食品中有机化学论文

pr o , So (M 9, e m rox f FL oin idan ecr � 2012 Elsevier Ltd. All rights reserved. s) deri rbonyl . Sever vity in wla, Ch , & N es, 200 sidered Antioxidants function at each stage of lipid oxidation via differ- ent mechanisms, hence it is crucial to apply the correct knowledge in order to select antioxidants. Likewise, it is advised to implement more than one assay when evaluating the antioxidative capacity of food components, since a single assay cannot determine the effec- system and reaction temperature and time with a view to identify- ing those combinations which give the best performance in terms of overall antioxidant capacity. 2. Materials and methods 2.1. Chemicals and reagents Fructose, 1,1-diphenyl-2-picryl-hydrazyl (DPPH), ferric chloride (FeCl3), 2,20-azobis(2-methylpropionamidine) di-hydrochloride ⇑ Corresponding author. Address: Department of Food Technology, Faculty of Applied Sciences, Cape Peninsula University of Technology, P.O. Box 1906, Bellville 7535, South Africa. Tel.: +27 (0)21 953 8746; fax: +27 (0)21 959 6095. Food Chemistry 137 (2013) 92–98 Contents lists available at Food Che lse E-mail address: vhanganil@cput.ac.za (L.N. Vhangani). nent. Moreover, several mechanisms of their antioxidant activity have been identified, including radical chain-breaking activity, me- tal chelation, decomposition of hydrogen peroxide and scavenging of reactive oxygen species (Gu et al., 2010). In addition, with grow- ing evidence of the role of natural food antioxidants in prevention of certain diseases, this has led to the development of a number of assays to determine their antioxidant capacity in food products. Presently the chain-breaking activity of antioxidants naturally present in food products is considered a significant parameter determining their dietary value (Ak & Gulcin, 2008). lesterol whereby the inhibitory potential of the antioxidant is as- sessed by subjecting these substrates to natural or accelerated oxidation conditions. In addition, antioxidant capacity methods feature a substrate, oxidant, initiator, intermediates and final prod- ucts (Zalueta et al., 2009). Measurement of any one of these can be used to assess antioxidant activity. However, in this study the focus was on the indirect HAT and ET methods. Therefore, the aim of the study was to measure four indices of the antioxidant activity, as well as BI of MRPs derived from model sugar-amino acid systems as a function of sugar type, pH of the 1. Introduction Maillard reaction products (MRP packaging and storage via amino-ca are common to many food systems these MRPs possess antioxidant acti they are formed or are added to (Cha Maillard, Billaud, Chow, Ordonaud Henares, Delgado-Andrade, & Moral antioxidant character, MRPs are con 0308-8146/$ - see front matter � 2012 Elsevier Ltd. A http://dx.doi.org/10.1016/j.foodchem.2012.09.030 ved during processing, compound interaction al authors proved that food products in which ander, & Sharma, 2009; icholas, 2007; Rufian- 9). As a result of their a value-added compo- tiveness of all antioxidant types (Apak et al., 2007). Furthermore, these methods are classified according to their inactivation mech- anism. They are divided into two categories, namely indirect and direct methods. Indirect methods measure the ability of a molecule to reduce a stable, artificial free radical by means of hydrogen donation or electron transfer (Laguerre, Lecomte, & Villeneuve, 2007). Hence, indirect methods are further sub-divided into hydro- gen atom transfer (HAT) and electron transfer (ET) reaction-based methods (Zalueta, Esteve, & Frigola, 2009). Direct methods, on the other hand, utilize oxidizable substrates such as lipids, DNA or cho- Radical scavenging Antioxidant (p < 0.05) reduction in pH, BI, DPPH-RS, PRS and RP than FL model systems, with no considerable differ- ences (p > 0.05) in HRS activity. Antioxidant activity of Maillard reaction from fructose–lysine and ribose–lysine m Lusani Norah Vhangani ⇑, Jessy Van Wyk Department of Food Technology, Cape Peninsula University of Technology, Bellville 7535 a r t i c l e i n f o Article history: Received 27 January 2012 Received in revised form 1 August 2012 Accepted 5 September 2012 Available online 16 September 2012 Keywords: Maillard reaction products Browning a b s t r a c t Maillard reaction products (FL) model systems at pH (BI) and pH reduction wer RS), peroxyl (PRS), and hyd oxidant activity. The pH o increased. This reduction c of HRS activity, the antiox while that of RL systems d journal homepage: www.e ll rights reserved. oducts (MRPs) derived del systems uth Africa RPs) were prepared from aqueous ribose–lysine (RL) and fructose–lysine heated at 60, 80 and 120 �C for 15, 60 and 120 min. Browning intensity onitored throughout the reaction. 1,1-Diphenyl-2-picryl-hydrazyl (DPPH- yl radical scavenging (HRS) and reducing power (RP) measured their anti- and RL system decreased (p < 0.05) as reaction temperatures and times cided with the increase (p < 0.05) in BI for all MRPs. With the exception t activity of FL increased (p < 0.05) with increased reaction temperature, eased (p < 0.05). Concerning sugar reactivity, RL systems exhibited higher SciVerse ScienceDirect mistry vier .com/locate / foodchem slight modifications. A 5 ml aliquot of 0.1 mM PGR was mixed with chloride; 1 mM EDTA; 1 mM ascorbic acid; 30 mM deoxyribose; buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide. The reac- od C (ABAP), ethylenediaminetetraacetic acid (EDTA) and trichloroace- tic acid (TCA) were obtained from Merck (Modderfontein, South Africa). Ribose, lysine, pyrogallol red (PGR), thiobarbituric acid (TBA) were obtained from Sigma (Aston Manor, South Africa) and deoxyribose from separations (Randburg, South Africa). All the chemicals used in this study were of analytical grade and chemical reagents were prepared according to standard analytical proce- dures. Prepared reagents were stored under conditions that pre- vented deterioration or contamination. The water used in the study was ultrapure water purified with a Milli-Q water purifica- tion system (Millipore, Microsep, Bellville, SA). 2.2. Preparation of the sugar-amino acid model Maillard reaction products (MRPs) Sugar-amino acid MRPs were prepared following the procedure described by Kim and Lee (2008) with slight modifications. Equi- molar (0.8 M) amounts of sugars (fructose and ribose) and amino acid (lysine) were each dissolved in 100 ml of 0.1 M Tris(hydroxy- methyl) aminomethane/hydrochloric acid buffer at pH 9. Equal volumes (50 ml) of each sugar and an amino acid were mixed to make up 100 ml solutions at final concentrations of 0.4 M. The solutions were transferred into 200 ml media bottles and heated at 60, 80 and 121 �C for 15, 60 and 120 min at each temperature. In total, eighteen model systems were generated. The resultant MRPs were immediately cooled down in an ice bath. A portion of the MRPs was retained for pH measurement, while the remainder was freeze-dried and stored at �18 �C until analysis. The powder was reconstituted before use to the required concentrations with Milli-Q water. 2.3. pH measurement The pH was measured using an 827 Lab pH metre (Metrohm, Switzerland) calibrated with buffer solutions of pH 4.0 and 7.0, respectively. 2.4. Measurement of browning intensity The method for determining the browning intensity (BI) of MRPs was according to Kim and Lee (2008). MRP samples were di- luted 400-fold in phosphate buffered-saline at pH 7.4. The BI was measured at a wavelength of 420 nm in a spectrophotometer (Lambda 25, Perkin Elmer, Singapore). 2.5. Determination of 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical scavenging activity of MRPs DPPH radical scavenging (DPPH-RS) of MRPs was determined according to the method described by Lertittikul, Benjakul, and Tanaka (2007). A 0.4 ml aliquot of each MRP sample (500-fold dilu- tion) was added to 2 ml of a 0.12 mM DPPH solution in ethanol. The mixture was vortexed (Vortex Genie 2, Scientific Industry Inc, USA) and allowed to stand at room temperature in the dark for 30 min. The absorbance of the reaction mixture was measured at 517 nm in a spectrophotometer (Lambda 25, Perkin Elmer, Sin- gapore). The control was prepared in the same manner, with the MRP solution substituted with Milli-Q water. The DPPH-RS radical scavenging activity was calculated according to the following equation: % DPPH-RS ¼ ð1� ðAsample ð517 nmÞ=Acontrol ð517 nmÞÞ � 100 L.N. Vhangani, J. Van Wyk / Fo where: Asample (517nm) is the absorbance of 0.4 ml MRP and 2 ml DPPH in absolute ethanol and Acontrol (517nm) is the absorbance of 0.4 ml MRP and 2 ml absolute ethanol. tion mixtures were incubated in a temperature-controlled water bath at 50 �C (Memmert, Germany) for 30 min, followed by the addition of 2.5 ml of 10% trichloroacetic acid. The reaction mix- tures were centrifuged (Coulter, Beckman, USA) at 1,650�g for 10 min at 23 �C. A 2.5 ml aliquot of the supernatant was added to 1 ml of Milli-Q water and 0.5 ml of 0.1% ferric chloride. The control was prepared in similar manner, except that MRPs were replaced with Milli-Q water. After 10 min, the absorbance of the reaction mix was measured at 700 nm in a spectrophotometer (Lambda 25, Perkin Elmer, Singapore). The RP was expressed as an increase in absorbance at 700 nm. 2.9. Statistical analysis All data were subjected to One-way analysis of variance (ANO- VA). Significant differences among means of replicates (n = 2) were and 20 mM hydrogen peroxide. The resultant solution was incu- bated at 37 �C for 90 min. After incubation, 2 ml of 2% TCA and 2 ml of 1% TBA were added. The reaction mixture was heated in a boiling water bath (100 �C) (Memmert, Germany) for 15 min. The absorbance of the pink colour that developed was measured at 532 nm, using a spectrophotometer (Lambda 25, Perkin Elmer, Singapore). The control was prepared in the same manner, but in- stead of the sample solution, pure Milli-Q water was used. The per- centage of HRS activity was calculated as follows: % HRS ¼ ½ðAcontrol � AsampleÞ=Acontrol� � 100 where Acontrol is the absorbance of the control at 532 nm and Asample is the absorbance of the sample at 532 nm. 2.8. Determination of reducing power of MRPs The reducing power (RP) of MRP samples was determined according to the methods of Lertittikul et al. (2007) and Jayanthi and Lalitha (2011) with slight modifications. One ml of MRP sam- ple (500-fold dilution) was mixed with 2.5 ml of 0.2 M Phosphate 0.5 ml MRP sample in a 1:500 (w/v) ratio to Milli-Q water and 0.015 ml of 600 mM ABAP, followed by incubation at 37 �C for 2 h in a water bath (Memmert, Lasec, Germany). At the end of incu- bation, the reaction mixture was cooled in an ice bath and the absorbance measured at 540 nm. For control samples, Milli-Q water was used instead of the MRP samples. The PRS activity was calculated according to the following equation: % PRS ¼ ½ðAcontrol � AsampleÞ=Acontrol� � 100 where Acontrol is the absorbance of the control at 540 nm and Asample is the absorbance of the sample at 540 nm. 2.7. Determination of hydroxyl radical scavenging activity of MRPs Hydroxyl radical scavenging (HRS) activity of MRPs was deter- mined according to the method of Chawla et al. (2009). A 1 ml ali- quot MRP sample (500-fold dilution) was added to 1 ml of 0.1 M phosphate buffer pH 7.4 containing the following: 1 mM ferric 2.6. Determination of the peroxyl radical scavenging activity of MRPs Peroxyl radical scavenging (PRS) of MRPs was determined according to the method of Sachindra and Bhaskar (2008) with hemistry 137 (2013) 92–98 93 determined by Duncan’s multiple range tests using SPSS 19.0 for Windows�. The level of confidence required for significance was selected at p 6 0.05. 3. Results and discussion 3.1. pH reduction The Maillard reaction is characterized by reactants consump- tion, formation of initial, intermediate and complex brown poly- mers. Depending on the set parameters, the reaction rates and end-products will differ. During the MR, the pH of the model sys- tem is crucial since the initial condensation step is facilitated by higher pH values (Lertittikul et al., 2007). In this study, the change in pH values during the Maillard reaction was monitored to evalu- ate its significance during the MR. Fig. 1 shows the reduction in the pH of all model systems as a function of heating temperature and time combinations. One-way ANOVAs with Duncan’s multiple range tests showed that the decrease in pH observed as the reac- tion temperature and time increased was significant (p < 0.05) for 94 L.N. Vhangani, J. Van Wyk / Food C each model system. This showed that higher reaction temperature and time combinations resulted in the highest pH reduction. The results of this study are in accordance with the findings of Kim and Lee (2009) and Gu et al. (2010) who also observed a decrease in pH when the heating temperature–time of both peptide–amino acid and casein–glucose model systems increased. The decrease in pH during the MR is due to the formation of acetic and formic acid (Fig. 2). Fig. 1 also shows the effect of sugar type on the pH reduc- tion of different model systems. Based on the ANOVA and Duncan’s multiple range tests, the sugar type had a significant effect on pH reduction. RL model systems exhibited significantly higher (p < 0.05) formation of acids compared to FL models systems, with the exception of MRPs formed at 121 �C for 120 min, while there were no significant differences (p > 0.05) observed between RL and FL model systems. It is well known that sugar type affects the rate of the MR, for example pentoses react more readily than hexoses (Hwang, Kim, Woo, Lee, & Jeong, 2011). Therefore, the MR is expected to be more pronounced with RL than FL model sys- tems, and thus the formation of substantial amounts of acid in RL systems resulting in lower pH values. These findings were also in agreement with the statement made by Liu, Yang, Jin, Hsu, and Chen (2008) and Lan et al. (2010), who assumed that the amount of acids formed is highly dependent on the co-existence of an ami- no and a carbonyl group. Therefore, the type of reactant participat- ing in the MR will have an effect on the amount of organic acids formed (pH reduction) as was observed in this study. 3.2. Development of browning Development of a brown colour is a non-specific index used to assess the extent and the rate to which the MR has taken place Time (min) 0 20 40 60 80 100 120 140 pH 4 6 8 10 FL 60 FL 80 FL 121 RL 60 RL 80 RL 121 Fig. 1. Change in pH values of Maillard reaction products (MRPs) as a function of reaction temperature and times. Each point on the curve represents mean ± SD of replicates (n = 2). ANOVA and Duncan’s multiple range tests were performed. (Laroque et al., 2008). In the first stage of the MR, the carbonyl group reacts with the amino group giving rise to colourless com- pounds which do not absorb in the visible spectrum. Further pro- gress of the MR involves the production of high molecular weight compounds, termed melanoidins with a characteristic absorbance maximum at 420 nm (Delgado-Andrade, Seiquer, Haro, Castellano, & Navaro, 2010). In the present study, the brown colour was used as an indicator of the stages of the MR and its possible link to antioxidative capacity. Fig. 3 shows browning intensity as a function of reaction temperature and time. The results of ANO- VAs and Duncan’s multiple range tests indicated that the differ- ences observed in the browning intensity was significant (p < 0.05) for RL model systems produced at different reaction tem- peratures and times. Moreover, the BI of RL model systems in- creased with an increase in reaction temperature and time. However, with regards to FL model systems there were no signifi- cant differences (p > 0.05) observed between model systems pro- duced at the three reaction times at 60 �C. However, at the higher temperatures of 80 and 121 �C, significant differences (p < 0.05) among the model systems were observed as a function of reaction time. Moreover, similar to the trend observed during pH monitoring, RL model systems exhibited significantly higher (p < 0.05) BI than their corresponding FL model systems. This fur- ther confirms sugar type as an important factor affecting the MR. 3.3. DPPH radical scavenging The stable DPPH-RS radical scavenging assay is based on hydro- gen atom transfer and has been used widely for determining pri- mary antioxidant activity. This assay determines free radical scavenging activity of all antioxidants, including those in pure form, as well as components of food products and plant and fruit extracts (Ak & Gulcin, 2008). To evaluate the free radical scaveng- ing properties of model MRP systems, these were allowed to react with stable DPPH radicals in solution. The degree of discolouration of the DPPH solution indicated the radical scavenging potential of MRPs. It manifested as a colour change from purple to yellow on acceptance of a hydrogen atom from MRPs to form a stable DPPH-Hmolecule (Sharma & Bhat, 2009). Table 1 shows the radical scavenging activity of MRPs expressed as percentage radical scav- enging activity. As the temperature increased from 60 to 80 �C a non-significant (p > 0.05) increase in DPPH-RS activity of most FL model systems was observed, with the exceptions of MRPs pro- duced at 15 and 120 min. However, at a temperature of 121 �C a significant increase (p < 0.05) was observed with an increase in reaction time for FL model systems. With reference to RL model systems produced at 60 and 80 �C, reaction times did not have a significant effect (p > 0.0.5) on DPPH- RS activity. However, as the temperature increased from 60 to 80 �C, the radical scavenging activity increased significantly (p < 0.05). A significant decrease (p < 0.05) in radical scavenging activity was observed as the reaction times increased from 15 to 120 min at 121 �C, while a similar trend was observed when the pH decreased drastically at 121 �C (Fig. 1). Hence, a possible expla- nation for the decrease in DPPH-RS activity as the reaction times increased at 121 �C are the observations of Knol, Linssen, and Van Boekel (2010) who found that a reduction in pH slows down the MR, thus lowering the radical scavenging potential of resultant MRPs. When comparing the effect of sugar type, RL model systems exhibited significantly higher (p < 0.05) radical scavenging activity than corresponding FL model systems, with the exception of model systems produced at 121 �C, where they exhibited similar radical hemistry 137 (2013) 92–98 scavenging activity. The results obtained at 60 and 80 �C further confirm the higher reactivity of ribose in terms of the antioxidant activity of MRPs (Hwang et al., 2011). The higher reactivity of od C L.N. Vhangani, J. Van Wyk / Fo ribose could also have resulted in the MR progressing so rapidly at 121 �C, leading to MRPs with such complexity that its effectivity as radical scavengers decreased (Amarowicz, 2009). 3.4. Peroxyl radical scavenging activity (PRS) Competitive techniques are widely employed to test the reac- tivity of antioxidants towards free radicals (Sachindra & Bhaskar, Fig. 2. Schematic representation of the formation of formic acid and acetic acid during t acid to the corresponding N-glycosylconjugate (2) followed by rearrangement to the Am deoxy2,3-pentodiulose (6) formed via 1,2 enolisation and 2,3 enolisation, respectively, y et al. (2005). Time (min) 0 20 40 60 80 100 120 140 A bs or ba nc e (4 20 n m ) 0 1 2 3 4 5 FL 60 FL 80 FL 121 RL 60 RL 80 RL 121 Fig. 3. Browning intensity (BI) of Maillard reaction products (MRPs) as a function of reaction temperatures and times. Each point on the curve represents mean ± SD of replicates (n = 2). ANOVA and Duncan’s multiple range tests were performed. hemistry 137 (2013) 92–98 95 2008). One such assay measures PRS activity of anti