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Lipid microencapsulation allows slow release of organic acids and natural identical flavors along the swine

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

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Lipid microencapsulation allows slow release of organic acids and natural identical flavors along the swine intestine 1,2

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 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

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 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

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 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, or both, that they undergo upon entering the duode- num. This could be overcome by microencapsulating the active compounds in a matrix that could dissolve as it passes along the intestine.

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Slow release of microencapsulated additives

487

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

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 pro- teins 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 l’Ambiente (CERZOO), where the study was conducted from 10 until 25 Sep- tember 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, Universita` Cattolica del Sacro Cuore, Pia- cenza, 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 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 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 supple- mented 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 Vet- agro S.r.l. (Reggio Emilia, Italy; Production authoriza- tion α 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.

Item

Experimental diet 1

CTRL

PB

NPB

%, as-fed basis

Ingredient Corn

Chemical composition, % of DM

25.4

25.4

25.4

Barley

10.5

10.5

10.5

Flaked barley

20.7

20.7

20.7

Soybean oil

3.5

3.5

3.5

Sweet dried whey

5.0

5.0

5.0

Wheat bran

10.2

9.8

9.8

Soybean meal (44%)

17.0

17.0

17.0

Potato protein 2

3.5

3.5

3.5

Limestone CaCO 3

0.4

0.4

0.4

Calcium sulphate (CaSO 4 )

0.6

0.6

0.6

Monocalcium phosphate (Ca(H 2 PO 4 ) 2 )

1.6

1.6

1.6

Sodium chloride (NaCl)

0.30

0.30

0.30

DL-Methionine

0.16

0.16

0.16

L-Lysine HCl

0.4

0.4

0.4

L-Threonine

0.16

0.16

0.16

L-Tryptophan

0.04

0.04

0.04

Vitamin/mineral premix 3

0.5

0.5

0.5

Micro-encapsulated blend

0.4

Nonmicroencapsulated blend

0.4

DM, %

90.86

90.86

90.96

CP

19.30

19.08

19.16

Ether extract

6.74

6.76

7.02

Crude fiber

5.01

4.96

4.97

Ash

6.84

6.61

6.62

Starch

39.61

37.99

37.95

Nutritive value, 4 MJ/kg of DM DE

16.07

16.07

16.07

NE

11.51

11.51

11.51

1 Control 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 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; vita- min D 3 , 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; CoCO 3 3Co(OH) 2H 2 O,

480

g; FeCO 3 , 300 mg; Ca(IO 3 ) 2 , 1.8 mg; MnO 2 , 48 mg; CuSO 4 5H 2 O,

120

mg; Na 2 SeO 3 , 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 ± 0.96°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. Bon- ico, Piacenza, Italy), by stunning with a captive bolt followed by complete bleeding. Immediately after death, the stomach, cranial jeju- num, caudal jejunum, ileum, cecum, and colon (at the

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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.

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).

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-

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 (H 2 with approximately 4 to 10% CO 2 ; 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.

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Slow release of microencapsulated additives

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Slow release of microencapsulated additives 489 Figure 1. Sorbic acid concentrations in gastrointestinal tracts of pigs

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,b In 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

lin was not detected ( < 0.66 nmol/g) from the cranial Figure 2. Vanillin concentrations in

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-

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490

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

Piva et al.

 

Total

short

chain

Gastrointestinal

 

Acetic

Propionic

Iso-butyric

n-butyric

Iso-valeric

Valeric

Lactic

fatty

tract

Treatment 1

pH

Ammonia

acid

acid

acid

acid

acid

acid

acid

acids 2

 

mol/g of DM

 
 

Stomach 3

Control

3.61

20.70

0.68

0.01

0.08

b

0.01

ND 4

ND

5.23

0.78

PB

3.48

16.33

0.65

0.00

0.03

a

0.00

ND

ND

2.63

0.69

NPB

3.66

15.97

0.84

0.00

0.01

a

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

0.14

b

ND

ND

ND

7.33

b

1.37

PB

5.15

36.04

2.01

ND

0.06

a

0.02

ND

ND

4.88

ab

2.00

NPB

5.34

35.90

1.72

0.01

0.01

a

0.01

ND

ND

3.42

a

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

5.31

a

35.59

1.74

ND

0.10

ND

ND

ND

12.58

b

1.76

PB

5.31

a

41.81

2.19

0.00

0.08

ND

ND

ND

15.04

b

2.27

NPB

6.10

b

32.52

3.36

0.01

0.05

ND

ND

ND

3.07

a

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

1.12

a

0.01

0.04

0.01

a

ND

ND

7.20

1.24

PB

5.09

50.96

0.98

a

0.01

0.04

0.01

a

ND

ND

8.36

0.98

NPB

6.07

54.14

6.31

b

0.04

0.05

0.26

b

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

0.35

a

0.64

b

17.12

PB

5.47

28.08

12.39

7.06

0.05

3.66

0.03

0.65

b

0.36

ab

23.85

NPB

5.27

21.69

13.87

6.83

0.02

3.55

0.03

0.25

a

0.15

a

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

24.80

b

13.72

b

0.21

b

5.62

0.15

b

1.01

c

0.22

b

45.71

b

PB

5.63

3.32

13.61

a

7.32

a

0.09

a

3.90

0.05

a

0.61

b

0.07

ab

25.58

ab

NPB

5.51

3.95

12.41

a

6.08

a

0.08

a

3.55

0.05

a

0.26

a

0.05

a

20.07

a

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

ac Within a column, means without a common superscript letter differ ( P < 0.05). 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 (Pen˜ alver 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).

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Slow release of microencapsulated additives 491 Figure 3. Lactic acid bacteria (LAB) microbial plate counts in
Slow release of microencapsulated additives 491 Figure 3. Lactic acid bacteria (LAB) microbial plate counts in

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,b In 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 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,b In 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

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

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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-

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.

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

Slow release of microencapsulated additives

493

Pen˜ alver, 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.

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.

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