A Novel Biodegradable Green Poly(L-Aspartic Acid-Citric Acid) Copolymer for Antimicrobial Applications

8/9/2019 A Novel Biodegradable Green Poly(L-Aspartic Acid-Citric Acid) Copolymer for Antimicrobial Applications

http://slidepdf.com/reader/full/a-novel-biodegradable-green-polyl-aspartic-acid-citric-acid-copolymer-for 1/6

O R I G I N A L P A P E R

A Novel Biodegradable Green Poly(L-Aspartic Acid-Citric Acid)Copolymer for Antimicrobial Applications

N. Mithil Kumar • K. Varaprasad •

K. Madhusudana Rao • A. Suresh Babu •

M. Srinivasulu • S. Venkata Naidu

Published online: 24 August 2011

Springer Science+Business Media, LLC 2011

Abstract Poly (L-aspartic acid-citric acid) green copoly-

mers were developed using thermal polymerization of aspartic acid (ASP) and citric acid (CA) followed by direct

bulk melt condensation technique. Antibacterial properties

of copolymer of aspartic acid based were investigated as a

function of citric acid content. This study is focused on the

microorganism inhibition performance of aspartic acid

based copolymers. Results showed that inhibition proper-

ties increase with increasing citric acid content. Charac-

terization of obtained copolymers was carried out with the

help of infrared absorption spectra (FTIR), x-ray diffrac-

tion (XRD), differential scanning calorimetry (DSC) and

thermo gravimetric analysis (TGA). The antibacterial

activity of copolymers against bacteria like E-coli, Bacillusand pseudomonas was investigated. The copolymers

showed excellent antimicrobial activities against three

types of microorganisms. Overall studies indicated that the

above copolymers possess a broad wound dressing activity

against above three types of bacteria and may be useful as

antibacterial agents.

Keywords Antibacterial activity Green copolymers

and melt condensation technique

Introduction

Green polymers are competing with synthetic non

degradable polymers today, due to increasing oil prices and

widely used in tissue engineering, drug delivery [1],

medical and pharmaceutical industries in recent years due

to their excellent biodegradability, non-toxic and capable

of chemical modifications [2]. These versatile multi-func-

tional polymers possess much useful biological function-

ality due to conformational states, hydrogen bonding and

polyanionic nature [3]. These green polymers are produced

without any environmental pollution and can be degraded

completely by microbes and epiphytes after being used

[4–7], which are suitable for various industrial medical

applications [8] including use as a material component in

dialysis membranes, drug delivery and agricultural appli-

cations to replace many non-biodegradable polymers in

use. The recent break through is made by products based on

the polyaspartates. Poly asparitic acid is one of the bio-

degradable water soluble polymer. This antiscalant does

not attack the colored metals, they are more environmen-

tally acceptable than polymers/polyphosphonates [9].

N. Mithil Kumar (&) S. Venkata Naidu (&)

Synthetic Polymer Laboratory-II, Department of Polymer

Science & Technology, Sri Krishnadevaraya University,

Anantapur 515055, Andhra Pradesh, India

e-mail: [email protected]

S. Venkata Naidu

e-mail: [email protected]

K. Varaprasad (&)

Synthetic Polymer Laboratory-I, Department of Polymer Science

& Technology, Sri Krishnadevaraya University,

Anantapur 515055, Andhra Pradesh, Indiae-mail: [email protected]

K. Madhusudana Rao

Ion Exchange (IND) Ltd, Hyderabad, Andhra Pradesh, India

A. Suresh Babu

Department of Physics, Rayalaseema University,

Karnool 518002, Andhra Pradesh, India

M. Srinivasulu

Department of Microbiology, Sri Krishnadevaraya University,

Anantapur 515055, Andhra Pradesh, India

1 3

J Polym Environ (2012) 20:17–22

DOI 10.1007/s10924-011-0335-z



During the last few decades, a number of investigations

were conducted to study the influence of biodegradable

green copolymer on water treatment [10] as well as various

bacteria [11]. Their high purity offers good processing

performance, optimal physical, chemical and mechanical

properties [12]. Further they are used in several areas such

as medical devices, health care products, water purification

systems, hospitals, food packaging and food storage etc.Consequently, biocidal polymers have received much

attention in recent years [13]. In the present study the

author attempted to synthesize such biocidal and biode-

gradable green copolymer by the addition of aliphatic tri-

carboxylic acid monomer (CA) to aspartic acid transforms

the polymerization from a solid state to a melt state reac-

tion between 165 and 260 C temperature.

Polyaspartic acid, a non-toxic and biodegradable anio-

nic polypeptide, is widely used as an effective dispersant,

inhibitor for both scale deposit and metallic corrosion and

also as dispersants in water treatment [14], paint and paper

processing [15] without disturbing the taste of water andthe quality and purity of water. As they are water soluble

polymers, they can be used as detergent builders, floccu-

lants, thickeners, emulsifiers, baby diapers and perme-

ability modifiers in oil field operations [16]. It is readily

water soluble in aqueous alkali solution [14]. These water

soluble copolymers were synthesized by using the citric

acid (CA) and aspartic acid (ASP) monomers, which are

used as descalling agents in water treatment system,

laundry detergents for fabrics, dishwashing detergents for

glassware [17], dental tartar and plaque formation inhib-

itor [18].

Recently, citric acid based biodegradable polymers were

developed and studied their chemical, physical, and surface

chemical properties [19]. They are used in a number of

applications, such as tissue engineering applications. The

material properties are influenced by citric acid monomer

ratio [20]. The copolymers are useful for antibacterial

agents [21]. Poly carboxylic acid (PCA), 1, 2, 3, 4-butane

tetracarboxylic acid (BTCA), and citric acid (CA) are the

most favorable compounds to inhibit the microorganisms.

Although BTCA is very effective, it is very costly.

Therefore CA is more feasible antimicrobial agent for

cotton fabric, textile and wound dressing agents [11].

Keeping in view of the above, CA is selected, which has

multifunctional monomer to provide valuable pendant

functionality [22]. In connection with this we have devel-

oped biodegradable water soluble green copolymer (poly

(ASP-CA)) as a new antimicrobial/wound dressing agent.

We used CA in combination with ASP to improve anti-

bacterial properties [21].

Experimental Section

Materials

L-Aspartic acid (ASP) (Product No 014881 assay 99%) was

purchased from SRL, Mumbai, India. Citric acid (CA)

(Product No 87984, Minimum assay 99%) and Calcium

chloride (Product No 20070) were purchased from S.D.

Fine chemicals, Mumbai, India, without further purifica-

tion. Nutrient agar was obtained from Himedia Chemicals

(Mumbai, India). The Department of Botany (Sri Krish-nadevaraya University, Anantapur, India) has provided

bacterial cultures of the organisms.

Synthesis of Poly (ASP-CA) Copolymer

Copolymers were synthesized using the following proce-

dure. Different molar ratios of both ASP and CA were mixed

together to form the copolymer as shown in Table 1.The

reactants (ASP and CA) were placed in a ceramic bowl and

the mixture of monomers was first heated up to 165 C for

16 h in a oven (Baheti Enterprises, Hyderabad, India), then

heated to 200 C for about 30 min till the reaction mass

melted partially. Further the temperature was increased to

reach to 250 C and maintained the reaction for 69 h con-

tinuously, until the mass attained the dark brownish colour.

Finally, temperature was increased to 260 C for 5 h to get

complete conversion of polymer which was conformed from

DSC. The synthesized copolymer was washed with dimethyl

formamide (DMF) and then with distilled water for several

times (10 times) at ambient temperature till polymer was

free from impurities. Theyield of the product is 80–95%. The

same procedure was followed for the synthesis of

poly(aspartic acid) homopolymer.

Table 1 Feed composition of biodegradable water soluble polymers and antibacterial inhibition zones

Polymer code Feed composition of homo and copolymers Antibacterial inhibition zone

L-Aspartic acid (mM) Citric acid (mM) E-coli (cm) Bacillus (cm) Pseudomonas (cm)

PASP 75.13 _ _ _ _

P(ASP-CA1) 75.13 13.01 0.6 0.6 0.7

P(ASP-CA2) 75.13 39.03 0.9 1.0 0.8

18 J Polym Environ (2012) 20:17–22

1 3



Characterization of Prepared Copolymer

FTIR Spectroscopy

FTIR spectra were recorded on a MB3000 Model, the

resolution being 16 cm-1, using KBr pellets (1 mg samples

as 400 mg KBr). FTIR spectrophotometer was used to

identify the poly (ASP-CA) copolymer formation. Torecord the FTIR spectra of copolymer, the samples were

completely dried in an oven (Baheti Enterprises, Hydera-

bad, India) at 90 C for 45 min. These samples were read

between 500 and 4,000 cm_1 on a MB3000 Model, ABB

company (Horizon software) FTIR spectrometer (Quebec,

Canada) using the KBr disk method.

X-ray Diffraction

The X-ray diffraction (XRD) method was used to identify

the formation of copolymer network. These measurements

were carried out for dried and finely powdered samples ona Rikagu diffractometer (Cu radiation, k 0.1,546 nm)

running at 40 kV and 40 mA.

Differential Scanning Calorimetry (DSC)

Differential scanning calorimetry of poly(ASP) and poly

(ASP-CA) were studied by using a SDT Q 600 DSC

instrument (T.A. Instruments-water LLC, Newcastle, DE

19720, USA) under the following operational conditions:

sample weight 10 mg, heating rate 20 C/min, and nitrogen

flow (100 mL/min).

Thermogravimetric Analysis (TGA)

The thermal analysis of poly (ASP) and poly (ASP-CA)

were evaluated on a SDT Q 600 TGA instrument (T.A.

Instruments-water LLC, Newcastle, DE 19720, USA) at a

heating rate of 20 C/min under a constant nitrogen flow

(100 mL/min). The samples were run from 30 C to

700 C.

Antibacterial Activity

Nutrient agar medium was prepared by mixing peptone

(5.0 g), beef extract (3.0 g), and sodium chloride (NaCl)

(5.0 g) in 1,000 mL distilled water and the pH was

adjusted to 7.0. Finally, agar (15.0 g) was added to the

solution. The agar medium was sterilized in an autoclave at

a pressure of 15 lbs for 30 min at 150 C. This medium

was transferred into sterilized Petri dishes in a laminar air

flow chamber. After solidification of the media, Esche-

richia coli, Bacillus and Pseudomonas culture (50 lL) was

spread on the solid surface of the media. One drop of

polymer solution (20 mg/10 mL distilled water with alkali

treatment) was added into the inoculated Petri dish using

50 lL tip and incubated for 2 days at 37 C in the incu-

bation chamber.

Results and Discussions

A current need for biodegradable green materials for use

as scaffold for tissue growth, drug delivery and other

biomedical applications has also fostered interest in

poly(aspartic acid) and related polymers. Poly (aspartic

acid) has potential biomedical applications including use as

a material components in drug delivery, artificial skin and

orthopedic implants [8]. In view of the above, the study of

these polymers for an antimicrobial application has been

motivated. Synthesis of copolymer is by melt polycon-

densation, a well-defined process of melting functional

amines and carboxylic acids for creating polymer net-

works. Tri-functional citric acid can react with amines toform amides without any catalysis [23]. The non-solubility

in water confirmed the formations of copolymer. But it can

be dissolved in the presence of aqueous alkali solution as

alkali groups break the amide bonds [23]. These are all eco

friendly with environmental systems and it can be degraded

completely by microbes and epiphytes after being used

[24]. The resultant copolymers were characterized by fol-

lowing analysis.

The copolymers were characterized by Fourier Trans-

form infrared spectroscopy, X-ray diffraction, differential

scanning calorimetry and thermo gravimetric analysis. The

synthesized copolymer, Pure ASP and CT were also char-

acterized by FTIR analysis. In the FTIR spectrum (Fig. 1),

The Peaks at 1,704 cm-1 and 3,005 cm-1 are observed

which corresponding to carbonyl and –OH (intermolecular

hydrogen bond) stretching [25] respectively. The spectra of

Fig. 1 FTIR spectra of (a) aspartic acid (ASP), (b) citric acid (CA)

and (c) poly (aspartic acid-citric acid) P(ASP-CA)

J Polym Environ (2012) 20:17–22 19

1 3



CA have shown a broad peak around 3,389 cm-1 which

indicates OH stretching. In the spectra two prominent peaks

at 1,714 and 1,390 cm-1 are observed from the asymmet-

rical and symmetrical stretching of COO– groups respec-

tively [26]. Similar bands are observed in the case of

copolymer spectra, but the bands appear at shifted positions

attributed to the copolymeric formation. The FTIR spec-

troscopy has proved to be a useful tool for studies of copolymer network (interactions of polymers) [25, 27]. In

the present investigation, the –OH and –COO– stretching

occurs at 3,490 and 1,726 cm-1 respectively. The increas-

ing in band intensity confirms the copolymer network

formation.

The XRD pattern gives the supporting information of

the formation of copolymer network. The XRD patterns of

the homopolymer and copolymer are presented in Fig. 2.

From the pattern of homopolymer it can be observed that

there are two broad peaks at16.26o and 28.75o corre-

sponding to the homopolymer peak, while in the XRD

pattern of copolymer there are two peaks at 17.28o and29.98o respectively. However, XRD pattern of copolymer

shows higher intensity peaks than homopolymer. This is

due to incorporation of citric acid which has flexible

(amorphous) nature than homopolymer.

Thermal properties of copolymer not only provide their

physical characteristics but also give information about the

components present in the copolymers. DSC of homo-

polymer, copolymers have shown 362.62, 357.31 and

337.37 C (glass transition temperature) respectively

which is shown in Fig. 3a. As the CA content of copolymer

is increased, reduction in glass transition temperature is

observed. This is due to the effect of incorporation of CA

units (aliphatic) which provide more flexibility and chain

length to the copolymers. Similar observation was reported

by N. Mithil Kumar et al. [27]. Comparing the DSC curves

obtained for the mixture containing same proportion of

ASP and CT to that of DSC curve obtained for P(ASP-

CT2) copolymer with the same monomer ratio. It is

observed that the peak obtained for copolymer is more thanASP-CT mixture with out polymerization.

TGA analysis of the samples exhibited final degradation

86.27, 96.26, and 98.88% at 615 C (respectively) shown

in Fig. 4. The above studies indicate that CA added

copolymers have reduced glass transition temperature, due

Fig. 2 X-rd spectra of poly(aspartic acid)(PASP) and) poly(aspartic

acid-citric acid) P(ASP-CA)

Fig. 3 (a) DSC spectras of homopolymer(PASP) and copolymers

[P(ASP-CA1), P(ASP-CA2)] (b) physical mixture of copolymer

[P(ASP-CA2)]

Fig. 4 TGA spectras of homopolymer(PASP) and copolymers

[P(ASP-CA1), P(ASP-CA2)]

20 J Polym Environ (2012) 20:17–22

1 3



to lower thermal stability of CA amounts with respect to

aspartic acid homo-polymer. With homopolymer (aspartic

acid as monomer), copolymer with less monomer ratio of

CA and copolymer with higher monomer ratio of CA have

also exhibited similar trends in both DSC and TGA studies.

In addition, more weight loss is also found due to the

presence of CA in copolymers.

Antibacterial Activity

In the present study, the existence of CA chains throughout

the copolymer has not only regulated the copolymer net-

works but also influenced in controlling the growth of

microorganisms. The main criteria in selecting CA as

comonomer in the preparation of copolymer is due to its

dissociation, functionality, solubility, location and number

of crosslinks and also due to its lower acidity nature that

enhances its use in antibacterial application [28].

The main aim of this study was to develop a new anti-

microbial/wound dressing agent. These antimicrobialagents are non-toxic materials which can be used system-

ically as an alterative to conventional systemic antibiotic,

antiviral and antifungal therapies that do not incur the

development of resistance by the target pathogens. In

ancient times honey pastes, plant fibers, and animal fats

were used as wound dressing materials. Nowadays, new

biopolymers have been in use as a wound dressing mate-

rials to achieve the highest rate of healing and the best

aesthetic repair of the wound due to having extraordinary

properties which enhance the healing process of a wound

[29]. To improve further their applicability in antimicro-

bial/wound/burn dressing, the present work involves in

developing citric acid green copolymer materials contain-

ing aspartic acid (amino acid) and citric acid (metabolic

and antimicrobial).This combinational approach enhances

their antibacterial efficacy and opens a new era in antimi-

crobial materials. For this purpose, green poly(ASP-

CA)copolymers are developed following procedure, men-

tioned in experimental method. This kind P(ASP-CA2) of

copolymers are biodegradable and water-soluble, making

them useful in particular aqueous compositions, such as

antimicrobial formulations.

These antimicrobial formulations may enhance the

efficiency and selectivity of currently used antimicrobial

agents, while decreasing associated environmental hazardsbecause antimicrobial polymers are generally nonvolatile

and biodegradable. This important property makes the

material to be used in areas of medicine as a means to fight

infection, in the food industry to prevent bacterial con-

tamination, and in water sanitation to inhibit the growth of

microorganisms in drinking water [30].

The antimicrobial activity of different molar weight

ratios of CA copolymers were examined against gram

negative and gram-positive bacteria. Generally, all non

water soluble copolymers showed insignificant activity

against the studied microorganisms. Meanwhile, all soluble

copolymer (in presence of alkali treatment) proved effec-tive against the tested microorganisms, but growth inhibi-

tory effects varied from one another [31]. Keeping this in

view, new biodegradable water soluble copolymers (in

presence of alkali treatment) were developed for anti

bacterial activity against Bacillus, E -coli and Pseudomonas

as shown in Fig. 5a, b and c respectively. The experiments

were performed for antibacterial activity (a) with homo-

polymer (aspartic acid as monomer) (b) copolymer with

less monomer ratio of CA and (c) copolymer with higher

monomer ratio of CA. Significant changes in the activity of

biodegradable water soluble green copolymers prepared at

different weight ratios of citric acid against the microor-

ganism were noticed. The results indicate that incorporated

amount of CA copolymers exhibited greater reduction of

E -coli, Bacillus and Pseudomonas growth compared to the

homopolymer (Table 1). The higher amount of monomer

CA copolymer showed enormous decrease in the growth of

Fig. 5 Antibacterial activity of (a) homopolymer(PASP), (b) P(ASP-CA1) and (c) P(ASP-CA2) copolymer

J Polym Environ (2012) 20:17–22 21

1 3



E -coli, Bacillus and Pseudomonas than homopolymer of

aspartic acid. The mode of antibacterial action is dependent

on the copolymer chain length, the presence of more

amounts of the CA and also number of carboxylic acid

groups [19]. Copolymer with higher monomer ratio of CA

copolymer showed 100% inhibition growth while other

copolymer showed less inhibition growth of E -coli,

Bacillus and Pseudomonas. The reason for this is two timesof more amount of CA suppress the growth of bacteria

when compared to less amount of CA in CA copolymers.

Copolymers of citric acid could significantly inhibit the

growth of micro-organisms within 48 h. The soluble green

copolymer bearing CA moiety is the most effective one,

against both gram-negative and gram-positive bacteria. The

copolymers from CA chains could interact sulfur contain-

ing intracellular proteins in bacteria and kill them.

Conclusions

From the results, the co-polymers prepared with higher

amount of citric acid exhibited better antibacterial activity

and were more effective against gram-positive and gram-

negative bacteria. The antibacterial activity of the

copolymer was enhanced further by increasing citric acid

concentrations.

Acknowledgments N. Mithil Kumar thanks the University Grants

Commission (UGC), Government of India, New Delhi for the finan-

cial support is the from meritorious fellowship.

References

1. Hater W (1998) Corrosion 98:213–222

2. Kim Ho H, Yeo Guw D, Yi Yil W. United States Patent 5548035

3. Ho G-H, Ho T-I, Hsieh K-H, Su Y-C, Lin P-Y, Yang J (2006)

J Chin Chem Soc 53:1363–1384

4. Ross RJ, Low KC (1997) Mater Perform 36:53–57

5. Low KC, Wheeler AP, Koskan LP (1996) Adv Chem Ser

248:99–111

6. Burke EM, Guo Y, Colon L, Rahima M, Veis A, Nancollas GH

(2000) Col Surf B 17:49–57

7. Roweton S, Huang SJ, Swift G (1997) Environ Polym Degrad

5:175–181

8. Cao H, Ma X, Sun S, Su H, Tan T, Alya AS, Abdel-Mohsena

AM, Hebeisha A (2010) Polym Bull 64:623–632

9. Miksic BA, Kharshan MA, Furman AY (2005) Presented at the

European symposium on corrosion inhibitors (10 SEIC), Ferrara,

Italy, September ctp 83

10. Swift G (1998) Polym Degra Stabil 59:19–24

11. Xue1 Z, He J-X, Zhan Y-Z (2009) China textile, 5

12. Jyothi AN (2010) Compos Interfaces 17:165–174

13. Shi R, Chen D, Liu Q, Wu Y, Xu X, Zhang L, Tian W (2009) Int

J Mol Sci 10:4223–4256

14. Alford DD, Wheeler HP, Pettigrew CA (1994) J Environ. Polym

Degrad 2:225–236

15. Wang X, Lee BI, Mann L (2002) Colloids Surf A Physicochem

Eng Aspects 202:71–80

16. Sanjay K, Singh RA (2001) Gross 786:2–40

17. Wood LL, Bayer AG (1996) United States Patent 5540863, 1–4

18. Namazi H, Adeli M (2003) Eur Polym J 39:1491–1500

19. Djordjevic I, Choudhury NR, Dutta NK, Kumar S (2005) Poly-

mer 50:1682–1691

20. Yang J, Webb AR, Ameer GA (2004) Adv Mater 16:511–516

21. Abd EI-Rehim HA, Mostafa TB, ABd EI-Monem B (2005)

J Macromole Sci 42:853–867

22. Tran T, Zhang Y, Gyawali D, Yang J (2009) Recent Pat Biomed

Eng 2:216–222

23. Kim SII, Son CM, Jeon YS, Kim J-H (2009) Bull Korean Chem

Soc 30:1–6

24. ZhenHua Q, YongChang C, XiuRong W, Cheng S, YunJie L,

ChongFang MA (2008) Sci chin Ser B Chem 51:695–699

25. Nita LE, Chiriac AP, Bercea M (2010) Iordana Neamtu. Colloids

Surf A Physicochem Eng Aspects 374:1–154

26. Wang A, Yin H, Lu H, Xue J, Ren M, Jiang T (2009)

25:12736–12741

27. Mithil Kumar N, Varaprasad K, Ramachanra Reddy G, Siva

Mohan Reddy G, Siva Barathi Y, Venkata Subba Reddy G,

Venkata Naidu S (2011) J Polym Environ 19:225–229

28. Orhan M, Kut D, Gunesoglu C (2009) J Appl Poly Sci

111:344–1352

29. Zahedi P, Rezaeian I, Ranaei-Siadat S-O, Jafari S-H, Supaphol P

(2010) Polym Adv Technol 21:77–95

30. Orhan M, Kut D, Gunesoglu C (2009) J Appl Poly Sci

111:1344–1352

31. Abd El-rehim HA, Mostafa TB, El-monem Bashind ABD (2005)

J Macro Sci Part A Pure Appl Chem 42:853–867

22 J Polym Environ (2012) 20:17–22

1 3

Documents

A Novel Biodegradable Green Poly(L-Aspartic Acid-Citric Acid) Copolymer for Antimicrobial Applications