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Functional properties of nisin–carbohydrate conjugates formed by radiation induced Maillard reaction
Radiation Physics and Chemistry 81 (2012) 1917–1922
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Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Functional properties of nisin–carbohydrate conjugates formed by radiation induced Maillard reaction Shobita R. Muppalla a, Rahul Sonavale b, Surinder P. Chawla a,n, Arun Sharma a a b
Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India Rajiv Gandhi Institute of IT and Biotechnology, Pune Satara Road, Katraj-Dhankawadi, Pune 411043, India
H I G H L I G H T S c c c
Nisin–carbohydrate conjugates were prepared using radiation induced Maillard reaction. Conjugation of nisin with dextran/glucose resulted in improvement of antibacterial spectrum. Conjugates of nisin with dextran/glucose had significant radical scavenging activity.
a r t i c l e i n f o
abstract
Article history: Received 2 May 2012 Accepted 17 July 2012 Available online 24 July 2012
Nisin–carbohydrate conjugates were prepared by irradiating nisin either with glucose or dextran. Increase in browning and formation of intermediate products was observed with a concomitant decrease in free amino and reducing sugar groups indicating occurrence of the Maillard reaction catalyzed by irradiation. Nisin–carbohydrate conjugates showed a broad spectrum antibacterial activity against Gram negative bacteria (Escherichia coli, Pseudomonas fluorescence) as well as Gram positive bacteria (Staphylococcus aureus, Bacillus cereus). Results of antioxidant assays, including that of DPPH radical-scavenging activity and reducing power, showed that the nisin–dextran conjugates possessed better antioxidant potential than nisin–glucose conjugate. These results suggested that it was possible to enhance the functional properties of nisin by preparing radiation induced conjugates suitable for application in food industry. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Nisin Maillard reaction Conjugates Radiation Antibacterial activity
1. Introduction Nisin is a bacteriocin, an antimicrobial peptide produced by several strains of Lactococcus lactis and recognized as GRAS by the United States Food and Drug Administration as stated in the Code of Federal Regulations (CFR Section 184.1538). Due to its antibacterial activity, attempts have been made to use it as a natural preservative in foods. Nisin has been used to preserve salad dressings (Delves-Broughton et al., 1996), canned foods (Thomas et al., 2000) or meat (Cutter and Siragusa, 1995). Nisin inhibits several Gram-positive bacteria such as Listeria spp., Staphylococcus spp. but does not inactivate a majority of Gram-negative bacteria. However, it has been reported to be efficient against Gramnegative bacteria when used together with chelating agents (EDTA) that affects bacterial cell membrane lipopolysaccharide component (Cutter and Siragusa, 1995). Enhanced antimicrobial activity and
n Corresponding author. Tel.: þ91 22 25593296/25595374; fax: þ 91 22 25505151/25519613. E-mail address: [email protected] (S.P. Chawla).
0969-806X/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radphyschem.2012.07.009
improved functional properties make nisin a very desirable food additive which can be used in a vast variety of food systems under different conditions. A number of studies to improve protein functionality have involved chemical modification, such as alkylation, esterification, amidination, deamidination, covalent attachment of carbohydrates and fatty acids, thiol-disulfide exchange and enzymatic modification (Kato et al., 1988; Scaman et al., 2006). Among the various modifications applied to food proteins, covalent coupling of carbohydrates through the Maillard-type reaction appears to produce marked changes in the functional properties (Nakamura et al., 1991). Maillard reaction is classified as non-enzymatic browning and involves conjugation of reducing sugars with amino acids or proteins that progresses into a complex network of reaction products that are collectively known as the Maillard reaction products (MRPs) upon heating (Ho, 1996). MRPs have been shown to have antioxidant activity in both the chemical model and food systems (Alaiz et al., 1996; Wijewickreme and Kitts, 1997). Novel technologies that involve non-thermal processes have been investigated as full or partial alternatives to conventional heat treatment. Guan et al. (2010) reported the
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characteristics of MRPs produced by low frequency ultrasound in a glycine–maltose model system and their antioxidant activities. Similarly, pulsed electric field treatment was used to promote the Maillard reaction in glycine–glucose solution (Wang et al., 2011). Using electromagnetic waves in chemical synthesis can significantly reduce the reaction time and improve yield, selectivity and purity of the product, compared with conventional heating methods (Cai et al., 2004). There is a growing scientific interest in the influence of irradiation processes on synthesis of compounds with improved properties. In this reaction, no other chemical reagents are introduced and there is no need to control temperature, environment or additives. The majority of chemical changes caused due to radiation are similar to those of other processing methods. Effect of radiation processing on safety has been repeatedly evaluated by a number of independent expert groups. The expert study group concluded that food irradiated at any dose appropriate to achieve the intended technological objective is safe to consume (WHO, 1999). Non-enzymatic browning in gamma-irradiated aqueous solutions of different carbohydrates with amino acids or amino group containing compounds and their antioxidant potentials have been reported (Chawla et al., 2007, 2009; Oh et al., 2006; Rao et al., 2011). One of the major advantages of the radiation induced Maillard reaction is that cytotoxic compounds like 5-hydroxymethyl furfurals are not formed (Oh et al., 2006; Rao et al., 2011). The objective of present study was to investigate radiation induced conjugation of nisin with dextran and glucose. The antibacterial and antioxidant activities of these conjugates were also determined.
The fluorescence intensity was measured at an excitation wavelength of 365 nm and emission wavelength of 440 nm using a fluorescence spectrophotometer.
2. Material and methods
Electron-donating ability of radiation induced MRPs was determined by employing a DPPH radical scavenging assay (Yamaguchi et al., 1998). Superoxide anion scavenging activity of MRPs was determined according to the method described by Liu et al. (1997). Decreased absorbance of the reaction mixture indicated increased radical scavenging activity. The radical scavenging activity of DPPH and superoxide anion was calculated using the following formula: Radical scavenging activity % ¼[(A0 As)/A0] 100. where A0 is the absorbance of control and As is the absorbance of sample.
2.1. Chemicals Nisin, glucose, dextran (MW: 60,000–90,000) and microbiological media were purchased from HiMedia (Mumbai India). 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade and procured from Qualigens Fine Chemicals (Mumbai, India) or S. D. Fine Chemicals (Mumbai, India).
2.4. Determination of free amino group and reducing sugar content Free amino group content was determined by the ninhydrin method described by Doi et al. (1981). The reducing sugar measurement was done by the dinitrosalicyclic acid method (Miller, 1959).
2.5. Antibacterial activity Antibacterial activity was assessed against S. aureus, B. cereus, P. fluorescence and E. coli. The cultures were inoculated in nutrient broth and incubated for 18 h at 37 1C on a shaker incubator. The cells were collected by centrifugation at 10,000 rpm at 4 1C for 20 min. After collection, the cells were washed with phosphate buffer saline (PBS, pH 7.2) twice. Final concentration was fixed at 105 cells/ml of PBS. Initial counts were estimated by the spread plating method on nutrient agar. Pure nisin and its conjugates were added at different concentrations of nisin (IU) to the tubes containing bacterial cells. Tubes were kept at room temperature for 24 h and final counts were estimated by the plating method.
2.6. Determination of radical scavenging activity
2.2. Microorganisms Escherichia coli JM109, Pseudomonas fluorescens (lab isolate), Staphylococcus aureus ATCC 6538P, Bacillus cereus MTCC 470 cultures were maintained at 4 1C. The long-term storage of cultures was done in 20% glycerol (v/v) at 20 1C. The isolates were subcultured twice before inoculation. 2.3. Preparation and spectrophotometric analyses of nisin– carbohydrate conjugates Nisin:glucose, nisin:dextran powder were taken in 1:5 ratios (w/w) and dissolved in 0.02 M HCl. The solutions were irradiated at 40 kGy using Gamma Cell 5000 (Board of Radioisotope Technology (BRIT), India) at a dose rate of 6 kGy/h at ambient temperature. Dosimetry was performed using a cerric–cerrous dosimeter calibrated against Fricke’s dosimeter. Dosimetry intercomparison was carried out with the National Standards established by Radiological Physics and Advisory Division (RP & AD), Bhabha Atomic Research Centre (BARC), Mumbai, India. Spectrophotometric changes were monitored according to the method described by Chawla et al. (2009). UV absorbance (284 nm) and browning (420 nm) were analyzed after appropriate dilutions. Fluorescence of samples was determined after 100-fold dilution.
2.7. Determination of reducing power Reducing power was determined by the method described by Yen and Duh (1993). Appropriately diluted sample (1 ml) was added to 2.5 ml of phosphate buffer (200 mM, pH 6.6) followed by 2.5 ml of 1% potassium ferricyanide. The reaction mixture was incubated for 20 min in a water bath at 50 1C. After incubation, 2.5 ml of 10% trichloroacetic acid was added, followed by centrifugation at 3000 rpm for 10 min. The upper layer (5 ml) was mixed with 5 ml distilled water and 1 ml of 0.1% ferric chloride. Absorbance of the resultant solution was measured at 700 nm. A high absorbance was indicative of strong reducing power.
2.8. Statistical analysis All results given in the figures are mean 7standard deviation. Differences between the variables were tested for significance by a one-way ANOVA with Tukey’s post-test using a GraphPad InStat version 3.05 for window 95, GraphPad Software, San Diego California, USA, http://www.graphpad.com. Differences at po0.05 were considered to be significant.
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3. Results and discussion
3.2. Reduction in amino group and reducing sugar content
In present study, the formation of the Maillard reaction products (MRPs) by gamma irradiation of aqueous solution of nisin with glucose/dextran was investigated.
Changes in free amino group and reducing sugar content of MRPs after irradiation are depicted in Fig. 2. Decrease in free amino and reducing sugar groups in MRP samples was observed when compared to unirradiated samples. These results suggested that NH2 group of amino acids in nisin is covalently attached to carbonyl group to form glycated product resulting in formation of MRPs. This indicated that radiation catalyzed the interaction between free amino groups of nisin and carbonyl group via glycation process. From the results, it is obvious that the decrease in free amino group and carbonyl group was in accordance with the increase in browning at 420 nm (Fig. 1b). Reduction in reducing sugar content during heat-induced Maillard reaction in fructose/lysine (Ajandouz et al., 2001) and radiation induced Maillard reaction in whey protein dispersion (Chawla et al., 2009) has been reported. The Maillard reaction involves a condensation reaction between the carbonyl group of a reducing sugar (dextran/gluocose) with an available amine group; in this case nisin, forming a Schiff base with release of water. The Schiff base that is formed subsequently cyclizes to the corresponding Nglycosylamine, which then undergoes an irreversible Amadori rearrangement to produce the Amadori product. Subsequently, degradation of the Amadori product occurs through a series of reaction forming the Maillard reaction products . Extent of reduction was more in case of nisin–dextran as compared to the nisin–glucose system. The differential reactivity of different carbohydrates observed might be explained in terms of differences in conformations, sizes, or solubility of these molecules. In case of the heat induced Maillard reaction, a linear correlation between the degree of substitution and the saccharide size has been reported (Nacka et al., 1998). The conjugation of proteins with small carbohydrate molecules such as glucose or lactose is liable to react with most lysyl residues exposed outside and to result in insoluble aggregates due to the progressive side reaction. To improve the functional properties of proteins, therefore, their conjugation with polysaccharides, but not with oligosaccharides, is desirable for industrial applications.
3.1. Formation of nisin–carbohydrate conjugates There are a number of reports of structural and chemical modification of protein with reducing sugar, following the Maillard reaction resulting in characteristic changes like fluorescence and browning. Effect of gamma irradiation (40 kGy) on UV absorbance, browning and fluorescence are shown in Fig. 1. The formation of nisin–carbohydrate MRPs through radiation induced the Maillard reaction was confirmed by increase in UV absorbance of solutions (Fig. 1a). The Maillard reaction is associated with the development of UV-absorbing intermediate compounds, prior to generation of brown pigments. UV-absorbing intermediate compounds are also formed prior to radiation-induced MRPs (Chawla et al., 2007). It can be seen that the browning intensity for nisin– dextran and nisin–glucose solutions also increased with the absorbed dose (Fig. 1b). Development of brown color, due to the formation of chromophores, has been widely studied in different model systems, and studies on melanoidin formation have been summarized (Rizzi, 1997). In the final stage of the browning reaction, the intermediates polymerize and colored polymers are formed. These findings suggest that irradiation leads to nonenzymatic browning reactions, similar to those induced by heating. The Maillard reaction is also associated with the development of fluorescent compounds. In the present study, formation of fluorescent compounds was observed in irradiated nisin–glucose and nisin–dextran solution suggesting formation of MRPs (Fig. 1c). Similar increases in UV absorbance, browning and fluorescence have been reported in sugar–amino acid solution, whey protein dispersion (Chawla et al., 2007, 2009) and chitosan– glucose solutions (Rao et al., 2011). It can be seen that UV absorbance, browning and fluorescence were greater in case of nisin–dextran conjugate when compared to that in case of nisin– glucose conjugate. Edimecheva et al. (2005) have reported the process of rupture of glycosidic bond is main reaction observed during radiolysis of disaccharides and polysaccharides. Radiation leads to breakage of glycosidic bonds in dextran and so more number of carbonyl groups are available for formation of MRPs.
Nisin is an effective bactericidal agent against Gram positive bacteria including strains of Lactococcus, Streptococcus, Staphylococcus, Micrococcus, Pediococcus, Lactobacillus, Listeria and Mycobacterium.
Fig. 1. Spectrometric analyses of nisin–carbohydrate conjugates formed by the radiation induced Maillard reaction.: (a) UV absorbance and browning and (b) fluorescence.
Fig. 2. Reduction in amino/reducing sugar group in nisin–carbohydrate conjugates by radiation treatment.
3.3. Antibacterial activity of nisin–carbohydrate conjugates
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Gram-positive spore formers like Bacillus and Clostridium spp. are particularly susceptible to nisin, with spores being more sensitive than vegetative cells (Delves-Broughton et al., 1996). The effect of nisin on the target bacteria in vegetative cells is exerted at the cytoplasmic membrane. Nisin forms pores that disrupt the proton motive force and the pH equilibrium causing leakage of ions and hydrolysis of ATP resulting in cell death. Normally, Gram-negative cells are resistant to nisin due to lipopolysaccharidic (LPS) composition at its outer layer which acts as a barrier to the action of the nisin on the cytoplasmic membrane. In the present study, nisin showed good activity against S. aureus and B. cereus (Fig. 3). Nisin-conjugates also showed activity against these bacteria and their minimum bactericidal concentrations (MBC) were less than nisin. Abdullah et al. (2010) reported the reduction in nisin’s antibacterial activity against various Gram-positive bacteria as a result of glycation with glucose. Most of the investigations have shown that the modified enzymes retain their antimicrobial properties in spite of loss of some lytic activity (Ibrahim et al., 1991). The antimicrobial effects of lysozyme–dextran conjugate were reported to be almost same as those of lysozyme for S. aureus and were more lethal than those of lysozyme for B. cereus (Nakamura et al., 1991). As expected, nisin did not show any activity against E. coli and P. fluorescens, whereas, both nisin–dextran and nisin–glucose conjugates showed antibacterial activity against these Gram-negative bacteria. The MBC obtained
was high for Gram negative bacteria than for S. aureus or B. cereus. Cutter and Siragusa (1995) demonstrated that the combination of nisin with chelators showed antibacterial activity against E. coli and Salmonella spp. in buffer. With regard to the MRPs, it has been reported that the MRPs destabilize the outer membrane and inhibit the growth of bacterial cells, due to their excellent surfactant properties (Nakamura et al., 1991). The lysozyme–dextran MRPs were found to be effective against E. coli in cheese curd during storage period (Amiri et al., 2008). Similarly, the conjugates of chitosan with soy protein, b-lacto globulin and glucosamine are all reported to enhance bactericidal action (Chung et al., 2005; Miralles et al., 2007; Usui et al., 2004). Nisin–dextran MRPs showed better antibacterial activity than nisin–glucose MRPs which may be due to better antioxidant activity as discussed in later part. The antimicrobial activity of MRPs is supposed to be due to the interference with the uptake of serine, glucose, and oxygen (Einarsson, 1987), inhibition of the carbohydrate catabolizing enzymes of the microorganisms (Lanciotti et al., 1999) or their potential antioxidant activities (Mattila and Sandholm, 1989). 3.4. Radical scavenging activity The DPPH radical is scavenged by an antioxidant in the system by donation of hydrogen to form a stable DPPH-H molecule. The color changes from purple to yellow by acceptance of a hydrogen
Fig. 3. Antibacterial activity of nisin–carbohydrate conjugates against Gram positive and Gram negative bacteria. The results shown are mean 7 SD of three independent experiments.
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disaccharides differ and that reaction products obtained from monosaccharides are different from those obtained from disaccharides (Kato et al., 1989). The radical-scavenging activity of the conjugates can be attributed to the advanced Maillard reaction product melanoidins, which show high antioxidant capacity. Radical-scavenging activity correlated well with browning intensity. UV absorbance, browning and fluorescence changes are good indirect indices to monitor the formation of MRPs with free radical-scavenging activity (Morales and Jimenez-Perez, 2001). 3.5. Reducing power
Fig. 4. Radical scavenging activity of nisin–carbohydrate conjugates formed by the radiation induced Maillard reaction. The results shown are mean 7 SD of three independent experiments.
The ability of MRPs to act as reducing agents through the donation of electrons to form more stable products was measured by the reducing power method. This method measures the ability of reducing the Fe3 þ –ferricyanide complex to the ferrous form. The reducing power of nisin-MRPs (Fig. 5) showed similar trends with those of radical scavenging activity. Nisin–dextran exhibited greater reducing power than the nisin–glucose and nisin. Hydroxyl groups of MRPs play a role in the reducing activity (Yoshimura et al., 1997). Studies have indicated that the antioxidant effect is related to the development of reductones that are terminators of free radical chain reactions (Shon et al., 2003).
4. Conclusion In present study, novel conjugates based on the nisin and carbohydrates (dextran/glucose) were developed using the radiation induced Maillard reaction. Browning and fluorescence of the irradiated solution indicated the presence of the Maillard reaction products. The major effect was extension of nisin activity towards Gram negative bacteria along with improved antioxidant activity. Thus, nisin–dextran and nisin–glucose MRPs were endowed with a broad spectrum antibacterial and antioxidant activities by radiation induced the Maillard reaction, and can be developed as a promising additive for food preservation. References
Fig. 5. Reducing power of nisin–carbohydrate conjugates formed by the radiation induced Maillard reaction. The results shown are mean 7 SD of three independent experiments.
atom from antioxidants and it becomes a stable diamagnetic molecule. Superoxide radicals contribute to the oxidation of food constituent, especially fats. Although they do not directly initiate lipid oxidation, superoxide radical anions are precursors of highly reactive hydroxyl radical, which contributes to lipid peroxidation in food systems. Thus superoxide anion scavenging activity indirectly contributes to antioxidant potential. The results of DPPH radical and superoxide scavenging activities of nisin MRPs were shown in Fig. 4. Nisin had negligible activity whereas nisinMRPs showed strong DPPH radical and superoxide radical scavenging activity. These findings suggest that Maillard reaction induced conjugation has the potential to improve radical scavenging potential of nisin–carbohydrate conjugates. At the same concentration used, MRP samples derived from glucose showed a lower radical-scavenging activity. MRPs derived from dextran showed 67% and 56% DPPH and superoxide radical scavenging activity. Benjakul et al. (2005) reported that MRPs derived from galactose, possessed greater DPPH radical-scavenging activity than those prepared from fructose and glucose. Several investigations have shown that the reaction mechanisms of monosaccharides and
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Radiation Physics and Chemistry journal homepage: www.elsevier.com/locate/radphyschem
Functional properties of nisin–carbohydrate conjugates formed by radiation induced Maillard reaction Shobita R. Muppalla a, Rahul Sonavale b, Surinder P. Chawla a,n, Arun Sharma a a b
Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085, India Rajiv Gandhi Institute of IT and Biotechnology, Pune Satara Road, Katraj-Dhankawadi, Pune 411043, India
H I G H L I G H T S c c c
Nisin–carbohydrate conjugates were prepared using radiation induced Maillard reaction. Conjugation of nisin with dextran/glucose resulted in improvement of antibacterial spectrum. Conjugates of nisin with dextran/glucose had significant radical scavenging activity.
a r t i c l e i n f o
abstract
Article history: Received 2 May 2012 Accepted 17 July 2012 Available online 24 July 2012
Nisin–carbohydrate conjugates were prepared by irradiating nisin either with glucose or dextran. Increase in browning and formation of intermediate products was observed with a concomitant decrease in free amino and reducing sugar groups indicating occurrence of the Maillard reaction catalyzed by irradiation. Nisin–carbohydrate conjugates showed a broad spectrum antibacterial activity against Gram negative bacteria (Escherichia coli, Pseudomonas fluorescence) as well as Gram positive bacteria (Staphylococcus aureus, Bacillus cereus). Results of antioxidant assays, including that of DPPH radical-scavenging activity and reducing power, showed that the nisin–dextran conjugates possessed better antioxidant potential than nisin–glucose conjugate. These results suggested that it was possible to enhance the functional properties of nisin by preparing radiation induced conjugates suitable for application in food industry. & 2012 Elsevier Ltd. All rights reserved.
Keywords: Nisin Maillard reaction Conjugates Radiation Antibacterial activity
1. Introduction Nisin is a bacteriocin, an antimicrobial peptide produced by several strains of Lactococcus lactis and recognized as GRAS by the United States Food and Drug Administration as stated in the Code of Federal Regulations (CFR Section 184.1538). Due to its antibacterial activity, attempts have been made to use it as a natural preservative in foods. Nisin has been used to preserve salad dressings (Delves-Broughton et al., 1996), canned foods (Thomas et al., 2000) or meat (Cutter and Siragusa, 1995). Nisin inhibits several Gram-positive bacteria such as Listeria spp., Staphylococcus spp. but does not inactivate a majority of Gram-negative bacteria. However, it has been reported to be efficient against Gramnegative bacteria when used together with chelating agents (EDTA) that affects bacterial cell membrane lipopolysaccharide component (Cutter and Siragusa, 1995). Enhanced antimicrobial activity and
n Corresponding author. Tel.: þ91 22 25593296/25595374; fax: þ 91 22 25505151/25519613. E-mail address: [email protected] (S.P. Chawla).
0969-806X/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.radphyschem.2012.07.009
improved functional properties make nisin a very desirable food additive which can be used in a vast variety of food systems under different conditions. A number of studies to improve protein functionality have involved chemical modification, such as alkylation, esterification, amidination, deamidination, covalent attachment of carbohydrates and fatty acids, thiol-disulfide exchange and enzymatic modification (Kato et al., 1988; Scaman et al., 2006). Among the various modifications applied to food proteins, covalent coupling of carbohydrates through the Maillard-type reaction appears to produce marked changes in the functional properties (Nakamura et al., 1991). Maillard reaction is classified as non-enzymatic browning and involves conjugation of reducing sugars with amino acids or proteins that progresses into a complex network of reaction products that are collectively known as the Maillard reaction products (MRPs) upon heating (Ho, 1996). MRPs have been shown to have antioxidant activity in both the chemical model and food systems (Alaiz et al., 1996; Wijewickreme and Kitts, 1997). Novel technologies that involve non-thermal processes have been investigated as full or partial alternatives to conventional heat treatment. Guan et al. (2010) reported the
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characteristics of MRPs produced by low frequency ultrasound in a glycine–maltose model system and their antioxidant activities. Similarly, pulsed electric field treatment was used to promote the Maillard reaction in glycine–glucose solution (Wang et al., 2011). Using electromagnetic waves in chemical synthesis can significantly reduce the reaction time and improve yield, selectivity and purity of the product, compared with conventional heating methods (Cai et al., 2004). There is a growing scientific interest in the influence of irradiation processes on synthesis of compounds with improved properties. In this reaction, no other chemical reagents are introduced and there is no need to control temperature, environment or additives. The majority of chemical changes caused due to radiation are similar to those of other processing methods. Effect of radiation processing on safety has been repeatedly evaluated by a number of independent expert groups. The expert study group concluded that food irradiated at any dose appropriate to achieve the intended technological objective is safe to consume (WHO, 1999). Non-enzymatic browning in gamma-irradiated aqueous solutions of different carbohydrates with amino acids or amino group containing compounds and their antioxidant potentials have been reported (Chawla et al., 2007, 2009; Oh et al., 2006; Rao et al., 2011). One of the major advantages of the radiation induced Maillard reaction is that cytotoxic compounds like 5-hydroxymethyl furfurals are not formed (Oh et al., 2006; Rao et al., 2011). The objective of present study was to investigate radiation induced conjugation of nisin with dextran and glucose. The antibacterial and antioxidant activities of these conjugates were also determined.
The fluorescence intensity was measured at an excitation wavelength of 365 nm and emission wavelength of 440 nm using a fluorescence spectrophotometer.
2. Material and methods
Electron-donating ability of radiation induced MRPs was determined by employing a DPPH radical scavenging assay (Yamaguchi et al., 1998). Superoxide anion scavenging activity of MRPs was determined according to the method described by Liu et al. (1997). Decreased absorbance of the reaction mixture indicated increased radical scavenging activity. The radical scavenging activity of DPPH and superoxide anion was calculated using the following formula: Radical scavenging activity % ¼[(A0 As)/A0] 100. where A0 is the absorbance of control and As is the absorbance of sample.
2.1. Chemicals Nisin, glucose, dextran (MW: 60,000–90,000) and microbiological media were purchased from HiMedia (Mumbai India). 1, 1-Diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade and procured from Qualigens Fine Chemicals (Mumbai, India) or S. D. Fine Chemicals (Mumbai, India).
2.4. Determination of free amino group and reducing sugar content Free amino group content was determined by the ninhydrin method described by Doi et al. (1981). The reducing sugar measurement was done by the dinitrosalicyclic acid method (Miller, 1959).
2.5. Antibacterial activity Antibacterial activity was assessed against S. aureus, B. cereus, P. fluorescence and E. coli. The cultures were inoculated in nutrient broth and incubated for 18 h at 37 1C on a shaker incubator. The cells were collected by centrifugation at 10,000 rpm at 4 1C for 20 min. After collection, the cells were washed with phosphate buffer saline (PBS, pH 7.2) twice. Final concentration was fixed at 105 cells/ml of PBS. Initial counts were estimated by the spread plating method on nutrient agar. Pure nisin and its conjugates were added at different concentrations of nisin (IU) to the tubes containing bacterial cells. Tubes were kept at room temperature for 24 h and final counts were estimated by the plating method.
2.6. Determination of radical scavenging activity
2.2. Microorganisms Escherichia coli JM109, Pseudomonas fluorescens (lab isolate), Staphylococcus aureus ATCC 6538P, Bacillus cereus MTCC 470 cultures were maintained at 4 1C. The long-term storage of cultures was done in 20% glycerol (v/v) at 20 1C. The isolates were subcultured twice before inoculation. 2.3. Preparation and spectrophotometric analyses of nisin– carbohydrate conjugates Nisin:glucose, nisin:dextran powder were taken in 1:5 ratios (w/w) and dissolved in 0.02 M HCl. The solutions were irradiated at 40 kGy using Gamma Cell 5000 (Board of Radioisotope Technology (BRIT), India) at a dose rate of 6 kGy/h at ambient temperature. Dosimetry was performed using a cerric–cerrous dosimeter calibrated against Fricke’s dosimeter. Dosimetry intercomparison was carried out with the National Standards established by Radiological Physics and Advisory Division (RP & AD), Bhabha Atomic Research Centre (BARC), Mumbai, India. Spectrophotometric changes were monitored according to the method described by Chawla et al. (2009). UV absorbance (284 nm) and browning (420 nm) were analyzed after appropriate dilutions. Fluorescence of samples was determined after 100-fold dilution.
2.7. Determination of reducing power Reducing power was determined by the method described by Yen and Duh (1993). Appropriately diluted sample (1 ml) was added to 2.5 ml of phosphate buffer (200 mM, pH 6.6) followed by 2.5 ml of 1% potassium ferricyanide. The reaction mixture was incubated for 20 min in a water bath at 50 1C. After incubation, 2.5 ml of 10% trichloroacetic acid was added, followed by centrifugation at 3000 rpm for 10 min. The upper layer (5 ml) was mixed with 5 ml distilled water and 1 ml of 0.1% ferric chloride. Absorbance of the resultant solution was measured at 700 nm. A high absorbance was indicative of strong reducing power.
2.8. Statistical analysis All results given in the figures are mean 7standard deviation. Differences between the variables were tested for significance by a one-way ANOVA with Tukey’s post-test using a GraphPad InStat version 3.05 for window 95, GraphPad Software, San Diego California, USA, http://www.graphpad.com. Differences at po0.05 were considered to be significant.
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3. Results and discussion
3.2. Reduction in amino group and reducing sugar content
In present study, the formation of the Maillard reaction products (MRPs) by gamma irradiation of aqueous solution of nisin with glucose/dextran was investigated.
Changes in free amino group and reducing sugar content of MRPs after irradiation are depicted in Fig. 2. Decrease in free amino and reducing sugar groups in MRP samples was observed when compared to unirradiated samples. These results suggested that NH2 group of amino acids in nisin is covalently attached to carbonyl group to form glycated product resulting in formation of MRPs. This indicated that radiation catalyzed the interaction between free amino groups of nisin and carbonyl group via glycation process. From the results, it is obvious that the decrease in free amino group and carbonyl group was in accordance with the increase in browning at 420 nm (Fig. 1b). Reduction in reducing sugar content during heat-induced Maillard reaction in fructose/lysine (Ajandouz et al., 2001) and radiation induced Maillard reaction in whey protein dispersion (Chawla et al., 2009) has been reported. The Maillard reaction involves a condensation reaction between the carbonyl group of a reducing sugar (dextran/gluocose) with an available amine group; in this case nisin, forming a Schiff base with release of water. The Schiff base that is formed subsequently cyclizes to the corresponding Nglycosylamine, which then undergoes an irreversible Amadori rearrangement to produce the Amadori product. Subsequently, degradation of the Amadori product occurs through a series of reaction forming the Maillard reaction products . Extent of reduction was more in case of nisin–dextran as compared to the nisin–glucose system. The differential reactivity of different carbohydrates observed might be explained in terms of differences in conformations, sizes, or solubility of these molecules. In case of the heat induced Maillard reaction, a linear correlation between the degree of substitution and the saccharide size has been reported (Nacka et al., 1998). The conjugation of proteins with small carbohydrate molecules such as glucose or lactose is liable to react with most lysyl residues exposed outside and to result in insoluble aggregates due to the progressive side reaction. To improve the functional properties of proteins, therefore, their conjugation with polysaccharides, but not with oligosaccharides, is desirable for industrial applications.
3.1. Formation of nisin–carbohydrate conjugates There are a number of reports of structural and chemical modification of protein with reducing sugar, following the Maillard reaction resulting in characteristic changes like fluorescence and browning. Effect of gamma irradiation (40 kGy) on UV absorbance, browning and fluorescence are shown in Fig. 1. The formation of nisin–carbohydrate MRPs through radiation induced the Maillard reaction was confirmed by increase in UV absorbance of solutions (Fig. 1a). The Maillard reaction is associated with the development of UV-absorbing intermediate compounds, prior to generation of brown pigments. UV-absorbing intermediate compounds are also formed prior to radiation-induced MRPs (Chawla et al., 2007). It can be seen that the browning intensity for nisin– dextran and nisin–glucose solutions also increased with the absorbed dose (Fig. 1b). Development of brown color, due to the formation of chromophores, has been widely studied in different model systems, and studies on melanoidin formation have been summarized (Rizzi, 1997). In the final stage of the browning reaction, the intermediates polymerize and colored polymers are formed. These findings suggest that irradiation leads to nonenzymatic browning reactions, similar to those induced by heating. The Maillard reaction is also associated with the development of fluorescent compounds. In the present study, formation of fluorescent compounds was observed in irradiated nisin–glucose and nisin–dextran solution suggesting formation of MRPs (Fig. 1c). Similar increases in UV absorbance, browning and fluorescence have been reported in sugar–amino acid solution, whey protein dispersion (Chawla et al., 2007, 2009) and chitosan– glucose solutions (Rao et al., 2011). It can be seen that UV absorbance, browning and fluorescence were greater in case of nisin–dextran conjugate when compared to that in case of nisin– glucose conjugate. Edimecheva et al. (2005) have reported the process of rupture of glycosidic bond is main reaction observed during radiolysis of disaccharides and polysaccharides. Radiation leads to breakage of glycosidic bonds in dextran and so more number of carbonyl groups are available for formation of MRPs.
Nisin is an effective bactericidal agent against Gram positive bacteria including strains of Lactococcus, Streptococcus, Staphylococcus, Micrococcus, Pediococcus, Lactobacillus, Listeria and Mycobacterium.
Fig. 1. Spectrometric analyses of nisin–carbohydrate conjugates formed by the radiation induced Maillard reaction.: (a) UV absorbance and browning and (b) fluorescence.
Fig. 2. Reduction in amino/reducing sugar group in nisin–carbohydrate conjugates by radiation treatment.
3.3. Antibacterial activity of nisin–carbohydrate conjugates
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Gram-positive spore formers like Bacillus and Clostridium spp. are particularly susceptible to nisin, with spores being more sensitive than vegetative cells (Delves-Broughton et al., 1996). The effect of nisin on the target bacteria in vegetative cells is exerted at the cytoplasmic membrane. Nisin forms pores that disrupt the proton motive force and the pH equilibrium causing leakage of ions and hydrolysis of ATP resulting in cell death. Normally, Gram-negative cells are resistant to nisin due to lipopolysaccharidic (LPS) composition at its outer layer which acts as a barrier to the action of the nisin on the cytoplasmic membrane. In the present study, nisin showed good activity against S. aureus and B. cereus (Fig. 3). Nisin-conjugates also showed activity against these bacteria and their minimum bactericidal concentrations (MBC) were less than nisin. Abdullah et al. (2010) reported the reduction in nisin’s antibacterial activity against various Gram-positive bacteria as a result of glycation with glucose. Most of the investigations have shown that the modified enzymes retain their antimicrobial properties in spite of loss of some lytic activity (Ibrahim et al., 1991). The antimicrobial effects of lysozyme–dextran conjugate were reported to be almost same as those of lysozyme for S. aureus and were more lethal than those of lysozyme for B. cereus (Nakamura et al., 1991). As expected, nisin did not show any activity against E. coli and P. fluorescens, whereas, both nisin–dextran and nisin–glucose conjugates showed antibacterial activity against these Gram-negative bacteria. The MBC obtained
was high for Gram negative bacteria than for S. aureus or B. cereus. Cutter and Siragusa (1995) demonstrated that the combination of nisin with chelators showed antibacterial activity against E. coli and Salmonella spp. in buffer. With regard to the MRPs, it has been reported that the MRPs destabilize the outer membrane and inhibit the growth of bacterial cells, due to their excellent surfactant properties (Nakamura et al., 1991). The lysozyme–dextran MRPs were found to be effective against E. coli in cheese curd during storage period (Amiri et al., 2008). Similarly, the conjugates of chitosan with soy protein, b-lacto globulin and glucosamine are all reported to enhance bactericidal action (Chung et al., 2005; Miralles et al., 2007; Usui et al., 2004). Nisin–dextran MRPs showed better antibacterial activity than nisin–glucose MRPs which may be due to better antioxidant activity as discussed in later part. The antimicrobial activity of MRPs is supposed to be due to the interference with the uptake of serine, glucose, and oxygen (Einarsson, 1987), inhibition of the carbohydrate catabolizing enzymes of the microorganisms (Lanciotti et al., 1999) or their potential antioxidant activities (Mattila and Sandholm, 1989). 3.4. Radical scavenging activity The DPPH radical is scavenged by an antioxidant in the system by donation of hydrogen to form a stable DPPH-H molecule. The color changes from purple to yellow by acceptance of a hydrogen
Fig. 3. Antibacterial activity of nisin–carbohydrate conjugates against Gram positive and Gram negative bacteria. The results shown are mean 7 SD of three independent experiments.
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disaccharides differ and that reaction products obtained from monosaccharides are different from those obtained from disaccharides (Kato et al., 1989). The radical-scavenging activity of the conjugates can be attributed to the advanced Maillard reaction product melanoidins, which show high antioxidant capacity. Radical-scavenging activity correlated well with browning intensity. UV absorbance, browning and fluorescence changes are good indirect indices to monitor the formation of MRPs with free radical-scavenging activity (Morales and Jimenez-Perez, 2001). 3.5. Reducing power
Fig. 4. Radical scavenging activity of nisin–carbohydrate conjugates formed by the radiation induced Maillard reaction. The results shown are mean 7 SD of three independent experiments.
The ability of MRPs to act as reducing agents through the donation of electrons to form more stable products was measured by the reducing power method. This method measures the ability of reducing the Fe3 þ –ferricyanide complex to the ferrous form. The reducing power of nisin-MRPs (Fig. 5) showed similar trends with those of radical scavenging activity. Nisin–dextran exhibited greater reducing power than the nisin–glucose and nisin. Hydroxyl groups of MRPs play a role in the reducing activity (Yoshimura et al., 1997). Studies have indicated that the antioxidant effect is related to the development of reductones that are terminators of free radical chain reactions (Shon et al., 2003).
4. Conclusion In present study, novel conjugates based on the nisin and carbohydrates (dextran/glucose) were developed using the radiation induced Maillard reaction. Browning and fluorescence of the irradiated solution indicated the presence of the Maillard reaction products. The major effect was extension of nisin activity towards Gram negative bacteria along with improved antioxidant activity. Thus, nisin–dextran and nisin–glucose MRPs were endowed with a broad spectrum antibacterial and antioxidant activities by radiation induced the Maillard reaction, and can be developed as a promising additive for food preservation. References
Fig. 5. Reducing power of nisin–carbohydrate conjugates formed by the radiation induced Maillard reaction. The results shown are mean 7 SD of three independent experiments.
atom from antioxidants and it becomes a stable diamagnetic molecule. Superoxide radicals contribute to the oxidation of food constituent, especially fats. Although they do not directly initiate lipid oxidation, superoxide radical anions are precursors of highly reactive hydroxyl radical, which contributes to lipid peroxidation in food systems. Thus superoxide anion scavenging activity indirectly contributes to antioxidant potential. The results of DPPH radical and superoxide scavenging activities of nisin MRPs were shown in Fig. 4. Nisin had negligible activity whereas nisinMRPs showed strong DPPH radical and superoxide radical scavenging activity. These findings suggest that Maillard reaction induced conjugation has the potential to improve radical scavenging potential of nisin–carbohydrate conjugates. At the same concentration used, MRP samples derived from glucose showed a lower radical-scavenging activity. MRPs derived from dextran showed 67% and 56% DPPH and superoxide radical scavenging activity. Benjakul et al. (2005) reported that MRPs derived from galactose, possessed greater DPPH radical-scavenging activity than those prepared from fructose and glucose. Several investigations have shown that the reaction mechanisms of monosaccharides and
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