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Shelf-life of fresh noodles as affected by chitosan and its Maillard reaction products
ARTICLE IN PRESS
LWT 40 (2007) 1287–1291 www.elsevier.com/locate/lwt
Shelf-life of fresh noodles as affected by chitosan and its Maillard reaction products Jin-ru Huanga, Chung-yi Huanga, Yao-wen Huangb,, Rong-hui Chenc a
Department of Food Science, National I-Lan University, I-Lan, Taiwan, ROC Department of Food Science and Technology, University of Georgia, Athens, GA 30602, USA c Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, ROC
b
Received 4 November 2005; received in revised form 4 August 2006; accepted 8 August 2006
Abstract The effects of Maillard reaction products (MRPs) prepared from chitosan and xylose on the shelf-life of fresh noodles were studied. A model system consisting of chitosan and xylose (1:1.5, w/w) dispersed in distilled water with pH values of 5, 6, 7, 9 was used. The systems were heated to 95 1C for up to 60 h. The development of brown color was more pronounced in the model system with a higher initial pH of the water. The antibacterial activity of chitosan increased at the beginning of the Maillard reaction, and the minimum inhibition concentration (MIC) of chitosan against Bacillus subtilis CCRC 10258 decreased to 50 mg/ml of medium from 250 mg as a result of the Maillard reaction. Gram-positive bacteria were more sensitive to chitosan or its MRPs than Gram-negative bacteria. MRP of chitosan/xylose showed a bactericidal effect against Bacillus subtilis CCRC 10258, while chitosan showed a bacteriostatic effect prior to the occurrence of the Maillard reaction. Addition of 0.05 g/100 ml chitosan (in 0.5 ml/100 ml acetic acid) to fresh noodle formulation resulted in an extension of its shelf-life for 6 more days when stored at 4 1C. However, addition of MRP resulted in the longer shelf-life lasting 14 days when stored at 4 1C. r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Chitosan; Xylose; Maillard reaction product (MRP); Shelf-life; Bacteriostatic; Preservative; Fresh noodle
1. Introduction Chitosan is a linear polyamine (Tanaka, Huang, Chiu, Shoichiro, & Takeshi, 1993) and a natural nontoxic biopolymer (Hong, Na, Shin, & Lsamuel, 2002) derived by the alkaline deacetylation of chitin, a major component of the shells of crustacean, such as shrimp, crawfish and crab. Most of the commercial high molecular weight hydrocolloids or polysaccharides used in industries are neutral or polyanionic. Chitosan, a cationic polyelectrolyte, is one of few exceptions. The unique physical, chemical, and biological properties of the positively charged chitosan in acidic solution may have an enormous potential in cosmetic, medical, and food-related industries. In pharmaceutical fields, chitosan has been used as an antimicrobial agent as well as an encapsulation substance Corresponding author. Tel.: +7065421092; fax: +7065421050.
E-mail address: [email protected] (Y.-w. Huang).
for drug ingredients (El Ghaouth, Grenier, & Asselin, 1992; Vorlop & Klein, 1981). Chitosan have been studied for the application on treatment of cheddar cheese whey (Savant & Torres, 2000) and surimi wash water (Wibowo, Torres, Savant, & Velazquez de la Cruz, 2004). A recent study demonstrated that the complex process involved in determining the antimicrobial activity of chitosan on raw oysters due to the effects of chitosan differ depending on the species of microbial contaminant (Chhabra, Huang, Frank, & Gates, 2006). The Maillard reaction, also called the nonenzymatic browning reaction, is a reaction between amino groups and reducing compounds. In food systems, the amino compounds are mostly free or protein-bound amino acids while the reducing sugars are one of the most commonly prevalent chemical compounds that exist during food processing and preservation. Therefore, there has been a growing interest in the physiological aspects of the Maillard reaction in food and biological systems. Most
0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2006.08.004
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J.-r. Huang et al. / LWT 40 (2007) 1287–1291
studies of MRP on bacteria have been done in microbial growth media. These studies show that MRP has the ability to stimulate microbial growth (Einarsson, Snygg, & Eriksson, 1983). It was even found that the growth of Aeropyrum pernix was severely inhibited in a medium containing reducing sugars and tryptone due to the formation of Maillard reaction products. The rate of the Maillard browning reaction was markedly enhanced under aerobic conditions, and the addition of Maillard reaction products to the culture medium caused fatal growth inhibition. It is identified that MRP may inhibit microbial growth (Kim & Lee, 2003). Maillard reactions in association with various technological, chemical, and physical aspects have been studied and reported (Lingnert & Eriksson, 1981). However, no report on the effect of Maillard reaction products (MRPs) on bacteria, especially those found in foods, is available. Since chitosan is likely to be involved in the Maillard reaction due to its amino groups, the objectives of this investigation were to determine the antibacterial activity of chitosan during its Maillard reaction with xylose, and to evaluate the antibacterial activity of chitosan before and after the reaction with xylose. MRPs have shown an effect on the shelf-life during storage of kamaboko in the earlier report (Huang & Chen, 1997). In this study, model systems consisting of chitosan and xylose were formulated. The effect of the Maillard reaction on the addition of chitosan and its MRP to fresh noodles in the extension of its shelflife was also studied.
2. Materials and methods 2.1. The Maillard reaction of chitosan with xylose Chitosan (D.D.83 g/100 g; Lee-Ton LTD. Taipei, Taiwan) and D-xylose (Tokyo Kasei Ltd., Japan) were used for creation of the Maillard reaction. First, chitosan was ground using an A11 basic analytical mill (IKA, South Carolina, USA) with a 250 mm screen. Then the model system was prepared by dispersing chitosan powder in a saturated xylose solution. The pH of the solution was adjusted to 5, 6, 7, and 9 using 1 mol/l hydrochloric acid or 1 mol/l sodium hydroxide. The ratio of chitosan to xylose was 1:1.5 by weight. The freeze-dried samples were then crushed to a fine powder. For the formation of Maillard reaction products, 10 g of the sample was placed in a glass vial (3.0 cm i.d. 8.0 cm) and sealed with a screw cap and then heated to 95 1C for up to 60 h. Samples were withdrawn at given time intervals, and cooled in an ice-water-bath. The extent of the reaction was monitored by the absorbance of a 10-fold diluted solution at 420 nm. Chitosan, prepared in a 0.5 ml/100 ml acetic acid solution, subjected to the Maillard reaction was referred to as browned chitosan. These were sterilized using Acrodisc syringe filter units (0.22 mm, Gelman Science, Ann Arbor, MI, USA) and used immediately after preparation.
2.2. Microorganisms Both Gram-positive and Gram-negative bacterial strains were used in this study. The G (+) bacterial strains used in this study were Staphylococcus aureus CCRC 12653, Bacillus cereus CCRC 10250, B. cereus CCRC 10258, B. cereus CCRC 10267, and Streptococcus faecalis CCRC 10066. The Gram-negative bacterial strains used in this study were Salmonella Typhimurium CCRC 12947, Escherichia coli ATCC 25922, Pseudomonas fluorescens ATCC 21541, and Vibrio parahaemolyticus CCRC 10806. All strains were provided by the Food Industry Research & Development Institute (Shin-chu, Taiwan). 2.3. Media The medium (pH 7.1) used for bacterial growth composed of 0.25 g/100 ml yeast extract, 0.1 g/100 ml glucose, 0.5 g/100 ml polypeptone, and 1.5 g/100 ml agar (Nissui Seiyaku Ltd., Tokyo, Japan). Three grams per one hundred milliliters of NaCl was added to the above medium for the Vibrio strain. Liquid versions of the above media were employed as the broth culture, and the dilution of inocula was made with sterile 0.85 g/100 ml of NaCl solution. 2.4. Determination of minimum inhibitory concentration (MIC) For determination of MIC, the agar dilution method was used (Islam, Itakura, & Motohiro, 1984). Diluted 20 h broth culture of bacterium, with approximately 107 cfu/ml, was plated on the agar plates containing an appropriate concentration of chitosan or MRP of chitosan. The plates were incubated for 20 h at 37 1C for all organisms with the exception of the Pseudomonas florescence plates that were incubated at 30 1C for 20 h. The MIC was defined as the lowest concentration of chitosan required to completely inhibit bacterial growth after incubation at 37 1C for 20 h. The extent of growth inhibition was observed with the naked eye. 2.5. Determination of lethal effect of MRP The B. cereus CCRC 10258 inoculum (0.5 ml of 108 cfu/ml) was added to a mixture of 9 ml broth medium and 0.5 ml of MRP chitosan solution. Each tube contained a different concentration of MRP. The tubes were then incubated at 37 1C for 20 h. The total viable cell numbers in each concentration of MRP were determined by the standard plate count method (AOAC, 1997). 2.6. Application of MRP to fresh-noodle formulation Fresh noodles were produced at the CGPRDI Baking Training Department in Taipei, Taiwan. Wheat flour was mixed first with water and salt and then a 0.05 g/100 ml
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MRP in 0.5 g/100 ml acetic acid was added. After mixing thoroughly, the dough was flatted, divided, heated for 15 min at 95 1C, cooled down to 25 1C, and formed into fresh noodles. Samples were stored at 4 1C. Total viable cell numbers of fresh noodles were measured by the standard plate count method.
3. Results and discussion 3.1. Development of brown color Xylose was used as a reducing sugar for the Maillard reaction throughout the entire study since MRP obtained with xylose has been reported to possess relatively potent antibacterial activity (Einarsson et al., 1983). The development of the brown color during the Maillard reaction caused by chitosan and xylose at various initial pH values is shown in Fig. 1. The rate of brown color development varied as the initial pH changed with the greater formation of brown color observed in systems at a higher initial pH value. However, there was no significant difference observed between pH 5 and pH 6. As shown in Fig. 1, there is a linear relationship between the brown color development and the length of heating time for pH value of 9. However, the induction period of approximately 20 h was observed for model systems with the initial pH values of 5, 6, and 7. As seen in Fig. 1, chitosan plays a role in the Maillard reaction in the presence of glucose since chitosan per se did not produce any brown color without the presence of glucose. These findings are similar to the results found by Tanaka et al. (1993). Development of brown color followed the zeroorder reaction, and the reaction rate was faster as the initial pH increased.
6
Absorbance (at 420nm)
5 4 3 2 1 0 0
10
20
30 40 Heating Time (h)
50
60
1289
3.2. Effect of the Maillard reaction on MIC The effect of Millard reaction on antibacterial activity of chitosan was followed by the result of MIC against Bacillus subtilis CCRC 10258. The MIC value (mg of chitosan/ml of agar medium) was presented as the lowest concentration that completely inhibited formation of visible growth (agar dilution method). The MIC value of chitosan per se used in this study was 250 mg/ml, which was in good agreement with the value reported by Islam et al. (1984). As the Maillard reaction occurred when chitosan and xylose were heated under 95 1C, the antibacterial activity of MRP increased at the initial stage and then decreased at the latter stage. Table 1 shows that the MIC of MRP decreased from 250 to 50 mg/ml in the presence of xylose at pH 6 after 10 h. On the other hand, the MIC of chitosan without xylose did not change when heated to 95 1C which suggests chitosan is fairly heat stable. In model systems with higher initial pH values, the increase of antibacterial activity during the initial stage of the Maillard reaction was not significantly different from that observed between pH values of 5 and 6 (Fig. 1). Results illustrate that changes in the antibacterial activity during the Maillard reaction is not necessarily attributable to the formation of melanoidins. Thus, the brown color, one of the easiest measurable factors of the Maillard reaction, is not a good indicator for the antibacterial activity of MRP of chitosan. 3.3. Sensitivity of bacterial strains to chitosan and its MRP In this study, we reported on a strengthening of antibacterial activity of chitosan by means of the Maillard reaction, MIC values of MRP of chitosan for other bacteria besides Bacillus subtilis CCRC 10258 were also determined. Table 2 shows the MIC value of chitosan and its MRP in the agar dilution method against various strains of bacteria. MRP used in this experiment was prepared by heating chitosan with xylose to 95 1C for 10 h and pH 6, which has the most potent growth inhibition against B. subtilis CCRC 10258. Table 2 shows that the MIC values varied among the different bacteria used in this study. Chitosan did not have antibacterial activity against Gram-negative bacteria used in this study. The effect of chitosan on Gram-positive bacteria studied in this experiment varied, and this finding is similar to the results of Tanaka et al. (1993). Furthermore, the sensitivity of B. subtilis varied considerably between B. subtilis CCRC 10258 and B. subtilis CCRC 10267. It was further revealed that the antibacterial activity of chitosan was more or less lost in the progress of the Maillard reaction (Huang & Chen, 1997).
70
Fig. 1. Absorbance changes (at 420 nm) of MRP preparation with chitosan and xylose over times at 95 1C at different pH values. () pH 5; ( ) pH 6; (m) pH 7; (E) pH 9.
3.4. Lethal effect of chitosan and its MRP The lethal effect of chitosan and its MRP on B. subtilis CCRC 10258 is shown in Fig. 2. It is evident from Fig. 2
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Table 1 Minimum inhibition concentration (MICa) of chitosan in Maillard reaction product on Bacillus subtilis CCRC 10258 prepared at 95 1C as affected by heating time and pH value Initial pH
Heating time (h)
5 6 7 9
0
5
10
15
20
25
30
40
50
60
250 250 250 250
150 150 175 175
100 50 150 150
100 100 150 200
125 150 175 250
175 175 200 250
175 175 200 250
200 200 250 250
250 250 250 250
250 250 250 250
Chitosan:xylose ¼ 1:1.5 (w/w). a mg/ml of medium. Table 2 Effect of chitosan and its MRP on different bacteria strains Strain
MIC (mg/ml of medium) Chitosan
Article I —b Section 1.01 Salmonella Typhimurium CCRC 12947 Alteromonas putrefacieus IAM 12098 — Article II — Escherichia coli ATCC 25922 Pseudomonas fluorescens ATCC 21541 — Vibrio parahaemolyticus CCRC 10806 — Staphylococcus aureus CCRC 12653 325 Bacillus cereus CCRC 10250 450 Bacillus subtilis CCRC 10258 250 Bacillus subtilis CCRC 10267 275 Streptococcus faecalis CCRC 10066 475
MRPa — — — — — 200 150 50 225 400
3.5. MRP as a preservation substance for fresh noodle
a
Prepared by heating chitosan and xylose at pH 6 and at 95 1C for 10 h. Antibacterial activity was not detected under the conditions used in this study. b
9 8 7 6 log CFU/ml
that chitosan has little influence on B. subtilis CCRC 10258 as long as the lethal effect is concerned, suggesting that chitosan is bacteriostatic rather than bactericidal at least for this bacterium. And this is contrary to the findings of Tanaka et al. (1993), who reported that chitosan was bactericidal against the Bacillus strains tested. On the other hand, the Maillard reaction of the chitosan with xylose at 95 1C for 10 h at pH 6, showed the antibacterial action of chitosan against B. subtilis turned to be bactericidal. Bacillus subtilis CCRC 10258 in this experiment could not survive in a culture medium containing 250 mg of MRP/ml. The antibacterial action of chitosan and its MRP need to be further investigated.
5 4 3 2 1
Because fresh noodles have a high water activity and a neutral pH, their shelf-life is relatively short. Bacillus organisms are bacteria often associated with spoilage of high water activity products, such as fresh noodles. Therefore, the application of chitosan to fresh noodles as a preservative could be useful. As shown in Fig. 3, the increase in viable bacterial cell numbers in fresh noodles prepared with and without 0.05 g/ 100 ml chitosan (in 0.5 ml/100 ml acetic acid) during storage at 4 1C. Viable cell numbers of fresh-noodle during the first 2 days of storage at 4 1C was less than 100/g. The results indicate that most of the vegetative cells were killed by heat treatment of 95 1C for 15 min. The shelf-life of fresh noodles prepared without chitosan was about 8 days at 4 1C. The addition of 0.05 g/100 ml chitosan extended shelflife by 6 days. The shelf-life of fresh noodles was extended by the application of 0.05 g/100 ml MRP prepared by heating with xylose at 95 1C for 10 h and at pH 6 for 14 days at 4 1C. Also, the addition of MRP to fresh noodles caused a slight discoloration of the product.
0 0
50
100 150 200 250 chitosan concentration ug / ml of medium
300
Fig. 2. Inhibitory effect of chitosan and MRP of different concentration on Bacillus subtilis CCRC 10258. The MRP were prepared by heating chitosan and xylose at pH 6 to 95 1C for 10 h. Results are the means7standard deviation of the three replications) () chitosan; (m) MRP.
4. Conclusion In conclusion, the antibacterial activity of chitosan was revealed to be further enhanced by its Maillard reaction at 95 1C for 10 h and at pH 6 with xylose. MRP was bactericidal to B. subtilis CCRC 10258 and its addition
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9 8 7
log CFU/ml
6 5 4 3 2 1 0 0
5
10
15 20 Storage time (Days)
25
30
Fig. 3. Changes in viable bacteria count on fresh noodles as affected by chitosan and MRP at 4 1C. Results were the means7standard deviation of the three replications () control/0.5 ml/100 ml acetic acid; ( ) 0.05 g/ 100 ml chitosan in 0.5 ml/100 ml acetic acid; (m) 0.05 g/100 ml MRP in 0.5 ml/100 ml acetic acid.
to fresh noodle at a level of 0.05/100 g led to a longer shelflife. References AOAC Research Institute. (1997). The referee, inside lab management (pp. 35). Association of Official Analytical Chemists, Washington, DC. Chhabra, P., Huang, Y. W., Frank, J. F., Chminlewski, R., & Gates, K. (2006). Fate of Staphylococcus aureus, Salmonella enterica Serovar
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Typhimurium, and Vibrio vulnificus in raw oysters treated with chitosan. Journal of Food Protection, 69, 1600–1604. Einarsson, H., Snygg, B. G., & Eriksson, C. (1983). Inhibition of bacterial growth by Maillard reaction products. Journal of Agriculture and Food Chemistry, 31, 1043–1047. El Ghaouth, A., Grenier, J., & Asselin, A. (1992). Antifungal activity of chitosan on two postharvest pathogens of strawberry fruits. Phytopathology, 82, 398–402. Hong, K. N., Na, Y. P., Shin, H., & Lsamuel, P. M. (2002). Antibacterial activity of chitosans and chitosan oligomers with different molecular weights. International Journal of Food Microbiology, 74, 65–72. Huang, J. R., & Chen, R. H. (1997). The bactericide effects on chitosanglucose Maillard reaction products obtained for different reaction times and the preservative effects of chitosan for kamaboko. Journal of Food Science, 24, 458–468. Islam, N. M., Itakura, T., & Motohiro, T. (1984). Antibacterial spectra and minimum inhibition concentration of clupine and salmine. Nippon Suisan Gakkaishi, 50, 1705–1708. Kim, K. W., & Lee, S. B. (2003). Inhibitory effect of Maillard reaction products on growth of the aerobic Mmrine hyperthermophilic archaeon Aeropyrum pernix. Applied and Enviromental Microbiology, 69, 4325–4328. Lingnert, H., & Eriksson, C. E. (1981). Antioxidative effect of Maillard reaction products. Progressive Food and Nutrition Science, 5, 453–466. Savant, V. D., & Torres, J. A. (2000). Chitosan-based coagulating agents for treatment of cheddar cheese whey. Biotechnology Progress, 16, 1091–1097. Tanaka, M., Huang, J. R., Chiu, W. K., Shoichiro, I., & Takeshi, K. (1993). Effect of the Maillard reaction on functional properties of chitosan. Nippon Suisan Gakkaishi, 59, 1915–1921. Vorlop, K. D., & Klein, J. (1981). Formation of spherical chitosan biocatalysts by ionotropic gelation. Biotechnology Letter, 3, 9–14. Wibowo, S., Torres, J. A., Savant, V. D., & Velazquez de la Cruz, G. (2004). Effect of chitosan concentration, reaction time and different molecular weight and degree of deacetylation on protein adsorption from surimi wash water. 2004 Institute of Food Technologists Annual Meeting Technical Program Abstracts No. 63-2 (p. 151).
LWT 40 (2007) 1287–1291 www.elsevier.com/locate/lwt
Shelf-life of fresh noodles as affected by chitosan and its Maillard reaction products Jin-ru Huanga, Chung-yi Huanga, Yao-wen Huangb,, Rong-hui Chenc a
Department of Food Science, National I-Lan University, I-Lan, Taiwan, ROC Department of Food Science and Technology, University of Georgia, Athens, GA 30602, USA c Department of Food Science, National Taiwan Ocean University, Keelung, Taiwan, ROC
b
Received 4 November 2005; received in revised form 4 August 2006; accepted 8 August 2006
Abstract The effects of Maillard reaction products (MRPs) prepared from chitosan and xylose on the shelf-life of fresh noodles were studied. A model system consisting of chitosan and xylose (1:1.5, w/w) dispersed in distilled water with pH values of 5, 6, 7, 9 was used. The systems were heated to 95 1C for up to 60 h. The development of brown color was more pronounced in the model system with a higher initial pH of the water. The antibacterial activity of chitosan increased at the beginning of the Maillard reaction, and the minimum inhibition concentration (MIC) of chitosan against Bacillus subtilis CCRC 10258 decreased to 50 mg/ml of medium from 250 mg as a result of the Maillard reaction. Gram-positive bacteria were more sensitive to chitosan or its MRPs than Gram-negative bacteria. MRP of chitosan/xylose showed a bactericidal effect against Bacillus subtilis CCRC 10258, while chitosan showed a bacteriostatic effect prior to the occurrence of the Maillard reaction. Addition of 0.05 g/100 ml chitosan (in 0.5 ml/100 ml acetic acid) to fresh noodle formulation resulted in an extension of its shelf-life for 6 more days when stored at 4 1C. However, addition of MRP resulted in the longer shelf-life lasting 14 days when stored at 4 1C. r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. Keywords: Chitosan; Xylose; Maillard reaction product (MRP); Shelf-life; Bacteriostatic; Preservative; Fresh noodle
1. Introduction Chitosan is a linear polyamine (Tanaka, Huang, Chiu, Shoichiro, & Takeshi, 1993) and a natural nontoxic biopolymer (Hong, Na, Shin, & Lsamuel, 2002) derived by the alkaline deacetylation of chitin, a major component of the shells of crustacean, such as shrimp, crawfish and crab. Most of the commercial high molecular weight hydrocolloids or polysaccharides used in industries are neutral or polyanionic. Chitosan, a cationic polyelectrolyte, is one of few exceptions. The unique physical, chemical, and biological properties of the positively charged chitosan in acidic solution may have an enormous potential in cosmetic, medical, and food-related industries. In pharmaceutical fields, chitosan has been used as an antimicrobial agent as well as an encapsulation substance Corresponding author. Tel.: +7065421092; fax: +7065421050.
E-mail address: [email protected] (Y.-w. Huang).
for drug ingredients (El Ghaouth, Grenier, & Asselin, 1992; Vorlop & Klein, 1981). Chitosan have been studied for the application on treatment of cheddar cheese whey (Savant & Torres, 2000) and surimi wash water (Wibowo, Torres, Savant, & Velazquez de la Cruz, 2004). A recent study demonstrated that the complex process involved in determining the antimicrobial activity of chitosan on raw oysters due to the effects of chitosan differ depending on the species of microbial contaminant (Chhabra, Huang, Frank, & Gates, 2006). The Maillard reaction, also called the nonenzymatic browning reaction, is a reaction between amino groups and reducing compounds. In food systems, the amino compounds are mostly free or protein-bound amino acids while the reducing sugars are one of the most commonly prevalent chemical compounds that exist during food processing and preservation. Therefore, there has been a growing interest in the physiological aspects of the Maillard reaction in food and biological systems. Most
0023-6438/$30.00 r 2006 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.lwt.2006.08.004
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studies of MRP on bacteria have been done in microbial growth media. These studies show that MRP has the ability to stimulate microbial growth (Einarsson, Snygg, & Eriksson, 1983). It was even found that the growth of Aeropyrum pernix was severely inhibited in a medium containing reducing sugars and tryptone due to the formation of Maillard reaction products. The rate of the Maillard browning reaction was markedly enhanced under aerobic conditions, and the addition of Maillard reaction products to the culture medium caused fatal growth inhibition. It is identified that MRP may inhibit microbial growth (Kim & Lee, 2003). Maillard reactions in association with various technological, chemical, and physical aspects have been studied and reported (Lingnert & Eriksson, 1981). However, no report on the effect of Maillard reaction products (MRPs) on bacteria, especially those found in foods, is available. Since chitosan is likely to be involved in the Maillard reaction due to its amino groups, the objectives of this investigation were to determine the antibacterial activity of chitosan during its Maillard reaction with xylose, and to evaluate the antibacterial activity of chitosan before and after the reaction with xylose. MRPs have shown an effect on the shelf-life during storage of kamaboko in the earlier report (Huang & Chen, 1997). In this study, model systems consisting of chitosan and xylose were formulated. The effect of the Maillard reaction on the addition of chitosan and its MRP to fresh noodles in the extension of its shelflife was also studied.
2. Materials and methods 2.1. The Maillard reaction of chitosan with xylose Chitosan (D.D.83 g/100 g; Lee-Ton LTD. Taipei, Taiwan) and D-xylose (Tokyo Kasei Ltd., Japan) were used for creation of the Maillard reaction. First, chitosan was ground using an A11 basic analytical mill (IKA, South Carolina, USA) with a 250 mm screen. Then the model system was prepared by dispersing chitosan powder in a saturated xylose solution. The pH of the solution was adjusted to 5, 6, 7, and 9 using 1 mol/l hydrochloric acid or 1 mol/l sodium hydroxide. The ratio of chitosan to xylose was 1:1.5 by weight. The freeze-dried samples were then crushed to a fine powder. For the formation of Maillard reaction products, 10 g of the sample was placed in a glass vial (3.0 cm i.d. 8.0 cm) and sealed with a screw cap and then heated to 95 1C for up to 60 h. Samples were withdrawn at given time intervals, and cooled in an ice-water-bath. The extent of the reaction was monitored by the absorbance of a 10-fold diluted solution at 420 nm. Chitosan, prepared in a 0.5 ml/100 ml acetic acid solution, subjected to the Maillard reaction was referred to as browned chitosan. These were sterilized using Acrodisc syringe filter units (0.22 mm, Gelman Science, Ann Arbor, MI, USA) and used immediately after preparation.
2.2. Microorganisms Both Gram-positive and Gram-negative bacterial strains were used in this study. The G (+) bacterial strains used in this study were Staphylococcus aureus CCRC 12653, Bacillus cereus CCRC 10250, B. cereus CCRC 10258, B. cereus CCRC 10267, and Streptococcus faecalis CCRC 10066. The Gram-negative bacterial strains used in this study were Salmonella Typhimurium CCRC 12947, Escherichia coli ATCC 25922, Pseudomonas fluorescens ATCC 21541, and Vibrio parahaemolyticus CCRC 10806. All strains were provided by the Food Industry Research & Development Institute (Shin-chu, Taiwan). 2.3. Media The medium (pH 7.1) used for bacterial growth composed of 0.25 g/100 ml yeast extract, 0.1 g/100 ml glucose, 0.5 g/100 ml polypeptone, and 1.5 g/100 ml agar (Nissui Seiyaku Ltd., Tokyo, Japan). Three grams per one hundred milliliters of NaCl was added to the above medium for the Vibrio strain. Liquid versions of the above media were employed as the broth culture, and the dilution of inocula was made with sterile 0.85 g/100 ml of NaCl solution. 2.4. Determination of minimum inhibitory concentration (MIC) For determination of MIC, the agar dilution method was used (Islam, Itakura, & Motohiro, 1984). Diluted 20 h broth culture of bacterium, with approximately 107 cfu/ml, was plated on the agar plates containing an appropriate concentration of chitosan or MRP of chitosan. The plates were incubated for 20 h at 37 1C for all organisms with the exception of the Pseudomonas florescence plates that were incubated at 30 1C for 20 h. The MIC was defined as the lowest concentration of chitosan required to completely inhibit bacterial growth after incubation at 37 1C for 20 h. The extent of growth inhibition was observed with the naked eye. 2.5. Determination of lethal effect of MRP The B. cereus CCRC 10258 inoculum (0.5 ml of 108 cfu/ml) was added to a mixture of 9 ml broth medium and 0.5 ml of MRP chitosan solution. Each tube contained a different concentration of MRP. The tubes were then incubated at 37 1C for 20 h. The total viable cell numbers in each concentration of MRP were determined by the standard plate count method (AOAC, 1997). 2.6. Application of MRP to fresh-noodle formulation Fresh noodles were produced at the CGPRDI Baking Training Department in Taipei, Taiwan. Wheat flour was mixed first with water and salt and then a 0.05 g/100 ml
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MRP in 0.5 g/100 ml acetic acid was added. After mixing thoroughly, the dough was flatted, divided, heated for 15 min at 95 1C, cooled down to 25 1C, and formed into fresh noodles. Samples were stored at 4 1C. Total viable cell numbers of fresh noodles were measured by the standard plate count method.
3. Results and discussion 3.1. Development of brown color Xylose was used as a reducing sugar for the Maillard reaction throughout the entire study since MRP obtained with xylose has been reported to possess relatively potent antibacterial activity (Einarsson et al., 1983). The development of the brown color during the Maillard reaction caused by chitosan and xylose at various initial pH values is shown in Fig. 1. The rate of brown color development varied as the initial pH changed with the greater formation of brown color observed in systems at a higher initial pH value. However, there was no significant difference observed between pH 5 and pH 6. As shown in Fig. 1, there is a linear relationship between the brown color development and the length of heating time for pH value of 9. However, the induction period of approximately 20 h was observed for model systems with the initial pH values of 5, 6, and 7. As seen in Fig. 1, chitosan plays a role in the Maillard reaction in the presence of glucose since chitosan per se did not produce any brown color without the presence of glucose. These findings are similar to the results found by Tanaka et al. (1993). Development of brown color followed the zeroorder reaction, and the reaction rate was faster as the initial pH increased.
6
Absorbance (at 420nm)
5 4 3 2 1 0 0
10
20
30 40 Heating Time (h)
50
60
1289
3.2. Effect of the Maillard reaction on MIC The effect of Millard reaction on antibacterial activity of chitosan was followed by the result of MIC against Bacillus subtilis CCRC 10258. The MIC value (mg of chitosan/ml of agar medium) was presented as the lowest concentration that completely inhibited formation of visible growth (agar dilution method). The MIC value of chitosan per se used in this study was 250 mg/ml, which was in good agreement with the value reported by Islam et al. (1984). As the Maillard reaction occurred when chitosan and xylose were heated under 95 1C, the antibacterial activity of MRP increased at the initial stage and then decreased at the latter stage. Table 1 shows that the MIC of MRP decreased from 250 to 50 mg/ml in the presence of xylose at pH 6 after 10 h. On the other hand, the MIC of chitosan without xylose did not change when heated to 95 1C which suggests chitosan is fairly heat stable. In model systems with higher initial pH values, the increase of antibacterial activity during the initial stage of the Maillard reaction was not significantly different from that observed between pH values of 5 and 6 (Fig. 1). Results illustrate that changes in the antibacterial activity during the Maillard reaction is not necessarily attributable to the formation of melanoidins. Thus, the brown color, one of the easiest measurable factors of the Maillard reaction, is not a good indicator for the antibacterial activity of MRP of chitosan. 3.3. Sensitivity of bacterial strains to chitosan and its MRP In this study, we reported on a strengthening of antibacterial activity of chitosan by means of the Maillard reaction, MIC values of MRP of chitosan for other bacteria besides Bacillus subtilis CCRC 10258 were also determined. Table 2 shows the MIC value of chitosan and its MRP in the agar dilution method against various strains of bacteria. MRP used in this experiment was prepared by heating chitosan with xylose to 95 1C for 10 h and pH 6, which has the most potent growth inhibition against B. subtilis CCRC 10258. Table 2 shows that the MIC values varied among the different bacteria used in this study. Chitosan did not have antibacterial activity against Gram-negative bacteria used in this study. The effect of chitosan on Gram-positive bacteria studied in this experiment varied, and this finding is similar to the results of Tanaka et al. (1993). Furthermore, the sensitivity of B. subtilis varied considerably between B. subtilis CCRC 10258 and B. subtilis CCRC 10267. It was further revealed that the antibacterial activity of chitosan was more or less lost in the progress of the Maillard reaction (Huang & Chen, 1997).
70
Fig. 1. Absorbance changes (at 420 nm) of MRP preparation with chitosan and xylose over times at 95 1C at different pH values. () pH 5; ( ) pH 6; (m) pH 7; (E) pH 9.
3.4. Lethal effect of chitosan and its MRP The lethal effect of chitosan and its MRP on B. subtilis CCRC 10258 is shown in Fig. 2. It is evident from Fig. 2
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Table 1 Minimum inhibition concentration (MICa) of chitosan in Maillard reaction product on Bacillus subtilis CCRC 10258 prepared at 95 1C as affected by heating time and pH value Initial pH
Heating time (h)
5 6 7 9
0
5
10
15
20
25
30
40
50
60
250 250 250 250
150 150 175 175
100 50 150 150
100 100 150 200
125 150 175 250
175 175 200 250
175 175 200 250
200 200 250 250
250 250 250 250
250 250 250 250
Chitosan:xylose ¼ 1:1.5 (w/w). a mg/ml of medium. Table 2 Effect of chitosan and its MRP on different bacteria strains Strain
MIC (mg/ml of medium) Chitosan
Article I —b Section 1.01 Salmonella Typhimurium CCRC 12947 Alteromonas putrefacieus IAM 12098 — Article II — Escherichia coli ATCC 25922 Pseudomonas fluorescens ATCC 21541 — Vibrio parahaemolyticus CCRC 10806 — Staphylococcus aureus CCRC 12653 325 Bacillus cereus CCRC 10250 450 Bacillus subtilis CCRC 10258 250 Bacillus subtilis CCRC 10267 275 Streptococcus faecalis CCRC 10066 475
MRPa — — — — — 200 150 50 225 400
3.5. MRP as a preservation substance for fresh noodle
a
Prepared by heating chitosan and xylose at pH 6 and at 95 1C for 10 h. Antibacterial activity was not detected under the conditions used in this study. b
9 8 7 6 log CFU/ml
that chitosan has little influence on B. subtilis CCRC 10258 as long as the lethal effect is concerned, suggesting that chitosan is bacteriostatic rather than bactericidal at least for this bacterium. And this is contrary to the findings of Tanaka et al. (1993), who reported that chitosan was bactericidal against the Bacillus strains tested. On the other hand, the Maillard reaction of the chitosan with xylose at 95 1C for 10 h at pH 6, showed the antibacterial action of chitosan against B. subtilis turned to be bactericidal. Bacillus subtilis CCRC 10258 in this experiment could not survive in a culture medium containing 250 mg of MRP/ml. The antibacterial action of chitosan and its MRP need to be further investigated.
5 4 3 2 1
Because fresh noodles have a high water activity and a neutral pH, their shelf-life is relatively short. Bacillus organisms are bacteria often associated with spoilage of high water activity products, such as fresh noodles. Therefore, the application of chitosan to fresh noodles as a preservative could be useful. As shown in Fig. 3, the increase in viable bacterial cell numbers in fresh noodles prepared with and without 0.05 g/ 100 ml chitosan (in 0.5 ml/100 ml acetic acid) during storage at 4 1C. Viable cell numbers of fresh-noodle during the first 2 days of storage at 4 1C was less than 100/g. The results indicate that most of the vegetative cells were killed by heat treatment of 95 1C for 15 min. The shelf-life of fresh noodles prepared without chitosan was about 8 days at 4 1C. The addition of 0.05 g/100 ml chitosan extended shelflife by 6 days. The shelf-life of fresh noodles was extended by the application of 0.05 g/100 ml MRP prepared by heating with xylose at 95 1C for 10 h and at pH 6 for 14 days at 4 1C. Also, the addition of MRP to fresh noodles caused a slight discoloration of the product.
0 0
50
100 150 200 250 chitosan concentration ug / ml of medium
300
Fig. 2. Inhibitory effect of chitosan and MRP of different concentration on Bacillus subtilis CCRC 10258. The MRP were prepared by heating chitosan and xylose at pH 6 to 95 1C for 10 h. Results are the means7standard deviation of the three replications) () chitosan; (m) MRP.
4. Conclusion In conclusion, the antibacterial activity of chitosan was revealed to be further enhanced by its Maillard reaction at 95 1C for 10 h and at pH 6 with xylose. MRP was bactericidal to B. subtilis CCRC 10258 and its addition
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9 8 7
log CFU/ml
6 5 4 3 2 1 0 0
5
10
15 20 Storage time (Days)
25
30
Fig. 3. Changes in viable bacteria count on fresh noodles as affected by chitosan and MRP at 4 1C. Results were the means7standard deviation of the three replications () control/0.5 ml/100 ml acetic acid; ( ) 0.05 g/ 100 ml chitosan in 0.5 ml/100 ml acetic acid; (m) 0.05 g/100 ml MRP in 0.5 ml/100 ml acetic acid.
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