Carbohydrate Food Chemistry & Analysis PDF

CARBOHYDRATE BWD 22303 FOOD CHEMISTRY & ANALYSIS  Objectives: 8.1 Enzymatic determination of starch. 8.2 Browning and non browning reaction, caramelization 8.3 Physical and enzymatic methods used to measure sucrose in food products. 8.4 Crude fibre, acid detergent fibre and neutral deterge...

CARBOHYDRATE BWD 22303 FOOD CHEMISTRY & ANALYSIS  Objectives: 8.1 Enzymatic determination of starch. 8.2 Browning and non browning reaction, caramelization 8.3 Physical and enzymatic methods used to measure sucrose in food products. 8.4 Crude fibre, acid detergent fibre and neutral detergent fibre determinations 8.5 AOAC and Englyst-Cumming methods for determination of dietary fibre  INTRODUCTION Carbohydrate are the most abundant and widely distributed food component in nature (55-60%) of the caloric food in human diet. Most forms of carbs are composed of C,H, O in the ratio 1:2:1 respectively. General formula (CH2O)n where n represent numbers of time the ratio is repeated. Digestible carbs provide an important source of energy. Indigestible carbs do not provide energy known as dietary fiber.  Glycoproteins: carbs molecules covalently attached to proteins. Glycolipids: carbs molecules covalently attached to lipids. Introduction…contd  Individual molecules can be classified according to the number of monomers they contain. ❖ monosaccharide – e.g. glucose, fructose, galactose. ❖ Disaccharide – maltose, sucrose, lactose. ❖ Oligosaccharides – e.g. raffinose, stachyose. ❖ Polysaccharides – e.g. starch (amylose, amylopectin) Classification  Simple sugar Water soluble crystalline compounds. An aliphatic aldehydes or ketones which contain one carbonyl group and one/more hydroxyl group as its reactive centers Natural mono have either 5 (pentose) or 6 (hexoses) C atoms. Reactive centers of mono are the carbonyl and hydroxyl groups. Monosaccharide  https://www.youtube.com/watch?v=lumT3cDeB2E (Fischer to Howarth) Structure of monosaccharide/simple sugar https://www.youtube.com/watch?v=5QIBUp1LUDg Drawing monosaccharides (Fischer)  Low MW polymers of mono, 20. At least 20% of carb in nature is in the form of polysaccharides. Homopolysaccharide-contain all the same monomer (e.g. starch, glycogen, cellulose, dextrins). Heteropolysaccharides-can contain more han one type of monomer (e.g. D-galacturonic acid, methylester, hemicellulose, gums). Starch is the only polysacc that human can digest and use as energy source. *other polysacc are indigestible. Polysaccharide  amylose starch Structure of polysaccharides  Nutritional labelling – inform consumer about nutritional content Standard of identity – composition conform to government regulation Food processing – efficiency depends on carbohydrate type and concentration Food Quality – physicochemical properties such as sweetness, appearance, texture and stability depend on carbohydrates type and concentration Importance of Analysis Food % Food % Milk 4.78 Rice 30.00 Yogurt 5.60 Dates 48.5 Fruit juice 6.12 Bread 52.20 Apple 12.39 Honey 75.10 Grape 16.11 Flour 80 – 85 Potato 19.80 Brown sugar 86.60 Soy bean 25.50 Table sugar 99.40 Carbohydrate content of selected foods  Very important to get accurate and consistent results. Due to the complex nature of food, sample must be purified to remove possible interferences that might affect analysis. Preparation techniques are related to the specific product analyzed and the specific carbs being determined. Aqueous solutions (e.g. fruit juice, syrup, honey) usually require very little preparation prior to analysis. Solid sample (e.g. nut, cereals, bread, fruit and vegetables) are physically associated chemically bound to other component. Therefore necessary to isolate before it can be analyzed. Sample preparation  Isolation technique - foods are dried under vacuum (to prevent thermal degradation) then ground to fine powder (to enhance solvent extraction) Finally, extract low MW carbs from foods by boiling defatted samples with 80% alcohol solution. Mono- and oligo saccharides are soluble in alcoholic solution. *but polysac are insoluble. Soluble components can be separated from insoluble component by filtering the boiled solution and collect filtrate. Then, components were dried and weighed to determine concentration. Contd…  Soluble component also contain various other small molecules (e.g. enzymes, amino acid, metals, organic acid, pigments, vitamins) that interferes with subsequent analysis. Compounds may be colored or produce turbidity thus interferes with endpoints determinations and spectroscopic analysis. Must remove these components prior to carb analysis. Achieved by treating solution with clarifying agents or use ion-exchange resins. Contd…  Clarifying agents: heavy metal salts e.g. lead acetate. - Form insoluble complexes with interfering substances; remove complexes formed by filtration/centrifugation - must not precipitate any carbs in the solution; caused underestimation. Ion-exchange: - many mono- and oligo- are non-polar charged molecules; therefore can be separated from charged molecules by passing it through ion-exchange columns. - non-polar molecules removed by passing through column of non-polar stationary phase. Contd…  Analysis of Mono- and Oligosaccharides: -Chemical method: ✓ Titration: Lane Eynon method, ✓ Gravimetric: Munson-walker method ✓ Colorimetric : Somogyi-Nelson method, Anthrone method, Phenol-Sulphuric method, DNS method. -Biochemical method : Enzymatic method -Optical method/Physical method: Refractive Index; Polarimetry -Chromatographic method Analytical Methods  Determination of nutrient polysaccharides -Blue-Value method (Amylose Content) Determination of structural polysaccharides: -Crude/Dietary Fiber. Total Carbohydrate: calculation by difference Analytical Methods  Based on the fact that mono- and oligo- are reducing sugars which can react with other components to yield precipitates or colored complexes that can be quantified. *Reducing sugar definition- sugar possess free aldehyde or ketone group in their structure react with reducing agents (Fehling’s reagent). Non-reducing carbs are hydrolyzed to make them as reducing then measured using the same methods. Analysis for Mono- and Oligosaccharides  Use to determine reducing sugar concentration in sample. Principle: - Based on reaction of reducing sugar with copper sulphate - followed by reaction with alkaline tartrate/* salt or ester of the organic compound tartaric acid, a dicarboxylic acid. - Mixture boiled for specific time, - followed by addition of methylene blue, - colored solution titrated until decoloration of the indicator I. Lane-Eynon Method I. Lane-Eynon Method  Procedure: - Carb solution from burette titrated into a flask containing known amount of boiling copper sulfate solution (mixed Fehling’s solution) and methylene blue indicator. - Air excluded from reaction mixture by keeping liquid boiling throughout titration process. - Reducing sugars in the solution will react with copper sulfate, converted to insoluble cuprous oxide. - Once all copper sulfate in solution has reacted, indicator change color from blue to colorless. - volume of sugar solution required to reach end point recorded. Application: Measure reducing sugar in honey and other high reducing sugar syrup. I. Lane-Eynon Method  Problem: - Reaction not stoichiometric, need to prepare calibration curve. Disadvantages: - results depend on precise reaction times, temperatures and reagent concentrations. * Parameters must be carefully controlled. - cannot distinguish between different types of reducing sugar. - cannot directly determine concentration of non-reducing sugars. - susceptible to interference from other types of molecules that act act as reducing agents. I. Lane-Eynon Method  SELF-READING II. Munson-Walker Method  Use to determine amounts of reducing sugars. Principle: - Based on reduction of Cu2+ ions (cuprous oxide) to Cu+ ions by reducing sugars. - Cu+ ions then reduce an arsenomolybdate reagent*. - Reduction of arsenomolybdate complex produces an intense, stable blue color measured spectrophotometrically. *prepared by reacting ammonium molybdate and sodium arsenate in sulphuric acid) III. Somogyi-Nelson Method  - Oxidation by cupric to cuprous by reducing sugars, with concomitsnt reduction of arsenomolybdate complex, - Produces stable blue colored solution. - Solution absorbances measured at 500 or 520 nm. - Requires preparation of standard curve. - Reducing sugar + Cu2+ → Cu+ + oxidized sugar Application: for samples with low concentration of carbohydrates. III. Somogyi-Nelson Method  SELF-READING II. Anthrone Method  Use to determine total concentration of carbs (total sugars) present in foods. Principle: - Carbs sensitive to strong acids and high T. - Under these conditions, series of complex reaction take place. - Simple dehydration reaction produces various furan derivatives which then condense with each other and other products to produce brown & black substances. - Also condense with phenol produces colored compounds. V. Phenol-Sulphuric Acid Method/Dubois method  Procedure: - Clear aqueous carbs solution pipetted into a test-tube, - Then, aqueous phenol solution and sulphuric acid were added. - Contents are mixed. - Sulphuric acid causes all non-reducing sugars to be converted to reducing sugars. - Solution turns to yellow-orange color as a result of interaction between carbs and phenol. - Absorbance measured at 490 nm. - Proportional to carbs concentration in sample. V. Phenol-Sulphuric Acid Method /Dubois method  Disadvantages: - Method is non-stoichiometric, need to prepare std curve. Advantages: - Simple, rapid, sensitive, specific for carbs and widely applied. - Inexpensive reagent, available and stable. - Color produced is stable and results are reproducible. - Under proper condition, method is accurate to ±2%. V. Phenol-Sulphuric Acid Method /Dubois method  SELF-READING VI. Dinitrosalicylic Method  SELF-READING + Refractive index, polarimetry, IR and density are common physical method that have been used to determine carbs concentration in food. Rely on changes in some physicochemical characteristics as its carb concentration varies. Suitable for pure carbs syrups, clarified solutions; sometimes require conversion table. Physical Analysis  Methods are rapid, highly specific and sensitive to low concentration Little sample preparation 2 methods commonly used: - Total change method - Kinetic method Enzymatic method  Total change method- allowing the reaction to go to completion and measuring the concentration of the product, which is proportional to the concentration of the initial substrate. -Glucose is converted to glucose-6-phosphate (G6P) by hexakinase and ATP; G-6-P+ + NADP+ → gluconate-6-phosphate + NADPH + H+ - The amount of NADPH formed is proportional to the concentration of G6P in the sample - and can be measured spectrophotometrically at 340 nm. Enzymatic method  Kinetic method- measuring the initial rate of the enzyme catalyzed reaction because the rate is proportional to the substrate concentration. -Glucose is oxidized by glucose peroxidase to form gluconic acid and hydrogen peroxide; which then reacts with dye in the presence of peroxidase to give a stable colored product that can be quantified spectrophotometrically. Enzymatic method  Analysis of Starch - In a natural form, starch exists as water-insoluble granules - Consists of two glucose homopolysaccharides o Amylose (500-2000 glucose units, linear) o Amylopectin (>1,000,000 glucose units, extensively branched. o Have different physicochemical properties. o Important to determine concentration of each individual component, as well as the overall starch concentration. - In processed foods, starch undergoes changes because of processing treatments involved (e.g. heating) Analysis of polysaccharides  Analysis of Starch - In a natural form, starch exists as water-insoluble granules - Consists of two glucose homopolysaccharides o Amylose (500-2000 glucose units, linear) o Amylopectin (>1,000,000 glucose units, extensively branched. o Have different physicochemical properties. o Important to determine concentration of each individual component, as well as the overall starch concentration. - In processed foods, starch undergoes changes because of processing treatments involved (e.g. heating) Analysis of polysaccharides  Starch content cannot be determined directly: contained within a structurally and chemically complex food matrix. In particular, starch present in semi-crystalline form (granular or retrograded starch) inaccessible to chemical reagents used for determination. Necessary to isolate starch from other components prior to starch analysis. In natural form e.g. in legumes, cereals or tubers, starch granules are separated by drying, grinding, steeping in water, filtration and centrifugation. Starch granules water-insolubility and relatively high density (1500 kg/m3) makes it separated from other water-soluble and less-dense materials. Sample preparation  In processed food, samples are normally dried, ground and dispersed in hot 80% ethanol solutions. Monos- and oligos- are soluble in ethanol solution, while starch insoluble. Hence, starch sperated from sugars by filtering/centrifuging the solution. For semi-crystalline starch (difficult to extract), sample dispersed in water and heated until starch gelatinizes (>65 C). Add perchloric acid or calcium chloride prior to heating facilitates solubilization. Sample preparation  SELF-READING Blue-Value (Amylose content) method  CHO content can be determined by calculating the % remaining after all the other components have been measured: % CHO= 100 – (% moisture + % protein + % of lipid + % mineral) Can lead to erroneous results due to experimental errors in any of the other methods. Calculation by difference  Most of chemical techniques for carb analysis are based on reduction of reducing sugars with chemical reagents to yield precipitates or colored complexes, which are quantitate by solubilization, by titration or by sectrophotometric determination. Some methods are non-stoichiometric, therefore requires std curve. Problems in accuracy when mixtures of sugars being determined. No universal procedure for polysaccharides analysis. Isolation must precede/’first thing to do’ measurement. SUMMARY  Introduction: generally defined as lignin plus plant polysaccharides e.g. cellulose, hemicellulose and pectin that cannot be digested by human enzymes. Some types of starch (resistant starch) are also indigestible by human enzymes – can be analyzed as dietary fibre. Divided into 2 categories: water soluble and water insoluble. - Insoluble fibres e.g. cellulose, hemicellulose and lignin - Soluble fibres e.g. gum and pectin. Crude/Dietary Fibre Determination  Index of feeding value of poultry and stock feed. Evaluating the efficiency of milling and separating bran fibre from endosperm. Determination of succulence of fresh vegetables and fruits Importance of fibre determination  Crude method gives an estimate of indigestible fibre in food It is determined by sequential extraction of a defatted sample or by selective precipitation after enzymatic solubilisation. Measured gravimetrically by weighing the mass of indigestible polysaccharides remain after digestible carbohydrate, lipids and proteins are selectively solubilized by chemicals or enzymes. Indigestible materials are then collected. Analytical methods  Liquid removal - Food sample analyzed is dried, ground to a fine powder and then lipids are removed by solvent extraction. Protein removal – Proteins are broken down and solubilized using enzymes, strong acid or strong alkali solutions. The resulting amino acids are then separated from insoluble fibre by filtration or from total fibre by precipitation with ethanol. Starch removal – starch is gelatinized and then broken down and solubilized by enzymes, strong acid or strong alkali. Glucose produced is separated from insoluble fibre via precipitation with ethanol. Sample preparation  Food with significant amount of resistant starch and lignin will show high fibre content If sugar is not extracted from sugar-rich-food prior to fibre analysis, fibre content will be underestimated. Incorporation of proteolytic enzyme in both AOAC and Englyst Cummings procedures to digest protein allows some fibre to be solubilized which then affect the movement of some of the insoluble fibre fraction into the soluble fibre fraction. Factors affecting Fibre Determination Accuracy Crude Fibre Principle : Digestible CHO, lipid, protein are selectively solubilized by chemical and/or enzymes. Indigestible carbohydrate is collected by filtration; residue is dried, weighed, ashed and quantified gravimetrically. A. Gravimetric Method  Procedures: - Crude fibre was determined by sequential extraction of sample with 1.25% H2SO4 and 1.25% NaOH. Sample digested by H2SO4 to hydrolyze CHO and protein. Reacted with NaOH to saponify fatty acid. - Insoluble residue is collected by filtration then dried, weighed, A grams. Ashing conducted, cooled and weighed to correct mineral contamination, B grams. - Crude fibre content = (A – B) grams - Crude fibre determined consist mainly of cellulose and lignin. - Pectin and hemicellulose are solubilized – cannot be detected. I. Crude Fibre  Principle: - Duplicate sample are enzymatically digested with - amylase, amyloglucosidase and protease to remove starch and protein. - Insoluble fibre is collected by filtration. - Soluble fibre is precipitated by bringing to 78% ethanol and collected by filtration. - The filtered fibre residues are washed with ethanol and acetone, oven-dried and weighed. - One duplicate is analyzed for protein and the other is incinerated to determine ash content. -Fibre = residue weight - (weight of protein+ash) II. Dietary Fibre – AOAC method  Fibre is eqal to the sum of all non-starch monosaccharide plus lignin. Monosaccharide are measured indirectly by colorimetric or chromatographic methods. A. Chemical Method  Principle: - Defatted food sample is heated in water to gelatinized the starch. - Enzymes like pancreatin and pullanase are then added to digest starch and protein. Fibre is precipitated by adding 100% ethanol, separated by centrifugation, washed and dried. - The remaining non-starch polysaccharides are hydrolysed by sulphuric acid to liberate monosaccharides. - Neutral sugars are determined by GC and uronic acids are determined colorimetrically. -Fibre = residue weight - (weight of protein+ash) III. Englyst-Cummings method  Methods selected for crude fibre analysis determined the accuracy of measurement/result achieved. Gravimetric and chemical methods commonly used to determine total, soluble and insoluble fibre content of foods, give comparable estimates of fibre content for most foods. Gravimetric method overestimates foods rich in simple sugars (glucose, fructose and sucrose) e.g. in dry fruits because sugars are trapped in precipitate formed when ethanol is added. SUMMARY  EC method does not determine lignin and resistant starch, therefore gives low fibre content for foods high in these substances (e.g. corn flakes or cereal brans). Gravimetric and EC methods both use proteolytic enzymes to digest protein- can cause some of the fibre to be broken down and solubilized, which would increase soluble fibre detected. There are also differences in time, equipment, chemicals and level of technical skill required to carry out each analysis that should be considered when selecting the most appropriate technique for a particular application. SUMMARY  Must remember all methods name + principal + what is being measured + troubleshooting + precaution + advantages/disadvantages. Some measurement that involved mechanism (chemical equation) and calculation; please memorize. Also, reactions that involves chemical reagents, try to memorize the names (the most significant ones) and their function. For all analytical techniques Application of Enzymes in Starch Processing  Browning is a common color change seen in food during pre-preparation, processing or storage of food It occurs in varying degrees in some food The color produced range from cream, pale yellow to dark brown and red Browning reactions observed in food may be classified as enzymatic or non-enzymatic browning Browning and non browning reaction, caramelization Enzymatic Browning https://www.youtube.com/watch?v=36K4iEuqQzo Enzymatic Browning Enzymatic Browning -Prevention Enzymatic Browning -Prevention Non-Enzymatic Browning Non-Enzymatic Browning Food products with Maillard rxn Food products with Maillard rxn Food products with Maillard rxn Browning rxn in meat Caramelization Caramelization Classes of caramel  Options: https://slideplayer.com/slide/10956555/  Crude fibre, acid detergent fibre and neutral detergent fibre determinations  Crude fibre, acid detergent fibre and neutral detergent fibre determinations  Crude fibre, acid detergent fibre and neutral detergent fibre determinations 1. Detergent Analysis 2. Enzymatic Analysis Flowchart Crude Fiber Determination 1. Standing method Crude Fiber Determination 1. Standing method Crude Fiber Determination 1. Standing method 2. Filtration method https://www.slideserve.com/sbass/determination-of-crude-fiber-powerpoint- ppt-presentation Analysis with automated instruments  BWD 20303 FOOD ANALYSIS 1 DURATION : 3 HOURS INSTRUCTION : All objectives questions 3 February 2025 2.30pm ALL THE BEST!! YOU CAN DO IT, SO PROVE IT.