Livestock Science 227 (2019) 90–96

Contents lists available at ScienceDirect

Livestock Science
journal homepage: www.elsevier.com/locate/livsci

Lactobacillus-based fermentation product and lactose level in the feed for T

weanling pigs: Effects on intestinal morphology, microbiota, gas emission,
and targeted intestinal coliforms
Y.D. Jeonga,1, H.S. Kob,1, A. Hosseindoustb, Y.H. Choia, B.J. Chaeb, D.J. Yua, E.S. Choa, K.H. Choa,
⁎
S.M. Shimb, C.S. Rab, J.W. Choib, A. Jangb, Y.I. Kimc, J.S. Kimb,
a
Swine Division, National Institute of Animal Science, Rural Development Administration, Cheonan 31000, Republic of Korea
b
College of Animal Life Sciences, Kangwon National University, Chuncheon, 24341, Republic of Korea
c
CTC Bio, Inc., Seoul, 138-858, Republic of Korea

A R T I C LE I N FO A B S T R A C T

Keywords: A 2 × 2 factorial arrangement was used to investigate the interactions between lactose levels (100 g/kg versus
Lactose 200 g/kg) and Lactobacillus-based fermentation product (FP; 0 and 1 × 1011 CFU Lactobacillus casei/kg) on
Lactobacillus weanling piglet performance and nutrient digestibility. Two hundred and forty weaned piglets (24 days old,
Fermentation product 7.05 kg live weight) were blocked on the basis of live weight and assigned to one of four dietary treatments with
Gas emission
six replicates and 10 piglets per replicate in two phases (phase 1, from d 1 to 14; phase 2, from d 14 to 28).
Microbiota
Volatile fatty acid concentrations and bacterial counts were performed in ileal, cecal, and colonic contents. In
Weanling pigs
phase Ⅰ, ADG was increased (P < 0.01) in pigs fed FP. The digestibility of DM and GE were greater (P < 0.01) in
pigs offered high lactose level. The interactions between lactose level and FP were significant for the con-
centration of ileal acetate (P < 0.05) and propionate (P < 0.01). The concentration of propionate in the cecum
and colon, as well as butyrate in the ileum, were greater (P < 0.05) in high lactose treatment. Dietary sup-
plementation of FP decreased acetate level in the cecum. The emission of total organic carbon and ammonia in
pigs feces were decreased (P < 0.01) in both high lactose and FP-supplemented diets. The interactions between
lactose and FP were significant (P < 0.05) for the population of Salmonella spp. in the ileum. The pH of colon
digesta was lower (P < 0.05) in pigs offered high lactose level. The abundance of Lactobacillus spp. was increased
(P < 0.05) only in the cecum digesta in pigs offered high lactose diet. In phase Ⅱ, there was a significant
improvement in the feed:gain in pigs fed the diet with high lactose content. The overall result showed that ADG
and ADFI were increased in pigs in FP treatment. The greater digestibility of DM was observed in pigs offered FP.
The concentration of butyric acid in the colon was increased in pigs fed FP. The supplementation of FP decreased
ileal and cecal Escherichia coli. This study demonstrates that dietary FP improved the growth performance and
DM digestibility of weanling pigs. When the dietary FP, and the lactose level were combined there was no
evidence of an interaction on growth performance. However, both lactose and FP decreased the emissions of
total organic carbon and ammonium.

1. Introduction
In intensive rearing systems, weaned piglets are subjected to en-
vironmental and dietary stressors that impair growth performance and
disrupt the normal equilibrium of intestinal microbiota (Giang et al.,
2010; Leliveld et al., 2013; Hosseindoust et al., 2017). Maintaining a
constant environment within this intestinal ecosystem may play an
important role in determining the health status of the host (Lalles et al.,
2007). A sudden change in the diet from milk to solids at weaning
renders piglets particularly susceptible to enteric pathogens and diar-
rhea (Lalles et al., 2007; Liu et al., 2015). Nutritional strategies using
compounds such as fermentation product (FP) and lactose can be used
to facilitate the enhancement of the digestive process and gastro-
intestinal microbiota.
Lactose has previously been described as a feed ingredient that
improves the gut health of weaning piglets by altering the gut

⁎
Correspondence to: Department of Animal Life Science, Kangwon National University, Chuncheon, 24341, Republic of Korea.
E-mail address: kjs896@kangwon.ac.kr (J.S. Kim).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.livsci.2019.06.018
Received 3 December 2018; Received in revised form 18 June 2019; Accepted 25 June 2019
Available online 26 June 2019
1871-1413/ © 2019 Elsevier B.V. All rights reserved.
Y.D. Jeong, et al. Livestock Science 227 (2019) 90–96

environment in favor of Lactobacillus spp. (Dillon et al., 2010). A large Table 1

proportion of lactose remains undigested in the small intestine and is Formula and chemical composition of experimental diets (as-fed basis).
simply fermented to lactic acid mainly by lactobacilli in the colon Lactose (%) Phase I Phase II
(Giang et al., 2010). Higher levels of dietary lactose in weaned pig 10 20
cause a decrease in the cecum and colon pH, and a concomitant in-
Ingredients (%)
crease in volatile fatty acids (VFA) and lactobacilli (Pierce et al., 2006;
Corn 45.82 33.45 51.16
Pieper et al., 2010). Fish meal (60% CP) 5 5 3
Lactobacillus species are common inhabitants of the intestine that Whey powder 15 15 5
can adhere to mucosal surfaces of the gastrointestinal tract while car- Soybean meal (44% CP) 27.1 29.52 28.5
rying out the fermentative activity. Their characteristic adherence to Soy oil 4.01 3.99 2.59
Choline chloride (50%) 0.05 0.05 0.05
intestinal surfaces may inhibit the adhesion of pathogens (Choi et al.,
MCP 0.44 0.45 0.65
2011; Messaoudi et al., 2013). Generally, all Lactobacillus species have Limestone 0.72 0.71 0.83
the capability of reducing pH by producing lactic acid as the end pro- Salt 0.2 0.2 0.2
duct of carbohydrate fermentation, resulting in the suppression of pa- Mineral premixa 0.15 0.15 0.15
Vitamins premixb 0.2 0.2 0.2
thogenic bacteria (Pringsulaka et al., 2015). However, some Lactoba-
Zinc oxide 0.3 0.3 0.3
cillus strains have additional abilities to produce anti-microbial peptides Purified lactose 0.25 10.25 6.75
and a variety of other metabolites that make them more specific anti- L-Lys (98%) 0.53 0.49 0.44
pathogenic agents (Cotter et al., 2005; Alcantara et al., 2016). Ad- DL−Met (98%) 0.23 0.24 0.18
ministration of a certain Lactobacillus spp. with anti-pathogenic speci- Calculated composition (%)
ME (kcal/kg) 3400 3400 3350
fications may positively stabilize the microbial population in intestine.
CP 21.0 21.0 19.5
Previous investigations have reported that Lactobacillus-based probio- Calcium 0.8 0.8 0.7
tics such as Lactobacillus brevis, Lactobacillus acidophilus, Lactobacillus Available phosphorus 0.4 0.4 0.33
reuteri, and Lactobacillus plantarum have the capability of controlling SID Lys 1.35 1.35 1.23
Escherichia coli colonization in the intestine of weaned pigs (Giang et al., SID Met+Cys 0.74 0.74 0.42
Lactose 10 20 10
2010; Choi et al., 2011; Liu et al., 2015). In addition, the fecal noxious Analysed composition (%)
gas emission may be associated with the nutrient digestibility. The CP 21.11 21.03 19.63
greater nutrient digestibility provides less substrate for microbial fer- Lysine 1.51 1.52 1.35
mentation in colon and consequently decrease the emission of total Methinoine 0.51 0.51 0.47
Calcium 0.82 0.81 0.77
mercaptans, ammonium, and hydrogen sulfide in weaned pigs feed a
Total phosphorus 0.80 0.78 0.76
Lactobacillus-based probiotic (Zhao and Kim, 2015).
Patterns of microbial colonization during weaning have accordingly CP, crude protein; MCP, mono calcium phosphate; ME, metabolizable energy;
been recognized as a long-lasting determinant of intestinal microbiota SID, standardized ileal digestible.
a
during the subsequent growth period (leliveld et al., 2013; Lalles et al., Supplied per kilogram of diet: 16,000 IU vitamin A, 3000 IU vitamin D3, 40
2007). For weaning piglets, there is a possibility that lactose could be IU vitamin E, 5.0 mg vitamin K3, 5.0 mg vitamin B1, 20 mg vitamin B2, 4 mg
used to manipulate populations of Lactobacillus spp. in the distal di- vitamin B6, 0.08 mg vitamin B12, 40 mg pantothenic acid, 75 mg niacin,
gestive tract via inoculation of preferential Lactobacillus strains. The 0.15 mg biotin, 0.65 mg folic acid.
b
Supplied per kilogram of diet: 45 mg Fe as ferrous sulfate, 0.25 mg Co
objective of the research reported here was to investigate the effects, or
cobalt sulfate, 50 mg Cu as copper sulfate, 15 mg Mn as manganese oxide,
interactions of lactose level in the presence of Lactobacillus-based FP on
25 mg Zn as zinc oxide, 0.35 mg I as potassium iodide, 0.13 mg Se as sodium
growth performance, intestinal microbiota, morphology, volatile fatty selenite.
acids and the amount of emitted gas in weanling pigs.
casei, 37 °C and pH 6.8 respectively. After 7 d of fermentation, the FP
2. Material and methods was dried in a forced-air drying oven at 40 °C for 72 h and mixed to-
gether. The final microbial count of FP was 1.0 × 1011 FU/g for L. casei.
The project underwent proper ethical standards and the experi- The FP at pH 6.8 contained 0.15 mg/g butyrate, 0.66 mg/g lactate,
ments (KW-170519-1) were approved by the Institutional Animal Care 25.1% protein, and 92.2% DM.
and Use Committee of Kangwon National University, Chuncheon,
Republic of Korea.
2.3. Animals, diets, and management
2.1. Fermentation product
A total of 240 weaned piglets (Landrace × Yorkshire × Duroc; in-
The FP (Lactobacillus casei) used in the current experiment was itial body weight (BW ± SD): 7.05 ± 0.2 kg; 24 ± 3 days of age) of
provided by a commercial company (CTC Bio, Inc.). L. casei (CLW-011) mixed sex were randomly allotted to four treatments on the basis of
was isolated from Korean kimchi. The isolated L. casei was maintained body weight and sex. There were six replicates pens in each treatment
in the laboratory at −80 °C as a stock culture. A culture broth (CB) with 10 pigs per pen. Dietary treatments included FP level (0, and 1 g
medium containing 6% corn steep liquor, 4% molasses, 0.5% KH2PO4 FP containing 1 × 1011 CFU L. casei/kg diet), and lactose levels (100 or
and 0.25% K2HPO4 in distilled water was prepared and autoclaved 200 g/kg). The FP used herein contained L. casei, and the dietary lactose
before being used. was provided by adding pure lactose and whey powder. The treatment
diets were fed in a meal form in 2 phases (d 0 to 14, phase Ⅰ; and d 15 to
2.2. Preparation of fermented product 28, phase Ⅱ). Diets for phases Ⅰ and Ⅱ were formulated to contain
14.23 MJ/kg ME and 13.5 and 12.3 g/kg SID lysine respectively
The microbes grown on CB were used as a starter to produce the FP (Table 1). All diets met or exceeded the nutrient requirements as sug-
by solid substrate fermentation (SSF) process. Maize and soybean meal gested by the NRC (2012). These experiments were conducted at the
(1:1) was used as the substrate and water was added to maintain a 30% facility of Kangwon National University farm and the piglets were
moisture content followed by pasteurization. Then the substrate (13 kg) housed in slotted and concrete floor pens with a pen size of
was inoculated with 2 L of starter and fermented for 7 d. The tem- 1.90 m × 3.0 m. All pens were equipped with a self-feeder and nipple
perature and pH maintained during fermentation were as follows: L. drinker to allow ad libitum access to feed and water.

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Y.D. Jeong, et al.
2.4. Experimental procedures, measurements, and analyses
Individual weanling piglet weight and feed intake from each pen
were recorded at the beginning of the experiment and at the end of
every phase to calculate average daily gain (ADG), average daily feed
intake (ADFI) and gain to feed ratio (G: F). To evaluate the effects of
dietary treatments on the apparent total tract digestibility (ATTD) of
nutrients, 0.25% chromic oxide (as an indigestible marker) was added
to all two phases (Phase Ⅰ, 0 to 14 days; and phase Ⅱ, 15 to 28 days)
diets. Pigs were fed diets mixed with chromic oxide from d 7 to 14, and
21 to 28, and fecal grab samples were collected from each pen on the
last 3 d of each experiment to determine the ATTD of DM, gross energy
(GE), and CP. The fecal samples were pooled within the pen and dried
in a forced air oven at 60 °C for 72 h, and ground in a Wiley mill
(Thomas Model 4 Wiley Mill, Thomas Scientific, Swedesboro, NJ) using
a 1-mm screen and used for chemical analysis.
To study the effects of dietary treatments on small intestinal mor-
phology, pH and the microbiota of ileal and cecal digesta, re-
presentative piglets from each group (2 piglets per replicate; one male
and one female) reflecting the average BW of the pen were selected and
sacrificed by electrocution at d 14 and 28 of each phase. The digesta
from the ileum and cecum were collected in sterile plastic bottles for
microbial analysis. The samples collected for microbial analysis
(Lactobacillus, E. coli, and Salmonella) were immediately placed on ice
until analyses were conducted. The samples of the intestinal segment
from the region of the duodenum (20 cm distal to the pyloric valve),
jejunum (middle jejunum), and ileum (30 cm proximal to the ileal
cecum) were collected after removing the content and flushing with
physiological saline. The samples were then submerged in a fixative
solution (0.1 M collidine buffer, pH 7.3) containing 3% glutaraldehyde,
2% paraformaldehyde and 1.5% acrolein and then brought to the la-
boratory to study the morphological changes.
2.5. Chemical analyses
Experimental diets and excreta samples were analyzed in triplicate
for DM (Method 930.15) and CP (Method 990.03) using AOAC (2007)
methods. The gross energy of diets and feces were measured by a bomb
calorimeter (Model 1261, Parr Instrument Co., Moline, IL), and chro-
mium concentration was determined with an automated spectro-
photometer (Jasco V-650; Jasco Corp., Tokyo, Japan) according to the
procedure of Fenton and Fenton (1979). The fecal gas emission para-
meters studied were soluble total organic carbon (TOC) and ammonium
nitrogen (NH4–N). Analyses were performed in accordance with the
standard methods (APHA, 1995). On d 14 and 28, about 250 g fresh
fecal samples were collected per pen via rectal massage and were stored
in 2.8-L sealed plastic boxes in duplicate. The samples were permitted
to ferment for a period of 7 d at 25 °C. In order to capture the gas
discharged from the plastic box, absorption tanks (500 mL of 1 M H2SO4
and 1 L of 1 M NaOH) were sequentially connected to the outlet line for
capturing carbon compound, and NH3. Liquor samples were collected
using the installed syringe to analyze the TOCs and NH4-N levels. The
TOC was analyzed with a Shimadzu TOC analyzer (Model TOC-500),
while NH4–N was analyzed with an auto analyzer (Quick Chem 8000,
LACHAT).
2.6. Microbial analyses
The microbial assay of the ileum, cecum and colon digesta was
carried out by culturing in different media as suggested by
Kim et al. (2017) and Gheisar et al. (2016). One gram of the composite
cecum or ileum digesta was diluted with 9 ml of 1% peptone broth
(Becton, Dickinson and Co., Franklin Lakes, NJ) and then homogenized.
Viable counts of bacteria in the samples were then conducted by plating
serial 10-fold dilutions. The microbial groups enumerated were Lacto-
bacillus spp. (using Man, Rogosa and Sharpe (MRS) agar + 0.200 g/l
92
Livestock Science 227 (2019) 90–96
NaN3 + 0.500 g/L L-cystine hydrochloride monohydrate), coliforms
(violet red bile agar), and Salmonella spp. (XLT4 agar). The MRS agar
(No. 288130), violet red bile agar (No. 216695), XLT4 (No. 212123)
were purchased from Difco Laboratories (Detroit, MI). The MRS and
violet red bile agar plates were incubated for 48 h at 39 °C, under
anaerobic condition. The XLT4 agar tubes were incubated for 48 h at
42 °C. The bacterial concentrations were transformed (log) before sta-
tistical analysis.
2.7. Volatile fatty acids
To measure the concentration of VFA, fecal samples were collected
(d 14 and 28) directly from the anus of 1 randomly selected pig in each
pen to minimize the air contact. These samples were immediately
sealed in vinyl bags and placed on ice. Fecal VFA concentrations were
measured by gas chromatography (HP 6890 Plus; Hewlett Packard,
Houston, TX) using a modified method by Porter and Murray (2001).
2.8. Small intestinal morphology
Three cross-sections for each intestinal sample were prepared after
staining with azure A and eosin using standard paraffin embedding
procedures (Kim et al., 2017). A total of ten intact, well-oriented crypt-
villus units were selected in triplicate for each intestinal cross-section.
Villus height was measured from the tip of the villi to the villus-crypt
junction, and crypt depth was defined as the depth of the invagination
between adjacent villi. All morphological measurements (villus height
and crypt depth) were made in 10-μm increments by using an image
processing and analysis system (Optimus version 6.5 software, Media
Cybergenetics, North Reading, MA).
2.9. Statistical analysis
Data generated in the experiment was analyzed as a completely
randomized design using repeated measures in a 2 × 2 factorial ar-
rangement. The model included FP, lactose level, and the FP × lactose
level interaction. Initial pen weight was tested as a covariate and phase
period was the repeated term in the overall model. Pens were con-
sidered the experimental unit for growth performance, and piglets were
experimental units for measuring the digestibility of nutrients and all
intestinal samplings including digesta pH, microbiota, VFA, gas emis-
sion, and intestinal morphology. The main effects of FP, and lactose and
their interaction were determined by repeated measurement options of
the mixed procedure of SAS statistical program (SAS Inst., Inc., Cary,
NC, US). P-values ≤0.05 were considered statistically significant.
3. Results
3.1. Growth performance
There was no mortality during the study (data not shown). There
were no interactions between FP, and lactose level for any of perfor-
mance parameters (Table 2). In phase Ⅰ, ADG, ADFI and feed:gain were
not affected. ADG was increased (P < 0.01) in pigs fed FP. In addition,
feed:gain did not differ in pigs offered FP. In phase Ⅱ, there was a
significant improvement in the feed:gain in pigs fed the diet with high
lactose content. No differences for ADG, ADFI or feed:gain were de-
tected in FP treatment. The overall result showed that ADG and ADFI
were increased in pigs in FP treatment, however, feed:gain was not
affected.
3.2. Digestibility of nutrients
Results for the digestibility of nutrients are presented in Table 3.
There was no dietary lactose × FP interaction for nutrients utilization
response. In phase Ⅰ, the digestibility of DM and GE were greater in pigs
Y.D. Jeong, et al. Livestock Science 227 (2019) 90–96

Table 2 Table 4
Effects of dietary lactose level and supplementation of Lactobacillus-based fer- Effects of dietary lactose level and supplementation of Lactobacillus-based fer-
mentation product (1 × 1011 cfu L. casei/kg diet) on growth performance in mentation product (1 × 1011 cfu L. casei/kg diet) on volatile fatty acids (VFA)
weanling pigs. concentration in the digesta of the different part of digestive tract (mg/l).
Fermentation 0 1 × 1011 cfu SEM P-value Fermentation 0 1 × 1011 cfu SEM P-value3
product (FP) product (FP)
Lactose levela (L) Low High Low High FP L FP × L Lactose levela Low High Low High FP L FP × L
(L)
Phase Ⅰ(d 0–14)
ADG, g 291 311 322 325 6.1 0.001 0.07 0.18 Phase Ⅰ(d 14)
ADFI, g 433 439 461 453 12 0.09 0.96 0.57 Ileum
F/G 1.49 1.41 1.43 1.39 0.04 0.29 0.12 0.59 Acetate 67.1 79.3 74.3 76.3 1.9 0.30 0.002 0.02
Phase II (d 15–28) Propionate 34.7 39.9 52.5 34.7 3.4 0.08 0.09 0.004
ADG, g 366 379 395 390 16 0.22 0.77 0.58 Butyrate 17.6 19.7 16.9 20.2 0.9 0.89 0.008 0.54
ADFI, g 572 575 619 588 19 0.13 0.47 0.38 Total VFA 129.3 138.1 143.7 131.1 4.8 0.34 0.56 0.086
F/G 1.57 1.52 1.57 1.51 0.03 0.85 0.041 0.75 Cecum
d 0–35 Acetate 753 820 669 710 36 0.016 0.16 0.72
ADG, g 327 341 356 351 7 0.011 0.54 0.21 Propionate 584 609 576 597 10 0.34 0.031 0.87
ADFI, g 502 507 540 521 8 0.005 0.37 0.15 Butyrate 396 406 373 397 9 0.11 0.08 0.46
F/G 1.54 1.49 1.52 1.48 0.02 0.52 0.06 0.70 Total VFA 1731 1835 1618 1704 41 0.021 0.046 0.26
Colon
ADG, average daily gain; ADFI, average daily feed intake; F/G, feed to gain Acetate 1278 1412 1274 1403 51 0.89 0.019 0.96
ratio. Propionate 791 842 781 838 14 0.60 0.001 0.83
a
Phase Ⅰ high (20%), low (10%); phase II, high (10%), low (10%). Butyrate 651 691 655 683 16 0.92 0.05 0.72
Total VFA 2716 2942 2711 2926 64 0.73 0.001 0.62
Phase II (d 28)
Table 3 Ileum
Effects of dietary lactose level and supplementation of Lactobacillus-based fer- Acetate 94.5 98.5 93.1 88.8 5.3 0.31 0.98 0.45
mentation product (1 × 1011 cfu L. casei/kg diet) on the total tract apparent Propionate 28.8 28.3 27.7 26.7 3.5 0.54 0.97 0.76
digestibility (%) of nutrients. Butyrate 20.3 18.6 20.7 21.5 2.8 0.45 0.74 0.79
Total VFA 143.6 145.2 141.3 137.2 9 0.41 0.88 0.78
Fermentation 0 1 × 1011 cfu SEM P-value Cecum
product (FP) Acetate 726 713 617 621 28 0.001 0.85 0.96
Lactose levela (L) Low High Low High FP L FP × L Propionate 443 444 507 492 40 0.09 0.59 0.58
Butyrate 347 274 299 395 43 0.27 0.97 0.11
Phase Ⅰ(d 0–14) Total VFA 1514 1433 1423 1506 71 0.84 0.92 0.08
Dry matter 82.5 83.6 83.2 84.1 0.31 0.08 0.006 0.79 Colon
Gross energy 80.7 81.5 81.2 81.7 0.19 0.06 0.003 0.30 Acetate 874 836 845 841 88 0.99 0.72 0.94
Crude protein 78.0 78.8 78.3 78.9 0.39 0.55 0.08 0.78 Propionate 531 520 568 529 56 0.66 0.64 0.78
Phase II (d 15–28) Butyrate 548 524 667 622 51 0.045 0.48 0.80
Dry matter 80.4 80.8 81.4 81.5 0.29 0.010 0.39 0.61 Total VFA 1952 1880 2078 1994 146 0.41 0.59 0.81
Gross energy 78.7 79.5 79.4 79.7 0.38 0.27 0.20 0.54
Crude protein 74.9 74.7 75.4 75.0 0.39 0.30 0.78 0.49 a
Phase Ⅰ high (20%), low (10%); phase II, high (10%), low (10%).
a
Phase Ⅰ high (20%), low (10%); phase II, high (10%), low (10%).
In phase Ⅱ, the concentrations of acetate, propionate, butyrate, and
total VFA were not affected by the supplementation of lactose or FP.
offered high lactose level. The FP did not affect the digestibility of CP,
Dietary supplementation of FP decreased the cecal acetate, however, no
however, the digestibility of DM and GE tented to be higher compared
differences have been observed in the concentration of propionate,
with pigs fed the non-supplemented diet. In phase Ⅱ, digestibility
butyrate, and total VFA. No significant differences were found in
analysis showed no differences between lactose treatments. The greater
acetate, propionate, butyrate, and total VFA contents in the cecum
digestibility of DM was observed in pigs offered FP, while no difference
between high and low dietary lactose levels. The concentration of bu-
was observed for the digestibility of GE and CP between FP treatments.
tyrate in the colon was increased in pigs fed FP, however, there were no
differences in the concentration of acetate, propionate, and total VFA.
3.3. Volatile fatty acids concentration Dietary supplementation of lactose had no effect on the concentration
of acetate, propionate, butyrate, and total VFA in the colon.
The interactions between lactose and FP were insignificant except
for the concentration of ileal acetate and propionate in phase Ⅰ
(Table 4). In phase Ⅰ, the concentration of acetate and butyrate in the 3.4. Gas emission
cecum were greater in high lactose treatment (P < 0.01). No difference
was observed in the concentration of propionate and total VFA in the In phase Ⅰ, the emission of TOC and ammonia in pigs feces were
ileum of pigs fed different lactose levels. There was no difference in the decreased in both high lactose and FP -supplemented diets (table 5).
acetate, propionate, butyrate, and total VFA concentrations in pigs of- There was no dietary lactose × FP interaction. In phase Ⅱ, no changes
fered FP. The concentration of propionate and total VFA in the cecum were observed in the volume of TOC and NH3 between lactose treat-
were greater in high lactose treatment (P < 0.05). Dietary supple- ments. The fecal rate of TOC was decreased in pigs fed FP. However,
mentation of FP decreased acetate and total VFA in the cecum. The there were no effects of FP on fecal NH3 emission.
concentration of acetate and propionate was not different in the cecum
of pigs fed dietary lactose. No difference was observed in the con- 3.5. Intestinal pH and microbiota
centration of propionate and butyrate in the cecum of pigs fed FP. The
concentration of acetate (P < 0.05), propionate (P < 0.01), butyrate Results for the intestinal pH and microbiota are presented in
(P < 0.05), and total VFA (P < 0.01) in the colon were greater in high Table 6. The interactions between lactose and FP were insignificant
lactose treatment. No difference was observed in the concentration of except for the population of Salmonella spp. In phase Ⅰ, the pH of digesta
acetate, propionate, butyrate, and total VFA in pigs fed fermented in ileum was lower in pigs fed high lactose level. The number of E. coli
product. and Lactobacillus spp. in the ileum did not differ in the FP and lactose

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Y.D. Jeong, et al. Livestock Science 227 (2019) 90–96

Table 5 treatments. The FP and high lactose treatments showed a lower popu-
Effects of dietary lactose level and supplementation of Lactobacillus-based fer- lation of Salmonella spp. in ileum. The pH of digesta in the cecum were
mentation product (1 × 1011 cfu L. casei/kg diet) on total fecal noxious gas not affected by the addition of FP and lactose. The abundance of Lac-
emission (g/m2). tobacillus spp. in the cecum was markedly increased (P < 0.05) in pigs
Fermentation 0 1 × 1011 cfu SEM P-value fed high lactose level. The FP treatment did not change the population
product (FP) of Lactobacillus spp. The supplementation of FP decreased the cecal E.
Lactose levela (L) Low High Low High FP L FP × L coli, however, no difference was observed in the population of Salmo-
Phase Ⅰ(d 14)
nella spp. between the treatments. A lower pH was observed in the
TOC 2168 1405 1377 990 156 0.002 0.001 0.22 colon of pigs fed high lactose level, however, the pH of colon was not
NH3 53.1 32.8 31.5 28.6 4.7 0.021 0.012 0.08 affected by the addition of FP. The number of Lactobacillus spp., E. coli,
Phase II (d 28) and Salmonella spp. in the colon did not differ between the treatments.
TOC 892 734 657 578 81 0.027 0.16 0.63
In phase Ⅱ, the pH, Lactobacillus spp., and Salmonella spp. in ileal
NH3 41.7 38.1 39.8 29.2 4.2 0.22 0.11 0.41
digesta did not differ between the treatments. The supplementation of
TOC, total organic carbon. FP decreased ileal E. coli population. There were no effects of dietary FP
a
Phase Ⅰ, high (20%), low (10%); phase II, high (10%), low (10%). and lactose on the pH, and the population of Lactobacillus spp. and
Salmonella spp. in the cecum. Dietary supplementation of FP decreased
Table 6 the population of E. coli in the cecum. There were no effects of dietary
Effects of dietary lactose level and supplementation of Lactobacillus-based fer- lactose and FP on the pH and the population of Lactobacillus spp., E. coli
mentation product (1 × 1011 cfu L. casei/kg diet) on pH and bacterial number g and Salmonella spp. in the colon.
(log gene copy number/g) in the fresh digesta of the different part of digestive
tract. 3.6. Small intestine morphology
Fermentation 0 1 × 1011 cfu SEM P-value
product (FP) The intestine morphological data, including villus heights, crypt
Lactose levela (L) Low High Low High FP L FP × L depth, and their ratio, in different sections of small intestine, are pre-
Phase Ⅰ(d 14)
sented in Table 7. There was no dietary lactose × FP interaction. In
Ileum phase Ⅰ, the villus height in jejunum did not change in pigs fed FP-
pH 6.59 6.45 6.47 6.25 0.09 0.09 0.05 0.68 supplemented or high lactose diets. In phase Ⅱ, no difference was found
Lactobacillus 7.56 7.81 7.72 8.16 0.25 0.31 0.18 0.69 in villus height, crypt depth, and VH/CD in the duodenum, jejunum,
spp.
and ileum among the treatments.
E. coli spp. 4.30 4.16 4.28 3.98 0.24 0.69 0.37 0.76
Salmonella 3.94 3.53 3.45 3.44 0.10 0.042 0.008 0.05
spp. 4. Discussion
Cecum
pH 5.56 5.14 5.18 5.27 0.13 0.34 0.21 0.06 The variable responses of weaned pigs to different levels of dietary
Lactobacillus 9.02 9.52 9.31 9.63 0.15 0.19 0.012 0.53
spp.
lactose emphasize the need for a more comprehensive understanding of
E. coli spp. 5.50 4.94 4.64 4.46 0.2 0.003 0.08 0.37 the underlying mechanisms. Even though there were no differences in
Salmonella 4.52 4.40 4.52 4.28 0.11 0.62 0.13 0.61 the ADFI and ADG of pigs from d 0 to 14 after weaning, the digestibility
spp. of DM and GE differed in lactose-supplemented diets. This may indicate
Colon
that the higher fermentation observed with lactose do not increase the
pH 5.38 5.09 5.04 4.85 0.12 0.077 0.03 0.67
Lactobacillus 8.14 8.57 8.32 8.36 0.24 0.93 0.34 0.42 efficiency of energy utilization, as has previously been reported in
spp. studies where the consumption of higher lactose level (170 vs 270 g/kg)
E. coli spp. 6.26 6.15 6.10 6.07 0.21 0.57 0.74 0.86 did not improve growth performance (Lynch et al., 2007). However, it
Salmonella 4.24 4.21 4.19 4.04 0.16 0.48 0.56 0.69 is thought that microbiota-derived VFA is beneficial by decreasing gut
spp.
Phase II (d 28)
mucosal permeability (Fukuda et al., 2011) and delaying the coloni-
Ileum zation of pathogenic bacteria (Pierce et al., 2006). The results of this
pH 6.57 6.49 6.54 6.45 0.09 0.72 0.38 0.93 experiment also indicated that high lactose levels tended to have ben-
Lactobacillus 7.41 7.49 7.59 7.69 0.10 0.06 0.36 0.91 eficial effects on the feed:gain ratio of the weaned piglet. This may be
spp.
partially explained by the importance of dietary lactose with respect to
E. coli spp. 4.71 4.46 4.33 4.38 0.11 0.013 0.23 0.08
Salmonella 4.20 4.17 4.08 4.29 0.22 0.99 0.68 0.60 the gut health of weaning pigs (Peirce et al., 2006; Kim et al., 2010).
spp. The non-significant performance results obtained in the present study
Cecum should therefore probably not be viewed as a reason for under-
pH 5.02 4.73 4.96 5.33 0.19 0.16 0.82 0.10 estimating the role of lactose in weaned piglets, particularly given that
Lactobacillus 8.35 8.14 8.64 8.44 0.23 0.21 0.37 0.98
spp.
we did not provide a lactose-free treatment, which would have enabled
E. coli spp. 5.66 5.71 5.41 5.35 0.18 0.021 0.92 0.59 us to undertake a with and without lactose inclusion comparison.
Salmonella 4.58 4.44 4.46 4.42 0.11 0.68 0.58 0.77 Furthermore, the inclusion of 10% lactose may provide benefits similar
spp. to those obtained with 20% dietary lactose in the first phase.
Colon
Lactobacillus species, including those of L. casei, comprise a very
pH 5.43 5.38 5.27 5.19 0.10 0.09 0.53 0.87
Lactobacillus 7.18 7.14 7.24 7.15 0.31 0.91 0.83 0.93 important element of the probiotic lactic acid bacteria group that has a
spp. high milk fermentation capacity (Alcantara et al., 2016). Results from
E. coli spp. 5.81 5.83 5.51 5.64 0.18 0.07 0.58 0.70 the present study demonstrate that the dietary FP improved ADG. One
Salmonella 4.21 4.14 4.34 4.20 0.14 0.51 0.48 0.81 possible reason for this finding may be the increase in nutrient digest-
spp.
ibility and ADFI. Our results are consistent with several reports showing
a
Phase Ⅰ high (20%), low (10%); phase II, high (10%), low (10%). the improvement in the growth performance of pigs fed diets supple-
mented with Lactobacillus spp. (Giang et al., 2010; Liu et al., 2015; Kim
et al., 2017). In contrast, the daily live weight gain and feed intake of
weaning pigs were not improved in response to dietary multispecies
lactobacilli supplementation (Lahteinen et al., 2015). Such variable

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Y.D. Jeong, et al. Livestock Science 227 (2019) 90–96

Table 7
Effects of dietary lactose level and supplementation of Lactobacillus-based fermentation product (1 × 1011 cfu L. casei/kg diet) on small intestinal morphology.
Fermentation product (FP) 0 1 × 1011 cfu SEM P-value
Lactose levela (L) Low High Low High FP L FP × L

Phase Ⅰ(d 14)

Duodenum
Villus height (μm) 473 479 487 529 19 0.55 0.62 0.27
Crypt depth (μm) 326 303 302 311 13 0.13 0.23 0.89
VH/CD 1.48 1.59 1.62 1.71 0.08 0.11 0.23 0.38
Jejunum
Villus height (μm) 380 418 423 428 14 0.07 0.09 0.13
Crypt depth (μm) 243 289 291 294 13 0.47 0.50 0.32
VH/CD 1.58 1.46 1.46 1.48 0.07 0.08 0.15 0.28
Ileum
Villus height (μm) 332 334 330 335 16 0.46 0.36 0.74
Crypt depth (μm) 216 231 211 218 12 0.67 0.53 0.63
VH/CD 1.57 1.46 1.57 1.55 0.10 0.97 0.84 0.94
Phase II (d 28)
Duodenum
Villus height (μm) 476 528 535 543 27 0.19 0.28 0.43
Crypt depth (μm) 333 350 332 354 13.9 0.91 0.17 0.84
VH/CD 1.43 1.52 1.63 1.53 0.08 0.22 0.93 0.27
Jejunum
Villus height (μm) 539 569 555 607 23 0.26 0.10 0.63
Crypt depth (μm) 301 304 357 327 23 0.11 0.56 0.49
VH/CD 1.89 1.94 1.55 1.87 0.14 0.17 0.20 0.36
Ileum
Villus height (μm) 295 304 309 315 24 0.63 0.77 0.95
Crypt depth (μm) 186 214 223 217 19 0.31 0.57 0.39
VH/CD 1.67 1.43 1.39 1.5 0.13 0.43 0.61 0.20

VH/CD, Villus height to crypt depth ratio.

a
Phase Ⅰ high (20%), low (10%); phase II, high (10%), low (10%).

results can probably be ascribed to variations in the level, type, and
strain of the supplementary probiotics, farm hygiene, diet composition,
feed forms, and interactions of dietary feed ingredients.
In phase Ⅰ, an increase in the DM digestibility of pigs offered the
high lactose-supplemented diet might be related to the proportionally
higher ratio of lactose (lower ash level) in the high-lactose diets and
that lactose rapidly disappears in intestine either through digestion or
fermentation. Previous study on lactose levels has shown that the DM
digestibility of pigs offered diets containing a high level (250 g/kg) of
lactose was enhanced compared with pigs that received a diet con-
taining a low level (150 g/kg) of lactose (Kim et al., 2010). The non-
significant digestibility, together with the non-significant results re-
lating to intestinal morphology and microbiota in phase Ⅱ, indicate that
dietary lactose does not have long-lasting effects on the digestibility of
nutrients.
Although DM, GE, and CP digestibility did not differ in FP-fed pigs
during phase Ⅰ, a higher DM digestibility was observed in phase Ⅱ,
which may be positively associated with an alteration in the intestinal
microbiota. The modification of microbial population structure and a
tendency for increased jejunal villus height in response to dietary FP are
most likely to explain the greater digestibility of DM.
In the present study, the higher dietary lactose level was associated
with the higher concentrations of acetic acid and propionic acid in the
colon. This relationship is likely to be related to the fact that enhanced
dietary lactose levels promote the growth of Lactobacillus spp. and en-
hance the fermentation rate, thereby increasing the levels of volatile
fatty acids (VFA). Furthermore, the lower concentration of fecal am-
monia may indicate that carbohydrates rather than proteins were the
main fermentation substrates. Several studies have investigated the
relationship between the amount of fermentable carbohydrates in the
diet and stimulation of microbial fermentation in the foregut and
hindgut of pigs (Pierce et al., 2006; Giang et al., 2010). However, the
lower ammonia emission observed in response to the provision of high
lactose diets was found to be unrelated to any effect on coliform growth
in the ileum or colon. Our observations regarding ammonia emission,
apart from confirming the effect of fermentable carbohydrates in
decreasing fecal ammonia emission, also indicate the decreased N
emission of FP-supplemented pigs. This is assumed to be at least partly
due to a decrease in pathogens in the intestinal contents.
Piglets fed a diet containing high lactose level had lower digesta pH
in the colon, with a tendency toward a lower ileal pH. The decrease in
colon pH as a result of high dietary lactose may be due to the higher
availability of lactose in the colon as a food source for Lactobacillus spp.
to produce VFA through fermentation. The reduction of lactase secre-
tion in the weanling pig increases the quantity of undigested lactose
reaching the colon (Kim et al., 1978). In this study, the high lactose
level decreased the colon pH, showing that some lactose was indeed
reaching the colon. The relationship between lactobacilli and VFA
production when a high dietary level of lactose is provided has pre-
viously been confirmed (Pierce et al., 2006). The activation of fer-
mentation by accelerating the breakdown of lactose in the GI tract may
explain the lower pH via an increase in the rate of VFA production.
Given the reported anti-pathogenic competition effect of
Lactobacillus spp. on specific pathogenic bacteria (Pieper et al., 2010), it
is possible that providing lactose as a Lactobacillus spp. growth stimu-
lator may affect commensal bacteria in the gut. In the present study,
which focuses exclusively on changes in bacterial populations, we re-
port that there was a tendency for increased Lactobacillus populations in
the cecal contents of piglets in response to high-lactose dietary sup-
plementation. The addition of lactose (20% in the diet) tended to de-
crease cecal E. coli populations during the first phase. This is consistent
with the findings of Dillon et al. (2010), who also observed a lower
population of E. coli when the diet of newly weaned piglets was sup-
plemented with 25% lactose (compared with 15% lactose). The ob-
served tendency of lower ileal pH in piglets fed FP or high lactose diets,
together with the interactive effects of FP and lactose on Salmonella spp.
reduction in the ileum may be due to the higher rate of VFA production.
Lalles et al. (2007) have reported that higher VFA production inhibits
the proliferation of pathogenic bacteria in the intestine. The trophic
effects of the VFA have also been reported to protect the mucosal de-
fense barrier against invading pathogens (Williams et al., 2001). The
insignificant relationships relating to coliforms and salmonella

95
Y.D. Jeong, et al.
proliferation may indicate that, although a high dietary lactose content
influences the concentration of fermentation end products (acetate,
propionate, and butyrate), do not necessarily result in a decrease of
potentially pathogenic bacteria in the small intestine. Moreover, the
inconsistent results regarding Lactobacillus spp. populations during the
second phase appear to indicate that the same amount of lactose as a
prebiotic in all the diets provided a similar amount of substances for
bacterial proliferation. Similarly, a weak relationship exists between
lactose levels and dietary FP.
Weaned piglets appear to be particularly susceptible to intestinal
infection by pathogens such as coliforms due to the sudden change from
feeding on the mother sows milk to mash diets. Diarrhea occurring after
the weaning period can be caused by different factors, including the
presence of E. coli (Lalles et al., 2007; Liu et al., 2015). In the current
experiment, FP had the most notable effects against E. coli, as their
population in the ileum decreased markedly during phase Ⅰ and in the
cecum in both phases. The colonization of Lactobacillus spp. in the in-
testinal environment permits the activity of the inoculum under phy-
siological conditions and ensures that piglets are in a favorable condi-
tion to resist possible pathogenic intrusion. As there was no difference
in the number of Lactobacillus spp. between FP treatments, the reduc-
tion of coliform and salmonella population in FP-supplemented treat-
ment may be associated with the higher efficacy of L. casei from FP.
However, no synergistic effects pertaining to the combination of FP and
lactose was shown in increasing the population of Lactobacillus spp. It
can be due to the limited substrate for Lactobacillus spp. proliferation in
FP-supplemented treatment compared with high lactose treatment.
The changes in villus height and crypt depth in the duodenum, je-
junum, and ileum of the small intestine are indicative of gut health and
the digestive capacity of pigs. There is a direct correlation between
villus height and increased epithelial turnover (Lalles et al., 2007).
Although colon morphology was not examined, the findings relating to
small intestine morphology showed that the site of lactose fermentation
in the small intestine does not necessarily contribute to the integrity of
villi.
In conclusion, the results obtained in the present study indicate that
FP may influence the colonization of coliforms in the ileum regardless
of dietary lactose levels. Depleted pathogen populations may be asso-
ciated with greater growth performance. The lack of significant results
in phase Ⅱ indicates that a high lactose level in phase Ⅰ did not have
long-lasting effects on the intestinal microbiota. Although we detected
no interactive effects between dietary lactose level and FP, we postulate
that dietary lactose level could be reduced to 10% in order to lower the
price of diets when using FP.
Acknowledgements
This work was carried out with the support of "Cooperative
Research Program for Agriculture Science and Technology
Development (Project No. PJ01201602 and PJ01160301)", and RDA
Fellowship Program of Rural Development Administration (National
Institute of Animal Science), Republic of Korea.
Declaration of interest
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.livsci.2019.06.018.
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