Published December 8, 2014

Lipid microencapsulation allows slow release of organic acids and natural

identical flavors along the swine intestine1,2
A. Piva,*3,4 V. Pizzamiglio,* M. Morlacchini, M. Tedeschi,4 and G. Piva
*DIMORFIPA, Universita` di Bologna, 40064 Ozzano Emilia, Bologna, Italy;
CERZOO, S. Bonico, 29100 Piacenza, Italy; Vetagro s.r.l., 42100 Reggio Emilia, Italy; and
ISAN, Facolta` di Agraria, Universita` Cattolica del Sacro Cuore, 29100 Piacenza, Italy

ABSTRACT: The purpose of the present work was

to investigate the in vivo concentrations of sorbic acid
and vanillin as markers of the fate of organic acids (OA)
and natural identical flavors (NIF) from a microencapsulated mixture and from the same mixture nonmicroencapsulated, and the possible consequences on the intestinal microbial fermentation. Fifteen weaned pigs
were selected from 3 dietary groups and were slaughtered at 29.5 0.27 kg of BW. Diets were (1) control;
(2) control supplemented with a blend of OA and NIF
microencapsulated with hydrogenated vegetable lipids
(protected blend, PB); and (3) control supplemented
with the same blend of OA and NIF mixed with the
same protective matrix in powdered form but without
the active ingredient coating (nonprotected blend,
NPB). Stomach, cranial jejunum, caudal jejunum, ileum, cecum, and colon were sampled to determine the
concentrations of sorbic acid and vanillin contained in
the blend and used as tracers. Sorbic acid and vanillin
were not detectable in pigs fed the control, and their

concentrations were not different in the stomach of PB

and NPB treatments. Pigs fed PB showed a gradual
decrease of the tracer concentrations along the intestinal tract, whereas pigs fed NPB showed a decline of
tracer concentration in the cranial jejunum and onwards, compared with the stomach concentrations. Sorbic acid and vanillin concentrations along the intestinal
tract were greater (P = 0.02) in pigs fed PB compared
with pigs fed NPB. Pigs fed PB had lower (P = 0.03)
coliforms in the caudal jejunum and the cecum than
pigs fed the control or NPB. Pigs fed the control or PB
had a greater (P = 0.03) lactic acid bacteria plate count
in the cecum than pigs fed NPB, which showed a reduction (P = 0.02) of lactic acid concentrations and greater
(P = 0.02) pH values in the caudal jejunum. The protective lipid matrix used for microencapsulation of the
OA and NIF blend allowed slow-release of both active
ingredients and prevented the immediate disappearance of such compounds upon exiting the stomach.

Key words: microencapsulation, natural identical flavor, organic acid, slow-release, swine
2007 American Society of Animal Science. All rights reserved.

INTRODUCTION

J. Anim. Sci. 2007. 85:486493

doi:10.2527/jas.2006-323

feed preservatives in foods and feeds (Frank, 1994) to

prevent spoilage. As such, feeding OA to farm animals,
especially pigs, is a widely accepted tool to control the
microbial balance in the stomach.
Some essential oils have antimicrobial properties
(Guenther, 1948; Boyle, 1955) that are attributed
mainly to phenolic components (Cosentino et al., 1999).
Because these natural compounds are classified as generally recognized as safe by the Food and Drug Administration (FDA, 2006), their use to prevent growth of
foodborne pathogens or spoilage organisms has gained
increasing interest. The inherent limitation of the effective dose of OA or botanicals in modulating intestinal
flora may reside in the prompt absorption, metabolism,
or both, that they undergo upon entering the duodenum. This could be overcome by microencapsulating
the active compounds in a matrix that could dissolve
as it passes along the intestine.

Following the ban of antibiotics as growth promoters

in the European Union (regulation No. 1831/2003/CE),
studies have been oriented to feeding strategies to prevent diet malabsorption, unbalanced intestinal fermentation, and diarrhea. Organic acids (OA) are used as

The authors are grateful to Terenzio Bertuzzi for the valuable

technical assistance. The study was supported by a grant from Vetagro S.r.l., Reggio Emilia, Italy.
2
Previously presented in abstract form: ASPA 10th biennal conference, Nov. 2730, 2005. Christchurch, New Zealand.
3
Corresponding author: andrea.piva@unibo.it
4
The study was conducted in 2001, and an EU patent (number
1391155B1) was issued in 2004; more patents are pending.
Received May 19, 2006.
Accepted September 23, 2006.

486

487

Slow release of microencapsulated additives

Microencapsulation can be used in a wide range of

applications, from delaying the absorption of drugs
(Piva et al., 1997) and protecting amino acids and proteins from rumen degradation (Noel, 2000) to providing
technological advantages in the handling of irritant or
corrosive products.
The purpose of the present work was to investigate
the in vivo concentrations of sorbic acid and vanillin as
markers of the fate of OA and natural identical flavors
(NIF) from a microencapsulated mixture and from the
same mixture nonmicroencapsulated, and the possible
consequences on the intestinal microbial fermentation.

MATERIALS AND METHODS

Animals and Diets
The current study was conducted in accordance with
the published guidelines for Good Laboratory Practices
(directives No. 88/320/EEC and No. 90/18/EEC), and
animal welfare and protection (directive No. 86/609/
EEC and Italian Law Act, Decreto Legislativo No. 116,
issued on January 27, 1992). The research farm Centro
Ricerche per la Zootecnia e lAmbiente (CERZOO),
where the study was conducted from 10 until 25 September 2001, is Good Laboratory Practices-certified and
is authorized to perform animal studies according to
Section 12 of Act No. 116, indicated above, by the Italian
Ministry of Health (Ministerial Decretory No. 253/95A, issued on 18 August 1995). In addition, the ethical
committee of the ISAN (Institute of Food Science and
Nutrition, Universita` Cattolica del Sacro Cuore, Piacenza, Italy) reviewed and approved the experimental
protocol.
Seventy-five piglets (77 d of age; Goland Duroc;
initial BW 23.1 3.5 kg), supplied by Vailati Facchini
farms (husbandry code 035 CR 004, Crema, Italy), were
allotted to the following 3 dietary treatments (Table 1)
for 15 d (1) control diet; (2) control plus a protected
blend (PB), which consisted of 4 g of OA/kg (fumaric,
760 mg/kg; malic, 360 mg/kg; citric, 360 mg/kg; sorbic,
440 mg/kg) and NIF (vanillin, 23 mg/kg; thymol, 11 mg/
kg; directive 70/524/CE; Regulation No. 1831/2003/CE)
microencapsulated in a protective matrix of hydrogenated vegetable lipids (C12:0, 0.15%; C14:0, 1.38%;
C16:0, 60.46%; C18:0, 37.25%; C20:0, 0.42%; all values
on an as-fed basis); and (3) control plus a nonprotected
blend (NPB), which consisted of the same OA and NIF
blends that were not microencapsulated but mixed with
the powdered protective matrix. The NPB was supplemented with the same lipid mixture and quantity to
compensate for the lipid supply of the protective matrix
of the blend in treatment PB. The microencapsulated
blend of OA and NIF, PB (Piva and Tedeschi, 2004;
European Patent No. 1391155B1), was supplied by Vetagro S.r.l. (Reggio Emilia, Italy; Production authorization IT000002RE). Sorbic acid and vanillin were both
present in PB and NPB to be used as markers to be
tracked by HPLC along the gastrointestinal tract.

Table 1. Ingredients and chemical composition of experimental diets fed to pigs

Experimental diet1
Item

CTRL

PB

NPB

%, as-fed basis
Ingredient
Corn
Barley
Flaked barley
Soybean oil
Sweet dried whey
Wheat bran
Soybean meal (44%)
Potato protein2
Limestone CaCO3
Calcium sulphate (CaSO4)
Monocalcium phosphate (Ca(H2PO4)2)
Sodium chloride (NaCl)
DL-Methionine
L-Lysine HCl
L-Threonine
L-Tryptophan
Vitamin/mineral premix3
Micro-encapsulated blend
Nonmicroencapsulated blend

25.4
10.5
20.7
3.5
5.0
10.2
17.0
3.5
0.4
0.6
1.6
0.30
0.16
0.4
0.16
0.04
0.5

25.4
10.5
20.7
3.5
5.0
9.8
17.0
3.5
0.4
0.6
1.6
0.30
0.16
0.4
0.16
0.04
0.5
0.4

25.4
10.5
20.7
3.5
5.0
9.8
17.0
3.5
0.4
0.6
1.6
0.30
0.16
0.4
0.16
0.04
0.5

0.4

Chemical composition, % of DM
DM, %
CP
Ether extract
Crude fiber
Ash
Starch

90.86
19.30
6.74
5.01
6.84
39.61

90.86
19.08
6.76
4.96
6.61
37.99

90.96
19.16
7.02
4.97
6.62
37.95

Nutritive value,4 MJ/kg of DM

DE
NE

16.07
11.51

16.07
11.51

16.07
11.51

1
Control diet; PB = control diet supplemented with microencapsulated blend of organic acids and natural identical flavors; and NPB =
control diet supplemented with the same blend of organic acids and
natural identical flavors without the protective matrix coating the
active ingredients.
2
Protastar, Kalmi Italia, Desenzano del Garda (BS), Italy.
3
Provided (per kg of diet, as-fed basis): vitamin A, 18,000 IU; vitamin D3, 2,400 IU; vitamin E, 98 IU; thiamine, 3 mg; riboflavin, 7.2
mg; pyridoxine, 6 mg; pantothenic acid, 24 mg; biotin, 240 g; ascorbic
acid, 90 mg; menadione, 4.8 mg; niacin, 30 mg; cyanocobalamin, 36
g; folic acid, 1.8 mg; choline chloride, 480 mg; CoCO33Co(OH)2H2O,
480 g; FeCO3, 300 mg; Ca(IO3)2, 1.8 mg; MnO2, 48 mg; CuSO45H2O,
120 mg; Na2SeO3, 120 g; and ZnO, 240 mg.
4
DE according to Whittemore (1980); NE according to Noblet et al.
(1994).

All piglets were kept in flat-deck cages (5 pens/dietary

treatment; 5 pigs/pen) and always had free access to
feed and water for the whole period until slaughter.
Throughout the study, pigs were kept in a controlled
room temperature (27.4 0.96C) and natural lighting
(September, 12 h of light/d). At 92 d of age, 1 animal
(29.5 0.27 kg of BW) from each pen was removed and
within less than 30 min after removal was killed under
supervision of the veterinarian at the CERZOO (S. Bonico, Piacenza, Italy), by stunning with a captive bolt
followed by complete bleeding.
Immediately after death, the stomach, cranial jejunum, caudal jejunum, ileum, cecum, and colon (at the

488

Piva et al.

sigmoid flexure) were sampled (the contents were

drained and collected after excision of each gastrointestinal section) to determine the presence of sorbic acid
and vanillin in the digesta. Samples from the caudal
jejunum and cecum were used to enumerate lactic acid
bacteria and coliforms, as described below. Samples
for sorbic acid, vanillin, short chain fatty acids, and
ammonia analyses were immediately stored at 20C;
samples for pH determination and microbial counts
were immediately processed.

Chemical Analyses of Feed

and Intestinal Contents
Feed composition analyses (DM, ash, and starch; Table 1) were performed according to the methods of the
Italian Ministry of Agriculture and Forest (Suppl. 2,
1975); CP according to G.U. Series General n. 92
21.04.96; ether extract according directive CEE n. 84/
4/CEE 20.12.83; G.U. CE n. L15 18.01.84; and crude
fiber according to directive CEE n. 92/89 03.11.92. The
analyses of sorbic acid, vanillin, and short chain fatty
acids concentrations, and pH were performed on the
intestinal contents.
Sorbic acid was analyzed by HPLC (PU-980, Jasco
Corp., Tokyo, Japan) using a Lichrospher 100, 5-m,
RP-C18 column (125 4 mm i.d.; Merck & Co. Inc.,
Whitehouse Station, NJ), eluted from the column with
water:methanol (75:25, vol:vol) in 7.4 min, at a flow
rate of 1 mL/min, registering the absorbance at 245 nm
(UV-1575, Jasco Corp.). Before injection, 50 g of each
gastrointestinal contents were added to 5 mL of trichloroacetic acid (5%, vol:vol), centrifuged (8,000 g for
10 min at 4C), and filtered. The filtrate (20 mL) was
extracted using a steam distillation in a Kjeldahl tube
for 12 min after adding 10 mL of HCl (3 mol/L), and
then 1 mL of the distilled portion was filtered through
a 0.45-m syringe filter (25 mm, nylon membrane;
Millipore Corporation, Bedford, MA). Using an autosampler (AS-1555, Jasco Corp.), samples were injected into a fixed, 30-L loop for loading into the column. The limit of detection for sorbic acid was 0.45
nmol/g of content for the gastrointestinal tracts samples. The recovery for sorbic acid was 96.1 2.4%.
Vanillin was analyzed by HPLC (PU-980, Jasco
Corp.) using a Lichrospher 100, 5-m, RP-C18 column,
as described above, and eluted from the column with
water:acetonitrile (82:18, vol:vol) in 4.8 min, at a flow
rate of 1 mL/min, registering the absorbance at 295 nm
(UV-1575, Jasco Corp.). Before injection, 50 g of the
gastrointestinal contents was added to 5 mL of trichloroacetic acid (5%, vol:vol), centrifuged (8,000 g for 10
min at 4C), and filtered. Then, 1 mL of the filtrate
was diluted to 10 mL with distilled water and filtered
through a 0.45-m syringe filter, as described above,
and analyzed using the autosampler and injection loop
described above. The limit of detection for vanillin was
0.66 nmol/g of content of stomach and cranial and cau-

dal jejunum, and 2.63 nmol/g of content of ileum, cecum,

and colon. The recovery for vanillin was 91.1 1.8%.
Ammonia in intestinal contents was measured with
an enzymatic kit for ammonia analysis (R-Biopharm
GmbH Italia, Milan, Italy) after protein precipitation,
as described previously, with trichloroacetic acid and
centrifugation (8,000 g) for 10 min at 4C. Short-chain
fatty acid and lactic acid concentrations were analyzed
by gas chromatography (Varian 3400, Varian Analytical Instruments, Sunnyvale, CA) using a Carbopack BDA/4% CW 2M, 80/120 packed column (Supelco, Sigma
Aldrich s.r.l., Milano, Italy). Before injection, the intestinal contents were centrifuged (6,000 g for 15 min
at 4C), and 2 mL of the supernatant were mixed with
1 mL of pivalic acid (98% pure), 1 mL of ossalic acid
(99.8% pure), and 250 L of formic acid (99% pure;
Fussel and McCailey, 1987).

Bacterial Counts
Serial 10-fold dilutions of 1 g of samples from caudal
jejunum and cecum were serially diluted and plated
onto Rogosa agar plates for lactic acid bacteria, and
Violet Red Bile agar (Oxoid Ltd., Basingstoke, Hampshire, UK) plates for coliforms. There were 5 replicates
per dietary treatment. Rogosa agar plates were incubated for 48 h at 39C under anaerobic conditions (H2
with approximately 4 to 10% CO2; BBL GasPak Plus
Anaerobic System Envelopes, BD, Sparks, MD). Violet
Red Bile agar plates were incubated for 24 h at 39C
under aerobic conditions.

Statistical Analyses
Data are reported as means SEM, and the level
of significance was P < 0.05. Sorbic acid and vanillin
concentrations in each gastrointestinal tract of animals
fed PB and NPB were compared by unpaired t-test;
sorbic and vanillin concentrations among gastrointestinal tracts of pigs within the same dietary treatment
were compared by 1-way ANOVA. Ammonia and shortchain fatty acid concentrations, pH, and microbial plate
counts within the same gastrointestinal site from the
3 dietary treatments (control, PB, and NPB) were compared, and significant differences among treatment
means were identified by ANOVA. When treatments
effects were detected, means were separated using
Newman-Keuls test. Data were analyzed using the program GraphPad Prism (GraphPad Software 4.00, San
Diego, CA).

RESULTS
Animal Health Status
No outward clinical conditions were observed during
study by the veterinarian in charge of animal welfare.
As such, no medical interventions or treatments were
performed and no piglets died during the study.

Slow release of microencapsulated additives

Figure 1. Sorbic acid concentrations in gastrointestinal

tracts of pigs fed the control diet, pigs fed the control diet
supplemented with a microencapsulated blend of organic
acids and natural identical flavors (PB, striped bars), and
pigs fed the control diet supplemented with the same
blend of organic acids and natural identical flavors with
the protective matrix powder but not coating the active
ingredients (NPB, black bars). In control-fed pigs, sorbic
acid was not detected in any section of the gastrointestinal
tract. Data are shown as means SEM (n = 5). a,bIn the
same segment of the gastrointestinal tract, different letters
indicate P < 0.05.

Chemical Analyses of Feed

and Intestinal Contents
No differences (P > 0.41) were detected among dietary
treatments for ingesta DM content within each gastrointestinal tract location. Sorbic acid was not detected
(<0.45 nmol/g) in gastrointestinal tract contents from
control pigs. The concentration of sorbic acid did not
differ (P = 0.61) in the stomach content of piglets fed
PB or NPB (7.76 1.14 vs. 7.00 0.87 mol/g of DM,
respectively). Conversely, sorbic acid concentration was
greater in every section of the intestine of pigs fed PB
than pigs fed NPB (P = 0.02). The NPB-fed pigs had
only traces of sorbic acid in cranial and caudal jejunum,
whereas it was not detectable in ileum, cecum, and
colon (Figure 1).
Vanillin was not detected (<0.66 nmol/g) in gastrointestinal tract contents of control pigs. Vanillin concentration did not differ (P = 0.65) in the stomach content
of pigs fed PB or NPB (153.8 20.80 vs. 136.4 31.03
nmol/g of DM, respectively). Pigs fed PB showed the
presence of vanillin in cranial and caudal jejunum
(73.34 19.23 vs. 84.74 33.24 nmol/g of DM, respectively). In pigs fed the NPB (Figure 2) treatment, vanillin was not detected (<0.66 nmol/g) from the cranial

489

Figure 2. Vanillin concentrations in gastrointestinal

tracts of pigs fed the control diet, pigs fed the control diet
supplemented with a microencapsulated blend of organic
acids and natural identical flavors (PB, striped bars), and
pigs fed the control diet supplemented with the same
blend of organic acids and natural identical flavors with
the protective matrix powder but not coating the active
ingredients (NPB, black bars). In control-fed pigs, vanillin
was not detected in any section of the gastrointestinal
tract. Data are shown as means SEM (n = 5).

jejunum to colon. No differences (P > 0.27) were reported

for ammonia concentration among treatments.
Control pigs had greater (P = 0.02) concentration of
total short chain fatty acids than pigs fed PB and NPB
in colon and greater concentration of iso-butyric acid
than pigs fed PB and NPB in stomach (P = 0.01), cranial
jejunum (P < 0.001), and colon (P < 0.001; Table 2).
Analyses of pH revealed greater (P = 0.02) values in
caudal jejunum for pigs fed NPB compared with control
pigs and those fed PB (Table 2).
Lactic acid concentration was lower (P < 0.02) in pigs
fed NPB than in controls in cranial and caudal jejunum,
cecum, and colon. Area under the curve (Ritschel, 1992)
for lactic acid calculated for the entire gastrointestinal
tract of pigs fed NPB was lower (P = 0.03) compared
with control pigs and those fed PB (14.73 vs. 30.05 and
29.99 mol/g of DM, respectively; pooled SEM = 4.12).
Lactic acid bacteria counts did not differ (Figure 3)
in caudal jejunum (P = 0.08), whereas a reduction (P =
0.03) was observed in the cecum of pigs fed NPB (9.41
vs. 10.14 and 10.08 log cfu/g of intestinal content in
control and PB, respectively; pooled SEM = 0.14). Conversely, microbial plate counts of coliforms (Figure 4)
were reduced (P < 0.03) by PB in caudal jejunum and
cecum compared with NPB and to control (caudal jejunum: 6.35 vs. 7.99, and 8.09 log cfu/g of intestinal con-

490

Piva et al.

Table 2. pH, ammonia, and molar proportions of short chain fatty acids in gastrointestinal tract samples from pigs
fed the experimental diets

Gastrointestinal
tract

Iso-valeric
acid

Valeric
acid

Lactic
acid

Total
short
chain
fatty
acids2

0.01
0.00
0.01
0.005
0.446

ND4
ND
ND
ND
ND

ND
ND
ND
ND
ND

5.23
2.63
2.63
0.742
0.068

0.78
0.69
0.88
0.157
0.734

0.14b
0.06a
0.01a
0.019
0.001

ND
0.02
0.01
ND
ND

ND
ND
ND
ND
ND

ND
ND
ND
ND
ND

7.33b
4.88ab
3.42a
0.792
0.021

1.37
2.00
1.84
0.283
0.417

ND
0.00
0.01
0.007
0.457

0.10
0.08
0.05
0.022
0.352

ND
ND
ND
ND
ND

ND
ND
ND
ND
ND

ND
ND
ND
ND
ND

12.58b
15.04b
3.07a
2.420
0.019

1.76
2.27
3.22
0.518
0.279

1.12a
0.98a
6.31b
0.324
0.001

0.01
0.01
0.04
0.017
0.325

0.04
0.04
0.05
0.006
0.764

0.01a
0.01a
0.26b
0.057
0.014

ND
ND
ND
ND
ND

ND
ND
ND
ND
ND

7.20
8.36
4.12
1.177
0.066

1.24
0.98
4.96
0.820
0.040

26.25
28.08
21.69
4.733
0.629

9.94
12.39
13.87
1.659
0.278

5.90
7.06
6.83
0.898
0.634

0.03
0.05
0.02
0.016
0.499

2.43
3.66
3.55
0.471
0.166

0.02
0.03
0.03
0.007
0.537

0.35a
0.65b
0.25a
0.083
0.022

0.64b
0.36ab
0.15a
0.057
0.007

17.12
23.85
24.11
2.956
0.274

1.28
3.32
3.95
1.252
0.380

24.80b
13.61a
12.41a
2.698
0.033

13.72b
7.32a
6.08a
1.648
0.015

0.21b
0.09a
0.08a
0.016
0.001

5.62
3.90
3.55
0.640
0.089

0.15b
0.05a
0.05a
0.007
0.001

1.01c
0.61b
0.26a
0.065
0.001

0.22b
0.07ab
0.05a
0.024
0.012

45.71b
25.58ab
20.07a
4.735
0.018

pH

Ammonia

Acetic
acid

Propionic
acid

Iso-butyric
acid

Control
PB
NPB

3.61
3.48
3.66
0.146
0.673

20.70
16.33
15.97
2.719
0.419

0.68
0.65
0.84
0.139
0.917

0.01
0.00
0.00
0.003
0.345

0.08b
0.03a
0.01a
0.013
0.010

Control
PB
NPB

4.97
5.15
5.34
0.257
0.320

34.57
36.04
35.90
7.301
0.988

1.23
2.01
1.72
0.262
0.213

ND
ND
0.01
0.001
0.001

Control
PB
NPB

5.31a
5.31a
6.10b
0.195
0.022

35.59
41.81
32.52
3.935
0.274

1.74
2.19
3.36
0.476
0.101

Control
PB
NPB

5.44
5.09
6.07
0.310
0.326

52.98
50.96
54.14
4.741
0.893

Control
PB
NPB

5.50
5.47
5.27
0.066
0.060

Control
PB
NPB

5.55
5.63
5.51
0.085
0.596

Treatment1

n-butyric
acid

mol/g of DM
Stomach3

Pooled SEM
P of the model, <
Cranial jejunum

Pooled SEM
P of the model, <
Caudal jejunum

Pooled SEM
P of the model, <
Ileum

Pooled SEM
P of the model, <
Cecum

Pooled SEM
P of the model, <
Colon

Pooled SEM
P of the model, <

Within a column, means without a common superscript letter differ (P < 0.05).
Control diet; PB = control diet supplemented with microencapsulated blend of organic acids and natural identical flavors; and NPB =
control diet supplemented with the same blend of organic acids and natural identical flavors with the protective matrix powder but not coating
the active ingredients.
2
Total short chain fatty acids not including lactic acid.
3
Data are shown as means (n = 5).
4
ND indicates not detectable.
ac
1

tent; pooled SEM = 0.195 cecum: 6.78, vs. 7.58, and

7.99 cfu/g of intestinal content; pooled SEM = 0.24; respectively).

DISCUSSION
The ban on antibiotics as growth promoters in the
European Union has forced careful consideration of the
fragile equilibrium between the intestinal microbial
balance and the fermentability of indigestible feed fractions. As quality and availability of feed raw materials
fluctuate, it has become necessary to investigate alternative methods to modulate the intestinal flora beyond
the stomach barrier.
Factors that can affect intestinal microbiota include
OA (Partanen and Mroz, 1999), NIF (Penalver et al.,

2005), enzymes (Kim et al., 2003), prebiotics (Gibson,

1998), and probiotics (Klaenhammer, 2000). Efficacy of
these appears to be associated to the environmental
bacterial challenge. Gastrointestinal epithelial changes
occurring in piglets at weaning could facilitate digestive
malfunction (Boudry et al., 2004), which is often associated with invasion of enterotoxigenic Escherichia coli.
As consequence, piglets are susceptible to diarrhea (Kyriakis, 1989). Feed-related measures may alleviate
symptoms of this disease (Melin and Wallgren, 2002).
Organic acids have been used to control the postweaning diarrhea and edema disease in piglets (Tsiloyiannis
et al., 2001a,b). Likewise, NIF such vanillin, carvacrol,
or thymol have been shown to exert antibacterial activity in food systems (Burt et al., 2005; Falcone et al.,
2005).

Slow release of microencapsulated additives

491

Figure 3. Lactic acid bacteria (LAB) microbial plate

counts in (a) caudal jejunum and (b) cecum. Samples from
pigs fed the control diet (white bars), pigs fed the control
diet supplemented with a microencapsulated blend of
organic acids and natural identical flavors (PB, striped
bars), and pigs fed the control diet supplemented with
the same blend or organic acids and natural identical
flavors with the protective matrix powder but not coating
the active ingredients (NPB, black bars). Data are shown
as means SEM (n = 5). a,bIn the same segment of the
gastrointestinal tract, different letters indicate P < 0.05.

Figure 4. Coliforms microbial plate counts in (a) caudal

jejunum and (b) cecum. Samples from pigs fed the control
diet (white bars), pigs fed the control diet supplemented
with a microencapsulated blend of organic acids and natural identical flavors (PB, striped bars), and pigs fed the
control diet supplemented with the same blend of organic
acids and natural identical flavors with the protective
matrix powder but not coating the active ingredients
(NPB, black bars). Data are shown as means SEM (n =
5). a,bIn the same segment of the gastrointestinal tract,
different letters indicate P < 0.05.

This study showed that sorbic acid and vanillin were

recovered from the gastrointestinal content without interfering background materials because they were not
present in the gastrointestinal fluids of control pigs.
Analyses of stomach contents showed that sorbic acid
and vanillin had equal concentrations regardless of
whether they were nonprotected or microencapsulated.
Pigs fed PB had no immediate disappearance of sorbic
acid and vanillin as observed for NPB fed pigs after the
stomach. Conversely, progressively lower concentrations of sorbic acid and vanillin were measured in the
cranial and caudal jejunum, likely due to the action of
digestive enzymes. The digesta 8 to 10 h after meal is

still present in small intestine (Piva et al., 1997), where

chemical and physical factors can degrade the lipid protective matrix and consequently the metabolism of the
released substances occurs. The protective matrix prevented sorbic acid from being metabolized and allowed
15% of the total sorbic acid detected in the stomach
content to reach the colon.
Piva et al. (1997) studied the absorption in gilts of
tryptophan and sulfamethazine in protected and nonprotected form and concluded that the protective matrix
delayed absorption without affecting total bioavailability. Sorbic acid data in the gastrointestinal content of
pigs fed PB suggested a slow release of the acid from

492

Piva et al.

the capsule. Progressively lower (P < 0.01) fractions of

the stomach sorbic acid concentration were recovered
along the gastrointestinal tract (44, 35, 22, 29, and 15%
for cranial jejunum, caudal jejunum, ileum, cecum, and
colon, respectively), whereas in pigs fed NPB, sorbic
acid concentration declined immediately after the stomach. Only 2% of sorbic acid in cranial and caudal jejunum could be measured, whereas in the subsequent
segments, sorbic acid was not detectable. The lipid matrix also delayed vanillin release as evidenced by 48
and 55% of stomach vanillin concentrations (P < 0.05)
being found in cranial and caudal jejunum, respectively.
The increased presence of sorbic acid in gastrointestinal tract compared with vanillin cannot be associated
with a lower water solubility (0.25% at 30C, wt/vol;
The Merck Index, 2001) compared with vanillin water
solubility (1% at 25C, wt/vol; Vanillin, 2005). Weak
acids with pKa > 3 (including sorbic acid with pKa of
4.76) are well absorbed (Baggot, 1977), and the ionized
form of the acid can pass through the intestinal mucosa.
Sorbic acid was absorbed at a fast degree in the cranial
jejunum of NPB-fed pigs, whereas the protection matrix
delayed sorbic acid disappearance and allowed it to
reach the subsequent intestinal sections with relevant
microbial activity. The antimicrobial role of OA is attributable to the capacity of their undissociated form
to freely diffuse across the semipermeable cell membrane of the microorganism into the cytoplasm (Partanen and Mroz, 1999) where pH is near 7 and weak acids
dissociate and depress the cellular enzymatic activity
and nutrient transport system (Lueck, 1980).
Sofos et al. (1985) reported a reduction of coliforms
count only in the duodenum of broilers fed diets supplemented with sorbic acid (0.04%). In our study similar
results were observed in jejunum and cecum of PB-fed
pigs, where the greater concentration of sorbic acid in
PB than NPB could explain the lower plate counts of coliforms.
Lactic acid bacteria plate counts tended to be reduced
in the jejunum (P = 0.08) and cecum (P = 0.006) of NPBfed pigs and might have accounted for reduced lactic
acid concentration and higher pH values in caudal jejunum of NPB-fed pigs. The same negative pattern was
observed by Canibe et al. (2005) when using 18 g/kg of
formic acid in growing pigs. Such disappearance of lactic acid production was not observed when pigs were
fed the microencapsulated blend.
We have found no references on synergistic effects
of OA and NIF on swine gastrointestinal microflora.
Proposed mechanisms of antibacterial action of NIF
include their action on the cell membrane (Burt, 2004),
the first barrier that OA encounter before entering the
bacterial cells. The increase in plasma membrane permeability due to NIF could help the entrance of OA in
the bacterial cell, where they can alter bacterial metabolism (Brul and Coote, 1999).
The protective lipid matrix used for microencapsulation of OA and NIF blend allowed slow-release of the
active ingredients, preventing the immediate disap-

pearance of such compounds upon exiting the stomach.

The longer permanence along the gastrointestinal tract
of active compounds allowed them to act synergistically
on the intestinal microflora and to reduce coliform
counts.

LITERATURE CITED
Baggot, J. D. 1977. Principles of Drug Disposition in Domestic Animals: The Basis of Veterinary Clinical Pharmacology. Saunders,
Philadelphia, PA.
Boudry, G., V. Peron, I. Le Huerou-Luron, J. P. Lalles, and B. Seve.
2004. Weaning induces both transient and long-lasting modifications of absorptive, secretory, and barrier properties of piglet
intestine. J. Nutr. 134:22562262.
Boyle, W. 1955. Spices and essential oils as preservatives. Am. Perfumer Essential Oil Rev. 66:2528.
Brul, S., and P. Coote. 1999. Preservative agents in food. Mode of
action and antimicrobial resistance mechanisms. Int. J. Food
Microbiol. 50:117.
Burt, S. 2004. Essential oils: Their antibacterial properties and potential applications in foodA review. Int. J. Food Microbiol.
94:223253.
Burt, S. A., R. Vlielander, H. P. Haagsman, and E. J. A. Veldhuizen.
2005. Increase in activity of essential oil components carvacrol
and thymol against Escherichia coli O157:H7 by addition of food
stabilizers. J. Food Prot. 68:919926.
Canibe, N., O. Hojberg, S. Hjsgaard, and B. B. Jensen. 2005. Feed
physical form and formic acid addition to the feed affect the
gastrointestinal ecology and growth performance of growing
pigs. J. Anim. Sci. 83:12871302.
Cosentino, S., C. I. G. Tuberoso, B. Pisano, M. Satta, V. Mascia, E.
Arzedi, and F. Palmas. 1999. In vitro antimicrobial activity and
chemical composition of Sardinian Thymus essential oils. Lett.
Appl. Microbiol. 29:130135.
Falcone, P., B. Speranza, M. A. Del Nobile, M. R. Corbo, and M.
Sinigaglia. 2005. A study on the antimicrobial activity of thymol
intended as a natural preservative. J. Food Prot. 68:16641670.
FDA. 2006. Food and drugs, 21CFR582. http://www.access.gpo.gov/
cgi-bin/cfrassemble.cgi?title=200221 Accessed Jul. 24, 2006.
Frank, K. 1994. Measures to preserve food and feeds from bacterial
damage. UE bersichten zur Tiererna Ehrung 22:149163.
Fussel, R. J., and D. V. McCailey. 1987. Determination of volatile fatty
acids (C2C5) and lactic acid in silage by gas-chromatography.
Analyst 112:12131216.
Gibson, G. R. 1998. Dietary modulation of the human gut microflora
using prebiotics. Br. J. Nutr. 80(Suppl. 2):209212.
Guenther, E. 1948. The Essential Oils. D. Van Nostrand, New
York, NY.
Kim, S. W., D. A. Knabe, K. J. Hong, and R. A. Easter. 2003. Use of
carbohydrases in corn soybean meal-based nursery diets. J.
Anim. Sci. 81:24962504.
Klaenhammer, T. R. 2000. Probiotic bacteria: Today and tomorrow.
J. Nutr. 130:415416.
Kyriakis, S. C. 1989. New aspects of the prevention and/or treatment
of the major stress induced diseases of the early weaned piglet.
Pig News Inf. 2:177181.
Lueck, E. 1980. Antimicrobial Food Additives: Characteristics, Uses,
Effects. Springer-Verlag, Berlin, Germany.
Melin, L., and P. Wallgren. 2002. Aspects on feed related prophylactic
measures aiming to prevent post weaning diarrhoea in pigs.
Acta Vet. Scand. 43:231245.
Noblet, J., H. Fortune, X. S. Shi, and S. Dubois. 1994. Prediction of
net energy value of feeds for growing pigs. J. Anim. Sci. 72(Suppl.
2):344354.
Noel, R. J. 2000. Official feed terms. Pages 187200 in Official Publication. Assoc. Am. Feed Control Officials Inc., West Lafayette, IN.
Partanen, K. H., and Z. Mroz. 1999. Organic acids for performance
enhancement in pig diets. Nutr. Res. Rev. 12:117145.

Slow release of microencapsulated additives

Penalver, P., B. Huerta, C. Borge, R. Astorga, R. Romero, and A.
Perea. 2005. Antimicrobial activity of five essential oils against
origin strains of the Enterobacteriaceae family. APMIS 113:16.
Piva, A., P. Anfossi, E. Meola, A. Pietri, A. Panciroli, T. Bertuzzi, and
A. Formigoni. 1997. Effect of micro-encapsulation on absorption
processes in the pig. Livest. Prod. Sci. 51:5361.
Piva, A., and M. Tedeschi, inventors. Vetagro s.r.l., Reggio Emilia,
Italy, assignee. February 25, 2004. Composition for use in animal
nutrition a controlled release matrix, process for preparing and
use thereof. European Patent No. 1391155 B1.
Ritschel, W. A. 1992. Bioavailability/bioequivalence of modified release drug delivery system: which pharmacokinetic parameters
to determine, single or multiple dose studies, pretests, conditions, and other aspects. Meth. Find. Exp. Clin. Pharmacol.
14:469482.

493

Sofos, J. N., D. J. Fagerberg, and C. L. Quarles. 1985. Effects of sorbic

acid feed fungistat on the intestinal microflora of floor-reared
broiler chickens. Poult. Sci. 64:832840.
The Merck Index. 2001. 13th ed. Merck & Co. Inc., Whitehouse Station, NJ.
Tsiloyiannis, V. K., S. C. Kyriakis, J. Vlemmas, and K. Sarris. 2001a.
The effect of organic acids on the control of porcine post-weaning
diarrhea. Res. Vet. Sci. 70:281285.
Tsiloyiannis, V. K., S. C. Kyriakis, J. Vlemmas, and K. Sarris. 2001b.
The effect of organic acids on the control of post-weaning oedema
disease of piglets. Res. Vet. Sci. 70:287293.
Vanillin. http://www.inchem.org/documents/sids/sids/121335.pdf Accessed Oct. 6, 2005.
Whittemore, C. T. 1980. The use of a computer model in determining
the nutrient requirement of pigs. Proc. Nutr. Soc. 39(Suppl.
2):205211.