Lipid microencapsulation allows slow release of organic acids and naturalidentical 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 wasto investigate the in vivo concentrations of sorbic acidand 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 pigswere 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 NIFmicroencapsulated with hydrogenated vegetable lipids(protected blend, PB); and (3) control supplementedwith the same blend of OA and NIF mixed with thesame protective matrix in powdered form but withoutthe active ingredient coating (nonprotected blend,NPB). Stomach, cranial jejunum, caudal jejunum, il-eum, cecum, and colon were sampled to determine theconcentrations of sorbic acid and vanillin contained inthe blend and used as tracers. Sorbic acid and vanillinwere not detectable in pigs fed the control, and their

Key words: microencapsulation, natural identical flavor, organic acid, slow-release, swine

2007 American Society of Animal Science. All rights reserved. J. Anim. Sci. 2007. 85:486493doi:10.2527/jas.2006-323

INTRODUCTION

Following the ban of antibiotics as growth promotersin 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

1The authors are grateful to Terenzio Bertuzzi for the valuabletechnical assistance. The study was supported by a grant from Veta-gro S.r.l., Reggio Emilia, Italy.

2Previously presented in abstract form: ASPA 10th biennal confer-ence, Nov. 2730, 2005. Christchurch, New Zealand.

3Corresponding author: andrea.piva@unibo.it4The 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

concentrations were not different in the stomach of PBand NPB treatments. Pigs fed PB showed a gradualdecrease of the tracer concentrations along the intesti-nal tract, whereas pigs fed NPB showed a decline oftracer concentration in the cranial jejunum and on-wards, compared with the stomach concentrations. Sor-bic acid and vanillin concentrations along the intestinaltract were greater (P = 0.02) in pigs fed PB comparedwith pigs fed NPB. Pigs fed PB had lower (P = 0.03)coliforms in the caudal jejunum and the cecum thanpigs fed the control or NPB. Pigs fed the control or PBhad a greater (P = 0.03) lactic acid bacteria plate countin 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 theOA and NIF blend allowed slow-release of both activeingredients and prevented the immediate disappear-ance of such compounds upon exiting the stomach.

feed preservatives in foods and feeds (Frank, 1994) toprevent spoilage. As such, feeding OA to farm animals,especially pigs, is a widely accepted tool to control themicrobial balance in the stomach.

Some essential oils have antimicrobial properties(Guenther, 1948; Boyle, 1955) that are attributedmainly 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 offoodborne pathogens or spoilage organisms has gainedincreasing interest. The inherent limitation of the effec-tive dose of OA or botanicals in modulating intestinalflora may reside in the prompt absorption, metabolism,or both, that they undergo upon entering the duode-num. This could be overcome by microencapsulatingthe active compounds in a matrix that could dissolveas it passes along the intestine.

Published December 8, 2014

Slow release of microencapsulated additives 487

Microencapsulation can be used in a wide range ofapplications, from delaying the absorption of drugs(Piva et al., 1997) and protecting amino acids and pro-teins from rumen degradation (Noel, 2000) to providingtechnological advantages in the handling of irritant orcorrosive products.

The purpose of the present work was to investigatethe in vivo concentrations of sorbic acid and vanillin asmarkers of the fate of OA and natural identical flavors(NIF) from a microencapsulated mixture and from thesame mixture nonmicroencapsulated, and the possibleconsequences on the intestinal microbial fermentation.

MATERIALS AND METHODS

Animals and Diets

The current study was conducted in accordance withthe published guidelines for Good Laboratory Practices(directives No. 88/320/EEC and No. 90/18/EEC), andanimal welfare and protection (directive No. 86/609/EEC and Italian Law Act, Decreto Legislativo No. 116,issued on January 27, 1992). The research farm CentroRicerche per la Zootecnia e lAmbiente (CERZOO),where the study was conducted from 10 until 25 Sep-tember 2001, is Good Laboratory Practices-certified andis authorized to perform animal studies according toSection 12 of Act No. 116, indicated above, by the ItalianMinistry of Health (Ministerial Decretory No. 253/95-A, issued on 18 August 1995). In addition, the ethicalcommittee of the ISAN (Institute of Food Science andNutrition, Universita` Cattolica del Sacro Cuore, Pia-cenza, Italy) reviewed and approved the experimentalprotocol.

Seventy-five piglets (77 d of age; Goland Duroc;initial BW 23.1 3.5 kg), supplied by Vailati Facchinifarms (husbandry code 035 CR 004, Crema, Italy), wereallotted to the following 3 dietary treatments (Table 1)for 15 d (1) control diet; (2) control plus a protectedblend (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 hydroge-nated vegetable lipids (C12:0, 0.15%; C14:0, 1.38%;C16:0, 60.46%; C18:0, 37.25%; C20:0, 0.42%; all valueson an as-fed basis); and (3) control plus a nonprotectedblend (NPB), which consisted of the same OA and NIFblends that were not microencapsulated but mixed withthe powdered protective matrix. The NPB was supple-mented with the same lipid mixture and quantity tocompensate for the lipid supply of the protective matrixof the blend in treatment PB. The microencapsulatedblend of OA and NIF, PB (Piva and Tedeschi, 2004;European Patent No. 1391155B1), was supplied by Vet-agro S.r.l. (Reggio Emilia, Italy; Production authoriza-tion IT000002RE). Sorbic acid and vanillin were bothpresent in PB and NPB to be used as markers to betracked by HPLC along the gastrointestinal tract.

Table 1. Ingredients and chemical composition of experi-mental diets fed to pigs

Experimental diet1

Item CTRL PB NPB

%, as-fed basisIngredientCorn 25.4 25.4 25.4Barley 10.5 10.5 10.5Flaked barley 20.7 20.7 20.7Soybean oil 3.5 3.5 3.5Sweet dried whey 5.0 5.0 5.0Wheat bran 10.2 9.8 9.8Soybean meal (44%) 17.0 17.0 17.0Potato protein2 3.5 3.5 3.5Limestone CaCO3 0.4 0.4 0.4Calcium sulphate (CaSO4) 0.6 0.6 0.6Monocalcium phosphate (Ca(H2PO4)2) 1.6 1.6 1.6Sodium chloride (NaCl) 0.30 0.30 0.30DL-Methionine 0.16 0.16 0.16L-Lysine HCl 0.4 0.4 0.4L-Threonine 0.16 0.16 0.16L-Tryptophan 0.04 0.04 0.04Vitamin/mineral premix3 0.5 0.5 0.5Micro-encapsulated blend 0.4 Nonmicroencapsulated blend 0.4

Chemical composition, % of DMDM, % 90.86 90.86 90.96CP 19.30 19.08 19.16Ether extract 6.74 6.76 7.02Crude fiber 5.01 4.96 4.97Ash 6.84 6.61 6.62Starch 39.61 37.99 37.95

Nutritive value,4 MJ/kg of DMDE 16.07 16.07 16.07NE 11.51 11.51 11.51

1Control diet; PB = control diet supplemented with microencapsu-lated blend of organic acids and natural identical flavors; and NPB =control diet supplemented with the same blend of organic acids andnatural identical flavors without the protective matrix coating theactive ingredients.

2Protastar, Kalmi Italia, Desenzano del Garda (BS), Italy.3Provided (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, 7.2mg; pyridoxine, 6 mg; pantothenic acid, 24 mg; biotin, 240g; ascorbicacid, 90 mg; menadione, 4.8 mg; niacin, 30 mg; cyanocobalamin, 36g; 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.

4DE according to Whittemore (1980); NE according to Noblet et al.(1994).

All piglets were kept in flat-deck cages (5 pens/dietarytreatment; 5 pigs/pen) and always had free access tofeed and water for the whole period until slaughter.Throughout the study, pigs were kept in a controlledroom 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 andwithin less than 30 min after removal was killed undersupervision of the veterinarian at the CERZOO (S. Bon-ico, Piacenza, Italy), by stunning with a captive boltfollowed by complete bleeding.

Immediately after death, the stomach, cranial jeju-num, caudal jejunum, ileum, cecum, and colon (at the

Piva et al.488

sigmoid flexure) were sampled (the contents weredrained and collected after excision of each gastrointes-tinal section) to determine the presence of sorbic acidand vanillin in the digesta. Samples from the caudaljejunum and cecum were used to enumerate lactic acidbacteria and coliforms, as described below. Samplesfor sorbic acid, vanillin, short chain fatty acids, andammonia analyses were immediately stored at 20C;samples for pH determination and microbial countswere immediately processed.

Chemical Analyses of Feedand Intestinal Contents

Feed composition analyses (DM, ash, and starch; Ta-ble 1) were performed according to the methods of theItalian Ministry of Agriculture and Forest (Suppl. 2,1975); CP according to G.U. Series General n. 9221.04.96; ether extract according directive CEE n. 84/4/CEE 20.12.83; G.U. CE n. L15 18.01.84; and crudefiber according to directive CEE n. 92/89 03.11.92. Theanalyses of sorbic acid, vanillin, and short chain fattyacids concentrations, and pH were performed on theintestinal contents.

Sorbic acid was analyzed by HPLC (PU-980, JascoCorp., 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 withwater:methanol (75:25, vol:vol) in 7.4 min, at a flowrate of 1 mL/min, registering the absorbance at 245 nm(UV-1575, Jasco Corp.). Before injection, 50 g of eachgastrointestinal contents were added to 5 mL of trichlo-roacetic acid (5%, vol:vol), centrifuged (8,000 g for10 min at 4C), and filtered. The filtrate (20 mL) wasextracted using a steam distillation in a Kjeldahl tubefor 12 min after adding 10 mL of HCl (3 mol/L), andthen 1 mL of the distilled portion was filtered througha 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 0.45nmol/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, JascoCorp.) using a Lichrospher 100, 5-m, RP-C18 column,as described above, and eluted from the column withwater:acetonitrile (82:18, vol:vol) in 4.8 min, at a flowrate of 1 mL/min, registering the absorbance at 295 nm(UV-1575, Jasco Corp.). Before injection, 50 g of thegastrointestinal contents was added to 5 mL of trichlo-roacetic acid (5%, vol:vol), centrifuged (8,000 g for 10min at 4C), and filtered. Then, 1 mL of the filtratewas diluted to 10 mL with distilled water and filteredthrough a 0.45-m syringe filter, as described above,and analyzed using the autosampler and injection loopdescribed above. The limit of detection for vanillin was0.66 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 withan enzymatic kit for ammonia analysis (R-BiopharmGmbH Italia, Milan, Italy) after protein precipitation,as described previously, with trichloroacetic acid andcentrifugation (8,000 g) for 10 min at 4C. Short-chainfatty acid and lactic acid concentrations were analyzedby gas chromatography (Varian 3400, Varian Analyti-cal Instruments, Sunnyvale, CA) using a Carbopack B-DA/4% CW 2M, 80/120 packed column (Supelco, SigmaAldrich s.r.l., Milano, Italy). Before injection, the intes-tinal contents were centrifuged (6,000 g for 15 minat 4C), and 2 mL of the supernatant were mixed with1 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 caudaljejunum and cecum were serially diluted and platedonto Rogosa agar plates for lactic acid bacteria, andViolet Red Bile agar (Oxoid Ltd., Basingstoke, Hamp-shire, UK) plates for coliforms. There were 5 replicatesper dietary treatment. Rogosa agar plates were incu-bated for 48 h at 39C under anaerobic conditions (H2with approximately 4 to 10% CO2; BBL GasPak PlusAnaerobic System Envelopes, BD, Sparks, MD). VioletRed Bile agar plates were incubated for 24 h at 39Cunder aerobic conditions.

Statistical Analyses

Data are reported as means SEM, and the levelof significance was P < 0.05. Sorbic acid and vanillinconcentrations in each gastrointestinal tract of animalsfed PB and NPB were compared by unpaired t-test;sorbic and vanillin concentrations among gastrointesti-nal tracts of pigs within the same dietary treatmentwere compared by 1-way ANOVA. Ammonia and short-chain fatty acid concentrations, pH, and microbial platecounts within the same gastrointestinal site from the3 dietary treatments (control, PB, and NPB) were com-pared, and significant differences among treatmentmeans were identified by ANOVA. When treatmentseffects were detected, means were separated usingNewman-Keuls test. Data were analyzed using the pro-gram GraphPad Prism (GraphPad Software 4.00, SanDiego, CA).

RESULTS

Animal Health Status

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

Slow release of microencapsulated additives 489

Figure 1. Sorbic acid concentrations in gastrointestinaltracts of pigs fed the control diet, pigs fed the control dietsupplementedwith amicroencapsulated blend of organicacids and natural identical flavors (PB, striped bars), andpigs fed the control diet supplemented with the sameblend of organic acids and natural identical flavors withthe protective matrix powder but not coating the activeingredients (NPB, black bars). In control-fed pigs, sorbicacidwas not detected in any section of the gastrointestinaltract. Data are shown as means SEM (n = 5). a,bIn thesame segment of the gastrointestinal tract, different lettersindicate P < 0.05.

Chemical Analyses of Feedand Intestinal Contents

No differences (P > 0.41) were detected among dietarytreatments for ingesta DM content within each gastro-intestinal tract location. Sorbic acid was not detected(

Piva et al.490

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

Totalshortchain

Gastrointestinal Acetic Propionic Iso-butyric n-butyric Iso-valeric Valeric Lactic fattytract 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 0.78PB 3.48 16.33 0.65 0.00 0.03a 0.00 ND ND 2.63 0.69NPB 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 0.157P 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 1.37PB 5.15 36.04 2.01 ND 0.06a 0.02 ND ND 4.88ab 2.00NPB 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 0.283P 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 1.76PB 5.31a 41.81 2.19 0.00 0.08 ND ND ND 15.04b 2.27NPB 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 0.518P 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 1.24PB 5.09 50.96 0.98a 0.01 0.04 0.01a ND ND 8.36 0.98NPB 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 0.820P 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 17.12PB 5.47 28.08 12.39 7.06 0.05 3.66 0.03 0.65b 0.36ab 23.85NPB 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 2.956P 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 4.735P of the model, < 0.596 0.380 0.033 0.015 0.001 0.089 0.001 0.001 0.012 0.018

acWithin a column, means without a common superscript letter differ (P < 0.05).1Control 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 coatingthe active ingredients.

2Total short chain fatty acids not including lactic acid.3Data are shown as means (n = 5).4ND indicates not detectable.

tent; pooled SEM = 0.195 cecum: 6.78, vs. 7.58, and7.99 cfu/g of intestinal content; pooled SEM = 0.24; re-spectively).

DISCUSSION

The ban on antibiotics as growth promoters in theEuropean Union has forced careful consideration of thefragile equilibrium between the intestinal microbialbalance and the fermentability of indigestible feed frac-tions. As quality and availability of feed raw materialsfluctuate, it has become necessary to investigate alter-native methods to modulate the intestinal flora beyondthe stomach barrier.

Factors that can affect intestinal microbiota includeOA (Partanen and Mroz, 1999), NIF (Penalver et al.,

2005), enzymes (Kim et al., 2003), prebiotics (Gibson,1998), and probiotics (Klaenhammer, 2000). Efficacy ofthese appears to be associated to the environmentalbacterial challenge. Gastrointestinal epithelial changesoccurring in piglets at weaning could facilitate digestivemalfunction (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 alleviatesymptoms of this disease (Melin and Wallgren, 2002).Organic acids have been used to control the postwean-ing diarrhea and edema disease in piglets (Tsiloyianniset 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).

Slow release of microencapsulated additives 491

Figure 3. Lactic acid bacteria (LAB) microbial platecounts in (a) caudal jejunum and (b) cecum. Samples frompigs fed the control diet (white bars), pigs fed the controldiet supplemented with a microencapsulated blend oforganic acids and natural identical flavors (PB, stripedbars), and pigs fed the control diet supplemented withthe same blend or organic acids and natural identicalflavors with the protective matrix powder but not coatingthe active ingredients (NPB, black bars). Data are shownas means SEM (n = 5). a,bIn the same segment of thegastrointestinal tract, different letters indicate P < 0.05.

This study showed that sorbic acid and vanillin wererecovered from the gastrointestinal content without in-terfering background materials because they were notpresent in the gastrointestinal fluids of control pigs.Analyses of stomach contents showed that sorbic acidand vanillin had equal concentrations regardless ofwhether they were nonprotected or microencapsulated.

Pigs fed PB had no immediate disappearance of sorbicacid and vanillin as observed for NPB fed pigs after thestomach. Conversely, progressively lower concentra-tions of sorbic acid and vanillin were measured in thecranial and caudal jejunum, likely due to the action ofdigestive enzymes. The digesta 8 to 10 h after meal is

Figure 4. Coliforms microbial plate counts in (a) caudaljejunum and (b) cecum. Samples from pigs fed the controldiet (white bars), pigs fed the control diet supplementedwith a microencapsulated blend of organic acids and nat-ural identical flavors (PB, striped bars), and pigs fed thecontrol diet supplementedwith the same blend of organicacids and natural identical flavors with the protectivematrix 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), wherechemical and physical factors can degrade the lipid pro-tective matrix and consequently the metabolism of thereleased substances occurs. The protective matrix pre-vented sorbic acid from being metabolized and allowed15% of the total sorbic acid detected in the stomachcontent to reach the colon.

Piva et al. (1997) studied the absorption in gilts oftryptophan and sulfamethazine in protected and non-protected form and concluded that the protective matrixdelayed absorption without affecting total bioavailabil-ity. Sorbic acid data in the gastrointestinal content ofpigs fed PB suggested a slow release of the acid from

Piva et al.492

the capsule. Progressively lower (P < 0.01) fractions ofthe stomach sorbic acid concentration were recoveredalong the gastrointestinal tract (44, 35, 22, 29, and 15%for cranial jejunum, caudal jejunum, ileum, cecum, andcolon, respectively), whereas in pigs fed NPB, sorbicacid concentration declined immediately after the stom-ach. Only 2% of sorbic acid in cranial and caudal jeju-num could be measured, whereas in the subsequentsegments, sorbic acid was not detectable. The lipid ma-trix also delayed vanillin release as evidenced by 48and 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 associatedwith a lower water solubility (0.25% at 30C, wt/vol;The Merck Index, 2001) compared with vanillin watersolubility (1% at 25C, wt/vol; Vanillin, 2005). Weakacids with pKa > 3 (including sorbic acid with pKa of4.76) are well absorbed (Baggot, 1977), and the ionizedform of the acid can pass through the intestinal mucosa.Sorbic acid was absorbed at a fast degree in the cranialjejunum of NPB-fed pigs, whereas the protection matrixdelayed sorbic acid disappearance and allowed it toreach the subsequent intestinal sections with relevantmicrobial activity. The antimicrobial role of OA is at-tributable to the capacity of their undissociated formto 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 acidsdissociate and depress the cellular enzymatic activityand nutrient transport system (Lueck, 1980).

Sofos et al. (1985) reported a reduction of coliformscount only in the duodenum of broilers fed diets supple-mented with sorbic acid (0.04%). In our study similarresults were observed in jejunum and cecum of PB-fedpigs, where the greater concentration of sorbic acid inPB than NPB could explain the lower plate counts of co-liforms.

Lactic acid bacteria plate counts tended to be reducedin the jejunum (P = 0.08) and cecum (P = 0.006) of NPB-fed pigs and might have accounted for reduced lacticacid concentration and higher pH values in caudal jeju-num of NPB-fed pigs. The same negative pattern wasobserved by Canibe et al. (2005) when using 18 g/kg offormic acid in growing pigs. Such disappearance of lac-tic acid production was not observed when pigs werefed the microencapsulated blend.

We have found no references on synergistic effectsof OA and NIF on swine gastrointestinal microflora.Proposed mechanisms of antibacterial action of NIFinclude their action on the cell membrane (Burt, 2004),the first barrier that OA encounter before entering thebacterial cells. The increase in plasma membrane per-meability due to NIF could help the entrance of OA inthe 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 theactive ingredients, preventing the immediate disap-

pearance of such compounds upon exiting the stomach.The longer permanence along the gastrointestinal tractof active compounds allowed them to act synergisticallyon the intestinal microflora and to reduce coliformcounts.

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