% sucrose; REU– = fermented wheat with L. reuteri TMW1.656 and 5% glucose + 5% fructose; LEV+ = fermented wheat with L. reuteri LTH5794 and 10% sucrose; LEV– = fermented wheat with L. reuteri LTH5794 and 5% glucose + 5% fructose.
2Lactobacillus reuteri R = L. reuteri strain TMW1.656, which produces the exopolysaccharide (EPS) reuteran in the presence of sucrose.
3Lactobacillus reuteri L = L. reuteri strain LTH5794, which produces the EPS levan in the presence of sucrose.
4DDGS = distiller’s dried grains with solubles.
5Supplied, per kilogram of diet, 7,500 IU vitamin A, 750 IU vitamin D, 50 IU vitamin E, 37.5 mg niacin, 15 mg pantothenic acid, 2.5 mg folacin, 5 mg riboflavin, 1.5 mg pyridoxine, 2.5 mg thiamine, 2,000 mg choline, 4 mg vitamin K, 0.25 mg biotin, and 0.02 mg vitamin B12.
6Supplied, per kilogram of diet, 125 mg Zn, 50 mg Cu, 75 mg Fe, 25 mg Mn, 0.5 mg I, and 0.3 mg Se.
7Sigma-Aldrich Co., St. Louis, MO; indigestible marker.
8Phase 1 and 2 diets were formulated to provide (as-fed basis) 2.5 and 2.4 Mcal NE/kg and 5.3 and 5.0 g standardized ileal digestible Lys/Mcal NE, respectively. Phase 1 and Phase 2 diets were fed from d 0 to 7 and d 8 to 21, respectively.

The initial wheat sourdough was prepared in the lab using ground wheat grain (Hard Red Spring) and water at the ratio of 1:1 (wt/vol), 10% sucrose (wt/wt), and cell cultures of either L. reuteri TMW1.656 or L. reuteri LTH5794 with approximately 107 cfu/g. The mixture was incubated at 37°C for 24 h. Subsequently, the fermented wheat was used as the first inoculum for additional wheat fermentation batches at the SRTC. Additional batches of ground wheat with equal amounts of water, 10% sucrose, and 10% initial sourdough were incubated at 37°C for 24 h. Only 10% of fermented wheat was used for backslopping the subsequent batches of wheat fermentation and the rest was incorporated in the diets. Cell counts and pH measurement were conducted daily for quality control of wheat grain fermentation. The seed sourdough was replaced by a new laboratory-grown cultures after 4 batches of backslopping the fermentation at SRTC.

The combination of 5% glucose and 5% fructose (wt/wt) was used in place of sucrose for unfermented wheat, chemically acidified wheat, and fermented wheat without added sucrose. The chemically acidified and unfermented wheat were mixed before feeding. Four parts lactic acid (80%) and 1 part glacial acetic acid (100%) were included in the mixture of ground wheat, water, 5% glucose, and 5% fructose (wt/wt) for the chemically acidified wheat. The unfermented wheat was simply made by combining ground wheat, water, 5% glucose, and 5% fructose (wt/wt). Experimental diets were freshly prepared by mixing 20% unfermented or fermented wheat with 80% feed base for Phase 1 diets and 50% unfermented or fermented wheat with 50% feed base for Phase 2 diets.

Sample and Data Collection

Feed added and leftovers were weighed daily at 0800 h. Individual pigs were weighed weekly to calculate ADFI, ADG, and feed efficiency (G:F). Freshly voided feces were collected from 0800 to 1600 h by grab sampling from pen floors on Days 5 and 6, 12 and 13, and 19 and 20 and stored at −20°C. Feces were then thawed, homogenized, subsampled, and freeze-dried.

Diarrheal incidence was assessed for each pig throughout the experiment. Fecal consistency was scored twice a day using a 5-point scoring system with 1 as firm (no diarrhea), 2 as soft, 3 as moist or solid–liquid, 4 as liquid (diarrhea), and 5 as watery/projectile diarrhea (severe diarrhea) as previously described (Madec et al., 2000).

At the end of the experiment on Day 20 and 21, pigs were euthanized using a captive bolt stunning technique. The abdomen was immediately opened and the digestive tract was clamped and removed. Digesta from the stomach, duodenum, jejunum, terminal ileum, colon, and cecum was collected in sterile containers and frozen at −20°C.

Two-centimeter segments of the duodenum, jejunum, and ileum were opened lengthwise, quickly rinsed in saline (0.9% NaCl), and fixed in scintillation tubes filled with 10% formalin. The tissues were eventually embedded in paraffin and sectioned. Slides were stained with hematoxylin and eosin. Villus height and crypt depth were measured using light microscopy with the AxioVision Imaging software (version 4.7.2; Carl Zeiss AG, Jena, Germany) and were averaged for 15 measurements from each intestinal segment.

Chemical Analyses

Samples of freeze-dried unfermented and fermented wheat, diets, and feces were ground through a 1-mm screen in a centrifugal mill (model ZM200; Retsch GmbH, Haan, Germany). Unfermented and fermented wheat were analyzed for N (method 984.13A-D), ADF inclusive of residual ash (method 973.18), ash (method 942.05), starch (assay kit STA-20; Sigma-Aldrich Co., St. Louis, MO), Ca (method 968.08), P (method 946.06), AA (method 982.30E), and chemically available Lys (method 975.44) as described by the AOAC (2006) and NDF (Holst, 1973). Diets were analyzed for DM (method 930.15; AOAC, 2006), GE using an adiabatic bomb calorimeter (model 5003; IKA-Werke GMBH & Co. KG, Staufen, Germany), CP (N × 6.25; method 988.05; AOAC, 2006), and ether extract, starch, crude fiber, ADF, NDF, AA, chemically available Lys, and TiO2 (Myers et al., 2004). Feces were analyzed for DM, GE, CP, and TiO2.

Based on results of chemical analyses, the apparent total tract digestibility (ATTD) of DM, GE, and CP and DE values were calculated using the marker concentration of feces relative to feed by the indicator method (Adeola, 2001). The NE value of diets was calculated based on DE value and chemical composition (CP, ether extract, starch, and ADF) using Eq. [5] of Noblet et al. (1994) and adopted by the NRC (2012). Volatile fatty acids were identified and measured using gas chromatography (model 3400; Varian, Walnut Creek, CA) with isocaproic acid as an internal standard.

Statistical Analyses

Data were analyzed using the MIXED procedure of SAS (version 9.2; SAS Inst. Inc., Cary, NC) with pig as the experimental unit. The fixed effect of diet and the random effect of block were included in the statistical model. Growth performance data were analyzed as repeated measures with week as the repeated term. Initial BW was included as a covariate to analyze growth performance. The effects of acid, fermentation, EPS, and fermentation with or without EPS were tested using single-degree-of-freedom contrasts. To test the hypotheses, P < 0.05 was considered significant.


RESULTS

Chemical Composition of Fermented and Unfermented Wheat Grain

With 10% addition of sucrose, fermented wheat after 24 h of fermentation contained 5.6 ± 1.0 g/kg reuteran and 3.2 ± 0.6 g/kg levan. Furthermore, fermented wheat contained other metabolites such as 80.7 ± 4.6 to 87.1 ± 5.8 mmol/kg lactate, 51.0 ± 4.2 to 57.3 ± 3.7 mmol/kg acetate, and 27.3 ± 4.9 to 36.2 ± 1.8 mmol/kg ethanol (Yang et al., 2015a). Fermented wheat grain contained 2% less CP on a DM basis than unfermented wheat, whereas chemically available Lys did not differ (Table 2). The Arg content was 0.18% lower whereas ornithine content was 0.1% greater in fermented wheat than in unfermented wheat. Crude fiber, ADF, and NDF were 0.5, 1.2, and 2.1% lower, respectively, in fermented wheat than in unfermented wheat.


View Full Table | Close Full ViewTable 2.

Analyzed nutrient content (%) of the ground wheat grain (DM basis)

 
Lactobacillus reuteri R3
L. reuteri L4
Variable CTRL1 ACD2 REU+ REU– LEV+ LEV–
Starch 58.1 59.4 63.5 59.4 56.7 58.1
CP 17.0 15.7 14.3 14.3 14.4 13.9
Crude fat 1.30 1.88 1.31 1.73 1.32 1.21
NDF 11.7 8.11 7.79 8.05 7.75 7.53
ADF 4.48 3.06 2.55 2.66 2.72 2.50
Crude fiber 2.66 2.15 1.85 1.87 1.90 1.90
Ash 2.34 2.51 2.22 2.53 2.47 2.55
P 0.42 0.44 0.41 0.42 0.40 0.41
Ca 0.10 0.40 0.36 0.44 0.43 0.45
Ornithine 0.00 0.00 0.10 0.10 0.11 0.10
Indispensable AA
    Arg 0.76 0.74 0.59 0.58 0.56 0.54
    His 0.39 0.37 0.34 0.34 0.34 0.34
    Ile 0.61 0.56 0.54 0.55 0.54 0.52
    Leu 1.20 1.09 1.03 1.01 1.01 0.99
    Lys 0.48 0.44 0.48 0.46 0.46 0.46
    Met 0.28 0.26 0.27 0.27 0.26 0.26
    Phe 0.83 0.79 0.72 0.73 0.71 0.71
    Thr 0.47 0.43 0.41 0.41 0.40 0.40
    Trp 0.17 0.17 0.15 0.15 0.15 0.14
    Val 0.73 0.66 0.65 0.64 0.64 0.63
Total AA 16.6 15.4 14.4 14.5 14.2 14.2
Available Lys 0.47 0.43 0.46 0.46 0.44 0.44
1CTRL = unfermented wheat.
2ACD = unfermented and chemically acidified wheat.
3Lactobacillus reuteri R = L. reuteri strain TMW1.656, which produces the exopolysaccharide (EPS) reuteran in the presence of sucrose. REU+ = fermented wheat with L. reuteri TMW1.656 and 10% sucrose; REU– = fermented wheat with L. reuteri TMW1.656 and 5% glucose + 5% fructose.
4Lactobacillus reuteri L = L. reuteri strain LTH5794, which produces the EPS levan in the presence of sucrose. LEV+ = fermented wheat with L. reuteri LTH5794 and 10% sucrose; LEV– = fermented wheat with L. reuteri LTH5794 and 5% glucose + 5% fructose.

Diet Digestibility

Feeding fermented wheat to weaned pigs reduced (P < 0.05; Table 3) ATTD of diet DM, GE, and CP by 0.7, 1.8, and 0.9%, respectively, during d 15 through 21 compared with feeding CTRL and ACD diets. Feeding fermented wheat did not affect diet DE and calculated NE values.


View Full Table | Close Full ViewTable 3.

Apparent total tract digestibility (ATTD) of nutrients, DE value, and calculated NE value (DM basis) of diets fed to weaned pigs1

 
Diet treatments
P-value4
Lactobacillus reuteri R2
L. reuteri L3
Variable CTRL ACD REU+ REU– LEV+ LEV– SEM Acid Ferm. Exopol Ferm.with EPS Ferm.without EPS
ATTD of DM, %
    d 0–7 84.0 82.8 83.4 82.0 81.1 82.1 2.24 0.589 0.381 0.879 0.473 0.428
    d 8–14 83.4 83.1 82.9 83.7 84.4 84.8 0.93 0.757 0.283 0.346 0.614 0.171
    d 15–21 85.7 85.1 85.3 84.1 84.7 84.6 0.54 0.282 0.033 0.110 0.277 0.009
ATTD of CP, %
    d 0–7 75.6 75.1 76.9 72.0 72.2 74.2 3.58 0.894 0.494 0.586 0.740 0.412
    d 8–14 81.4 79.8 79.3 79.9 80.2 82.8 1.76 0.407 0.964 0.186 0.494 0.556
    d 15–21 83.7 83.4 82.8 80.7 81.7 82.0 0.97 0.743 0.005 0.192 0.063 0.003
ATTD of GE, %
    d 0–7 81.3 79.0 80.3 78.8 77.7 78.5 2.96 0.435 0.473 0.862 0.574 0.501
    d 8–14 82.6 81.7 81.8 82.9 83.2 84.0 1.15 0.450 0.247 0.245 0.638 0.123
    d 15–21 85.7 84.8 85.0 84.0 84.1 84.3 0.60 0.113 0.021 0.333 0.115 0.014
Energy value, Mcal/kg
    DE 3.79 3.74 3.78 3.77 3.76 3.78 0.04 0.125 0.740 0.800 0.869 0.683
    Calculated NE 2.67 2.63 2.63 2.63 2.63 2.62 0.03 0.177 0.139 0.711 0.262 0.147
1Phase 1 and Phase 2 diets were fed from d 0 to 7 and 8 to 21, respectively. Least squares means were based on 6 pigs per dietary treatment. CTRL = unfermented wheat; ACD = unfermented and chemically acidified wheat; REU+ = fermented wheat with L. reuteri TMW1.656 and 10% sucrose; REU– = fermented wheat with L. reuteri TMW1.656 and 5% glucose + 5% fructose; LEV+ = fermented wheat with L. reuteri LTH5794 and 10% sucrose; LEV– = fermented wheat with L. reuteri LTH5794 and 5% glucose + 5% fructose.
2Lactobacillus reuteri R = L. reuteri strain TMW1.656, which produces the exopolysaccharide (EPS) reuteran in the presence of sucrose.
3Lactobacillus reuteri L = L. reuteri strain LTH5794, which produces the EPS levan in the presence of sucrose.
4Acid = acid addition (CTRL vs. ACD); Ferm. = fermentation (CTRL and ACD vs. REU+, REU–, LEV+, and LEV–); Exopol = fermentation with or without the EPS reuteran or levan (REU+ and LEV+ vs. REU– and LEV–); Ferm. with EPS = fermentation with EPS (CTRL and ACD vs. REU+ and LEV+); Ferm. without EPS = fermentation without EPS (CTRL and ACD vs. REU– and LEV–).

Growth Performance of Weaned Pigs

Diarrhea was not observed during the experiment (data not shown). Weaned pigs fed fermented wheat grain diets had 17% lower ADFI (P < 0.05; Table 4) than pigs fed CTRL and ACD diets during d 0 through 7 but did not differ during d 8 through 21. Pigs fed fermented wheat diets tended to have lower (P < 0.10) ADG and G:F than pigs fed CTRL and ACD during d 15 through 21. However, ADFI, ADG, and G:F did not differ between pigs fed unfermented and fermented wheat diets for the entire study.


View Full Table | Close Full ViewTable 4.

Growth performance of weaned pigs fed unfermented and fermented wheat grain with Lactobacillus reuteri1

 
Diet treatments
P-value4
L. reuteri R2
L. reuteri L3
Variable CTRL ACD REU+ REU– LEV+ LEV– SEM Acid Ferm. Exopol Ferm.with EPS Ferm.without EPS
ADFI, g
    d 0–7 154 172 133 153 132 126 32 0.335 0.024 0.625 0.028 0.081
    d 8–14 326 302 247 279 257 261 44 0.642 0.097 0.616 0.092 0.226
    d 15–21 535 510 512 511 499 496 51 0.664 0.601 0.960 0.668 0.633
    d 0–21 338 328 297 314 296 294 39 0.797 0.182 0.789 0.197 0.303
ADG, g
    d 0–7 76 121 87 99 58 76 33 0.036 0.151 0.326 0.086 0.453
    d 8–14 253 210 223 214 206 223 52 0.496 0.700 0.923 0.702 0.773
    d 15–21 444 462 413 408 368 420 42 0.670 0.056 0.424 0.042 0.198
    d 0–21 258 264 241 240 211 240 33 0.841 0.174 0.538 0.139 0.376
G:F
    d 0–7 0.48 0.69 0.64 0.61 0.43 0.61 0.11 0.061 0.865 0.364 0.540 0.747
    d 8–14 0.67 0.66 0.85 0.74 0.78 0.81 0.13 0.931 0.137 0.653 0.130 0.276
    d 15–21 0.84 0.93 0.81 0.79 0.75 0.86 0.09 0.245 0.066 0.350 0.042 0.248
    d 0–21 0.67 0.76 0.77 0.72 0.66 0.76 0.05 0.083 0.680 0.498 0.986 0.487
1The Phase 1 and Phase 2 diets were fed from d 0 to 7 and 8 to 21, respectively. Least squares means were based on 6 pigs per dietary treatment. CTRL = unfermented wheat; ACD = unfermented and chemically acidified wheat; REU+ = fermented wheat with L. reuteri TMW1.656 and 10% sucrose; REU– = fermented wheat with L. reuteri TMW1.656 and 5% glucose + 5% fructose; LEV+ = fermented wheat with L. reuteri LTH5794 and 10% sucrose; LEV– = fermented wheat with L. reuteri LTH5794 and 5% glucose + 5% fructose.
2Lactobacillus reuteri R = L. reuteri strain TMW1.656, which produces the exopolysaccharide (EPS) reuteran in the presence of sucrose.
3Lactobacillus reuteri L = L. reuteri strain LTH5794, which produces the EPS levan in the presence of sucrose.
4Acid = acid addition (CTRL vs. ACD); Ferm. = fermentation (CTRL and ACD vs. REU+, REU–, LEV+, and LEV–); Exopol = fermentation with or without the EPS reuteran or levan (REU+ and LEV+ vs. REU– and LEV–); Ferm. with EPS = fermentation with EPS (CTRL and ACD vs. REU+ and LEV+); Ferm. without EPS = fermentation without EPS (CTRL and ACD vs. REU– and LEV–).

Histomorphology of the Small Intestine

Villi in the duodenum and jejunum were 11% longer (P < 0.05; Table 5) in pigs fed fermented wheat without EPS compared with pigs fed fermented wheat with EPS, whereas crypts were 9% deeper in the duodenum (P < 0.05). Villus height and crypt depth in the duodenum and jejunum did not differ between pigs fed fermented diets and pigs fed unfermented diets. However, pigs fed unfermented wheat had 11% longer villi and 16% deeper crypts in the ileum than pigs fed fermented wheat diets (P < 0.05). The ratio of villus height to crypt depth did not differ in the small intestine of weaned pigs.


View Full Table | Close Full ViewTable 5.

Villus height, crypt depth (μm), and the ratio of villus height to crypt depth (V:C) in the duodenum, jejunum, and ileum of weaned pigs fed unfermented and fermented wheat-based diets1

 
Diet treatments
P-value4
Lactobacillus reuteri R2
L. reuteri L3
Variable CTRL ACD REU+ REU– LEV+ LEV– SEM Acid Ferm. Exopol Ferm.with EPS Ferm.without EPS
Duodenum
    Villus height 485 483 490 541 449 528 42.7 0.957 0.511 0.042 0.625 0.111
    Crypt depth 322 340 315 309 294 362 21.2 0.386 0.403 0.047 0.087 0.761
    V:C 1.51 1.43 1.56 1.74 1.56 1.46 0.12 0.509 0.164 0.680 0.311 0.159
Jejunum
    Villus height 492 497 453 511 460 514 36.5 0.893 0.667 0.039 0.155 0.484
    Crypt depth 231 237 243 233 234 260 12.0 0.612 0.277 0.353 0.628 0.163
    V:C 2.15 2.10 1.89 2.20 1.97 1.99 0.15 0.748 0.233 0.126 0.076 0.790
Ileum
    Villus height 373 374 325 319 328 355 20.9 0.960 0.003 0.455 0.004 0.022
    Crypt depth 212 227 184 181 180 193 24.5 0.557 0.027 0.749 0.037 0.072
    V:C 1.80 1.71 1.83 1.77 1.84 1.87 0.15 0.563 0.472 0.909 0.496 0.570
1Small intestinal tissues were collected after pig euthanasia on Day 21, fixed in 10% formalin, embedded in paraffin, sectioned, stained in hematoxylin and eosin, and measured under microscopy. Least squares means were based on 15 villi measured/segment per pig per dietary treatment. CTRL = unfermented wheat; ACD = unfermented and chemically acidified wheat; REU+ = fermented wheat with L. reuteri TMW1.656 and 10% sucrose; REU– = fermented wheat with L. reuteri TMW1.656 and 5% glucose + 5% fructose; LEV+ = fermented wheat with L. reuteri LTH5794 and 10% sucrose; LEV– = fermented wheat with L. reuteri LTH5794 and 5% glucose + 5% fructose.
2Lactobacillus reuteri R = L. reuteri strain TMW1.656, which produces the exopolysaccharide (EPS) reuteran in the presence of sucrose.
3Lactobacillus reuteri L = L. reuteri strain LTH5794, which produces the EPS levan in the presence of sucrose.
4Acid = acid addition (CTRL vs. ACD); Ferm. = fermentation (CTRL and ACD vs. REU+, REU–, LEV+, and LEV–); Exopol = fermentation with or without EPS reuteran or levan (REU+ and LEV+ vs. REU– and LEV–); Ferm. with EPS = fermentation with EPS (CTRL and ACD vs. REU+ and LEV+); Ferm. without EPS = fermentation without EPS (CTRL and ACD vs. REU– and LEV–).

VFAs in Ileal Digesta and Feces

Ileal digesta VFA did not differ among dietary treatments (Table 6). The concentrations of acetic, propionic, and branched-chain fatty acids and total VFA in feces increased by 33, 17, 30, and 27%, respectively (P < 0.05; Table 7), in pigs fed fermented wheat grain with EPS compared with those fed fermented diets without EPS. Fecal VFA concentrations did not differ between pigs fed unfermented and fermented wheat diets. The concentration of butyric acid in feces was not affected by dietary treatments.


View Full Table | Close Full ViewTable 6.

Concentrations and molar ratios of acetate, propionate, butyrate, branched-chain fatty acids, and total VFA (μmol/g ileal digesta) of ileal digesta resulting from feeding unfermented and fermented wheat-based diets to weaned pigs1

 
Diet treatments
P-value4
Lactobacillus reuteri R2
L. reuteri L3
Variable CTRL ACD REU+ REU– LEV+ LEV– SEM Acid Ferm. Exopol Ferm.with EPS Ferm.without EPS
Acetic acid 6.60 9.19 5.49 5.76 5.78 11.6 3.29 0.446 0.717 0.201 0.346 0.740
Propionic acid 0.17 0.68 0.44 0.31 0.41 0.41 0.26 0.067 0.843 0.721 0.996 0.724
Butyric acid 0.20 0.33 0.18 0.08 0.19 0.29 0.14 0.375 0.368 0.989 0.433 0.433
BCFA5 0.11 0.10 0.06 0.16 0.12 0.362 0.735 0.953 0.749 0.789
Total 7.09 10.4 6.44 6.24 6.65 12.6 3.72 0.394 0.741 0.285 0.418 0.802
Ratio, %:total VFA
    Acetic acid 94.5 91.0 87.1 93.0 87.8 93.5 4.50 0.455 0.394 0.078 0.112 0.875
    Propionic acid 1.87 5.22 6.57 4.29 5.87 3.35 2.54 0.204 0.355 0.188 0.154 0.879
    Butyric acid 2.17 2.56 2.96 1.31 2.67 1.91 1.07 0.728 0.817 0.121 0.561 0.322
    BCFA 0.76 1.04 0.77 0.39 0.67 0.281 0.682 0.147 0.286 0.703
    Acetic:propionic acid 18.0 18.4 16.4 16.0 17.3 23.7 7.20 0.961 0.975 0.528 0.806 0.762
    Acetic:butyric acid 32.8 30.3 24.2 36.0 36.0 33.0 9.76 0.797 0.898 0.536 0.834 0.681
1Digesta was collected at the end of the experiment, on Day 21, after pig euthanasia. Least squares means were based on 6 replicate pigs per dietary treatment. CTRL = unfermented wheat; ACD = unfermented and chemically acidified wheat; REU+ = fermented wheat with L. reuteri TMW1.656 and 10% sucrose; REU– = fermented wheat with L. reuteri TMW1.656 and 5% glucose + 5% fructose; LEV+ = fermented wheat with L. reuteri LTH5794 and 10% sucrose; LEV– = fermented wheat with L. reuteri LTH5794 and 5% glucose + 5% fructose.
2Lactobacillus reuteri R = L. reuteri strain TMW1.656, which produces the exopolysaccharide (EPS) reuteran in the presence of sucrose.
3Lactobacillus reuteri L = L. reuteri strain LTH5794, which produces the EPS levan in the presence of sucrose.
4Acid = acid addition (CTRL vs. ACD); Ferm. = fermentation (CTRL and ACD vs. REU+, REU–, LEV+, and LEV–); Exopol = fermentation with or without EPS reuteran or levan (REU+ and LEV+ vs. REU– and LEV–); Ferm. with EPS = fermentation with EPS (CTRL and ACD vs. REU+ and LEV+); Ferm. without EPS = fermentation without EPS (CTRL and ACD vs. REU– and LEV–).
5BCFA = branched-chain fatty acids, including isobutyrate, isovalerate, valerate, and caproic acid.

View Full Table | Close Full ViewTable 7.

Concentrations of acetate, propionate, butyrate, branched-chain fatty acids, and total VFA (μmol/g wet feces) of wet feces resulting from feeding unfermented and fermented wheat-based diets to weaned pigs1

 
Diet treatments
P-value4
Lactobacillus reuteri R2
L. reuteri L3
Variable CTRL ACD REU+ REU– LEV+ LEV– SEM Acid Ferm. Exopol Ferm.with EPS Ferm.without EPS
Acetic acid
    d 0–7 53.4 77.4 78.3 60.1 72.8 38.4 19.8 0.222 0.806 0.075 0.469 0.256
    d 8–14 39.8 48.7 45.0 29.8 62.3 35.3 10.4 0.401 0.857 0.008 0.213 0.124
    d 15–21 36.5 31.3 49.6 30.4 31.9 33.0 7.08 0.469 0.597 0.081 0.183 0.664
    d 0–21 43.2 52.4 57.6 40.1 55.8 36.4 7.72 0.231 0.941 0.001 0.106 0.082
Propionic acid
    d 0–7 16.2 23.7 21.0 19.2 21.9 14.2 3.78 0.051 0.698 0.092 0.578 0.228
    d 8–14 13.2 14.3 13.5 11.2 16.4 13.7 2.21 0.635 0.980 0.122 0.443 0.418
    d 15–21 13.5 12.8 15.3 12.0 11.8 12.6 1.53 0.664 0.808 0.258 0.718 0.436
    d 0–21 14.3 16.9 16.6 14.1 16.7 13.7 1.56 0.093 0.723 0.016 0.352 0.125
Butyric acid
    d 0–7 5.79 6.40 6.53 6.77 7.12 4.92 1.42 0.658 0.780 0.347 0.470 0.806
    d 8–14 4.88 4.94 5.29 5.34 4.88 4.58 1.07 0.955 0.861 0.869 0.815 0.945
    d 15–21 6.48 5.80 5.31 5.14 5.04 5.60 1.02 0.510 0.177 0.792 0.194 0.295
    d 0–21 5.71 5.71 5.71 5.75 5.63 5.08 0.66 0.998 0.673 0.585 0.928 0.523
BCFA5
    d 0–7 6.82 12.4 11.2 7.96 10.7 5.66 3.28 0.089 0.716 0.089 0.562 0.233
    d 8–14 13.7 20.9 20.3 10.7 24.1 19.7 6.41 0.277 0.722 0.134 0.288 0.647
    d 15–21 10.3 9.40 16.1 8.79 8.70 10.8 3.33 0.795 0.538 0.278 0.284 0.990
    d 0–21 10.3 14.2 15.9 9.16 14.8 12.3 2.93 0.177 0.658 0.028 0.135 0.461
Total
    d 0–7 86.9 124.2 120.4 99.3 117.3 67.4 24.5 0.128 0.765 0.055 0.446 0.208
    d 8–14 72.6 90.5 85.2 58.6 109.3 74.8 17.1 0.303 0.969 0.018 0.206 0.229
    d 15–21 68.6 60.9 88.0 57.9 58.7 63.4 11.2 0.501 0.746 0.120 0.288 0.609
    d 0–21 76.0 91.9 97.9 71.9 95.5 69.9 10.9 0.145 0.981 0.001 0.100 0.093
1The Phase 1 and Phase 2 diets were fed from Days 0 to 7 and Days 8 to 21, respectively. Least squares means were based on 6 pigs per dietary treatment. CTRL = unfermented wheat; ACD = unfermented and chemically acidified wheat; REU+ = fermented wheat with L. reuteri TMW1.656 and 10% sucrose; REU– = fermented wheat with L. reuteri TMW1.656 and 5% glucose + 5% fructose; LEV+ = fermented wheat with L. reuteri LTH5794 and 10% sucrose; LEV– = fermented wheat with L. reuteri LTH5794 and 5% glucose + 5% fructose.
2Lactobacillus reuteri R = L. reuteri strain TMW1.656, which produces the exopolysaccharide (EPS) reuteran in the presence of sucrose.
3Lactobacillus reuteri L = L. reuteri strain LTH5794, which produces the EPS levan in the presence of sucrose.
4Acid = acid addition (CTRL vs. ACD); Ferm. = fermentation (CTRL and ACD vs. REU+, REU–, LEV+, and LEV–); Exopol = fermentation with or without EPS reuteran or levan (REU+ and LEV+ vs. REU– and LEV–); Ferm. with EPS = fermentation with EPS (CTRL and ACD vs. REU+ and LEV+); Ferm. without EPS = fermentation without EPS (CTRL and ACD vs. REU– and LEV–).
5BCFA = branched-chain fatty acids, including isobutyrate, isovalerate, valerate, and caproic acid.


DISCUSSION

Advantages of Fermentation of Carbohydrate-Rich Ingredients in Swine Feeding

Feed fermentation is considered in swine production as an alternative for feed antibiotics (Stein and Kil, 2006; Canibe and Jensen, 2012). Fermentation of carbohydrate-rich ingredients is preferred to fermentation of complete feed. The consistent composition and low buffering capacity of cereals rapidly reduces pH and subsequently stabilizes pH (Missotten et al., 2010). Additionally, fermentation of grain increases nutrient availability by activation of a variety of intrinsic cereal enzymes, leading to degradation of fiber and antinutritional factors in cereals, particularly phytate. Furthermore, fermentation of grain prevents the loss of synthetic AA and protein degradation if complete feed is fermented (Moran et al., 2006; Missotten et al., 2010).

Chemical Changes in Fermented Wheat Grain with Lactobacillus reuteri

Lactobacillus reuteri is a symbiont of pigs that forms stable populations in the pars esophagus and also occurs in cereal fermentations (Walter, 2008; Su et al., 2012). Most strains of L. reuteri produce EPS from sucrose. The EPS formation supports biofilm formation by L. reuteri and is needed for colonization of the upper intestinal tract of mice (Walter et al., 2008). Feed fermentation with L. reuteri changed the nutrient content of wheat. During fermentation, the metabolic activities of L. reuteri and activity of cereal enzymes degrade carbohydrates, proteins, and other phenolic compounds in wheat grain. These breakdown products in turn serve as substrates for bacterial growth (Gänzle, 2014). In the present study, fermented wheat grain contained less CP, crude fiber, ADF, and NDF than unfermented wheat. Starch and fat content marginally varied.

Although total AA in fermented wheat grain was reduced, the content of Lys and available Lys was not affected. This finding is similar to observations that fermentation of wheat by lactobacilli converts Gln, Glu, and Arg but not Lys (Gänzle, 2014). The lower content of Arg and greater content of ornithine in fermented wheat resulted from the conversion of Arg into ornithine by L. reuteri via the arginine–deiminase pathway (Gänzle et al., 2007).

Digestibility of Nutrients in Fermented Feed

Wheat fermentation with L. reuteri offers several features similar to feed digestion in the stomach of pigs with the presence of lactic acid, organic acids, enzymes, and commensal bacteria L. reuteri. Feeding fermented wheat grain contributes to digestion by modifying raw feedstuffs before feeding, maintaining the low gastric pH, which is important to weaned pigs for protection against coliforms and denaturation of protein, especially when secretion of HCl in the stomach is limited after weaning (Brooks et al., 2001; Yen, 2001). Therefore, fermentation of cereal grains increased nutrient digestibility in previous studies (Shekib, 1994; Cho et al., 2013).

Despite these advantages of fermentation, feeding fermented wheat to weaned pigs did not increase nutrient digestibility in the present study. Similar to our results, fermented feed did not increase total tract digestibility of nutrients in growing pigs (Pedersen and Stein, 2010). Digestibility of nutrients can be affected by voluntary feed intake of pigs. The presence of feed in the digestive tract stimulates the secretion of pancreatic enzymes and thus helps increase digestibility (Makkink et al., 1994).

Effects of Fermented Feed on Growth Performance

Pigs fed fermented wheat grain in the present study had lower feed intake in the first week compared with pigs fed unfermented wheat. However, wheat fermentation did not affect ADFI, ADG, and G:F of pigs for the entire trial, similar to other studies. Pigs fed fermented liquid feed ate less and gained less weight than pigs fed unfermented liquid feed (Canibe and Jensen, 2003).

Low feed intake can be associated with palatability, which is influenced by pH, concentration of organic acids produced during fermentation, and the flavor and texture of the feed. High levels of acetic acid, butyric acid, and biogenic amines that resulted from spontaneous fermentation reduced palatability of pig feed (Beal et al., 2005; Canibe et al., 2010; Missotten et al., 2010). In the present study, production of reuteran and levan from sucrose by L. reuteri TMW1.656 and LTH5794, respectively, reduced sucrose and increased acetate in fermented wheat (Korakli et al., 2001). Thus, the sweet taste of fermented wheat grain consisting of reuteran or levan is reduced, and the grain is less palatable to young pigs. It explains the numerical decrease in feed intake of pigs fed fermented diets with reuteran or levan compared with those fed fermented diets without EPS.

Effects of Fermentation on Gut Morphology

Feeding fermented wheat grain did not influence intestinal morphology in the duodenum and jejunum but decreased villus height and crypt depth in the ileum of weaned pigs. However, pigs fed fermented diets without EPS had higher villi and deeper crypts than pigs fed fermented wheat with EPS in the duodenum, most likely due to greater feed intake. In line with our results, inclusion of fermented wheat in liquid diets increased villus height and the villus height–to–crypt depth ratio in the proximal small intestine of pigs during the first week after weaning (Scholten et al., 2002).

The greater increase in villus height and crypt depth in the proximal small intestine than in the distal small intestine may be related to the amount of available nutrients in the lumen (Vente-Spreeuwenberg et al., 2003). The presence of feed in the intestinal lumen of pigs has potential to stimulate cell proliferation, differentiation, and turnover, which then affects structure and functions of the intestinal epithelial layer. Hence, reduced feed intake at weaning is a major cause for villous atrophy in weaned pigs, because of not only the absence of feed in the gut but also energy deprivation (Pluske et al., 1997).

Effects of Fermentation on Gut Microbiota and VFA Concentrations

Fecal samples were used to characterize microbiota and measure VFA concentrations, because luminal microbial composition and fermentation in the colon was reflected in feces as turnover of colonic content (Eckburg et al., 2005; Gerritsen et al., 2011; Walker et al., 2011). The composition of fecal microbiota of weaned pigs, as described by Yang et al. (2015b), changed over time but 2 phyla, Bacteroidetes and Firmicutes, remained dominant throughout the study. Microbial diversity increased after weaning and was mostly attributable to the increased diversity of Firmicutes bacteria.

In the ileum, concentrations of individual and total VFA did not differ among dietary treatments, indicating lack of fermentation of EPS in the distal small intestine. This observation is similar to findings that fermentation of nondigestible oligosaccharides occurs in the large intestine, where bacterial diversity is greater than that in the ileum (Montagne et al., 2003; Niba et al., 2009).

The high proportion of acetate, followed by propionate, in fecal VFA is associated with the prevalence of bacteria in BacteroidesPrevotella group that mainly produce acetate and propionate from carbohydrate fermentation (Louis et al., 2007). The increase in concentrations of VFA in feces of pigs fed fermented wheat with EPS, reuteran or levan, compared with those in pigs fed fermented wheat without EPS indicates the effect of EPS on microbial fermentation in the hindgut. Because reuteran and levan are not digested in the small intestine, they become fermentable substrates for proliferation of Bacteroides in the distal gut, especially Bacteroides thetaiotaomicron (Sonnenburg et al., 2010). Bacteroides thetaiotaomicron fully metabolizes levan and partially degrades reuteran by enzymes and transport systems that are activated by upregulation of polysaccharide utilization loci specific for β-fructans and α-glucans utilization, in response to levan and reuteran, respectively, in the environment (Sonnenburg et al., 2010; van Bueren et al., 2015). Therefore, the present study demonstrated prebiotic effects of reuteran and levan in fermented wheat, as these EPS favored the growth of commensal flora B. thetaiotaomicron that increased VFA production in the hindgut and potentially benefits weaned pigs.

In conclusion, feeding fermented wheat grain with L. reuteri and reuteran or levan demonstrated the prebiotic effects of these exopolysaccharides on increased short-chain fatty acid production in the large intestine of weaned pigs. Fermented wheat without reuteran or levan increased villus height and crypt depth in the proximal small intestine over fermented wheat with exopolysaccharides, which is most likely related to feed intake. Digestibility of nutrients and growth performance of weaned pigs fed fermented wheat were not affected. Therefore, feeding fermented wheat with reuteran- or levan-producing L. reuteri may improve gut health of weaned pigs.

 

References

Footnotes


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