Lipid microencapsulation allows slow release of organic acids and natural identical

flavors along the swine intestine

A. Piva, V. Pizzamiglio, M. Morlacchini, M. Tedeschi and G. Piva

J ANIM SCI 2007, 85:486-493.

doi: 10.2527/jas.2006-323 originally published online October 13, 2006

The online version of this article, along with updated information and services, is located on
the World Wide Web at:
http://www.journalofanimalscience.org/content/85/2/486

www.asas.org

Downloaded from www.journalofanimalscience.org by guest on November 20, 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, Università di Bologna, 40064 Ozzano Emilia, Bologna, Italy;

†CERZOO, S. Bonico, 29100 Piacenza, Italy; ‡Vetagro s.r.l., 42100 Reggio Emilia, Italy; and
§ISAN, Facoltà di Agraria, Università 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 microencap-
sulated mixture and from the same mixture nonmicro-
encapsulated, and the possible consequences on the in-
testinal microbial fermentation. Fifteen weaned pigs
were selected from 3 dietary groups and were slaugh-
tered 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, il-
eum, 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 intesti-
nal tract, whereas pigs fed NPB showed a decline of
tracer concentration in the cranial jejunum and on-
wards, compared with the stomach concentrations. Sor-
bic 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 reduc-
tion (P = 0.02) of lactic acid concentrations and greater
(P = 0.02) pH values in the caudal jejunum. The protec-
tive lipid matrix used for microencapsulation of the
OA and NIF blend allowed slow-release of both active
ingredients and prevented the immediate disappear-
ance 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. J. Anim. Sci. 2007. 85:486–493
doi:10.2527/jas.2006-323

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

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 pre-
vent diet malabsorption, unbalanced intestinal fermen-
tation, and diarrhea. Organic acids (OA) are used as
1
The authors are grateful to Terenzio Bertuzzi for the valuable
technical assistance. The study was supported by a grant from Veta-
gro S.r.l., Reggio Emilia, Italy.
2
Previously presented in abstract form: ASPA 10th biennal confer-
ence, Nov. 27–30, 2005. Christchurch, New Zealand.
3
Corresponding author: andrea.piva@unibo.it
4 or both, that they undergo upon entering the duode-
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.
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 gen-
erally recognized as safe by the Food and Drug Adminis-
tration (FDA, 2006), their use to prevent growth of
foodborne pathogens or spoilage organisms has gained
increasing interest. The inherent limitation of the effec-
tive dose of OA or botanicals in modulating intestinal
flora may reside in the prompt absorption, metabolism,
num. This could be overcome by microencapsulating
the active compounds in a matrix that could dissolve
as it passes along the intestine.

486

Downloaded from www.journalofanimalscience.org by guest on November 20, 2014

 Slow release of microencapsulated additives 487
Microencapsulation can be used in a wide range of Table 1. Ingredients and chemical composition of experi-
applications, from delaying the absorption of drugs mental diets fed to pigs
(Piva et al., 1997) and protecting amino acids and pro-
Experimental diet1
teins from rumen degradation (Noel, 2000) to providing
technological advantages in the handling of irritant or Item CTRL PB NPB
corrosive products.
%, as-fed basis
The purpose of the present work was to investigate Ingredient
the in vivo concentrations of sorbic acid and vanillin as Corn 25.4 25.4 25.4
markers of the fate of OA and natural identical flavors Barley 10.5 10.5 10.5
(NIF) from a microencapsulated mixture and from the Flaked barley 20.7 20.7 20.7
Soybean oil 3.5 3.5 3.5
same mixture nonmicroencapsulated, and the possible Sweet dried whey 5.0 5.0 5.0
consequences on the intestinal microbial fermentation. Wheat bran 10.2 9.8 9.8
Soybean meal (44%) 17.0 17.0 17.0
MATERIALS AND METHODS Potato protein2 3.5 3.5 3.5
Limestone CaCO3 0.4 0.4 0.4
Calcium sulphate (CaSO4) 0.6 0.6 0.6
Animals and Diets Monocalcium phosphate (Ca(H2PO4)2) 1.6 1.6 1.6
Sodium chloride (NaCl) 0.30 0.30 0.30
The current study was conducted in accordance with DL-Methionine 0.16 0.16 0.16
the published guidelines for Good Laboratory Practices L-Lysine HCl 0.4 0.4 0.4
(directives No. 88/320/EEC and No. 90/18/EEC), and L-Threonine 0.16 0.16 0.16
L-Tryptophan 0.04 0.04 0.04
animal welfare and protection (directive No. 86/609/
Vitamin/mineral premix3 0.5 0.5 0.5
EEC and Italian Law Act, Decreto Legislativo No. 116, Micro-encapsulated blend — 0.4 —
issued on January 27, 1992). The research farm Centro Nonmicroencapsulated blend — — 0.4
Ricerche per la Zootecnia e l’Ambiente (CERZOO), Chemical composition, % of DM
where the study was conducted from 10 until 25 Sep- DM, % 90.86 90.86 90.96
tember 2001, is Good Laboratory Practices-certified and CP 19.30 19.08 19.16
is authorized to perform animal studies according to Ether extract 6.74 6.76 7.02
Crude fiber 5.01 4.96 4.97
Section 12 of Act No. 116, indicated above, by the Italian
Ash 6.84 6.61 6.62
Ministry of Health (Ministerial Decretory No. 253/95- Starch 39.61 37.99 37.95
A, issued on 18 August 1995). In addition, the ethical Nutritive value,4 MJ/kg of DM
committee of the ISAN (Institute of Food Science and DE 16.07 16.07 16.07
Nutrition, Università Cattolica del Sacro Cuore, Pia- NE 11.51 11.51 11.51
cenza, Italy) reviewed and approved the experimental 1
Control diet; PB = control diet supplemented with microencapsu-
protocol. lated blend of organic acids and natural identical flavors; and NPB =
Seventy-five piglets (77 d of age; Goland × Duroc; control diet supplemented with the same blend of organic acids and
natural identical flavors without the protective matrix coating the
initial BW 23.1 ± 3.5 kg), supplied by Vailati Facchini active ingredients.
farms (husbandry code 035 CR 004, Crema, Italy), were 2
Protastar, Kalmi Italia, Desenzano del Garda (BS), Italy.
3
allotted to the following 3 dietary treatments (Table 1) Provided (per kg of diet, as-fed basis): vitamin A, 18,000 IU; vita-
min D3, 2,400 IU; vitamin E, 98 IU; thiamine, 3 mg; riboflavin, 7.2
for 15 d (1) control diet; (2) control plus a protected mg; pyridoxine, 6 mg; pantothenic acid, 24 mg; biotin, 240 ␮g; ascorbic
blend (PB), which consisted of 4 g of OA/kg (fumaric, acid, 90 mg; menadione, 4.8 mg; niacin, 30 mg; cyanocobalamin, 36
760 mg/kg; malic, 360 mg/kg; citric, 360 mg/kg; sorbic, ␮g; folic acid, 1.8 mg; choline chloride, 480 mg; CoCO3ⴢ3Co(OH)ⴢ2H2O,
480 ␮g; FeCO3, 300 mg; Ca(IO3)2, 1.8 mg; MnO2, 48 mg; CuSO4ⴢ5H2O,
440 mg/kg) and NIF (vanillin, 23 mg/kg; thymol, 11 mg/ 120 mg; Na2SeO3, 120 ␮g; and ZnO, 240 mg.
kg; directive 70/524/CE; Regulation No. 1831/2003/CE) 4
DE according to Whittemore (1980); NE according to Noblet et al.
microencapsulated in a protective matrix of hydroge- (1994).
nated 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 All piglets were kept in flat-deck cages (5 pens/dietary
blend (NPB), which consisted of the same OA and NIF treatment; 5 pigs/pen) and always had free access to
blends that were not microencapsulated but mixed with feed and water for the whole period until slaughter.
the powdered protective matrix. The NPB was supple- Throughout the study, pigs were kept in a controlled
mented with the same lipid mixture and quantity to room temperature (27.4 ± 0.96°C) and natural lighting
compensate for the lipid supply of the protective matrix (September, 12 h of light/d). At 92 d of age, 1 animal
of the blend in treatment PB. The microencapsulated (29.5 ± 0.27 kg of BW) from each pen was removed and
blend of OA and NIF, PB (Piva and Tedeschi, 2004; within less than 30 min after removal was killed under
European Patent No. 1391155B1), was supplied by Vet- supervision of the veterinarian at the CERZOO (S. Bon-
agro S.r.l. (Reggio Emilia, Italy; Production authoriza- ico, Piacenza, Italy), by stunning with a captive bolt
tion α IT000002RE). Sorbic acid and vanillin were both followed by complete bleeding.
present in PB and NPB to be used as markers to be Immediately after death, the stomach, cranial jeju-
tracked by HPLC along the gastrointestinal tract. num, caudal jejunum, ileum, cecum, and colon (at the

Downloaded from www.journalofanimalscience.org by guest on November 20, 2014

488
sigmoid flexure) were sampled (the contents were
drained and collected after excision of each gastrointes-
tinal 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 −20°C;
samples for pH determination and microbial counts
were immediately processed.
Chemical Analyses of Feed
and Intestinal Contents
Feed composition analyses (DM, ash, and starch; Ta-
ble 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 trichlo-
roacetic acid (5%, vol:vol), centrifuged (8,000 × g for
10 min at 4°C), 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 au-
tosampler (AS-1555, Jasco Corp.), samples were in-
jected into a fixed, 30-␮L loop for loading into the col-
umn. The limit of detection for sorbic acid was 0.45
nmol/g of content for the gastrointestinal tracts sam-
ples. 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 trichlo-
roacetic acid (5%, vol:vol), centrifuged (8,000 × g for 10
min at 4°C), 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-
Downloaded from www.journalofanimalscience.org by guest on November 20, 2014
Piva et al.
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 4°C. Short-chain
fatty acid and lactic acid concentrations were analyzed
by gas chromatography (Varian 3400, Varian Analyti-
cal Instruments, Sunnyvale, CA) using a Carbopack B-
DA/4% CW 2M, 80/120 packed column (Supelco, Sigma
Aldrich s.r.l., Milano, Italy). Before injection, the intes-
tinal contents were centrifuged (6,000 × g for 15 min
at 4°C), 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, Hamp-
shire, UK) plates for coliforms. There were 5 replicates
per dietary treatment. Rogosa agar plates were incu-
bated for 48 h at 39°C 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 39°C
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 gastrointesti-
nal tracts of pigs within the same dietary treatment
were compared by 1-way ANOVA. Ammonia and short-
chain fatty acid concentrations, pH, and microbial plate
counts within the same gastrointestinal site from the
3 dietary treatments (control, PB, and NPB) were com-
pared, 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 pro-
gram 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 gastro-
intestinal 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 gastroin-
testinal tract contents of control pigs. Vanillin concen-
tration 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, respec-
tively). In pigs fed the NPB (Figure 2) treatment, vanil-
lin was not detected (<0.66 nmol/g) from the cranial
Downloaded from www.journalofanimalscience.org by guest on November 20, 2014
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). Con-
versely, 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 jeju-
num: 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
Total
short
chain
Gastrointestinal Acetic Propionic Iso-butyric n-butyric Iso-valeric Valeric Lactic fatty
tract Treatment1 pH Ammonia acid acid acid acid acid acid acid acids2

␮mol/g of DM
Stomach3 Control 3.61 20.70 0.68 0.01 0.08b 0.01 ND4 ND 5.23 0.78
PB 3.48 16.33 0.65 0.00 0.03a 0.00 ND ND 2.63 0.69
NPB 3.66 15.97 0.84 0.00 0.01a 0.01 ND ND 2.63 0.88
Pooled SEM 0.146 2.719 0.139 0.003 0.013 0.005 ND ND 0.742 0.157
P of the model, < 0.673 0.419 0.917 0.345 0.010 0.446 ND ND 0.068 0.734
Cranial jejunum Control 4.97 34.57 1.23 ND 0.14b ND ND ND 7.33b 1.37
PB 5.15 36.04 2.01 ND 0.06a 0.02 ND ND 4.88ab 2.00
NPB 5.34 35.90 1.72 0.01 0.01a 0.01 ND ND 3.42a 1.84
Pooled SEM 0.257 7.301 0.262 0.001 0.019 ND ND ND 0.792 0.283
P of the model, < 0.320 0.988 0.213 0.001 0.001 ND ND ND 0.021 0.417
Caudal jejunum Control 5.31a 35.59 1.74 ND 0.10 ND ND ND 12.58b 1.76
PB 5.31a 41.81 2.19 0.00 0.08 ND ND ND 15.04b 2.27
NPB 6.10b 32.52 3.36 0.01 0.05 ND ND ND 3.07a 3.22
Pooled SEM 0.195 3.935 0.476 0.007 0.022 ND ND ND 2.420 0.518
P of the model, < 0.022 0.274 0.101 0.457 0.352 ND ND ND 0.019 0.279
Ileum Control 5.44 52.98 1.12a 0.01 0.04 0.01a ND ND 7.20 1.24
PB 5.09 50.96 0.98a 0.01 0.04 0.01a ND ND 8.36 0.98
NPB 6.07 54.14 6.31b 0.04 0.05 0.26b ND ND 4.12 4.96
Pooled SEM 0.310 4.741 0.324 0.017 0.006 0.057 ND ND 1.177 0.820
P of the model, < 0.326 0.893 0.001 0.325 0.764 0.014 ND ND 0.066 0.040
Cecum Control 5.50 26.25 9.94 5.90 0.03 2.43 0.02 0.35a 0.64b 17.12
PB 5.47 28.08 12.39 7.06 0.05 3.66 0.03 0.65b 0.36ab 23.85
NPB 5.27 21.69 13.87 6.83 0.02 3.55 0.03 0.25a 0.15a 24.11
Pooled SEM 0.066 4.733 1.659 0.898 0.016 0.471 0.007 0.083 0.057 2.956
P of the model, < 0.060 0.629 0.278 0.634 0.499 0.166 0.537 0.022 0.007 0.274
Colon Control 5.55 1.28 24.80b 13.72b 0.21b 5.62 0.15b 1.01c 0.22b 45.71b
PB 5.63 3.32 13.61a 7.32a 0.09a 3.90 0.05a 0.61b 0.07ab 25.58ab
NPB 5.51 3.95 12.41a 6.08a 0.08a 3.55 0.05a 0.26a 0.05a 20.07a
Pooled SEM 0.085 1.252 2.698 1.648 0.016 0.640 0.007 0.065 0.024 4.735
P of the model, < 0.596 0.380 0.033 0.015 0.001 0.089 0.001 0.001 0.012 0.018

Within a column, means without a common superscript letter differ (P < 0.05).
a–c
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 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.

tent; pooled SEM = 0.195 cecum: 6.78, vs. 7.58, and
7.99 cfu/g of intestinal content; pooled SEM = 0.24; re-
spectively).
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 frac-
tions. As quality and availability of feed raw materials
fluctuate, it has become necessary to investigate alter-
native methods to modulate the intestinal flora beyond
the stomach barrier.
Factors that can affect intestinal microbiota include
OA (Partanen and Mroz, 1999), NIF (Peñalver 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 associ-
ated with invasion of enterotoxigenic Escherichia coli.
As consequence, piglets are susceptible to diarrhea (Ky-
riakis, 1989). Feed-related measures may alleviate
symptoms of this disease (Melin and Wallgren, 2002).
Organic acids have been used to control the postwean-
ing diarrhea and edema disease in piglets (Tsiloyiannis
et al., 2001a,b). Likewise, NIF such vanillin, carvacrol,
or thymol have been shown to exert antibacterial activ-
ity in food systems (Burt et al., 2005; Falcone et al.,
2005).

Downloaded from www.journalofanimalscience.org by guest on November 20, 2014

 Slow release of microencapsulated additives
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.
This study showed that sorbic acid and vanillin were
recovered from the gastrointestinal content without in-
terfering 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 concentra-
tions 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
Downloaded from www.journalofanimalscience.org by guest on November 20, 2014
491
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 nat-
ural 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.
still present in small intestine (Piva et al., 1997), where
chemical and physical factors can degrade the lipid pro-
tective matrix and consequently the metabolism of the
released substances occurs. The protective matrix pre-
vented 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 non-
protected form and concluded that the protective matrix
delayed absorption without affecting total bioavailabil-
ity. Sorbic acid data in the gastrointestinal content of
pigs fed PB suggested a slow release of the acid from
492
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 stom-
ach. Only 2% of sorbic acid in cranial and caudal jeju-
num could be measured, whereas in the subsequent
segments, sorbic acid was not detectable. The lipid ma-
trix 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 gastrointesti-
nal tract compared with vanillin cannot be associated
with a lower water solubility (0.25% at 30°C, wt/vol;
The Merck Index, 2001) compared with vanillin water
solubility (1% at 25°C, 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 at-
tributable to the capacity of their undissociated form
to freely diffuse across the semipermeable cell mem-
brane of the microorganism into the cytoplasm (Parta-
nen 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 supple-
mented 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 co-
liforms.
Lactic acid bacteria plate counts tended to be reduced
in the jejunum (P = 0.08) and cecum (P = 0.006) of NPB-
fed pigs and might have accounted for reduced lactic
acid concentration and higher pH values in caudal jeju-
num 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 lac-
tic 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 per-
meability due to NIF could help the entrance of OA in
the bacterial cell, where they can alter bacterial metab-
olism (Brul and Coote, 1999).
The protective lipid matrix used for microencapsula-
tion of OA and NIF blend allowed slow-release of the
active ingredients, preventing the immediate disap-
Downloaded from www.journalofanimalscience.org by guest on November 20, 2014
Piva et al.
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 Ani-
mals: 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 modifi-
cations of absorptive, secretory, and barrier properties of piglet
intestine. J. Nutr. 134:2256–2262.
Boyle, W. 1955. Spices and essential oils as preservatives. Am. Per-
fumer Essential Oil Rev. 66:25–28.
Brul, S., and P. Coote. 1999. Preservative agents in food. Mode of
action and antimicrobial resistance mechanisms. Int. J. Food
Microbiol. 50:1–17.
Burt, S. 2004. Essential oils: Their antibacterial properties and poten-
tial applications in food—A review. Int. J. Food Microbiol.
94:223–253.
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. 68:919–926.
Canibe, N., O. Hojberg, S. Højsgaard, 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. 83:1287–1302.
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. 29:130–135.
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. 68:1664–1670.
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 22:149–163.
Fussel, R. J., and D. V. McCailey. 1987. Determination of volatile fatty
acids (C2–C5) and lactic acid in silage by gas-chromatography.
Analyst 112:1213–1216.
Gibson, G. R. 1998. Dietary modulation of the human gut microflora
using prebiotics. Br. J. Nutr. 80(Suppl. 2):209–212.
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. 81:2496–2504.
Klaenhammer, T. R. 2000. Probiotic bacteria: Today and tomorrow.
J. Nutr. 130:415–416.
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. 2:177–181.
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. 43:231–245.
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):344–354.
Noel, R. J. 2000. Official feed terms. Pages 187–200 in Official Publica-
tion. 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. 12:117–145.
 Slow release of microencapsulated additives
Peñalver, 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 113:1–6.
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. 51:53–61.
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 re-
lease drug delivery system: which pharmacokinetic parameters
to determine, single or multiple dose studies, pretests, condi-
tions, and other aspects. Meth. Find. Exp. Clin. Pharmacol.
14:469–482.
Downloaded from www.journalofanimalscience.org by guest on November 20, 2014
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. 64:832–840.
The Merck Index. 2001. 13th ed. Merck & Co. Inc., Whitehouse Sta-
tion, 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. 70:281–285.
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. 70:287–293.
Vanillin. http://www.inchem.org/documents/sids/sids/121335.pdf Ac-
cessed 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):205–211.
References This article cites 22 articles, 6 of which you can access for free at:
http://www.journalofanimalscience.org/content/85/2/486#BIBL
Citations This article has been cited by 3 HighWire-hosted articles:
http://www.journalofanimalscience.org/content/85/2/486#otherarticles

Downloaded from www.journalofanimalscience.org by guest on November 20, 2014