a novel lactocin-based solid lipid nanoparticles for smart...

460

Tissue Engineering and Regenerative Medicine, Vol. 5, No. 3, pp 460-466 (2008)

｜Original Paper｜

A Novel Lactocin-Based Solid Lipid Nanoparticles for Smart Probiotic Nanofood : Rheological, Mucoadhesive and in vitro Release Properties

Dong-Myung Kim1*, Myungjune Chung2, Gee-Dong Lee3, Yong Kook Shin4 and Woo Kyu Jeon5

1*Nanofood Research Society of Seoul National University, Seoul, 151-742, Korea2Cellbiotech Co., Ltd, Seoul, 157-030, Korea

3Daegu Gyeongbuk Institute for Oriental Medicine Industry, Gyeongsan, 704-230, Korea4Chungbuk Health Industry Center, Chungbuk Technopark, Chungbukdo, 363-883, Korea

5Department of Gastroenterology, Kangbuk Samsung Hospital, Seoul, 110-746, Korea

(Received: July 5th, 2008; Accepted: July 23th, 2008)

Abstract : The aim of the present work was to prepare and evaluate oral mucoadhesive sustained release nanoparti-cles of lactocin(CBT-LP2) these nanoparticles could then be used to improve patient compliance by simplifying theadministration of lactocin, improving the therapeutic effect and reducing the dose related side effects. CBT-LP2 con-taining solid lipid nanoparticles(SLN) were prepared by ultrasonication using soybean lecithin as a stabilizing agent.The results showed that this method was reproducible, easily performed and led to efficient entrapment of CBT-LP2as well as formation of spherical particles ranging from 80-200 nm. In addition, process variables, including the effectof gliadin concentration and the effect of surfactant, were also evaluated with respect to the percent CBT-LP2 entrap-ment and percent yields. The maximum percent CBT-LP2 entrapment and percent yield were approximately 73 and88%, respectively. The sustained release behaviors of the gliadin nanoparticles and the smart probiotic nano-food(SPN) were evaluated in both phosphate buffered saline(pH 7.4) and simulated gastric fluid(pH 1.2) at 37±1oC.Their mucoadhesive properties were determined by in vitro and in vivo methods. The shelf life of prepared SPN wasdetermined by storage at various temperatures in simulated gastric fluid(pH 1.2) with and without added enzymes.

Key words: lactocin(CBT-LP2), smart probiotic nanofood(SPN), solid lipid nanoparticles(SLN), mucoadhesive,release nanoparticles

1. Introduction

Antimicrobial chemotherapeutic agents have been widely

used to control gastrointestinal infections. However, widespread

use of antibiotics is now being discouraged due to problems

including the emergence of drug resistant strains and chronic

toxicity.1 Antibiotics are often responsible for acute diarrhea as

they cause the loss of normal intestinal microbes in addition to

the protection they provide against pathogenic organisms.2 As

alternatives, probiotics such as lactobacilli and bifidobacterium,

or their derivates, have been administered. Indeed, some lactic

acid bacteria(LAB) have health-promoting attributes including

antimicrobial properties,3-6 immunomodulation,7-10 the ability to

alleviate lactose intolerance,11 antitumor characteristics,10,12,13

and hypocholesterolemic effects.14,15 These findings have

heightened the interest of nutrition, food and microbiology

scientists in the production of functional foods.

We isolated L. plantarum CBT-LP2 and L. plantarum CBT-

LP3 from kimchi, Pediococcus pentosaceus CBT-PP1 from

goat’s milk, and L. lactis CBT-P7 from cow’s milk. These

probiotics were cultured in the appropriate broth, condensed by

vacuum evaporation and mixed with equal doses of each

pathogenic strain. In this study, the antimicrobial effects of this

probiotic culture condensate mixture(PCCM, LACTOCIN-

W™) were evaluated using in vitro and in vivo models of food-

borne pathogens.16,17

Lactocin(CBT-LP2) is well absorbed from the GI tract, but its

systemic bioavailability(55%) is relatively low due to first pass

metabolism. It undergoes rapid biodegradation to produce

microbiologically active metabolites. Oral administration

represents the most convenient and common route of nutrition

delivery.18-21 However, the bioavailability of many nutrients

after oral administration is very low for a variety of reasons,

such as a short gastric residence time, instability in the GI tract or*Tel: +82-19-9164-4524; Fax: +82-2-2649-4524e-mail: [email protected] (Dong-Myung Kim)


461

lack of intestinal permeation of nutrients. The gastrointestinal

uptake of poor orally absorbed CBT-LP2 may be improved by

binding them to colloidal solid lipid nanoparticles(CBT-LP2-

SLN) as a smart probiotic nanofood(SPN) made with gliadin.

SPN can protect labile molecules from degradation in the GI

tract and might be able to transport non-absorbable molecules

into the systemic circulation.

This SPN has the ability to both control the release of and

protect the loaded CBT-LP2 against degradation. Moreover, the

small particle size allows them to penetrate the mucus layer and

thus bind to the underlying epithelium and/or adhere directly to

the mucus network.22 The adhesive interactions of the SPN

with the boundary layer may improve CBT-LP2 bioavailability

through a number of different mechanisms. SPN may enhance

the CBT-LP2 absorption rate by reducing the diffusion barriers

between CBT-LP2 and the site of action or absorption.23

Similarly, they may prolong the residence time in the GI tract.

Gliadin SPN has a strong adhesive capacity for the GI

mucosa, which may be due to the gliadin composition. The

neutral amino acids of gliadin can promote hydrogen-bonding

interactions with the mucosa whereas its lipophilic components

can interact with biological tissues through hydrophobic

interactions.24 The bioadhesive capacity of gliadin SPN has

also been evaluated when orally administered to animals.

Gliadin SPN shows great tropism for the upper gastrointestinal

regions, but its presence in other intestinal regions is very low.25

Since DM Kim initially introduced the term ‘nanofood’, which

means nanotechnology for food, and showed that encapsulated

materials can be protected from moisture, heat or other extreme

conditions, thus enhancing their stability, applications for this

nanofood technique have increased.18-21,23,26-33

In the present report, CBT-LP2 loaded CBT-LP2-SLN for

SPN was prepared by ultrasonication, and the sustained release

behavior and the physicochemical characteristics of the SPN

produced by this method were studied. The SPN produced high

bioavailability, a targeting effect, and mucoadhesive properties,

which suggest that oral administration would be possible.

2. Materials and Methods

2.1 MaterialsCBT-LP2 was procured as a gift from Cellbiotech Co., Ltd.,

Korea. Stearic acid(obtained from Sigma, USA) was used as

the lipid material for the CBT-LP2-SLN. Gliadin, purchased

from Sigma-Aldrich(USA), was used as the coating material

for the SPN. Soybean lecithin was obtained from Central Soya

Co., LTD., USA. All other reagents were of analytical grade.

2.2 Preparation of CBT-LP2-SLN and Gliadin SPNCBT-LP2, stearic acid and soybean lecithin were precisely

weighed with an electric balance(BP-121S, Sartorius Ltd.,

Germany) and were then dissolved in absolute ethanol in a

water bath at 70oC. An aqueous phase was prepared by

dissolving glycerin in distilled water. The resultant organic

solution was rapidly injected through an injection needle into

the stirred aqueous phase(80oC). The resulting suspension was

continually stirred at 80oC for 2 h. The CBT-LP2-SLN suspension

was then ultrasonicated for 300 s using the Ultrahomogenizer

(Heidolph Electro, Kelhaim Co., Ltd., Germany).

To obtain a sustained-release gliadin SPN, gliadin and CBT-

LP2-SLN were dissolved in 20 ml of ethanol:water(7:3 v/v)

and this solution was poured into stirred physiological saline

(0.9% NaCl w/v in water) containing 0.5% soybean lecithin as

a stabilizer. The ethanol was eliminated by evaporation under

reduced pressure(Buchi R-140, Switzerland) and the resulting

gliadin SPN was purified by centrifugation at 15,000 rpm for 1

h(Eltech Centrifuge, India). The supernatant was removed and

the pellets were resuspended in water. The suspension was

passed through a 0.45 mm pore size membrane filter and the

filtrate was centrifuged again. Finally, the nanoparticles were

freeze-dried using 5% glucose solution as a cryoprotector. Gliadin

SPN was hardened by the addition of 2 mg glutaraldehyde per

mg gliadin SPN and stirred for 2 h at room temperature before

purification and freeze-drying(Table 1).

2.3 Particle Size and MorphologyThe particle size, size distribution and zeta potential of CBT-

LP2-SLN and gliadin SPN were measured in a Zetasizer 3000

HS(Malvern Instrument Ltd., UK). The surface morphology

and internal structure of gliadin SPN were determined by

scanning electron microscopy(SEM). A thin film of the aqueous

dispersion of nanoparticles was applied on double-stick tape

over an aluminum stub and air dried to obtain a uniform layer of

particles. These particles were coated with gold to a thickness of

about 450 Å using Sputter gold coater(Fig. 1).

2.4 Percentage CBT-LP2 Entrapment and PercentageRecovery

The percentage CBT-LP2 entrapment and the percentage

recovery were determined using the following equations.23 The

appropriate amount of freeze dried gliadin SPN was digested

with a minimum amount of ethanolic solution(water:ethanol,

Dong-Myung Kim, Myungjune Chung, Gee-Dong Lee, Yong Kook Shin and Woo Kyu Jeon

462

7:3 v/v). The digested homogenates were centrifuged at 15,000

rpm for 30 min and the supernatant was analyzed for CBT-LP2

entrapment. The entrapped CBT-LP2 was measured at 760 nm

using the Shimadzu 1601 UV/Vis. spectrophotometer(Table 2).

% CBT-LP2 entrapment =

(Mass of CBT-LP2-SLN÷Mass of CBT

LP2 used in the formulation) × 100

% CBT-LP2-SLN recovery(% yield) =

(Concentration of CBT-LP2-SLN÷Concentration of CBT

LP2-SLN recovered) × 100

2.5 In vitro Drug Release StudiesVarious formulations of gliadin SPN were selected for in

vitro CBT-LP2 release studies.25,30,31 In vitro drug release of

CBT-LP2 from gliadin SPN was estimated by cell membrane

dialysis in two different media, phosphate-buffered saline(PBS,

pH 7.4)(Table 3) and simulated gastric fluid(SGF, pH 1.2)

(Table 4) at 37±1oC for 24 h under sink conditions. The

appropriate volume of gliadin SPN was added to the dialysis

tube with 100 ml of media and continuously stirred with a

magnetic stirrer at 37±1oC. After the appropriate time interval

(1 h), a 1 ml of sample was withdrawn and analyzed for CBT-

LP2 content at 760 nm in a Shimadzu 1601 UV/Vis. spectro-

photometer. An equal volume of fresh media preheated to 37oC

was added to replace the withdrawn sample.

2.6 In vitro Evaluation of Gastric Mucoadhesion ofGliadin SPN

Male Sprague-Dawley rats weighing 200-250 g were fasted

overnight but allowed free access to water. Their stomachs were

excised under anesthesia and then perfused with physiological

saline to remove the stomach contents. The cleaned stomach was

used immediately. A 100 mg gliadin SPN sample was suspended

in SGF(pH 1.2) and added to the cleaned stomach, which was

ligated and then incubated in physiological saline at 37oC for 30

Table 1. Formulation for the preparation of gliadin SPN.

CBT-LP2:gliadin (ratio) Formulation code(s)Volume (ml) of inner

ethanol:water phase (7:3 ratio)Volume of outer phase

(ml, saline)

1:0.5 GNP1 20 100

1:1 GNP2 20 100

1:2 GNP3 20 100

1:3 GNP4 20 100

GNP1 is gliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:0.5), GNP2 is gliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:1), GNP3 is gliadinSPN(CBT-LP2-SLN:gliadin ratio, 1:2) and GNP4 is gliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:3).

Figure 1. SEM photomicrograph of CBT-LP2-SLN (1a) and glia-din SPN (1b).

Table 2. Percentage CBT-LP2 entrapment and percentage recov-ery.

Formulationcode (s)

% CBT-LP2entrapment

% yield

GNP1 43.7±2. 3 63.6±1.7

GNP2 55.6±3.4 65.2±2.3

GNP3 73.1±1.3 88.7±2.8

GNP4 68.6±2.1 76.1±1.9

GNP1 is gliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:0.5), GNP2 isgliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:1), GNP3 is gliadinSPN(CBT-LP2-SLN:gliadin ratio, 1:2) and GNP4 is gliadinSPN(CBT-LP2-SLN:gliadin ratio, 1:3).


463

min. The liquid contents of the stomach were then removed by

injecting air and perfusing the stomach with SGF(pH 1.2) for 30

min at a flow rate of 1 ml per min. The stomach was cut open and

the nanoparticles that remained in the stomach were recovered

with SGF(pH 1.2). The final volume of washing solution was

mixed with 10 ml of ethanolic solution and incubated for 2 h

to allow complete digestion of the gliadin nanoparticles. After

filtration through 0.45 mm filter paper, the absorbance was

measured spectro- photometrically at 230.6 nm(gliadin) and

gastric mucoadhesion was determined as the percentage of

nanoparticles remaining in the stomach after perfusion.

2.7 In Vivo Evaluation of Gastric Mucoadhesion ofGliadin SPN

Sprague-Dawley rats(200-250 g) were fasted for 24 h but

were allowed free access to water. Gliadin SPN(100 mg) in

capsule form was administered to the rats using a gastric sonde.

Four hr after administration, the rats were sacrificed and the

stomach was removed and washed with SGF(pH 1.2) to recover

the remaining CBT-LP2-SLN. The amount of CBT-LP2-SLN

remaining in the stomach was determined as described above for

the in vitro methods. For X-ray studies, gliadin SPN was

prepared with barium sulfate as a contrast agent for the in vivo

studies. Albino rats free of detectable gastrointestinal diseases or

disorders were fasted overnight. Each subject then ingested the

CBT-LP2-SLN formulation together with 2 ml of water. The

intragastric behavior of the formulations was observed by taking

a series of X-ray photographs at suitable time intervals (Fig. 2)

beginning 30 min after dosing.

2.8 Stability studiesThe stability of gliadin SPN was evaluated in PBS(pH 7.4)

Table 3. In vitro CBT-LP2 release profile in PBS (pH 7.4) at37±1°C.

Time(h)

% cumulative CBT-LP2 released

GNP1 GNP2 GNP3 GNP4

1 8.3±1.2 9.1±2.3 5.2±2.1 5.0±2.2

2 12.9±3.2 9.9±2.4 5.7±2.3 6.8±2.3

3 13.4±2.2 11.6±3.0 6.1±1.8 8.1±2.6

4 15.2±1.3 13.9±1.6 6.9±2.3 11.7±1.3

5 18.7±3.0 16.7±1.9 8.3±2.6 14.2±2.9

6 21.2±2.9 17.2±2.4 10.5±1.6 17.6±1.5

7 25.8±2.3 19.1±3.3 19.7±2.4 18.5±2.4

8 26.2±1.8 19.9±2.1 21.8±3.2 20.7±3.6

24 65.7±2.3 58.2±2.6 63.4±1.6 49.7±2.3


Table 4. In vitro CBT-LP2 release profile in SGF (pH 1.2) at37±1°C.

Time(h)

% cumulative CBT-LP2 released

GNP1 GNP2 GNP3 GNP4

1 6.0±2.3 6.3±3.1 3.2±1.4 6.4±0.9

2 9.3±2.6 7.8±2.2 10.5±2.1 8.2±1.2

3 10.9±1.9 11.6±1.3 20.2±1.9 12.9±3.1

4 17.2±1.6 24.9±2.0 29.8±2.3 16.2±2.1

5 24.2±3.2 27.8±3.2 35.1±2.6 19.3±1.6

6 36.4±2.7 38.4±1.2 40.1±1. 623.1±2.1

7 41.2±3.4 43.2±1.7 42.2±2.3 25.7±3.1

8 54.3±1.8 49.8±3.1 53.9±1.9 38.9±2.1

24 73.2±2.7 68.2±1.6 62.3±2.5 52.6±1.9


Figure 2. X-ray photographs reveal the mucoadhesive properties ofthe gliadin SPN formulation. The black portion of the stomachshows the absence of gliadin SPN at time 0(A). The white patchesin the stomach show the presence of gliadin nanoparticles and indi-cate its mucoadhesive properties after 0.5 hour(B), 1.5 hour(C), 2hour(D) and 4.5 hour(E), or show the presence of gliadin SPN andindicate its mucoadhesive properties after 6 hour(F).


464

Table 5. Percentage gastric retention of CBT-LP2-SLN.

Formulationcode (s)

Average% gastric retention

In vitro In vivo

GNP1 74.9±2.7 62.4±2.9

GNP2 76.1±3.4 66.3±1.3

GNP3 81.5±2.3 69.7±2.4

GNP4 70.4±1.6 67.1±3.1


Table 6. Remaining percentage of gliadin content in different media.

Media % remaining of gliadin content

2 h 4 h 8 h 24 h

PBS(pH 7.4) 98.3±1.4 98.1±1.1 97.8±2.6 97.5±2.7

SGF(pH 1.2) 98.4±1.8 97.6±2.3 97.4±1.5 93.2±1.3

SGF(pH 1.2)+enzyme(pepsin) 96.3±0.9 95.8±1.2 92.9±3.1 71.4±2.2

GNP1 is gliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:0.5), GNP2 is gliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:1), GNP3 is gliadinSPN(CBT-LP2-SLN:gliadin ratio, 1:2) and GNP4 is gliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:3). PBS is phosphate buffered saline andSGF is simulated gastric fluid.

Table 7. Effect of storage on residual CBT-LP2 content.

Formulationcode (s)

Initial% residual CBT-LP2 content

10 days 20 days 30 days

0 8o RT 0 8o RT 0 8o RT 0 8o RT

CGNP1 100 100 100 97.9±2.1 98.7±1.9 98.6±2.2 93.9±2.9 94.3±3.6 91.8±1.3 83.4±1.5 89.2±4.3 88.1±1.6

CGNP2 100 100 100 98.9±25 98.5±3.9 95.0±5.6 93.3±5.7 94.8±1.4 91.2±3.4 82.9±2.4 88.6±4.2 89.3±4.2

CGNP3 100 100 100 99.6±5.9 98.1±4.9 97.4±2.1 91.7±5.2 91.4±1.8 90.1±3.2 84.6±5.7 85.8±7.1 88.9±5.7

CGNP4 100 100 100 97.4±5.4 97.7±3.6 95.2±2.9 95.5±2.4 94.3±4.8 92.4±3.4 81.1±3.5 87.2±5.8 90.4±4.8

GNP1 is gliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:0.5), GNP2 is gliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:1), GNP3 is gliadinSPN(CBT-LP2-SLN:gliadin ratio, 1:2) and GNP4 is gliadin SPN(CBT-LP2-SLN:gliadin ratio, 1:3).

and in SGF(pH 1.2) with or without pepsin. Ten milligram

formulations were incubated at 37±1oC with 20 ml of PBS(pH

7.4) and SGF(pH 1.2) with or without pepsin for a period of 2,

4, 8, and 24 h. At the specified time intervals, the suspension

was centrifuged at 15,000 rpm for 1 h, the supernatant was

removed, and the CBT-LP2-SLN was dissolved in ethanolic

solution(7:3 v/v, water:ethanol). The gliadin content was

determined by measuring the absorbance at 230.6 nm against

a blank(Table 6). To determine the shelf life, each formulation

was stored in amber-colored vials with rubber closer at room

temperature, 0oC and 8oC. The percentage residual drug

contents were then determined after 10, 20 and 30 d(Table 7).

3. Results and Discussion

The protein and polysaccharide from the aqueous phase can

be desolvated by pH or temperature change or by addition of

appropriate counter ions or other desolvating agents such as

ethanol and isopropanol. Desolvation deaggregates the protein

and causes the suspension to become colloidal and milky in

appearance. Gliadin SPN was prepared by the desolvation

method for macromolecules in this method, a solvent phase of

gliadin was added to a non-solvent phase, which has low

viscosity and high mixing capacity in all proportions. Addition

of the desolvating agent resulted in the desolvation of the

macromolecules. In a process commonly known as coaservation,

a new phase, the coaservate phase, was treated with a cross-

linking agent(glutaraldehyde) to produce CBT-LP2-SLN

macromolecules.24,25 The desolvation process can be regarded

as the gradual removal of solvent molecules from around

macromolecules in which the molecules “roll up” in the less

friendly environment. The protein macromolecules thus

become discrete, poorly solvated, rolled up molecules, which

produce colloidal particles. Gliadin represents a group of

polymorphic proteins extracted from gluten that are soluble in

ethanolic solution and show remarkably low solubility in water

except at extreme pH.20,23,28,30 Due to its physicochemical

properties, gliadin SPN can be prepared by the desolvation


465

method for macromolecules, using environmentally acceptable

solvents such as ethanol and water. These macromolecules

showed a high capacity for loading CBT-LP2 and were soluble

without further chemical or physical cross-linking treatment. In

the present system, the diffusion of ethanol(good solvent for

gliadin) from the gliadin solution into the aqueous medium

drastically reduced the solubility of gliadin solution, allowing

formation of nanoparticles in the solution. The particle sizes of

gliadin SPN ranged from 80-200 nm with a positive zeta

potential of 22.8 mV. The gliadin SPN was spherical with a

smooth surface(Fig. 1).

The percentage of CBT-LP2 entrapment in the CBT-LP2-

SLN at different gliadin concentrations was 23.7±2.3, 41.6±

3.4, 67.1±1.3, and 63.6±2.1. The release of CBT-LP2 mainly

depended on the gliadin concentration. The burst release of CBT-

LP2 from CBT-LP2-SLN at the initial stage resulted from the

dissolution of CBT-LP2 crystals on the surface of CBT-LP2-

SLN with increasing gliadin concentration, the release rate of

CBT-LP2 from CBT-LP2-SLN decreased drastically. The smaller

CBT-LP2-SLN particles released CBT-LP2 more rapidly than

the larger ones in PBS and SGF at the initial stage since the

smaller particles have a larger surface area.

Mucoadhesion involves various interactive forces between

mucoadhesive materials and the mucus surface, including

electrostatic attraction, hydrogen bonding, Van der Waals

forces, mechanical interpenetration, and entanglement.8,19 The

spectrophotometric method(λmax 230.6 nm) was used to measure

the in vitro mucoadhesive capacity of the formulations

developed. Table 5 shows the percentage of gastric retention of

gliadin SPN in the rat gastrointestinal mucosa. The adhesion

properties of CBT-LP2-SLN increased with increasing concen-

tration of gliadin, and better mucoadhesion was observed for

the GNP4 formulations (3%).

The disulfide bonds of GNP4 gliadin interact with the fucose

and sialic acid groups that are present in the gastric mucosa.

The mucolytic activity of thiols, such as N-acetyl cysteine,

result in disulfide exchange reactions between the mucin

glycoprotein in the mucus and the mucolytic agent. Due to the

exchange reaction, intra- and intermolecular disulfide bridges

within the glycoprotein structure are cleaved, leading to

breakdown of the mucus. As the mucolytic agent is covalently

bound to the mucin glycoprotein in mucus, other thiol-bearing

compounds, in particular polymers with thiols, should also

covalently bind to the mucus.20-25 Further, the in vivo bioadhesive

behavior was confirmed by taking X-ray photographs of the

CBT-LP2-SLN in the stomachs of rats(Figs. 2-7). To sharpen

the radio-graphical contrast in vivo, sufficient CBT-LP2 must

be enclosed in the CBT-LP2-SLN. To meet this opposing

requirement, CBT-LP2-SLN was prepared with a particle

density of 1.099/cm2 sub.

In the early stages within 30 min after dosing, the CBT-LP2-

SLN adhered to the stomach(Fig. 3). After 6 h, some CBT-LP2-

SLN remained since the bioadhesive system delayed the arrival

to the pylorus. The prolonged residence time of CBT-LP2-SLN in

the stomach might be explained by its mucoadhesive properties

as well as by a random emptying effect caused by the presence

of a multiple unit system. Due to their spreading over the

antrum, as the subunits approach the pylorus, they might pass

through the stomach individually and release CBT-LP2

concurrently in the gut, leading to decreased variability of CBT-

LP2 action among patients compared with that occurring in the

single unit dosage form.23 The results of this study suggest that

gliadin SPN could serve as a novel delivery device to improve

the bioavailability of CBT-LP2 and possibly other compounds

that are used to produce local and specific effects in the stomach

and are observed in the upper region of the stomach.30-33

In this study, we showed that lactocin resided in the stomach

for a longer period of time when it was administered in the form

of the mucoadhesive CBT-LP2-SLN than when administered

as a suspension or in a conventional system. The CBT-LP2

containing gliadin SPN also provided greater anti-bacterial

activity than the plain CBT-LP2 formulations.16,17,30-33

Acknowledgements: This study was supported by a

grant(ATC-10026108) from the Ministry of Knowledge

Economy(MKE) and a grant(07052 KFDA 137) from the

Korea Food & Drug Administration.

References

1. S Dundas, J Murphy, RL Soutar, et al., WTA Todd, Effec-tiveness of therapeutic plasma exchange in the 1996 LanarkshireEscherichia coli O157:H7 outbreak, Lancet, 354, 1327 (1999).

2. W Van der, Horstra DH, Wiegersma N, Effect of β-lactamantibiotics on the resistance of the digestive tract to colonization,J Infect Dis, 146, 417 (1982).

3. HS Gill, KJ Rutherfurd, Immune enhancement conferred byoral delivery of Lactobacillus rhamnosus HN001 in differentmilk based substrates, J Dairy Res, 68, 611 (2001).

4. SH Kim, SJ Yang, HC Koo, et al., Inhibitory activity ofBifidobacteria longum HY8001 against vero cytotoxin ofEscherichia coli O157:H7, J Food Protec, 64, 1667 (2001).

5. G Perdigon, MEN Marcias de, S Alvarez, et al., Prevention ofgastrointestinal infection using immunobiological methodswith milk fermented with Lactobacillus casei and Lactobacillus


466

acidophilus, J Dairy Res, 57, 255 (1990). 6. Q Shu, KJ Rutherfurd, SG Fenwick, et al., Dietary Bifidobac-

terium lactis (HN019) enhances resistance to oral Salmonellatyphimurium infection in mice, Microbiol Immunol, 44, 213(2000).

7. HS Gill, KJ Rutherfurd, J Prasad, et al., Enhancement of naturaland acquired immunity by Lactobacillus rhamnosus (HN001),Lactobacillus acidophilus (HN017) and Bifidobacterium lactis(HN019), Brit J Nutr, 83, 167 (2000).

8. HS Gill, KJ Rutherfurd, Immune enhancement conferred byoral delivery of Lactobacillus rhamnosus HN001 in differentmilk based substrates, J Dairy Res, 68, 611 (2001).

9. P Marteau, JC Rambaud, Potential of using lactic acid bacteriafor therapy and immunomodulation in man, FEMS MicrobiolRev, 12, 207 (1993).

10. C Matar, JC Valdez, M Medina, et al., Immunomodulatingeffects of milks fermented by Lactobacillus helveticus and itsnon-proteolytic variant, J Dairy Res, 68, 601 (2001).

11. R Fuller, Probiotics in man and animals, J Appl Bacteriol, 66,365 (1991).

12. J Kato, K Endo, S Rybka, Effects of oral administration ofLactobacillus casei on antitumor responses induced by tumourresection in mice, Int J Immunopharmacol, 16, 29 (1997).

13. JJ Rafter, The role of lactic acid bacteria in colon cancer prevention,Scand J Gastroenterol, 30, 497 (1995).

14. BZ De Rodas, SE Gilliland, CV Maxwell, Hypocholesterolemicaction of Lactobacillus acidophilus ATCC 43121 and calciumin swine with hypercholesterolemia induced by diet, J DairySci, 79, 2121 (1996).

15. M Fukushima, M Nakano, Effects of a mixture of organisms,Lactobacillus acidophilus or Streptococcus faecalis on chole-sterol metabolism in rats fed on a fat- and cholesterol-enricheddiet, Brit J Nutr, 76, 857 (1996).

16. D Kim, New probiotic application using probiotic culture conden-sate mixture (LACTOCIN-W) and tyndallized lactic acid bacteria(PROLAC-T), Agrofood, 12, 10 (2004).

17. D Kim, MJ Chung, A probiotics condensate mixture novel anti-microbial application, Nutrafoods, 3, 11 (2004).

18. DM Kim, Using freezing and drying techniques of emulsions forthe nanoencapsulation of fish oil to improve oxidation stability,Kor Nanoresearch Soc 13, 17 (2001).

19. DM Kim, JS Park, A continuous oxidation process for the regio-

selective oxidation of primary alcohol groups in chitin and chitosan,Korean Patent 2001-10.2002.0007851.

20. DM Kim, Preparation and characterization of biodegradable orenteric-coated nanospheres containing the protease inhibitorcamostat, Biomaterials, 28, 8 (2001).

21. DM Kim, HS Kwak, Nanofood materials and approachabledevelopment of nanofunctional dairy products, Kor Dairy FoodEngin, 1, 1 (2004).

22. C Durrer, JM Irache, F Puisieux, et al., Mucoadhesion of latexesII. Adsorption isotherms and desorption studies, Pharm Res, 11,680 (1994).

23. DM Kim, GD Lee, YK Shin., Oral mucoadhesive sustainedrelease nanoparticle coated probiotic nanofood, J Food Sci Nutr,11, 348 (2007).

24. MA Arangoa, G Ponchel, AM Orecchioni, et al., Bioadhesivepotential of gliadinsystems, Eur J Pharm Sci, 11, 333 (2000).

25. MA Arangoa, MA Companero, MJ Renedo, et al., Gliadinnanoparticles as carriers for the oral administration of lipophilicdrugs, Pharm Res, 11, 1521 (2001).

26. DM Kim, HS Kwak, Development of functional nanofood andits future, Kor Dairy Food Engin, 2, 1 (2004).

27. DM Kim, EJ Cha, Clinical analysis of lutein in taking HPMC-lutein nanoparticle (nanofood) and in taking raw or cookedvegetables, Adv Drug Deliv Rev, 47, 221 (2005).

28. DM Kim, GS Cho, Nanofood and its materials as nutrientdelivery system (NDS), Agric Chem Biotechnol, 49, 39 (2006).

29. DM Kim, GD Lee, Introduction to the technology, applications,products, markets, R&D, and perspectives of nanofoods in thefood industry, J Food Sci Nutr, 11, 348 (2006).

30. DM Kim, G.D Lee, YK Shin, Oral mucoadhesive sustainedrelease nanoparticle coated probiotic nanofood, Tissue EnginRegen Med, 4(4), 543 (2007).

31. DM Kim, JE Kim, GS Lee, MJ Chung, Method of preparingtriple-coated lactic acid bacteria, triple-coated lactic acid bacteriaprepared thereby and articles comprising the same, PCT/KR2008/004199 (2008).

32. DM Kim, H Baek, JH Kim, MJ Chung, Anticancer compositioncomprising a culture fluid of Lactobacillus casei as an effectivecomponent. PCT/KR2008/004375 (2008).

33. DM Kim, Target controlled nanofood using by smart probioticsolid lipid nanoparticles(SLN), Kor Dairy Food Engin, 15, 31(2007).

a novel lactocin-based solid lipid nanoparticles for smart...

Documents