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ISSN : 1229-1153(Print)
ISSN : 2465-9223(Online)
Journal of Food Hygiene and Safety Vol.32 No.5 pp.443-446

Polymer Hydrogels Formulated with Various Cross-Linkers for Food-Surface Application to Control Listeria monocytogenes

Sejeong Kim1,2, Hyemin Oh1,2, Heeyoung Lee1,2, Soomin Lee1,2, Jimyeong Ha1,2, Jeeyeon Lee1,2, Yukyoung Choi1,2, Yohan Yoon1,2*
1Risk Analysis Research Center, Sookmyung Women’s University, Seoul, Korea
2Department of Food and Nutrition, Sookmyung Women’s University, Seoul, Korea
Correspondence to: Yohan Yoon, Food microbiology lab., Dept. Food and Nutrition, Sookmyung Women’s University. 100, 47-gil, Cheongpa-ro, Youngsan-gu, Seoul 04310, Korea 82-2-2077-7585,
September 12, 2017 September 29, 2017 October 17, 2017


This study investigated the physical properties of polymers and antimicrobial activities of organic acids on Listeria monocytogenes to develop hydrogels. κ-carrageenan (1, 2, and 3%), carboxymethylcellulose (CMC; 1, 3, and 5%), and agar (1.5 and 3%) were mixed with cross-linkers (Na+, K+, Ca2+, and Al3+) or each other by stirring or heating to form cross-linkage, and their physical properties (hardness, elasticity, and swelling) were measured. The hydrogels formulated with organic acid (1, 3, and 5%) were analyzed by spot assay against L. monocytogenes. κ-carrageenan formed hydrogels with high hardness without other cross-linkers, but they had low elasticity. The elasticity was improved by mixing with other cross-linkers such as K+ or other polymer, especially in 3% κ-carrageenan. CMC hydrogel was formed by adding cross-linkers Al3+, Na+, or Ca2+, especially in 5% CMC. Thus, stickiness and swelling for selected hydrogel formulations (two of κ-carrageenan hydrogels and three of CMC hydrogels) were measured. Among the selected hydrogels, most of them showed appropriate hardness, but only 3% κ-carrageenan-contained hydrogels maintained their shapes from swelling. Hence, 3% κ-carrageenan+0.2% KCl and 3% κ-carrageenan+1% alginate+0.2% KCl+0.2% CaCl2 were selected to be formulated with lactic acid, and showed antilisterial activity. These results indicate that 3% κ-carrageenan hydrogels formulated with lactic acid can be used to control L. monocytogenes on food surface.


    Rural Development Administration


    Listeria monocytogenes is foodborne illness bacterium, causing abortion, still birth, meningitis, and sepsis1). The mortality rate of L. monocytogenes infection is about 20- 30%2), and the pathogen is usually isolated from food surfaces3). Thus, various technologies to inhibit L. monocytogenes have been developed, but the technology for nonthermal application is not very common.

    Hydrogel is three-dimensional structure constructed from cross-linking between one or more polymers4). The polymeric materials were hydrophilic, and thus, they can absorb water from environments and swell, but they are not dissolved in water due to their cross-linked network5). Because of this property, hydrogels were developed in various fields such as wound healing, drug delivery, tissue engineering, and cosmetics which apply hydrogels as vehicle or reservoirs for functional materials6-8). The hydrogel has different mechanical properties determined by polymer, cross-linker, or gelling conditions9).

    For applying hydrogels on food surface, natural materials are appropriate. κ-carrageenan, carboxymethylcellulose (CMC), agar, etc. should be appropriate for making hydrogels due to their nontoxicity, biodegradability, and renewability 10). Among various carrageenans (κ-, ι-, and λ- carrageenan), κ-carrageenan forms strong and rigid gels in the presence of potassium ions, and it is a common hydrophilic polysaccharide found in various seaweeds11,12). κ-carrageenan gels are formed when the polymers are heated and cooled, and the cooling rate affects mechanical properties of the gels13). CMC is another natural polysaccharide gelling in the presence of cation such as Na+, Ca2+, and Al3+14). Agar is also a natural polymer. Heating and subsequent cooling can induce gelation of the agar15).

    If hydrogels are functionalized by adding antimicrobial agents or antioxidants, the hydrogels can be non-thermal decontamination technology. Therefore, in this study the physical properties of the hydrogels produced from various polymers and antilisterial activities of the hydrogels formulated with organic acids were investigated to develop an antilisterial hydrogel.

    Materials and Methods

    Preparation of hydrogels

    κ-carrageenan was suspended in 6 mL of glycerol at 1, 2 and 3%, and distilled water (19 mL) with or without 0.2% KCl and CaCl2 was added to the mixtures to provide Ca+. After heating at 95°C for 10 min, the mixtures were poured onto petridish, and they were cooled at room temperature to form hydrogel16). CMC hydrogels were prepared according to a study by Lee et al. 17) and Roy et al.18). Briefly, CMC (1, 3, and 5%) and 3% alginate in glycerol were mixed with 0.2% tartaric acid (TTA)+dihydroxyaluminum aminoacetate (DDA) solution, 0.2% of CaCl2, or 0.2% NaCl to provide Al+, Ca+, or Na+, respectively, and stirred at room temperature until gels were formed. The gels were poured onto petridish, and followed by incubation at 42°C to dry the gels. To prepare agar hydrogels, agar powder (1.5 g or 3 g) was dissolved in 25 mL distilled water or glycerol to be mixed with 3% CMC and 3% CMC+0.2% (DAA+TTA), and the mixture was heated using microwave for 30 sec. The heated solution was poured onto petridish and cooled at room temperature for the gelation19).

    Measurement of hydrogel mechanical properties

    Hydrogels were cut into 2 cm × 2 cm, and the hydrogel pieces were analyzed by TA Express (Texture analyzer, Stable Micro Systems LTD., Surrey, UK) and hardness, elasticity and stickiness of hydrogels were calculated by Exponent Lite Express (Software, Stable Micro Systems LTD., Surrey, UK). For swelling measurement, 1 cm × 1 cm of the hydrogel pieces were prepared and weighed before and after placing the gels in 24-well plate containing 0.5 mL of distilled water for 24 h. Swelling conditions were determined by the weight changes of the gels.

    Spot assay of antimicrobials

    L. monocytogenes 1/2b, isolated from beef carcass in previous study20), was used in this study. The bacterial strain in tryptic soy broth (TSB; Becton, Dickinson and Company, Sparks, MD, USA) was subcultured in 14 mL TSB until the growth reached at log phase (OD600 = 0.7-1.0), and the culture was mixed with TSB+0.8% agar to be OD600 = 0.1. Three milliliter aliquots of the mixtures were poured on TSA, and left at room temperature until solidified. To prepare antilisterial hydrogels, the hydrogels (1 cm × 1 cm) were placed in 10 mL of 1, 3, and 5% lactic acid for 1 h, and the hydrogels were placed on the agar containing L. monocytogenes. After 24-h incubation at 30°C, the sizes (mm) of the clear zones were measured.

    Statistical analysis

    The study for antimicrobial activity of hydrogels was repeated three times. The data for the size of clear zones measuring antimicrobial activity of hydrogels were analyzed with the GLM procedure of SAS (version 9.2, SAS Institute, Cary, NC, USA). LS means among fixed effects were compared by pairwise t-test at alpha = 0.05.

    Results and Discussion

    In this study, hydrogels composed of natural polymer and its proper cross-linkers were prepared, and the physical properties of the hydrogels such as hardness, elasticity, stickiness and swelling were compared.

    Hardness of κ-carrageenan hydrogel increased as concentration of κ-carrageenan increased (Table 1). Addition of K+ into κ-carrageenan by adding 0.2% KCl increased the hardness of the hydrogel, but addition of 1% alginate did not increase the hardness of κ-carrageenan hydrogel (Table 1). Hydrogel was not formed by CMC itself (Table 1). However, CMC was cross-linked with cationic ions such as Na+, Ca2+, and Al3+ (Table 1). Especially, addition of 0.2% DAA+TTA for generating Al3+ into CMC constructed CMC hydrogels that had higher hardness than other cross-linkers (Table 1). Agar hydrogels could be simply formed by heat treatment, and the hardness of gels increased by adding CMC 3%+2% (DAA+TTA) (Table 1). Although hydrogels exhibit high hardness, if the elasticity of the hydrogels are not sufficiently high, hydrogels may be broken while they are applied. Hence, the elasticity of the hydrogels was further measured.

    Elasticity values of hydrogels made from various polymer and cross-linker matrixes are presented in Table 2. As the value is close to 0, the elasticity of the gel is higher. In general, the elasticity of κ-carrageenan hydrogels was much higher than those of CMC and agar hydrogels. The elasticity values of κ-carrageenan hydrogels generally decreased as cross-linkers were added. Although the elasticity decreased by adding cross-linkers, the combination of 3% κ-carrageenan with 0.2% KCl that had the highest hardness exhibited comparatively higher elasticity than other combinations, and the combination of 3% κ-carrageenan with 1% alginate+0.2% KCl+0.2% CaCl2 that had the second highest hardness also showed comparatively higher elasticity than other combinations (Table 2). As described above, CMC did not form hydrogel itself, and thus, adding crosslinkers into CMC were necessary. Among CMC hydrogels, a combination of 3% CMC with 3% alginate+0.2% NaCl had the most elastic properties, but it had low hardness. Among 5% CMC, combinations of 5% CMC with 0.2% (DDA+TTA), 3% alginate, and 3% alginate+0.2% NaCl had high hardness, and their elasticity values were −0.507, −0.363, and −0.464, respectively. Taken together of hardness and elasticity, 3% κ-carrageenan with 0.2% KCl, 3% κ-carrageenan with 1% alginate+0.2% KCl+0.2% CaCl2, 5% CMC+0.2% (DDA+TTA), 5% CMC+3% alginate, and 5% CMC+3% alginate+0.2% NaCl seemed appropriate hydrogels. Thus, they were further-analyzed for stickiness. Most elasticity values of agar hydrogels were further from 0. Hence, agar hydrogels were not considered for further analysis.

    As stickiness value is close to 0, the stickiness of hydrogel is low. Stickiness of 5% CMC-contained hydrogels was higher than 3% κ-carrageenan-contained hydrogels (Table 3). However, the 5% CMC-contained hydrogels were melted thoroughly during swelling test (Table 3). Hence, only 3% κ-carrageenan+0.2% KCl and 3% κ-carrageenan+1% alginate+ 0.2% KCl+0.2% CaCl2 were used to contain antimicrobial and examined for antimicrobial activity of the hydrogels. Both κ-carrageenan hydrogels showed increased antilisterial activity as lactic acid concentration increased (Table 4).

    In conclusion, κ-carrageenan can be used for hydrogel, especially at 3%, and the physical properties were improved by addition of 0.2% KCl or 1% alginate+0.2% KCl+0.2% CaCl2. In addition, formulating lactic acid into the hydrogels showed antilisterial activity, which may suggest other organic acids to be formulated into hydrogels. Therefore, the 3% κ- carrageenan hydrogels formulated with lactic acid can be used to control L. monocytogenes on food surface.


    This work was carried out with the support of “Cooperative Research Program for Agriculture Science & Technology Development (Project No. PJ0119932016)” Rural Development Administration, Republic of Korea.



    Hardness of κ-carrageenan, carboxymethyl cellulose (CMC) and agar hydrogels formulated with various cross-linkers

    1)No hydrogel formation

    Elasticity of κ-carrageenan, carboxymethyl cellulose (CMC) and agar hydrogels formulated with various cross-linkers

    1)No hydrogel formation

    Stickiness and swelling of hydrogels, selected according to hardness and elasticity of hydrogels

    1)gel weight was changed under 10%.
    2)gel weight was changed over 10% and shape was maintained.
    3)gel was melted thoroughly.

    The sizes (mm; mean ± S.D) of clear zones formed by κ-carrageenan hydrogels formulated with lactic acid against Listeria monocytogenes

    a-dmeans with different letters are significantly different (p < 0.05).
    A-Dmeans with different letters are significantly different (p < 0.05).


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