Step IIL: Fnol jon of Amadori Rearrangement Products In Step Ill, the ketoseamines are decomposed by 1,2-enolization or 2,3-enolization, The former pathway appears to be the more important one for the formation of brown color, whereas the latter results in the formation of flavor products. The 1,2-enolization pathway appears mainly t0 lead to browning but also contributes to formation of off-flavors through hydroxymethylfurfural, which may bea factor in causing off-flavors in stored, overheated, or dehydrated food products, The mechanism of this reaction is shown below 1.2 enolization 7 Arlite pit ¥2. yon —= | slow nd Sdeoxyosone ' L\ ‘Shydroxy methyl 2 furaldehyde | aT a " ae 16 2.3 eneaminal I deoxyosonese Readuetones ‘The intermediate products like dicarbonyl compounds (3 deoxyosone, 1 deoxyosone) of eduetones in the above scheme are very teactive. Compounds having two enolic hydroxyls in Conjugation with a carbonyl are known as reductones. Ascorbic acid is @ reductone, Generation of sedvetones through Maillard browning can account for some of the apparent increases in antioxidant properties of some Foods during processing and storage. Strecker Degradation ‘The dicarbonyl compounds will react with an amino acid to generate CO: and an aldehyde with ‘a chain length one carbon less than the amino acid. The nitrogen-containing fragment can rearrange and break down to generate ammonia that is transferred to other components of the system. Two fragments pth condense to form various pyrazines, which are potent flavor compounds found in coffee roasted huts, and other foods.Ho Acetaldehyde ‘The Strecker degradation: Reaction of u-dicarbonyls th amino acid iyIs with amino acids, Different important intermediate formed during this step R N. B OH ~y ee meee Tk OY coo ue Final Stages: Condensation and Polymerization ‘The compounds formed in the Maillard browning scheme presented so far are colorless. The initial condensation product and the Amadori compounds are colorless, Polymerization of carbonyls through the aldol condensation is generally accepted as a primary route for formation of polymeric. melanoidin pigments. Increased conjugation through dehydration will eventually lead 10 colored compounds. Concentration of reactants and dehydrating conditions will favor the reaction Factors affecting the Maillard Reaction “The rate of the Mallard reaction and the nature of the products formed depend on the chemical environment of food including the water activity (as), pH, and chemical composition of the food System, temperature being the most important factor “The reaction rate is significantly affected by the pH ofthe system; it generally increases with sr reducing sugar fas a great influence on Maillard reaction development. Pentoses (¢.8. pH. The type readily than hexoses (¢g. glucose), which, in tum, are more reactive than Fibose) react Thor ee of amino acids in the Maillard reaction is variable; Isine is the most disaccharides. Parti pars been shown that an increase in temperature increases the rate of Mallard reactive amino Acie: 7 Mand ratio of reducing sugar to amino acid have a significant impact on the browning. Concer itn inereases with increasing amino acid: reducing sugar ratios. Water activity reaction. Browning Toe a ctor influencing Maillard reaction development; thus, this reaction occurs ~ (p,) is another impo (ese ready in the mobility 0! ‘values from Hoods with high ay values. At high a, values, reactants are diluted, while at low a,. values reactants is limited, Numerous studies have demonstrated a browning rate maximum at 015 to 0.8 in dried and intermediate-moisture foodsCaramelizatio The formation of the caramel pigment can be consider. a nonenzymatic browning reaction in subjected to heat in the absence of water or ies of reactions occurs that finally leads to caramel formation the absence of nitrogenous compounds, When sugars are are heated in concentrated solution, a ser The initial stage is the formation of anhydo su glucose) and levoglucosan (1,6-anhydro-p-D- +69° and -67°, respectively igars, Glucose yields glucosan (1,2-anhydro-a-D= lucose); these have widely differing specific rotation, Caramelization of sucrose requires a temperature of about 200°C. At 160°C, sucrose melts and forms gluicose and fructose anhydride (levulosan). At 200% distinct stages well separated in time. The fist step requires 35 minutes of heating and involves a weight loss of 4.5 percent, corresponding to a loss of one molecule of water per molecule of sucrose This could involve formation of compounds such as isosacchrosan the reaction sequence consists of three 2'-dianhydro-a-D-glucopyranosy1-p-D-fructofuranose. (Isosacchosan) After an additional 55 minutes of heating, the weight loss amount to 9 percent and the pigment formed is named caramelan. This corresponds approximately to the following equation 2CiH2O)) - 4130 ——+ CalisOis uble in water and ethanol and has a bitter taste Its melting point is The pigment caramelan is soluble in water and ethanol and r 138°C. A further 55 minutes of heating leads to the formation of caramelen. This compound corresponds to a weight loss of about 14 percent, which is about eight molecules of water from three molecuiles of sucrose, as follows: 4CjgH2201) - SHO ——> CicHl0Oos faramelen is soluble in water only and melts at 154°C. Additional heating results in the format snot zy dark, nearly insoluble pigment of average molecular composition C\3sHixsOxo. This formation of a ve material is called humin or caramelin 1e typical earamel flavor is the result of a number of sugar fragmentation and dehydration PrT earamel flavor, namely, acetylformoin (4-hydroxy-2,3,5-hexane-trione) and 4-hydroxy-2,S- typical car dimethyl-3(2H)-furanone,A large number of and type of carbohydrat, calculating the percent f analytic ical techniques ES present in gues HAVE been developed Te ttt foods, The sarge 2 Pe4 9 measure ihe oa concentration ARer al the other compe OMEN Fa fod can be dete ponents have been measured. : 00 -% moisture =» moisture - % protein -% lipid -¥% mineral esulls due to experimental errors in any of the 'y measure the carbohydrate content for accurate Monosaccharides and Oligosaccharides Sample Preparation ‘The amount of preparation needed to prepare a sample for cart Ralite ofthe food being analyz. Agucos soators oe Fa ae ne Fequiire very little preparation prior to analysis. On the other hand, many foods contain carbohydrates that are physically associated or chemically bound to other components, eg. nuts, cereals, fruit, breads and vegetables. In these foods itis usualy necessary to isolate the carbohydrate from the resto the food before it can be analyzed. The precise method of carbohydrate isolation depends on the carbohydrate type, the food matrix iype and the purpose of analysis, however, thre are some [procedures that are common to many isolation techniques. For example. foods are usually dried under vacuum (to prevent thermal degradation), ground to a fine powder (to enhance solvent extraction) and then defatted by solvent extraction. ‘One of the most commonly used methods of extracting low molecular weight carbohydrates from foods is to boil a defatted sample with an 80% alcohol solution as show in the flow sheet ound to a flne powder and then extraction, Foods are dried under vacu defatted by solve t oil a defutted sample with an 80% alcohol solution. 4 ecosaccharies and oliesccharies ar sll alcohol aan paras proteins plexcaries and dear Mer ae sabe can be separate he insoluble ‘The soluble components can he separated from the insol components by filtering the boiled solution and collecting the Mtrate J ‘These two fractions can then be dried and weighed to determine their concentrations.Monosaccharides and oligosaccharides are soluble in alcoholic solutions, whereas polysaccharides and dietary fiber are insoluble, The soluble components can be separated from the insoluble components by filtering the boiled solution and collecting the filtrate (the part which passes through the filter) and the retentante (the part retained by the filter). These two fractions can then be died and weighed to determine their concentrations, In addition, to monosaccharides and cligosaceharides various other small molecules may also be present in the alcoholic extract that could interfere with the subsequent analysis.eg, amino acids, organic acids, pigments, vitamins minerals efc. It is usually necessary to remove these components prior to carrying out a carbohydrate analysis. This is commonly achieved by treating the solution with clarifying agents or by passing it through one o more ion-exchange resins + Clarifying agents. Water extracts of many foods contain substances that are colored or produce turbidity, and thus interfere with spectroscopic analysis or endpoint determinations. For this Feason solutions are usually clarified prior to analysis. The most commonly used clarifying agents are heavy metal salts (such as lead acetate) which form insoluble complexes with interfering substances that can be removed by filtration or centrifugation. Howevei important that the clarifying agent does not precipitate any of the carbohydrates from solution as this would cause an underestimation of the carbohydrate content. itis ‘+ Ton-exchange, Many monosaccharides and oligosaccharides are polar non-charged molecules and can therefore be separated from charged molecules by passing samples through ion- exchange columns. By using a combination of a positively and a negatively charged column it is possible to remove most charged contaminants. Non-polar molecules ean be removed by passing a solution through a column with a non-polar stationary phase. Thus proteins, amino acids, 1 carbohydrates prior to analysis nie acids, minerals and hydrophobic compounds can be separated from the Prior to analysis, the alcohol can be removed from the solutions by evaporation under vacuum So that an aqueous solution of sugars temains Method of analysis ethods Chromatographic and Flectrophoret omatography (TLC), Gas chromatography (GC) and High Performance Liquid chromatography basis of t column Carbohydrates can be separated on the basis of their partition coefficients, polarities or sizes, depending on the type of column used, HPLC is currently the most important chromatographie method for analyzing carbohydrates because it is capable of rapid, specific, sensitive and precise measurements, In addition, GC requires that the samples be volatile, which usually requires that they be derivitized, whereas in HPLC samples can often be analyzed directly2. Chemical methous ewe eben and oligosaccharides are te pmo cnc Teena ane he ame ea eon Nei methods are avaiable ore tre ton ae titrimetric and colotimetnie eae Mo of ese cane vied mo es etgeres se different types is given below deter a) Titration Methods The Lane-Eynon method is an example o! or Of reducing sugars in a sample. A buretc in ved can flask containing a known amount of a methylene blue indicator. The reducing sulfate present of determining the concentration is used to add the carbohydrate solution being analyzed to a boiling copper sulfate solution (Fehling’s solution) and sugars in the carbohydrate solution react with the copper the flask. Once all the copper sulfate in solution has reacted, any further addition of reducing sugars causes the indicator to change from blue to white. The volume of sugar solution Tequired to reach the end point is recorded. The reaction isnot stoichemetric, which means that itis Recessary to prepare a calibration curve by carrying out the experiment with a series of standard solutions of known carbohydrate concentration b) Colorimetric Methods : : concentration of the total sugars ina sample. Sugars react with the anthrone reagent under acidic conditions to yield a blue-green color. The sample is mixed with sulin act and the anthrone reagent and then boiled until the reaction is completed. The solution's the Allowed to cool and its absorbance is measured at 620 nm, There is a near relationship between the absorbance and the amount of sugar that was present inthe eriginal sample This method determines both reducing and non-reducing sugars betause of the presence of the strongly oxidizing sulfuric acid, Like the other methods it therefore itis necessary t0 prepare carbohydrate concentration ‘a calibration curve using a series of standards of known, cid method is an example of a colorimetric method that is widely Te Phen Sulfuric Acid mot oy af anouhdttes preset foods. A ler aque Used fo determi Mr yrates to be analyzed is placed in atst-tube, then phenol and sulfuric ae ed. The “solution turns a yellow-orange color as a result of the interaction acid ate det pohydrates and the phenol. The absorbance at 420 nm is proportional tothe between Hie Seoncentration initially in the sample. The sulfuric acid causes al! non-reducing carbohydrate coerted to reducing sugars, so that this method determines the total sugars Suga (oP ctrethod is non-soichemelric and so it iS necessary 10 prepare a calibration iealiare {a series of standards of known carbohydrate concentration,3. Enzymatic Methods Analytical Methods are rapid: rena’ based on enzymes rely on their ability to catalyze specific reactions. These determination of can oy sPesite and sensitive to low concentrations and are therefore ideal for iquid foods can be ana it fads. In addition, litle sample preparation is usually required ey feryias cy rectly, whereas solid foods have to be dissolved in water first. There are say kit cl carbohydrates, Kits Which can be purchased commercially to carry out analysis for specific Glue i oe ee to yield gluconic acid and peroxide in the presence of glucose oxidase and he oxidation of a chromogen (i¢., o-dianisidine) in the presence of H:O:, thereby enabling quantitative me: been established © Measurement spectrophotometrically when an appropriate standard curve has Es ene 4, Physical Methods Many different physical methods have been used to determine the carbohydrate concentration ‘of foods. These methods rely on their being a change in some physicochemical characteristic of a food ‘as its carbohydrate concentration varies. Commonly used methods include polarimetry, refractive index, IR, and density, a) Polarimetry ‘Molecules that contain an asymmetric carbon atom have the ability to rotate plane polarized jight, A polarimeter is a device that measures the angle that plane polarized light is rotated on passing through a solution. A polarimeter consists of a source of monochromatic light, a polarizer, a sample ‘cell of known length, and an analyzer to measure the angle of rotation. The extent of polarization is related to the concentration of the optically active molecules in solution by the equation a=[alle, where, «is the measured angle of rotation, (a) is the optical activity (which is a constant for each type of molecule), /is the pathlength of solution and c isthe concentration.b) Refractive Index € index of a material can be determir ned by measuring ce (i) ata boundary between it and ai nother material of This method is quick and simple to carry out and can be instruments. It is used routinely in industry to determine sugar Molasses, tomato products and jams. performed with simple hand-held concentrations of Syrups, honey,