J. Microbiol. Biotechnol. (2010), 20(4), 803–808doi: 10.4014/jmb.0911.11021First published online 30 January 2010

Effects of Ultra High Molecular Weight Poly-γ-glutamic Acid from Bacillussubtilis (chungkookjang) on Corneal Wound Healing

Bae, Sun-Ryang1, Chung Park

2, Jae-Chul Choi

2, Haryoung Poo

3, Chul-Joong Kim

4, and Moon-Hee Sung

2,5*

1College of Medicine, The Catholic University of Korea, Seoul 150-101, Korea2BioLeaders Corporation, Daejeon 305-500, Korea3Viral Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-806, Korea4College of Veterinary Medicine, Chungnam National University, Daejeon 305-764, Korea5Department of Bio and Nanochemistry, Kookmin University, Seoul 136-702, Korea

Received: November 19, 2009 / Accepted: January 9, 2010

Poly-γ-glutamic acid (γ-PGA) is a natural edible polypeptide

in which glutamate is polymerized via γ-amide linkages.

First, we assessed the eye irritancy potential of γ-PGA in

rabbits. Additionally, we studied the effects of γ-PGA on

corneal wound healing, due to the anti-inflammatory

properties and water retaining abilities of γ-PGA. In this

study, the effects of γ-PGA on corneal wound healing after

an alkali burn were evaluated. Thirty eyes wounded by

alkali burning in 30 white rabbits were divided into three

groups: group A was treated with 0.1% 5,000 kDa γ-PGA

for 2 days; group B was treated with 0.1% hyaluronic

acid; and group C was not treated, as a control. The area

of corneal epithelial defect was examined at 12, 24, 30, 36,

42, and 48 h after corneal alkali wounding to determine

initial wound healing. We found that γ-PGA promoted

corneal wound healing, compared with controls, and showed

similar effects to hyaluronic acid. These results indicate

that γ-PGA stimulates corneal wound healing by an

anti-inflammatory effect and enhancing cell migration

and cell proliferation. γ-PGA is a promising biomaterial

that may be a substitute for hyaluronic acid in corneal

wound healing treatment.

Keywords: Poly-γ-glutamic acid (γ-PGA), corneal wound

healing, hyaluronic acid

Poly-γ-glutamic acid (γ-PGA) is an edible and biodegradable

polymer of the amino acid glutamate (Fig. 1). The

molecular mass of γ-PGA ranges from 10 to 10,000 kDa,

depending on γ-PGA producer [2]. γ-PGA is synthesized

primarily by microorganisms [1, 20, 23], including Bacillus

subtilis, the microorganism involved in the fermentation of

chungkookjang, a traditional food in Korea [16]. γ-PGA is

a biodegradable, biocompatible, and water-soluble material,

and can therefore be used as a thickener, calcium absorption

stimulating agent, humectant, and drug delivery matrix

owing to its unique properties [4]. We have succeeded in

the industrial production of γ-PGA with high molecular

weight and in developing new functional applications in

foods and cosmetics. High molecular weight γ-PGA not

only has excellent moisture retaining abilities, but also

relieves allergies and inflammation by inhibiting hyaluronidase,

a penetrating factor that dissolves the cell walls by

unlinking the hyaluronic acid part of the cell wall or

membrane [30]. Recently, we reported that the oral

administration of high molecular weight γ-PGA showed

TLR4- and dendritic-cell-dependent antitumor effects [18,

19].

*Corresponding authorPhone: +82-2-910-4808; Fax: +82-2-910-8550;E-mail: smoonhee@kookmin.ac.kr

Fig. 1. Structures of poly-γ-glutamic acid (γ-PGA): Na salt form(left) and acid form (right).

804 Bae et al.

Cells such as keratocytes and polymorphonuclear leukocytes

(PMNs) are primarily involved in the scrape injury healing

process after corneal alkali burns [24, 25]. Keratocytes

and PMNs produce matrix metalloproteinases, such as

stromelysin, gelatinase, and collagenase, and different

proteases [14], because these enzymes dissolve collagen

fibers.

As such, protease inhibition is important in scrape injury;

various agents have been examined for the treatment of

scrape injury healing in the cornea [7]. Epithelial growth

factor (EGF), matrix metalloproteinase inhibitors, and

hyaluronic acid have been investigated as therapeutics and

their effects have been reported [3, 8, 28].

Hyaluronic acid has been studied as a therapeutic for

corneal alkali burns and corneal surface disease [12, 27,

32]. Hyaluronic acid is a disaccharide polymer that absorbs

water, and is present in the extracellular matrix where it

plays a role in cell movement, and is thus involved in

tissue expansion [31]. It has a different mechanism of

action from fibronectin or EGF, which heal scrape injury

by promoting epithelial movement [22]. Hyaluronic acid

protects the corneal epithelium and endothelium [26, 29],

stimulates epithelial movement and proliferation [15, 21],

and promotes hemidesmosome formation [9]. Endogenous

hyaluronic acid is produced by corneal epithelial cells,

keratocytes, corneal endothelial cells, iris pigment epithelial

cells, and lens epithelial cells [10, 11, 33].

There are various reports regarding agents with efficacy

after administration into eyes with induced corneal wounds,

of which the best known example is hyaluronic acid.

Several recent studies have indicated that hyaluronic acid

is involved in cell protection, cell movement, growth control,

cell division, and tissue morphogenesis. In the present

study, we investigated the effects of γ-PGA in a rabbit

corneal wound model.

MATERIALS AND METHODS

Preparation of Ultra High Molecular Weight Poly-γ-Glutamic

Acid

A 1% culture solution of Bacillus subtilis (chungkookjang) (KCTC

0697BP) was inoculated into 3 l of preparative Basic Medium of

γ-PGA [GS basic medium with 5% L-glutamic acid:glucose 5%,

(NH4)2SO4 1%, KH2PO4 0.27%, Na2HPO4·12H2O 0.42%, NaCl 0.05%,

MgSO4·7H2O 0.3%, vitamin solution 1 ml/l, pH 6.8], and cultured

with stirring at 150 rpm, an aeration rate of 1 vvm, at 37oC for 72 h.

After culturing, a filter press was used to eliminate fungal contaminants,

yielding a sample solution containing γ-PGA.

After adding 2 N sulfuric acid solution to the above sample

solution containing γ-PGA, the sample was left to stand at 10oC for

12 h to sediment the γ-PGA. After cleaning with a sufficient amount

of reverse osmosis (RO) water, γ-PGA was obtained using a

Nutsche filter. The γ-PGA had a molecular mass of 1-15,000 kDa,

and separate experiments were carried out on subfractions with a

range of molecular weights.

Molecular Mass Determination of Ultra High Molecular Weight

Poly-γ-Glutamic Acid

The molecular mass of γ-PGA was determined by gel permeation

chromatography (GPC). Briefly, PGA solution was diluted with

0.1 M NaNO3, and injected into the GPC equipped with a VicoGel

GMPWXL column (7.8 mm×30 cm; Viscotech, Houston, TX, U.S.A.),

which had been equilibrated with 0.1 M NaNO3, at 40oC and a flow

rate of 0.8 ml/min. γ-PGA was detected with a Viscotek LR25 Laser

Refractometer. Polyacrylamide was used as a standard material for

molecular mass determination. Additionally, viscosity was determined

using a viscometer (Brookfield DV+I Viscometer; Brookfield

Engineering, Middleboro, MA, U.S.A.) at 25oC.

We determined the molecular mass using a chemical modification

method, as previously described [30]. Amino groups modified with

FDNB were used for determination of the number of moles and

estimation of the average molecular size of γ-PGA, because every

molecule of γ-PGA had a single free amino group. Both the total

mole number of glutamate monomer and the number of FDNB-

modified glutamate contained in the hydrolysates of FDNB-modified

γ-PGA were assayed. The average number of glutamate units of γ-

PGA could be determined by dividing the glutamate number by the

FDNB-modified glutamate number.

Atomic force microscopy (AFM; XE-100; PSIA Inc., Santa

Clara, CA, U.S.A.) was used to analyze the ultra high molecular

weight γ-PGA, as described previously [30]. Static light scattering

(SLS) was also used for characterization of macromolecules in

solution. SLS makes use of the time-averaged intensity of scattered

light, from which the weight-average molecular weight and second

virial coefficient can be determined.

Eye Irritation Study in Rabbits

Eye irritation tests were performed to assess the irritancy potential

of the test material in the eyes of New Zealand White rabbits. The

method was designed to meet the OECD Guidelines for the Testing

of Chemicals No. 405 “Acute Eye Irritation/Corrosion” (adopted 24

April 2002) and the Guide to Quasi-Drug and Cosmetic Regulations

in Japan 2006 (February 2006). The ocular route was chosen

because it is a potential route of exposure in humans and has been

the route of choice, based on the method of Draize [13, 17]. Three

animals received 0.1 ml of γ-PGA solution into the conjunctival sac

of the right eye after gently pulling the lower eyelid away from the

eyeball, with the left eye serving as the untreated control. The upper

and lower lids were then gently held together for about 1 s, to

prevent loss of the test substance, and were then released.

The animals were observed for clinical signs once a day and

body weights were measured on day 1 (the day of application) and

day 5 during the observation period, using a balance (HW-60KGL;

A&D, Seoul, Korea). The treated eyes (right eyes) were examined

1, 24, 48, 72, and 96 h after administration of test substance. At

each observation time point, lesions of the cornea, iris, and conjunctivae

were scored separately using a numerical system, based on the

method of Draize [13]. The degree of eye irritation was evaluated

according to the method of Kay and Calandra [17], and the extent

was classified.

Establishment of the Rabbit Corneal Wound Model

An alkali burn caused by placing filter paper (5.5 mm in diameter,

wet with 1 N NaOH) on the cornea is an experimental model for

obstructing voluntary transparency recovery without complications,

EFFECTS OF γ-PGA ON CORNEAL WOUND HEALING 805

such as ulcer, perforation, or neovascularization, and allowing

observation of the alkali burn healing process, similar to that which

occurs clinically. Thus, it is possible to analyze healing of the

epithelium, stroma, and endothelium quantitatively after cornea

alkali burn using this model [5, 6].

General anesthesia was induced in 30 female New Zealand White

Rabbits, 2.5 kg in body mass, by intravenous administration of

pentobarbital, and the ocular surface was anesthetized using 0.5%

proparacaine. After inducing a cornea alkali burn in one eye by

touching the center of the cornea with a circular filter paper (5.5 mm

in diameter, wet with 1 N NaOH) for 60 s, balanced salt solution was

used for irrigation (BSS; Alcon, Fort Worth, TX, U.S.A.) for 120 s.

Treatment of Corneal Wounded Rabbit’s Eyes with γ-PGA

The eyes of 10 of the 20 rabbits in the healing group were treated

with 0.1% hyaluronic acid solution with a molecular mass of

1,200 kDa, whereas the remaining 10 were treated with 0.1% γ-

PGA solution with a molecular mass of 5,000 kDa four times a day.

A further 10 rabbits were treated with phosphate-buffered saline

(PBS) four times a day as a control group.

Assessment of Corneal Wound Healing by γ-PGA Application

The injured corneal epithelium area was stained with 2% fluorescein

solution immediately, 12, 24, 30, 36, 42, and 48 h after inducing

alkali burn injury, and photographs were taken using a Nikon D80

digital camera with a Micro-Nikkor 105 mm 1:4 lens (Nikon, Tokyo,

Japan). In this study, the healing group was compared with the

control group by measuring the area of the epithelial defect using

AutoCAD 2007 (Autodesk, Inc., San Rafael, CA, U.S.A.) and by

determining the healing rate of the epithelium by linear regression

analysis.

Seven days after alkali injury, seven rabbits in each group were

sacrificed to extract the cornea. Six corneas were fixed in 4%

paraformaldehyde, dehydrated in alcohol, and embedded in paraffin.

These specimens were cut into tissue sections and stained with

hematoxylin and eosin. Photographs of the corneal samples were

taken at 400× magnification with an optical microscope to measure

the cell numbers in the cornea. Cell numbers were determined in an

area of 1 mm2 in the central region of the scrape injury and in an

area of the same size around the scraped wound in the total corneal

stroma.

The rest of the cornea was fixed in 4% paraformaldehyde-2.5%

glutaraldehyde in phosphate buffer at 4oC for electron microscopy.

After embedding the tissue in epoxy resin, thermal polymerization

was performed at 60oC for 3 days. The embedded tissue was cut

into semi-thin sections 1 µm thick and stained with 1% toluidine

blue. The lesions were confirmed under an optical microscope, and

the samples were cut into very thin sections on an ultramicrotome

(Leica Microsystems, Wetzlar, Germany). The tissue was double-

stained with uranyl acetate and lead citrate, and then observed by

transmission electron microscopy (JEM-1010; JEOL, Tokyo, Japan).

Statistical Methods

Student’s t-test was used for comparison of the corneal wound

healing rate between the control, hyaluronic acid, and γ-PGA

groups, and the numbers of polymorphonuclear leukocytes (PMNs)

infiltrating the cornea were compared among the groups using

Wilcoxon’s rank sum test. All statistical analyses were performed

using SPSS 12.0. for Windows (SPSS Inc., Chicago, IL, U.S.A.),

and P values <0.05 were deemed to indicate statistical significance.

RESULTS AND DISCUSSION

Molecular Mass Determination of Ultra High Molecular

Weight γ-PGA

The molecular masses of the poly-γ-glutamic acid samples

were 500, 2,000, and 5,000 kDa, as determined by gel

permeation chromatography (GPC) with polyacrylamide

(10-9,000 kDa) as a standard. The viscosity of samples

increased with increasing molecular mass. Atomic force

microscopy (AFM) was also used to determine the

molecular mass. It is possible to calculate the molecular

mass of samples using this method, because it is directly

proportional to the molecular volume. It was confirmed

that the AFM measurement increased with increasing

molecular mass, and that it is possible to calculate the

molecular mass of high molecular weight γ-PGA using this

method on the basis of the number of glutamate monomers

generated by hydrolysis, and quantified with 1-fluoro-2,4-

Table 1. Molecular mass determination of poly-γ-glutamic acid.

GPC [kDa]a Chemical modification [kDa]b Light scattering [kDa]cAFM [nm3](GPC, kDa)d

Viscosity [cP]e

600 - -9.80×10

2

(470)18

2,300 1,600 1,1003.90×104

(1,900)76

5,000 - 2,9001.10×10

5

(6,000)228

- No data.a Determined using GPC (column: Viscotek GMPWXL).b

Determined based on the number of glutamate monomers generated by hydrolysis and with the free amino group quantified using 1-fluoro-2,4-

dinitrobenzene (FDNB).c Determined from the time-averaged intensity of scattered light.d Determined by atomic force microscopy.e Determined by viscometry.

806 Bae et al.

dinitrobenzene (FDNB). The absolute molecular mass of

high molecular weight γ-PGA was successfully determined

by static light scattering (SLS). As shown in Table 1, we

succeeded in determining the molecular mass of three

different types of γ-PGA with various methods, which

were then applied in animal experiments to confirm the

effects of high molecular weight γ-PGA (>5000 kDa).

Eye Irritation Test in Rabbits

This study was performed to evaluate the possible irritation

potential when a single dose of the test substance, γ-PGA

solution, was administered into the conjunctival sac in

three 16-week-old male NZW rabbits. Briefly, 0.1 ml of

γ-PGA solution was administered into the conjunctival sac

of the right eye after gently pulling the lower eyelid away

from the eyeball, with the left eye serving as an untreated

control. The cornea, iris, and conjunctiva were scanned 1,

24, 48, 72, and 96 h after administration. The eye irritation

related to the test substance was calculated according to

Table 2. Irritation scores of poly-γ-glutamic acid in an eyeirritation study.

Test group

No. of animals

Mean total score (MTS)MMTS

1 ha

24 h 48 h 72 h 96 h

γ-PGA 3 0 0 0 0 0 0

MMTS: Maximum mean total score.a Hour after application.

Fig. 2. Effects of ultra high molecular weight poly-γ-glutamic acid on corneal wound healing: (top) clinical signs during the observationperiod; (bottom) wound recovery (%).

EFFECTS OF γ-PGA ON CORNEAL WOUND HEALING 807

the method of Draize [13], and the degree of eye irritation

was classified according to the method of Kay and

Calandra [17]. No eye irritation of the cornea, iris, or

conjunctivae was observed in any of the animals at 1, 24,

48, 72, or 96 h after administration. The mean total score

(MTS) was recorded as 0 and evaluated as “non-irritating.”

There was no abnormal clinical sign, death, or body weight

change attributable to the test substance in any animal

during the observation period. Based on these results, it

was concluded that γ-PGA solution did not induce eye

irritation in rabbits under the conditions used in this study

(Table 2).

Effects of γ-PGA on Corneal Wound Healing in a

Rabbit Model

The early wound defect area was 23.83±0.79 mm2 in the

PBS group, 23.73±1.03 mm2 in the hyaluronic acid group,

and 23.77±0.67 mm2 in the γ-PGA group; there was no

significant difference between the three groups (P=0.957).

The wound healing rate was 0.466±0.059 mm2/h in the

PBS group, 0.490±0.055 mm2/h in the hyaluronic acid

group, and 0.531±0.076 mm2/h in the γ-PGA group. There

was no significant difference between the PBS and hyaluronic

acid groups (P=0.361) or between the hyaluronic acid

group and γ-PGA group (P=0.189). However, the healing

rate of the γ-PGA group was significantly faster than that

of the PBS group (P=0.048; Fig. 1 and 2).

By electron microscopy, we could not observe the

basement membrane after 7 days because there was severe

basement membrane injury in all three groups, so observation

of hemidesmosomes was impossible. During the initial

7 days, the wounds healed without infection or ulcer

formation.

As hyaluronic acid is degraded by hyaluronidase in the

living body, inhibition of hyaluronidase activation, which

leads to increased activation of hyaluronic acid, promotes

corneal wound healing. γ-PGA is known to indirectly

reduce inflammation by inhibiting hyaluronidase activation,

thus hindering hyaluronic acid degradation, although γ-

PGA is not made in the living body, unlike hyaluronic acid

[18]. Additionally, there was no ocular irritation associated

with its administration into the eyes, and in combination

with water, it had superior moisturizing capacity versus

hyaluronic acid as well as a moisturizer activation function

[30].

The results of the present study indicate that corneal

wound healing was significantly faster in the γ-PGA group

than in the control group. However, there was no significant

difference in healing rate between the hyaluronic acid and

control groups, which was similar to the results of previous

studies. It is possible that corneal wound healing can be

promoted directly, rather than indirectly, by inhibition of

hyaluronidase. Thus, γ-PGA is a possible new substitute

for hyaluronic acid for use in corneal wound healing.

Acknowledgments

This work was supported by the Seoul R&BD Program

(10580) and the Research Program 2008 of Kookmin

University in Korea.

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