J. Anim Sci. 2007. 85:486-493. doi:10.2527/jas.2006-323
© 2007 American Society of Animal Science
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;
and
CERZOO, S. Bonico, 29100 Piacenza, Italy;
and
Vetagro s.r.l., 42100 Reggio Emilia, Italy; and
and
ISAN, Facoltà di Agraria, Università Cattolica del Sacro Cuore, 29100 Piacenza, Italy
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Abstract
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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
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INTRODUCTION
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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 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.
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.
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MATERIALS AND METHODS
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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 l’Ambiente (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. 253/95-A, issued on 18 August 1995). In addition, the ethical committee of the ISAN (Institute of Food Science and Nutrition, Università Cattolica del Sacro Cuore, Piacenza, Italy) reviewed and approved the experimental protocol.
Seventy-five piglets (77 d of age; Goland x 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.
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 ± 0.96°C) 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 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 –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; 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-µm, RP-C18 column (125 x 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 x 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-µm syringe filter (25 mm, nylon membrane; Millipore Corporation, Bedford, MA). Using an autosampler (AS-1555, Jasco Corp.), samples were injected into a fixed, 30-µL 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-µm, 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 x 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-µm 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 caudal 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 x g) for 10 min at 4°C. Short-chain fatty acid and lactic acid concentrations were analyzed by gas chromatography (Varian 3400, Varian Analytical 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 intestinal contents were centrifuged (6,000 x 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 µL 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 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 gastrointestinal 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 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).
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RESULTS
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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.
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 µmol/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).
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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.
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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 jejunum to colon. No differences (P > 0.27) were reported for ammonia concentration among treatments.
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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).
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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).
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Table 2. pH, ammonia, and molar proportions of short chain fatty acids in gastrointestinal tract samples from pigs fed the experimental diets
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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 µmol/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 content; pooled SEM = 0.195 cecum: 6.78, vs. 7.58, and 7.99 cfu/g of intestinal content; pooled SEM = 0.24; respectively).
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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.
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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.
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DISCUSSION
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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 (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 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).
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 non-protected 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 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 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 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 NPB-fed 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 disappearance 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.
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Footnotes
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1 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. 27–30, 2005. Christchurch, New Zealand.
4 The study was conducted in 2001, and an EU patent (number 1391155B1) was issued in 2004; more patents are pending.
3 Corresponding author: andrea.piva{at}unibo.it
Received for publication May 19, 2006.
Accepted for publication September 23, 2006.
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Abstract
INTRODUCTION
