Food Chemistry
Elham Ranjbar Nedamani; Alireza Sadeghi Mahoonak; Mohammad Ghorbani; Charlotte Jacobsen; Vahid Khouri
Abstract
The aim of the present study was to use the Maillard reaction as a means to glycosylate protein hydrolysates obtained from Cajanus cajan and to evaluate the effects of this chemical modification on antioxidant and emulsifying properties. Chemical properties, amino acid composition, and molecular weight ...
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The aim of the present study was to use the Maillard reaction as a means to glycosylate protein hydrolysates obtained from Cajanus cajan and to evaluate the effects of this chemical modification on antioxidant and emulsifying properties. Chemical properties, amino acid composition, and molecular weight distribution of the hydrolysates were evaluated. Glucose, galactose, and maltodextrin in the ratios of 1:2, 1:1, and 2:1 (hydrolysate: sugar, dry weight basis) were used for glycosylation. Antioxidant activity was evaluated by 1, 1- diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and nitric oxide scavenging. The sonication technique was used to prepare the (oil/water) emulsions. The droplet size distribution and zeta potential of the emulsions were measured during 4 days of storage. Results showed that glycosylation by glucose in the ratio of 2:1 increased DPPH scavenging activity from 37.96% to 85.53% and nitric oxide inhibition activity from 14.50% to 54.83%. Although glycosylation improved emulsifying stability of glycosylated hydrolysates compared to non-glycosylated hydrolysates, no significant difference was observed between the three examined sugars.
Fakhri Shahidi; Farideh Tabatabaei Yazdi; Majid Nooshkam; Zahra Zareie; Fereshte Fallah
Abstract
Introduction: Lipid oxidation leads to the generation of off-flavors and potential toxic compounds. Synthetic antioxidants are frequently applied for inhibiting this reaction, however; there is a concern regarding to the potent toxic effects of synthetic antioxidants on human health. The non-enzymatic ...
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Introduction: Lipid oxidation leads to the generation of off-flavors and potential toxic compounds. Synthetic antioxidants are frequently applied for inhibiting this reaction, however; there is a concern regarding to the potent toxic effects of synthetic antioxidants on human health. The non-enzymatic glycosylation reaction (Maillard reaction) has been broadly used to ameliorate the biological and functional features of proteins and polysaccharides. The Maillard reaction produces products with versatile functions such as antioxidant, antimicrobial, antihypertensive, anti-browning, and prebiotic properties. In this regard, the Maillard reaction products (MRPs) can be used in the food industry to inhibit the oxidation reaction due to their superb antioxidant effect. In this study, chitosan was glycosylated with inulin, fructose, and glucose. Chitosan is a chitin derivative with cationic nature having antimicrobial, antioxidant, metal chelation, and film-forming features. Inulin is recognized as a prebiotic sugar with vast applications in food and pharmaceutical sciences. The purpose of this study was to chemically modify chitosan through the Maillard reaction in order to boost its antioxidant and antimicrobial properties. Materials and methods: Chitosan (0.5% w/v) was dissolved in 1.0% v/v acetic acid solution followed by stirring for 1.0 h at room temperature. Afterwards, sugars inulin, glucose, and fructose were separately added to the chitosan solution at final concentration of 1.0% w/v. The obtained solutions were then stirred until complete sugar dissolution. The pH of solution was adjusted to 6.07 by adding 2.0 M sodium hydroxide and then the chitosan-sugar Maillard conjugates were fabricated through autoclaving the solutions at 121 °C. Changes in pH after the reaction were measured using a pH meter. The extent of the Maillard reaction was estimated via measuring the absorbance of the conjugated solutions at 294 nm (the intermediate products) and 420 nm (final products). Fourier transform infrared (FTIR) spectroscopy at transmission mode and 400-4000 cm-1 was employed to evaluate the structural changes of chitosan upon conjugation. Antioxidant activity of the conjugates was evaluated based on the reducing power assay. One mL of the samples was charged with 1.0 mL of distilled water and 1.0 mL of potassium ferricyanide (1.0% w/v). The solution was mixed and incubated at 50 °C for 20 min. After adding 2.5 mL of tri-chloroacetic solution (10% w/v), the obtained solution was centrifuged at 5000 g for 5.0 min. Afterwards, 2.0 mL of the supernatant was mixed with 2.0 mL of distilled water and 1.0 mL of ferric chloride (0.1% w/v). The solution was stand for 10 min at ambient temperature and then its absorbance was recorded at 700 nm. Antimicrobial effect of the conjugates against pathogenic microorganisms (E. coli, S. aureus, B. subtilis, P. aeruginosa, A. niger, and C. albicans) was measured according to the minimum inhibitory (MIC) and microbiocidal (MBC) concentrations. SPSS software (version 21) and one-way ANOVA were applied for data analysis. Duncan’s multiple range test was employed to determine the differences between means. Results & discussion: The Maillard reaction led to a significant decrement in pH value of chitosan-saccharide systems, mainly due to the covalent coupling of amino groups of chitosan to carbonyl groups of reducing sugars in conjugation with the production of acetic and formic acids. The highest intermediate compounds (A 294nm) and lowest browning intensity (A 420nm) observed in chitosan-fructose conjugate, which was likely attributed to the lower reactivity of fructose. Chitosan-inulin conjugate presented the highest A 420nm and lowest intermediate-to-final ratio (A 294nm/A 420nm), probably due to the lower inulin molecules and subsequently carbonyl groups compared to fructose and glucose. These groups may react with amino groups of chitosan at initial reaction times, leading more conversion rate of the intermediate compounds to the final ones. FTIR spectra of the chitosan and conjugates revealed that absorbance peak at 1661 cm-1 in chitosan spectrum decreased and shifted to 1578 cm-1 (in chitosan-fructose conjugate), 1579 cm-1 (in chitosan-glucose conjugate), and 1580 cm-1 (in chitosan-inulin conjugate), indicating the stretching C-N group and -C=N group and the formation of Schiff base (-C=N) between reducing end of the saccharides and amino groups of chitosan. Reducing power of the chitosan-saccharide systems improved after the thermal process. Although, chitosan-glucose and chitosan-fructose conjugates had significantly higher reducing power than unconjugated counterparts, but chitosan-inulin conjugate showed non-significantly improved antioxidant activity compared to its non-heated mixture. Antioxidant activity of the Maillard conjugates was ascribed from the electron donating ability of their hydroxyl and pyrrole groups. The conjugates had lower MIC and MBC in comparison to their unconjugated pairs, except for chitosan-glucose conjugate, which showed no differences in MIC and MBC compared with its non-heated mixture. Antimicrobial property of the Maillard products, especially melanoidins has been attributed to their metal chelating features; melanoidins exert a bacteriostatic effect at low concentration and bactericidal effect at high levels through sequestering ionic iron from medium and magnesium from outer membrane, leading to the cell membranes destabilization. Additionally, antioxidant capacity, high surface activity, and inhibiting effect towards catabolic enzymes have been reported as another antimicrobial mechanisms of the Maillard products. In general, it can be concluded that chitosan-saccharide Maillard-based conjugates, particularly inulin-chitosan one could be used in the food sector as a novel prebiotic-based active bio-compound with antioxidant and antimicrobial features.
Fatemeh Azarikia; Soleiman Abbasi
Abstract
Introduction: Maillard reaction is a well-known technique for covalent coupling of protein–polysaccharide which is usually used to improve the functional properties of proteins. Conjugation by the dry heating method is occurred during Amadori rearrangement step in the Maillard reaction where amine ...
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Introduction: Maillard reaction is a well-known technique for covalent coupling of protein–polysaccharide which is usually used to improve the functional properties of proteins. Conjugation by the dry heating method is occurred during Amadori rearrangement step in the Maillard reaction where amine groups of the proteins are linked to the reducing end of the polysaccharides. This glycation process leads to the improvement of solubility, foaming and emulsifying properties of proteins (Liu et al., 2012). Protein-based emulsions are sensitive to pH and ionic strength alterations as well as heating and freezing–thawing processes. While, joining proteins to high molecular weight polysaccharides protects them against environmental stresses. A wide range of carbohydrates were already utilized to enhance the emulsifying properties of proteins–polysaccharides namely whey protein isolate–dextran (Akhtar et al., 2003), β-lactoglobulin–dextran (Wooster et al., 2006), sodium caseinate–maltodextrin (O’Regan et al., 2009), deamidated wheat protein–maltodextrin or glucose (Wong et al., 2011), whey protein isolate–pectin (Xu et al., 2012), yolk phosvitin–dextran (Chen et al., 2014), β-lactoglobulin–six-carbon monosaccharides (Cheetangdee et al., 2014), soy protein isolate–soy soluble polysaccharides (Yang et al., 2015), lysozyme–tragacanth (Koshani et al., 2015), and β-lactoglobulin–Persian gum (Golkar et al., 2015). However, based on the existing literature, it seems that conjugation of Iranian native gums and proteins needs to be more attended to show their potential applications. Therefore, in this study, conjugate formation between milk proteins (sodium caseinate and whey protein isolate) and soluble fraction of Iranian native gums (gum tragacanth and Persian gum) was optimized using response surface methodology (RSM) and the resulting conjugates were used in emulsion formulation in order to compare protein capability before and after being attached to the gums.
Materials and methods: Iranian native gums were pulverized and sieved (mesh size < 60) after being prepared from local herbal stores. To obtain protein–polysaccharide conjugates, dry heat treatment was accomplished at 60oC and 79% relative humidity at different protein:polysaccharide ratios and heating times. Free amino group content was determined by the ninhydrin method described by Doi et al. (1981). To measure color changes before and after Maillard reaction, Hunter Lab was used. Besides, possibility of stabilizing oil-in-water emulsions using the conjugates was studied. Emulsions were formed by addition of oil phase (4% w/w) into protein–polysaccharide conjugates solution (0.4% w/w) following by ultrasound treatment (amplitude of 100%, for 4 min). Samples were kept at 4 oC for 30 days after adjusting the pH at 3, 5 and 7 to compare the effect of protein–polysaccharide conjugates on their stability at different protein charges. For optimization of Maillard reaction using RSM (central composite design), protein:polysaccharide ratio and heating time were selected as independent variable and were studied at 5 levels. The dependent variables were the substitution degree of free amino groups and color change.
Results & Discussion: Based on our findings, the decrease of free amino group indicated that NH2 group of amino acids in milk proteins was covalently linked to carbonyl group of the gums. Comparing free amino group reduction of the samples containing soluble fraction of gum tragacanth and Persian gum also showed that Persian gum was more capable of forming covalent linkage with milk proteins than gum tragacanth; probably, due to the lower side branches of Persian, its molecular weight as well as its structural flexibility. Moreover, we believe the higher side branches of attached-tragacanthin molecules might prevent further attachment of protein to the other polysaccharide molecules via steric repulsion. According to the results of color measurement before and after dry Maillard reaction, conjugation led to reduction of L* and increase of a* and b*. In addition, higher heating time and protein:polysaccharide ratio caused progress of Maillard reaction (especially in the case of milk proteins–soluble fraction of Persian gum). Based on our findings, in dry Maillard reaction, the optimum heating time and protein:polysaccharide ratio were 8 days and 1:1.59 for whey protein isolate–soluble fraction of Persian gum, 7 h and 1:2 for sodium caseinate–soluble fraction of Persian gum, 13.64 days and 1:3 for whey protein isolate–soluble fraction of gum tragacanth, and 7.82 h and 1:3 for sodium caseinate–soluble fraction of gum tragacanth, respectively. Besides, the obtained Maillard reaction products did not cause complete stability of emulsions at pHs 3, 5 and 7. Our findings also declared that attachment of polysaccharides to proteins might negatively affect the proteins functionality, as emulsifier, possibly by hindering adsorption of hydrophobic groups of proteins to oil droplets. Furthermore, homogenization process during emulsion preparation using ultrasound could break the formed covalent bonds and polysaccharide structure leading to lower steric repulsion and viscosity.
Sareh Boostani; Mahmoud Aminlari; Marzieh Moosavi-Nasab; Mehrdad Niakosari; Gholam Reza Mesbahi
Abstract
Introduction : Soybean is an excellent plant protein source with diverse applications in food systems. Despite numerous commercial applications and rich nutritional properties of soybeans, soy proteins are sensitive to heat and other damaging agents during food processing which can limit their applications ...
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Introduction : Soybean is an excellent plant protein source with diverse applications in food systems. Despite numerous commercial applications and rich nutritional properties of soybeans, soy proteins are sensitive to heat and other damaging agents during food processing which can limit their applications in food industries. Maillard reaction includes a series of chemical reactions between the free carbonyl groups of carbohydrates and the un-protonated amino groups of proteins under mild experimental conditions. This is one of the most desirable approaches for applying in food systems, because of the safety of the procedure and the independency of adding extra chemicals. Natural occurring Maillard reaction can be a relatively safe and inexpensive method in order to improve functionalities of food proteins. The production of conjugates haspositive influences on food proteins functionality such as solubility, water holding capacity, emulsion activity and stability, foaming properties, thermal stability, whipping ability, textural and gelation properties and also reduce allergenicity of proteins. Due to the positive characteristics and reasonable price of both soy proteins and maltodextrin in food industries, the aim of current study was to enhance the heat stability and functional properties of soy proteins through glycosylation with maltodextrin. In addition, assessment of changes in protein properties as a function of incubation time were evaluatedMaterials and methods: Preparation of purified soy proteins (Acid precipitated soy proteins) was done by a multistage process of washing, centrifugation, dialysis and freeze drying. The resulting powder contained pure soy globulins. Conjugation of acid precipitated soy proteins with maltodextrin was performed according to the method described as follows: protein-polysaccharide at a weight ratio of 1: 3 were dissolved in 0.01 M phosphate buffer, at pH values of 8, and were incubated at ambient temperature for 1 hr. Solutions were frozen at –80°C and then freeze dried. Lyophilized powders were incubated at 60 °C for 1, 3, 5 and 7 days, under 79% of relative humidity provided by saturated KBr. For each treatment an un-conjugated (control) sample was prepared under the same conditions. The formation of protein-polysaccharide conjugates was confirmed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography (Sephadex G-100 chromatographic system was used to separate the conjugated proteins from the un conjugated samples). Determination of protein denaturation temperature changes were carried out using METTLER TA Q100-DSC thermal analyser.The emulsifying properties of the samples including emulsifying activity and emulsion stability were assessed according to the procedure established by Pearce and Kinsella. Protein solubility was measured by calculating the amount of nitrogen in the supernatant and total nitrogen content of the samples and reported as percentage of protein solubility at pH 3, 5, 7 and 9. Foaming properties of the samples including foaming capacity and foaming stabilitywere determined using calibrated measuring cylinderDiscussion & Results: When the heating duration is increased, wider and heavier molecular weight bands emerge near the top of the running gel of SDS-PAGE, and yet these were not observed in the control. As a result of conjugation, the protein-carbohydrate covalent binding occurs, producing heavier molecular weight species, and thus leading to its accumulation on top of the separating gel. Compared with un-modified soy proteins, the conjugated soy proteins eluted in the void volume of G-100 gel permeation chromatography column, suggesting increase in the size and molecular weight of soy proteins due to the covalent attachment of maltodextrin. According to differential scanning calorimetry (DSC) analysis, thermal stability of soy proteins was remarkably increased by conjugation with maltodextrin and maximum denaturation temperature was observed for the mixture incubated for 7 days. The improved thermal stability is manifested in increase in denaturation temperature of globular proteins, hence conjugation leads to significant improvement of soy proteins stability. Increase in thermal stability is the result of inclusion of the hydrophilic carbohydrate moiety to the surface of the proteins. Compared to control sample, the solubility, foaming characteristics and emulsifying properties were significantly improved by increasing incubation time. The protein solubility of conjugate remarkably increased at all pH’s compared with the un-conjugated proteins. Covalent links between hydrophilic maltodextrin and soy proteins could enhance the reaction tendency between proteins and water molecules under unfavorable conditions. Improvements in the emulsifying properties of the conjugated samples can be explained by the fact that there is a combination among the emulsifying activity of proteins and the stabilizing impacts of polysaccharides per molecule. Foaming capacity of proteins can be affected by the solubility of proteins. Furthermore, maltodextrin is a hydrophilic carbohydrate which can improve the stability of soy proteins foams by acting as a thickener, thus increasing the strength of bubbles. It should also be considered that functionality of proteins are frequently influenced by protein solubility, and this could also serve the understanding of why improvements occur in functional properties of conjugated proteins, compared to un-conjugated ones. The results indicate that physiochemical and functional properties of soy proteins were modified and improved by conjugation with maltodextrin.
Sareh Boostani; Mahmoud Aminlari; Marzieh Moosavi-Nasab; Mehrdad Niakosari; Gholam Reza Mesbahi
Abstract
Introduction: Engineering of food proteins with improved functional properties and higher resistance to heat is the main goal of food scientists. Food technologists are always seeking for innovative, simple and effective methods to manipulate proteins, hence natural modification has had the most attention ...
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Introduction: Engineering of food proteins with improved functional properties and higher resistance to heat is the main goal of food scientists. Food technologists are always seeking for innovative, simple and effective methods to manipulate proteins, hence natural modification has had the most attention in last decades. One of the natural ways used for protein modifications isMaillard grafting reaction. Maillard reaction as a consequence of covalentbindingbetween the available amino groups of the proteins and carbonyl containing moiety of the polysaccharides, causes a loss in free amino group content of the mixture that can be measured through different methods. Protein-polysaccharide hybrids, as a result of dry heating of two biopolymers mixture under controlled reaction conditions, cause the emergence of conjugates with novel functionalities.Selecting appropriate reaction conditions has a significant impact on the properties of the final conjugates. Among the factors affecting the degree of conjugation, temperature, pH and incubation time have considerable roles in determining the degree of conjugation. There are various methods by which conjugation degree can be assessed and factors such as accuracy, cost, accessibility and etc. must be considered when selecting a measuring method. Therefoe, in this study the effect of different Maillard reaction conditions on the conjugation degree of soy proteins-dextran heated mixtures have been investigated. In addition, the glycation degree was compared and reported by both OPA assay and UV absorbance methods.Materials and methods: Soy proteins–dextran conjugates were prepared as described later. First, soy proteins and dextran were mixed with phosphate buffer (0.1 M, pH: 8.5 and 7) and 1 to 4 ratio of protein to polysaccharide. After mixing and incubating at ambient temperature for some hours, solutions were frozen at –80 ℃ and freeze dried. Then, the lyophilized powder was incubated at 40 ℃ for 0, 4, 8, 24 hours and 2, 4, 6, 8, 12 days, at 60 ℃ for 0, 1, 2, 3, 4, 6, 8 days and at 80 ℃ for 0, 1, 2, 4, 8, 16, 24, 48 hours, under the 79 percent relative humidity in presence of saturated KBr. For each treatment a non conjugated sample was prepared in the exact same condition. Conjugation of proteins to polysaccharides was monitored by two methods (OPA assay and UV absorbance). Determining degree of glycation by OPA method was done as follows, in this procedure the level of available amino groups was estimated using the ortho- phthaldialdehyde (OPA) reagent, the absorbance was measured at 340 nm and degree of glycation was measured using a formula. To investigate the UV absorption of conjugated proteins, the samples were diluted using distilled water and the absorption was read using a UV-visible spectrophotometer.Results & Discussion: Covalent linkage between amino group of proteins and carbonyl group of polysaccharides causes depletion in the amount of available amino groups. The extent of soy proteins-dextran conjugation under various Maillard reaction conditions was evaluated by the reduction of available amino groups of proteins, the more reduction in amount of amino groups, the more conjugation between protein and polysaccharide. OPA results showed that, in the samples heated at 40 °C and 80 °C (at both pH 7 and 8.5), the amount of free amino groups slightly reduced compared to 60 °C heated samples. The disappearance of available amino groups at 60°C was faster than other temperatures and formation of conjugates in this temperature was more successful. A stepwise reduction in free amino group content observed with increasing incubation time. When soy proteins were incubated at pH 8.5 for 8 days at 60 °C, a considerable decrease in available amino group contents occurred. UV absorption results showed similar trend of changes in OPA method. Increasing UV absorption is due to the intermediate Maillard reaction products (MRP). Increasing incubation time, temperature and pH cause a significant increase in UV absorbance. Increasing UV absorption with increasing heating time indicates the fact that Maillard reaction products (MRP) formation is more favorable at alkaline pH. Data of UV absorption are a proper evidence for OPA assay results. Conclusion: As a conclusion, both OPA assay and UV absorption methods are cost effective, accurate and simple methods which can represent valuable information concerning the Maillard conjugation.
