Food Chemistry Module 1-4 PDF

TECHNOLOGICAL UNIVERSITY OF THE PHILIPPINES VISAYAS Capt. Sabi St., City of Talisay, Negros Occidental College of Engineering Technology Office of the College Dean LEARNING MODULE CHEM413B FOOD CHEMISTRY DEPARTMENT: BS CHEMISTRY COMPILED BY: ROSELYN DE LA CRUZ USERO Week 1 – 4 2024 This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION 2 LEARNING GUIDE Week No.: 1-4 TOPIC: Introduction to Food Chemistry and Principal Components of Food with focus on Carbohydrates and Proteins I. Introduction to Food Chemistry 1.1 Importance of Food chemistry 1.2 Analytical Approach to Food Chemistry 1.3 Strategic Research Fields 2. Principal Components of Food 2.1 Carbohydrates 2.1.1 1ntroduction 2.2.2 Monosaccharides 2.2.3 Oligosaccharides 2.2.4 Polysaccharides 2.2.4.1 Starch and Starch Degrading Enzymes 2.2.4.2 High Fructose Corn Syrup (HFCS) 2.2.4.3 Starch Hydrolyzates: Corn Sweeteners and Modified Starches 2.2.4.4 Glycogen 2.2.4.5 Cellulose, Pentosans/Hemicelluloses 2.2.4.6 Dietary Fiber 2.2.4.7 Lignins 2.2.4.8 Pectins and Gums 2.2.5 Physical Properties and Chemical Reactions 2.2.6 Functional Properties in Food 2.2.7 Test Methods 2.2 Proteins 2.2.1 Introduction 2.2.2 Amino Acid Composition and Structure 2.2.3 Protein Classification 2.2.4 Denaturation 2.2.5 Nonenzymic Browning 2.2.6 Chemical Changes 2.2.7 Functional Properties 2.2.8 Animal and Plant Proteins 2.2.9 Test Methods - Determination of Overall Protein Concentration 2.2.9 Test Methods - Protein Separation and Characterization This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 3 EXPECTED COMPETENCIES At the end of the lesson, you should have 1. A better understanding of the chemical aspects of food composition, chemistry behind the biochemical nature of food, its properties and how they are processed in the body; 2. described the structure of carbohydrates and proteins; 3. known the functional properties and chemical reactions of the major components of foods with focus on carbohydrates and proteins and the enzymatic reactions; and 4. learned the principles and method in the qualitative and quantitative tests for carbohydrates and proteins. CONTENTS/TECHNICAL INFORMATION I. Introduction to Food Chemistry 1.1 Importance of Food chemistry Food chemistry is a branch of chemistry that deals with the study of chemical processes and interactions between all biological and non-biological components of foods, the changes in foods taking place during processing and storage (Kumar, 2016). It provides techniques and ways either to enhance the changes in food like enhancement of fermentation by conversion of lactose to lactic acid by microorganisms or to prevent changes in foods like prevention of browning in apples and pears. Food chemistry allows for subjecting food materials to chemical scrutiny. It employs chemistry tools to analyze food items so that they transform to nutritious, safe and materials of commercial value. Instruments that are popular in the vicinity of chemistry are employed in food chemistry. The importance of food chemistry lies in its ability to counter the effects of decomposition and spoilage and extend the shelf life of foods (Kumar, 2016). Common methods of food preservation include salting, cooking, drying, refrigeration, canning, irradiation, dehydration, wood smoke, use of spices, pickling, fermentation etc. Flavors, preservatives, emulsifiers, thickeners, stabilizers, sweeteners, colors are some of the materials that are produced from food chemistry. A consideration of the development of these materials from their crude source through research, development, production, regulation and commerce; just proves how expansive is the importance of food chemistry. 1.2 Analytical Approach to Food Chemistry Food analysis chemistry is able to provide information about chemical composition, processing, quality control (QC) and contamination of foodstuffs, ensuring compliance with food and trade laws (Rocco, Aturki and Fanali, 2013). Thorough knowledge of analytical chemistry, chemical and microbial kinetics, thermodynamics, biochemistry, industrial microbiology, and This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 4 molecular biology is the key criteria to understand food chemistry better. Analytical chemistry is the branch of chemistry based on the qualitative and quantitative determination of compounds present in a sample under examination, therefore, through its application, it is possible to determine the quality of a product and/or its nutritional value, reveal adulterations, identify the presence of xenobiotic substances potentially harmful to human health (Gallo, 2020). Food chemistry is concerned also in the desirable and undesirable reactions which are controlled by a variety of physical and chemical parameters (Cheungand Mehta, 2015). Food chemistry helps to design and develop new food products for industries. The study of the component of various food substances could be used to initiate an array of chemical reactions that could lead to the formation of either new or improved food products. The improvement could be taste enhancement, new aroma, color, or increased shelf life. 1.3 Strategic Research Fields Food Proteins - Proteins are highly susceptible to chemical modifications during processing and storage, e.g. by oxidation, Maillard reactions (i.e. protein glycation), and by reactions with phenolic compounds. Proteins are key nutrients and it is critically important that the proteins in our diet are of high nutritional quality and are not damaged during processing and storage. o Development of new gentle biotechnologies to reduce or avoid those modifications of proteins that are responsible for food quality deterioration and improve protein functionality. o Understanding chemical mechanisms for protein modifications that allows for specific and tailored solutions to prevent or control protein modifications. Physical and Chemical Stability of Foods- the general approach is based on using combinations of model systems and real food systems for obtaining both specific information about and more generic understanding of kinetics and mechanisms of chemical and physical reactions that affect the stability of foods and beverages. A special focus area has been the development and use of Electron Spin Resonance spectroscopy (ESR) for studies of food systems and the role of radicals as reactive intermediates during processing and storage of foods and beverages. Food Innovation - Understanding the reaction kinetics and the thermodynamics of the enzyme-catalyzed processes in food and model systems using enzyme assays, spectroscopic or chromatographic techniques. o Reaction kinetics and thermodynamics of food enzymes. o Spectroscopic techniques for the study of structural properties and reactions of proteins, including absorption and fluorescence spectroscopy under high hydrostatic pressure. o Separation techniques in protein chemistry including chromatographic methods. o Characterisation of protein denaturation by differential scanning calorimetry. o Enzymatic extraction of food hydrocolloids by pressure tuning of reaction conditions Enzymology - Enzymes catalyze many chemical reactions, which can have a positive or negative effect on the food materials. Understanding the reaction kinetics and the thermodynamics This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 5 of the enzyme-catalyzed processes in food systems is mandatory in order to control the reaction and obtain the desired outcome. The underlying mechanism and relationship between the concentration of enzyme and substrate are the key factors in tuning enzymatic reactions like hydrolysis responsible for color, texture and flavor improvement or degradation. Enzyme activity can be assessed by both qualitatively and quantitatively by using various enzyme assays in combination with spectroscopic and chromatographic techniques. Functional Food Design- Food processing involves various physical and chemical changes that need to be controlled in order to obtain the desired food product. Knowing the underlying mechanisms and relationships between the process, functional properties like gelation, emulsification, foaming ability and water binding, and structure is highly important for a successful outcome. Since the structure of a food product is based on the physical or chemical properties of the individual components as well as interrelations, the processing needs to modify these properties to create the right functionality. Functional Food Design - focus on process-property-structure interactions in food materials, by coupling food processing technology and food chemistry. Tailoring functional properties due to well-characterized processes can be utilized in product development. o design unique foods with improved structure, flavor, or nutritional quality based on physicochemical understanding of interaction between food components and matrix effects. o anchor knowledge and application about functionality and properties together with product formulation and processing technologies in order to solve the world’s challenges concerning food quality and safety, public health and environmental sustainability. 2. Principal Components of Food 2.1 Carbohydrates 2.1.1 1ntroduction Carbohydrates are hydrates of carbon with the empirical formula Cm(H2O)n, structurally they are more accurately viewed as polyhydroxy aldehydes and ketones. Three chemical groups of CHO: a. monosaccharides (simple sugars) b. oligosaccharides c. polysaccharides. Carbohydrates in food can also be classified as simple or complex, with the difference between the two forms being the chemical structure and how quickly they are absorbed and digested. In animal organisms, the main sugar is glucose and the storage carbohydrate is glycogen; in milk, the main sugar is disaccharide lactose. In plant organisms, a wide variety of monosaccharides and oligosaccharides occur, storage polysaccharides (starch, and structural polysaccharides) such as cellulose and hemicellulose. 2.2.2 Monosaccharides The simplest sugars or monomeric units which cannot be hydrolyzed into smaller carbohydrates. Chemically, they are aldehydes or ketones with two or more hydroxyl groups. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 6 General chemical formula --- Cn(H2O)n. Monosaccharides range from 3-8 C atoms which constitute the backbone of the sugar. The C backbone is then appended with Hydrogen or Hydroxy groups. Three classifications according to different characteristics: a. Placement of the carbonyl group. b. Number of C atoms it contains. c. Chirality. If the carbonyl group is an aldehyde then the sugar is an aldose; if the carbohyl group is a ketone the sugar is a ketose. Most natural sugars are the D series. D and L structures are non-superimposable mirror images, named enantiomers. The main difference between L and D isomers is that the OH- group of the penultimate carbon is positioned on the right side of the D isomer whereas, in L isomer, it is located on the left side. Structures of monosaccharides Source: britannica.com The 3-C aldose is referred to as D or L-glyceraldehyde (2,3-dihydroxypropanal) and the keto version is dihydroxyacetone (dihydroxy- propanone). The most common sugars in food and nutritional chemistry are pentoses and hexoses. D-glucose is the most important monosaccharide and is derived from the simplest sugar, D-glyceraldehyde, an aldotriose. The designation of aldose and ketose sugars indicates the chemical character of the reducing form and can be the simple or open-chain formula of Fischer. Haworth representations were developed to give a more accurate spatial view of the molecule. Carbohydrates are either acyclic or exist as furanosides (f) or pyranosides (p). Haworth formula does not account for the actual bond angles, the modern conformational formulas moreaccurately represent the sugar molecule. Representation Fischer, Haworth, and conformational representations of α- and β- D-glucose structures Source: linkspringer.com This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 7 2.2.3 Oligosaccharides The reducing group of one monosaccharide can connect to one of the hydroxyl groups on another through glycosidic bond, to form disaccharides. More connections of glycosidic bonds will give rise to trisaccharides, tetrasaccharides, etc.,categorized as oligosaccharides. The glycosidic bond is the result of the condensation of the hydroxyl of the hemiacetal group of C-1 with the hydroxyl of an alcohol. When a glycosidic linkage is established only between the lactol groups of two monosaccharides, then a nonreducing disaccharide is formed, and when one lactol group and one alcoholic HO group are involved, a reducing disaccharide results (Belitz et al. 2009, Cheung and Meta, 2015). Monosaccharides bind together in condensation reactions to form glycosidic linkages Source: http://silt3.com/GlycosidesResearch/glycosidic-linkages. Oligosaccharides can be homologous or heterologous, and occur widely in plants, but can also be produced synthetically or via microbial fermentative and enzymatic processes. Primary oligosaccharides are those synthesized in vivo from a mono or oligosaccharide and a glycosyl donor by the action of a glycosyl transferase enzyme (Kandler and Hopf 1980; Eggleston and Côté 2003; deMan, et al., 2018). Secondary oligosaccharides are those formed in vivo or in vitro by hydrolysis of larger oligosaccharides, polysaccharides, glycoproteins, or glycolipids. Sucrose is the most common primary oligosaccharide in plants. It is a major sweetener, the largest commercial sources are from sugarcane or sugar beet. o In sucrose the reducing groups of the constituent monosaccharides (glucose and fructose) are linked by a glycosidic bond, thus it is one of the few non-reducing disaccharides. o Due to the unique carbonyl-to-carbonyl linkage, sucrose is highly labile in acid medium, and acid hydrolysis is more rapid than other di and oligosaccharides. o When sucrose is heated to 210 °C, partial decomposition takes place and caramel is formed. o Sucrose is highly soluble over a wide temperature range. This property makes sucrose an excellent ingredient for syrups and other sugar-containing foods. Lactose - the sugar in mammalian milk which is normally easily digested and converted to energy. Some individuals, however, lack the enzyme lactase, which hydrolyzes lactose and are, therefore, “lactose intolerant.” In such individuals the lactose is fermented in the lower gastrointestinal tract causing discomfort and diarrhea. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 8 o Lactose from cow’s milk is less sweet than sucrose and, unlike sucrose, is a reducing sugar. o Lactose is the only sugar in the milk of all mammalian species and does not occur elsewhere. It is also a major constituent of the dry matter of cow’s milk, as it represents close to 50% of the total solids. The lactose content of cow’s milk ranges from 4.4 to 5.2%, with an average of 4.8% expressed as anhydrous lactose, the human milk is higher, about 7.0%. o Lactose is a disaccharide of D-galactose and D-glucose. It is hydrolyzed by the enzyme β-D-galactosidase (lactase) or by dilute solutions of strong acids. Organic acids such as citric acid, which easily hydrolyze sucrose, are unable to hydrolyze lactose. Maltose - represents an important disaccharide found widely in plants, and is the basic building block of starch and glycogen polysaccharides. o When starch and glycogen polysaccharides are hydrolyzed, the primary degradation product is maltose disaccharide. For example, in brewing, starch is hydrolyzed by amylases to maltose (maltose has a characteristic flavor of malt) which is then available for hydrolysis to glucose by glucoamylase, with subsequent conversion to ethanol by yeast. The α-1 → 4 linkage is broken by amylases and maltases. o Maltose is a reducing disaccharide, shows mutarotation, is fermentable, and is easily soluble in water. Other glucose disaccharides o Isomaltose is a breakdown product during production of glucose from starch and dextran polysaccharides, found in honey. o Gentiobiose occurs as a glycoside in amygdalin, found various stone fruits. o Trehalose is found in yeast. o Sugars are important in the texture and appearance of foods, particularly confections, cakes and cookies. The equilibrium between dissolved and crystalline sugars in the cream centers of confections help control the texture. In cookies, sugar can exist in crystalline, glass, and dissolved forms. o In the crystalline form most sugars are single anhydrous anomers. When crystallized at temperatures below 50 °C, glucose crystalizes as an α-pyranose monohydrate. o Syrups of reducing sugars such as corn syrup (mostly glucose) are particularly resistant to crystallization. o Crystallization of sucrose from concentrated solutions produce high purity crystals. o Hard candy is produced by boiling an aqueous mixture of sucrose and glucose with flavors and colors to produce a glass which is a super-cooled liquid. o In cakes, the glucose syrup functions to retain moistness in the crumb and molding characteristics of fondants. Disaccharide Sugar Alcohols o The most common disaccharide alcohols include iso- malt, maltitol, lactitol, and hydrogenated starch hydrolyzates (HSH). o Maltitol is hydrogenated maltose and has the highest sweetness of the disaccharide polyols compared to sugar, gives no cooling effect in contrast to sorbitol and xylitol. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 9 Maltitol exhibits a very high viscosity in solution. Sorbitol and maltitol are derived from starch. o Human milk contains about 1 g/L of these oligosaccharides referred to as the bifidus factor with a beneficial effect on the intestinal flora of infants. o Fructooligosaccharides (FOSs) or “fructans” are also primary oligosaccharides based on sucrose and occur naturally as components of edible plants including banana, tomato, and onion (Spiegel et al. 1994, deMan, et al., 2018). FOSs are also manufactured commercially by the action of a fungal enzyme from Aspergillus niger, β- fructofuranosidase, on sucrose. 2.2.4 Polysaccharides Polysaccharides consist of more than ten monosaccharide units bound to each other by glycosidic linkages. Their complete acidic hydrolysis yields monosaccharides. Polysaccharides (glycans) can have only a type of sugar structural unit (homoglycans) or several types of sugar units (heteroglycans). 2.2.4.1 Starch and Starch Degrading Enzymes Starch is a hompolymer of D-glucose and is a storage carbohydrate in plants. It is a polymer of glucose linked to one another through the C1 oxygen, known as the glycosidic bond. This glycosidic bond is stable at high pH but hydrolyzes at low pH. At the end of the polymeric chain is the reducing end, a latent aldehyde group. Each granule contains millions of amylopectin molecules packed with a larger number of smaller amylose molecules. o The amylose polymer is composed of glucose units in a linear polymer while amylopectin is a highly branched polymer. Both are composed of α-D-glucose units. The helical organization of amylase and the branched structure of amylopectin Source: https://byjus.com/biology o Starches are mainly used to absorb water and produce viscous fluids, pastes and gels for control of texture in foods. The extent of gelatinization of starch in baked goods has a This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 10 major impact on product properties including texture, storage stability and rate of digestion. o More extensive hydrolysis of starch with acid or enzymes results in the formation of maltodextrins that are categorized by their chain length which is expressed as dextrose equivalents (DE) or degree of hydrolysis. DE is defined as the amount of reducing sugars present as dextrose and calculated as a % of the total dry matter. o DE is related to the average degree of polymerization (DP) in the maltodextrin by the equation: DE = 100/DP. The DE can also be considered as the % of reducing power that would come from pure glucose. The DE is inversely related to average MW of remaining polymers in the material. o There are basically four groups of starch-converting enzymes: (i) endoamylases; (ii) exoamylases; (iii) debranching enzymes; and (iv)transferases. Different enzymes involved in the degradation of starch. Source: researchgate.net Enzymatic conversion of starch Source: Tomasik and Horton (2012) This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 11 2.2.4.2 High Fructose Corn Syrup (HFCS) HFCS from corn starch is less expensive than sucrose. The major use of HFCS has been in beverages and soft- drinks. The production of HFCS from glucose syrup requires the use of ion-exchange chromatography to remove calcium which inhibits glucose isomerase. Chromatographic techniques are used to produce syrups with various proportions of fructose and glucose. Production of High Fructose Corn Syrup (https://www.foodscience) 2.2.4.3 Starch Hydrolyzates: Corn Sweeteners and Modified Starches o Starch can be hydrolyzed by acid or enzymes or by a combination of both. Corn is sprayed with HCl and heat applied and neutralized. The product is washed with water and dried. In lightly treated starches the granules remain intact but the starches cook more quickly and result in thinner and more clear and less viscous solutions. These starches are used for coatings, film formation and pan coating of nuts and candies. o Corn starch is an excellent starting material to produce strong, fast setting gels in products like jelly beans and processed cheese loafs. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 12 Table 1 Primary reactions in starch modification Source: deMan, et al., (2018) This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 13 Table 2. Starch modification reactions Source: deMan, et al., (2018) 2.2.4.4 Glycogen o Humans, animals, and fungi produce glycogen in muscle as an energy reserve. It is the polysaccharide used for storing carbohydrates in animal tissues and is a highly branched polysaccharide of glucose. Plays an important role in glucose cycle. o In humans, glycogen, hydrated with three or four parts of water, is produced and stored primarily in the cells of the liver and muscles. Muscle glycogen is converted into glucose by muscle cells, and liver glycogen coverts to glucose for use throughout the body. Glycogen is similar to starch in is readily rehydrolyzed to produce glucose for the muscle tissue on demand. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 14 o In the liver, when a meal containing carbohydrates or protein is eaten and digested, blood glucose levels rise, and the pancreas secretes insulin. Insulin acts on blood glucose in the liver to stimulate the action of several enzymes including glycogen synthase. Glycemic Index (GI) is a number associated with a particular type of food that indicates the food’s effect on a person’s blood glucose (also called blood sugar) level). 100 is the standard equivalent to pure glucose. The GI represents the rise in a person’s blood sugar level 2 h after consumption of the food, and is useful to understand who the body breaks down available carbohydrates in foods, i.e., starch. The GI is applied in the context and quantity of the food and the amount of carbohydrate in the food that is actually consumed. A related measure, the glycemic load (GL), factors this by multiplying the glycemic index of the food by the carbohydrate content of the serving. Glycemic index Charts often give only one value per food, but variations occur due to, for example, variety, ripeness, cooking methods, processing, and the length of food storage. Glycogen structure Source: Engelking (2015) 2.2.4.5 Cellulose, Pentosans/Hemicelluloses Cellulose - one of the most widely distributed compounds in nature generally existing as homologus polymers accompanied by other polysaccharides and lignins. The great strength, fibrous character, insolubility and inert characteristics of cellulose, integral to its skeletal function in plant cell walls are due to the ordered packing of the cellulose chains. The H bonds result in a high degree of crystallinity and dense structure This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 15 contributing to areas that do not absorb water effectively and exhibit a high degree of enzyme resistance. Cellulose and starch structures Source: Storz, Henning. (2014) Cellulose and modified celluloses are used in a wide range of foods to provide important physical characteristics including bulk replacements of digestible carbohydrates in low calorie foods. Unmodified cellulose and its derivatives are used to provide bulk, imbibe oils or flavors, or carriers to improve the flow characteristics of intense sweeteners or flavors. 2.2.4.6 Dietary Fiber 2.2.4.7 Lignins Polymeric natural products resulting from enzyme-initiated dehydrogenative polymerization of three primary recursors:trans-coniferyl,trans- sinapyl, and trans-p-coumaryl alcohol. Lignin is not a polysaccharide but a component of dietary fiber and an important constituent of plant tissues. Lignin is present in mature plant cells and provides mechanical support, conducts solutes, and provides resistance to microbial degradation. Lignin structure. Source: Eloide (2021) This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 16 2.2.4.8 Pectins and Gums Pectins and gums are important polysaccharides in foods because of their functional properties. They are widely used as gelling agents, thickeners, and stabilizers. They are constituents of plant tissue and are large, complex molecules whose exact nature is not certain. Sugar competes for water, thus making less water available to associate with the pectin molecules. This reduces the attractive forces between the pectin and water molecules. Gum arabic is a dried exudate from acacia trees. It is a neutral or slightly acidic salt of a complex polysaccharide containing calcium and magnesium. The molecule is made up of four sugars, L-arabinose, L-rhamnose, D-galactose, and D-glucuronic acid. It is one of the few gums that require high concentration to give increased viscosity and is used as crystallization inhibitor and emulsifier. Seaweed Polysaccharides include the agars, alginates, and carrageenans. Unlike most other gums, they are able to form gels under certain conditions. Carrageenan is obtained from red seaweeds, especially from Irish moss. It occurs as three main fractions, known as kappa, iota, and lambda carrageenan. Each is a galactose polymer containing varying amounts of negatively charged sulfate esters. Kappa carrageenan contains the smallest number of sulfate esters, and is the least negatively charged. It is able to form strong gels with potassium ions. Lambda carrageenan contains the largest number of sulfate groups and is too highly charged to form a gel. Iota carrageenan forms gels with calcium ions. 2.2.5 Physical Properties and Chemical Reactions Hygroscopicity of sugar in crystallized form is the ability of moisture uptake by sugars. The hygroscopicity varies and depends on the sugar structure, isomers present, and sugar purity (Belitz et al. 2009). Solubility. Monosaccharides tend to be soluble in polar solvent; they are also soluble to a small extent in ethanol but not soluble in organic solvents such as benzene, chloroform, and ether. When the crystal sugars cake together, the solubility will decrease. Sugar solution in high concentrations, e.g, glucose syrup, is used in baking industry to retain food moisture. The solubility of mono- and oligosaccharides in water is good. The physical and sensory properties of oligosaccharides are covered by monosaccharides since oligosaccharides consist of not more than 10 sugar molecules. While sugars form true solutions, polysaccharides form colloidal solutions and therefore, difficult to purify. The polysaccharides are tasteless and amorphous. Chemical Reactions Carbohydrates can perform wide chemical reactions with other compounds because besides having hydroxyl groups, some monosaccharides and sugars also have carbonyl groups available for reaction. Maillard Reaction- is categorized as nonenzymatic browning and plays an important role in improving appearance and taste of cooked food, particularly roasting, toasting, and baking food. The compounds that contribute to Maillard reaction were sugars and amino acids in water. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 17 The chemistry underlying the Maillard reaction is very complex. It encompasses not one reaction pathway but a whole network of various reactions. Source: https://foodresearchlab.com/academy Caramelization -is different from Maillard. In caramelization, sugar decomposition occurs at high temperature and produces brown color; this reaction does not involve proteins and produces different variety of compounds than Maillard, including organic acids, aldehydes, and ketones. Reaction is facilitated by the presence of an acid, base, or salt. Heating causes dehydration of the sugar molecule with introduction of double bonds or formation of anhydro rings. In Maillard browning, intermediates such as 3-deoxyosones and furans are formed. The unsaturated rings may condense to form useful, conjugated double-bond-containing, brown- colored polymers. Catalysts increase the reaction rate and are used to direct the reaction to specific types of caramel colors, solubilities, and acidities. Caramel served as colorant and flavor in cola, acidic beverages, baked goods, syrups, candies, and dry seasoning 2.2.6 Functional Properties in Food The most obvious sensory property of sugars such as glucose, fructose, and sucrose is their sweetness, which varies depending on the specific sugar. Lactose, which is commonly found as dominant sugars in milk, is the least sweet, whereas fructose is the sweetest sugar. Sugars can form solution because they are soluble in water and form syrups when the concentration in water is high. In syrup production, water is evaporated to lead to crystal formation, thus the molecular solutions are formed because of hydrogen-bond interchange. When sugar is placed in water, the water molecules immediately form hydrogen bonds with the sugar molecules, thus hydrating them and removing them from hydrogen bonds between the sugar crystals. At high concentrations, sugars act as a food preservative which prevents food spoilage by growth of microorganisms. This mechanism is related to reducing the water activity in food to a level below which bacterial growth cannot be supported. Mono- and polysaccharides in appropriate concentration or proportion contribute to the viscosity of foods, thus giving mouthfeel to foods. This functionality related the binding This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 18 capacity of the molecules particularly with water; when replaced by a chemically sweetening additive such as aspartame or saccharin. 2.2.7 Test Methods of Analysis for Carbohydrates a. Qualitative Methods 1). Molisch Test: used to detect the presence of carbohydrates in different samples. It can be used to detect the formation of carbohydrates as a by-product in different reactions and distinguish it from other biomolecules. Principle: The concentrated acid catalyzes the dehydration of sugars to form furfural (from pentoses) or hydroxymethyl furfural (from hexoses). Either of these aldehydes condenses with two molecules of naphthol to form a purple or violet colored complex at the interface of the acid and test layer. If the carbohydrate is poly- or disaccharide, a glycoprotein or glycolipid, the acid first hydrolyses it into component monosaccharides, which get dehydrated to form furfural or its derivatives. A green ring might be observed if any impurities are present in the reagent as they might interact with the α-naphthol and the acid. A rind ring is seen if a concentrated sugar solution is used. This might be due to the charring of the sugar due to the acid. Reaction in Molisch Test Reaction in Molisch Test Source: https://microbenotes.com/molisch-test/ https://microbenotes.com/molisch-test/ The formation of the purple colored ring occurs at the interface between the sulphuric acid and the test solution. The acid remains above the test solution as the acid is denser than the test solution. The absence of color indicates a negative result. Limitations of Molisch Test- trioses and tetroses do not have the necessary five carbon atoms for furfural formation, so they do not give a positive result for this reaction. Molisch test is This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 19 not a specific test for carbohydrates. Furfurals as such or furfural yielding substance, some organic acids like citric acids, lactic acid, oxalic acid, formic acid, etc. can give a positive result. 2). Benedict's Test: a qualitative test often used for the differentiation of carbohydrates (saccharides/sugars) into reducing and non-reducing types. Principle of Benedict’s Test. NaCO3 in the Benedict reagent increases the pH of the sample-reagent solution mixture. Under warm alkaline conditions reducing sugars are tautomerism to strong reducing agents, enediols. These enediols reduce the cupric ions (Cu 2+) (present as CuSO4) of Benedict reagent into cuprous ions (Cu+). The cuprous particles are present in form of insoluble Copper (I) oxide or cuprous oxide (Cu2O) which is of red color precipitate. The concentration of reducing sugar in the sample differs from the intensity and shade of the color of the reaction mixture. Color may vary from greenish to yellow to orange-red to brick- red. As the concentration of reducing sugar increases color gradually changes from greenish to yellowish to orange to brick-red. Any change in color from blue to green or yellow or orange or red within 3 minutes indicates a positive Benedict test i.e. presence of reducing sugar in the sample. For semiquantitative evaluation, the concentration of reducing sugar can be estimated based on the shade of developed colors. Reaction in Benedict’s Test Source: https://microbenotes.comt/ 3). Barfoed's Test - a chemical test used to detect the presence of monosaccharides which detects reducing monosaccharides in the presence of disaccharides. This reaction can be used for disaccharides, but the reaction would be very slow. Principle of Barfoed’s Test- the Barfoed reagent is made up of copper acetate in a dilute solution of acetic acid. Since acidic pH is unfavorable for reduction, monosaccharides, which are This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 20 strong reducing agents, react in about 1-2 min. The reducing disaccharides take a longer time of about 7-8 minutes, having first to get hydrolyzed in the acidic solution and react with the reagent. Once the reaction takes place, thin red precipitate forms at the bottom of the sides of the tube. The difference in the time of appearance of precipitate distinguish reducing monosaccharides from reducing disaccharides. Reaction in Barfoed’s Test Source: https://microbenotes.comt/ Limitation of Barfoed’s Test. This test cannot be used to detect sugar in samples containing Cl– ions, which might interfere with the reaction. If a higher concentration of disaccharides is present in a sample, it might give a positive result. 4). Bial's Test is a chemical test performed to detect the presence of pentoses and pentosans (derivatives of pentoses). A derivation of this test termed the Bial’s Orchintest is performed to detect the presence of RNA in solutions. Principle of Bial’s Test is based on the principle that under hydrolysis pentosans are hydrolyzed into pentoses. Further, pentoses are dehydrated to yield furfural, which in turn condense with orcinol to form a blue-green precipitate. In the presence of hexoses, hydroxyfurfural is formed instead of furfural which upon condensation with orcinol forms a muddy brown colored precipitate. The intensity of the precipitation is directly proportional to the concentration of the pentoses in the sample. The intensity of the color developed depends on the concentration of HCl, FeCl3, orcinol, and the duration of boiling. The concentration of the sugars is determined by measuring the absorbance of 620 nm wavelength in a spectrophotometer. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 21 Reaction in Bial’s Test Source: https://microbenotes.comt/ Limitations of Bial’s Test - On prolonged heating, glucoronates might also give a blue- green colored precipitate which might result in false-positive results. The color produced might be different with different sugars, and the concentration might not be proportional to the intensity at higher levels 5). Seliwanoff's or Resorcinol Test is used to differentiate between sugars that have a ketone group (ketose) and sugars that have an aldehyde group (aldoses). This test is a timed color reaction specific to ketohexoses. Principle of Seliwanoff’s test - the reagent of this test consists of resorcinol and concentrated HCl. The acid hydrolysis of polysaccharides and oligosaccharides yields simpler sugars. Ketoses are more rapidly dehydrated than aldoses. Ketoses undergo dehydration in the presence of concentrated acid to yield 5-hydroxymethyl furfural. The dehydrated ketose reacts with two equivalents of resorcinol in a series of condensation reactions to produce a complex (not a precipitate), termed xanthenoid, with deep cherry red color. Aldoses produce a faint pink to cherry red color if the test is prolonged. The product and reaction time of the oxidation reaction helps to distinguish between carbohydrates. Other carbohydrates like sucrose and inulin also give a positive result for this test as these are hydrolyzed by acid to give fructose. Uses of Seliwanoff’s test- Seliwanoff’s color reaction is used in the method for the colorimetric determination of fructose in fermentation media. A modified version of this test can be used for the determination of the concentration of ketoses in a given sample. Limitations of Seliwanoff’s test -the high concentration of glucose or other sugar may interfere by producing similar colored compounds with Seliwanoff’s reagent. Prolonged boiling can transform glucose to fructose by the catalytic action of acid and form cherry red-complex giving a false-positive result. This test is a generalized test and doesn’t distinguish between specific ketoses, and a separate test is required for the particular ketose sugar identification. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 22 Reaction in Seliwanoff’s Test Source: https://microbenotes.comt/ 6). Tollens’ test - is a chemical test used to differentiate reducing sugars from non- reducing sugars. This test is also called the silver mirror test based on the end product of this test. Principle of Tollens’ test – the Tollen’s reagent is the alkaline solution of silver nitrate (AgNO3) mixed with liquid ammonia (NH3), which results in the formation of a complex. The aqueous solution of silver nitrate forms a silver aqua complex where the water acts as a ligand. The aqua complexes are then converted into silver oxides (Ag2O) by the action of hydroxide ions. Silver oxide forms a brown precipitate, which is then dissolved by aqueous ammonia resulting in the formation of the [Ag(NH3)2]+ complex. This complex is the primary component of the Tollen’s reagent and is a strong oxidizing agent. The complex then oxidizes the aldehyde group present in some sugars to form a carboxylic acid. At the same time, the silver ions present in the reagent are reduced to metallic silver. The reduction of silver ions into metallic silver results in the formation of a silver mirror on the bottom and sides of the test tube. An α-hydroxy ketone gives a positive as the Tollen’s reagent oxidizes the α-hydroxy ketone into an aldehyde. Uses of Tollens’ test - routinely performed in chemical laboratories for the qualitative organic analysis, which distinguishes aldehydes from ketones. This test is also used for the differentiation of reducing sugars from non-reducing sugars. Limitations of Tollens’ test - Some carbohydrates that do not have an aldehyde group might give a positive result on Tollen’s test because of the isomerization of such sugars under alkaline conditions. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 23 Reaction Tollen’s test Reaction in Tollen’s Test Source: https://microbenotes.comt/ b. Quantitative Methods 1) Anthrone test is used to determine and quantify carbohydrates in a variety of samples such as milk, blood serum, and its variations. Principle- the concentrated acid in the Anthrone reagent first hydrolyzes carbohydrate into component monosaccharide if it is present in the form of free carbohydrate as poly- or monosaccharide or bound as in a glycoprotein or glycolipid. The concentrated acid also catalyzes the dehydration of monosaccharides, resulting in furfural (from pentoses) or hydroxyl furfural (from hexoses). Anthranol, the enol tautomer of anthrone, is the active form of the reagent, which combines with the carbohydrate furfural derivative to produce a green colour in dilute solutions and a blue colour in concentrated solutions, which can be detected colorimetrically. The blue - green solution shows absorption maximum at 620 nm. Reaction in Anthrone Test Source: https://microbenotes.comt/ Reactions:  Hydrolysis of polysaccharides to monosaccharide, Polysaccharide → Monosaccharides  Dehydration of monosaccharides to furfural, Monosaccharide → Furfural  Reaction of furfural with naphthol Furfural + Anthrone reagent (naphthol) → Blue-green complex. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 24 2). DNSA (Dinitrosalicylic acid) method - DNSA is an aromatic compound that reacts with react with reducing sugars to estimate the amount of carbohydrates. Principle- 3, 5- Dinitrosalicylic acid (DNSA) that forms 3-amino-5-nitrosalicylic acid (ANSA) when it combines with reducing sugars [with a free carbonyl group (C=O)]. The aldehyde functional group (in glucose) and the ketone functional group (in fructose) are both oxidised in this process. DNSA is reduced to ANSA, which under alkaline condition is transformed to a reddish brown colored complex. The absorbance is measured at 540 nm. Mechanism of DNSA (Dinitrosalicylic acid) method Source: https://biocyclopedia.com/index/biotechnology 3). Phenol sulphuric acid method- the portion of neutral sugar in oligosaccharides, proteoglycans, glycoproteins, and glycolipids is most commonly determined using this method. Principle - in the presence of a strong acid, carbohydrates (simple sugars, oligosaccharides, polysaccharides, and their derivatives) is dehydrated to hydroxymethy furfural that condense with phenol to produce stable yellow-gold compounds that could be spectrophotometrically quantified at 490 nm. Phenol sulphuric acid method 4). Advanced analysis of carbohydrates in food Nuclear Magnetic Resonance (NMR). Since the structure of the carbohydrate will determine its function, the use of NMR is becoming crucial for an accurate characterization of both the newly designed and natural carbohydrates. The application of NMR may provide information related to the structure, purity and safety of carbohydrate samples. Mass Spectrometry (MS) is a powerful technique used to identify chemical compounds based on their different mass-charge ratio (m/z). The chemical compound has to be ionized before entering the MS analyzer; electron impact ionization (EI), chemical ionization (CI), This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 25 electrospray ionization (ESI), plasma desorption ionization (PDI) and matrix laser desorption ionization (MALDI) are the most frequently used ionization techniques for analysis of organic compounds such as food carbohydrate. Gas chromatography- has been extensively applied to carbohydrates determination in foods. and has been mainly applied to study mono-, di- and tri-saccharides composition of foods. Concerning the detection methods, flame ionization detector (FID) is the most commonly used. More recently, the use of mass spectrometers as detectors has increased, providing advantages over the more traditional FIDs, mainly, the possibility of attaining a higher degree of certainty in the identification of carbohydrates, the determination of the molecular mass and on the reproducible fragmentation patterns. A common approach consists of using both, a GC- MS method to identify the carbohydrates and a GC-FID to carry out the quantification. High performance liquid chromatography - is at present the main analytical technique used for carbohydrates analysis. Compared to the other analytical techniques, HPLC is able to provide some additional advantages. Although less sensitive than GC, HPLC is faster, does not necessarily require sample derivatization, allows the analysis of larger carbohydrates and is more versatile due the different separation mechanisms and detectors that can be employed. Capillary electrophoresis - charged compounds are separated in dissolution according to their effective electrophoretic mobilities under the influence of an electric field. Modification of the carbohydrate structure through derivatization procedures can improve both sensitivity and selectivity of the CE separation. Among the derivatizing compounds to allow UV detection, phenylethylamine (200 nm), 4-amino-benzoic acid ethyl ester (306 nm) or 6-aminoquinolone (245 nm) can be cited as examples. pNitroaniline was used to allow UV detection of different hexoses in powdered milk and rice 31 syrup, using a blue light-emmiting diode as light source and detecting at 406 mn. The use of this reagent was justified considering its high solubility in water and molar absorptivity. Other detection methods that have been used together with CE to detect underivatized carbohydrates are electrochemicaldetectors or even Fourier transform infrared (FTIR) detection Multidimensional techniques, glycomics and others- multidimensional techniques along with hyphenated techniques for carbohydrates analysis together with new approaches for glycomics are presented. Sample complexity has lead to the development of new methods involving, in some cases, on-line sample preparation through microdialisis coupled to HPAEC and integrated pulsed electrochemical detection-mass spectrometry (IPED-MS) to solve in a fast and automatic way the complete analysis of carbohydrates. 2.2 Proteins 2.2.1 Introduction Proteins are present in all living things and have a key role in many biological processes. The nutritional energy value of proteins is 4 kcal/g. Amino acids are required building blocks for protein biosynthesis. Proteins contribute to the flavor of food, precursors for aroma compounds and colors formed during various processing of food. Proteins have ability to build or stabilize gels, foams, emulsions, and fibrillar structures, which is essential in certain food products. Proteins are polymers of some 21 different amino acids joined together by peptide bonds. Because of the variety of side chains that occur when these amino acids are linked together, the different proteins may have different chemical properties and widely different secondary and tertiary structures. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 26 2.2.2 Amino Acid Composition and Protein structure There are 20 amino acids forming the building blocks of most proteins. Each amino acid contains a primary amine and carboxylic acid group linked by peptide (amide) bonds formed between α-amino and α-carboxylic acid groups of neighboring amino acids in the polypeptide sequence. The amide linkage in proteins is a partial double bond. General structure of amino acid In the simplest case, R = H (aminoacetic acid or glycine). In other amino acids, R is an aliphatic, aromatic, or heterocyclic residue and may incorporate other functional groups. The amounts of these essential amino acids present in a protein and their availability deter- mine the nutritional quality of the protein. In general, animal proteins are of higher quality than plant proteins. Plant proteins can be upgraded nutritionally by judicious blending or bygenetic modification through plant breeding. An essential amino acid is an amino acid that cannot be synthesized de novo (from scratch) by the organism, and thus must be supplied in the diet. The nine amino acids humans cannot synthesize are phenylalanine, valine, threonine, try tophan, methionine, leucine, isoleucine, lysine, and histidine. Six other amino acids, are arginine, cysteine, glycine, gluta- mine, proline and tyrosine are considered conditionally essential in the human diet. Egg protein is one of the best quality proteins and is considered to have a biological value of 100. It is widely used as a standard, and protein efficiency ratio (PER) values sometimes use egg white as a standard. Cereal proteins are generally deficient in lysine and threonine, as indicated. Primary Structure - refers to the linear sequence in which the constituent amino acids are covalently linked through amide bonds, also known as peptide bonds. The primary structure is determined by its genetic code and post-translational covalent modifications. The primary structure of a protein determines the physicochemical and the functional properties of food protein. Secondary Structure- the primary structure gives the sequence of amino acids in a protein chain while the secondary structure reveals the arrangement of the chain in space. Secondary structure may result from aperiodic (random coil) or periodic structures. Tertiary Structure refers to the spatial arrangement attained when a linear protein chain with secondary structure segments folds further into a compact three-dimensional form. Formation of tertiary structure involves hydrophobic, electrostatic, van der Waals, and hydrogen bonding between various groups of proteins. The folding of the protein into a tertiary structure defines the size and shape of that protein. Quaternary Structure refers to the spatial arrangement of a protein when it contains more than one polypeptide chain, which are also referred to as subunits or oligomers. Formation of quaternary structure is primarily driven by the thermodynamic requirement to bury exposed hydrophobic surfaces of subunits. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 27 Table 3 Different Amino Acids Source: Cheung.,et al. (2015). Structures of different amino acids This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 28 Structure of Proteins Source: https://microbenotes.comt/ 2.2.3 Protein Classification- Proteins modified by enzyme or made complex with nonprotein components (prosthetic groups) are called conjugated (heteroproteins) proteins whereas modified proteins are known as homoproteins. Table 4. Classification of Proteins Source: Cheung.,et al. (2015). 2.2.4 Denaturation Protein denaturation refers to changes in the secondary and tertiary structure of the protein. The primary structure (the amino acid sequence) of a protein remains unchanged. The denaturation is a reversible or irreversible change of native conformation without cleavage of covalent bonds. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 29 Denaturation does not involve any chemical changes in the protein. Any treatment that cleaves hydrogen bridges or ionic or hydrophobic bonds is responsible for denaturation. This can be accomplished by: changing the temperature, adjusting the pH, increasing the interface area, or adding organic solvents, urea, salts, guanidine hydrochloride, or detergents such as sodium dodecyl sulfate. Denaturation is generally reversible when the peptide chain is stabilized in its unfolded state by the denaturing agent and the native conformation can be reestablished after removal of the agent. Irreversible denaturation occurs when the unfolded peptide chain is stabilized by interaction with other chains (as occurs, for instance, with egg proteins during boiling) Protein denaturation Source: researchgate.net 2.2.6 Nonenzymic Browning Non-enzymatic browning between reducing carbohydrates and amines, also known as the Maillard reaction (MR), is of crucial importance in food science where it significantly contributes to taste, aroma and color. It is now established that the MR takes place in vivo under physiological conditions, where non-enzymatic reactions between carbohydrates and proteins lead to irreversible protein modifications associated with a wide range of diseases, such as diabetes mellitus. Using nonenzymatic browning reaction, Maillard reaction is commonly invoked to account for abiotic chemical transformations of organic matter and is ubiquitous in baking, toasting and cooking of foods and in vivo in mammalian organisms. Nonenzymatic Browning The key difference between enzymatic and nonenzymatic browning is that the enzymatic browning involves enzymes such as polyphenol oxidase and catechol oxidase whereas the nonenzymatic browning does not involve any enzymatic activity. Food browning is the process of turning a food such as fruits and vegetables into a brown color due to the chemical reactions that take place in that food involving enzymes such as polyphenol oxidase and catechol oxidase. This has many implications for the food industry, especially regarding the cost. Nonenzymatic browning is the process of food browning due to a chemical reaction that not catalyzed by an enzyme. It produces brown pigments in food. It involves a chemical reaction This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 30 between the amine group of free amino acid and the carbonyl group of reducing sugar. There are two main types of this reaction as caramelization and Mallard reaction. Caramelization involves the pyrolysis of sugar useful in cooking to get a nutty flavor and brown color. Volatile chemicals release producing the characteristic caramel flavor. In Mallard reaction, a chemical reaction takes place between the amine group of free amino acid and the carbonyl group of reducing sugar. This reaction occurs with the addition of heat. The sugar reacts with the amino acid producing a variety of odors and flavors. This is responsible for the production of flavor after the food is cooked. It is important in producing artificial flavors for processed food. The type of amino acid that involves the reaction determines the flavor of the end product. Methods of preventing browning could consist of measures intended to slow reaction rates, such as control of moisture, temperature, or pH, or removal of an active intermediate. It is easier to use an inhibitor. One of the most effective inhibitors of browning is SO2 or sodium bisulfite. SO2 reacts with the degradation products of the amino sugars preventing these compounds from condensing into melanoidins. A serious drawback of the use of SO2 is that it reacts with thiamine and proteins, thereby reducing the nutritional value of foods. SO2destroys thiamine and is not permitted for use in foods containing this vitamin. 2.2.7 Chemical Changes Mild heat treatments in the presence of water can significantly improve the protein’s nutritional value in some cases. S-containing amino acids may be more available and antinutritional factors such as the trypsin inhibitors of soybeans may be deactivated. Excessive heat in the absence of water can be detrimental to protein quality; for example, in fish proteins, tryptophan, arginine, methionine, and lysine may be damaged. The Maillard reaction leads to the formation of brown pigments, or melanoidins, which are not well defined and may result in numerous flavor and odor compounds. The browning reaction may also result in the blocking of lysine. Lysine becomes unavailable when it is involved in the Amadori reaction, the first stage of browning. Light-induced oxidation of proteins has been shown to lead to off-flavors and destruction of essential amino acids in milk. 2.2.8 Functional Properties depend on physicochemical properties like shape, size, composition and sequence of amino acid, distribution of charges, structural levels of proteins, hydrophobicity/hydrophilicity ratio, molecular flexibility, and ability to react/interact with other components (like lipids, sugars, polysaccharides, salts, and minor components). Proteins can form networks and structures and provide essential amino acids. They interact with other components and improve quality attributes of foods Solubility of Proteins o Protein solubility provides useful information on foams, emulsions, and gels. Protein solubility is influenced by sequence and composition of amino acid, molecular weight, and conformation and content of polar and nonpolar groups in amino acids. o Ionic strength, type of solvent, pH, temperature, and processing conditions affect the This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 31 solubility of proteins. o At low ionic strengths, the solubility rises with increase in ionic strength. Neutral salts have a twofold effect on protein solubility. At low concentrations they increase the solubility (“salting in” effect) by suppressing the electrostatic protein-protein interaction (binding forces). Protein solubility is decreased (“salting out” effect) at higher salt concentrations due to the ion hydration tendency of the salts. o Solubility of various proteins decreases variously with temperature and time of heating. Water Holding Capacity of Proteins o The water holding capacity (WHC) of foods can be defined as the ability to hold its own and added water during the application of forces, pressing, centrifugation, or heating. WHC plays a major role in the formation of food texture, in comminuted meat products and baked doughs. o Water retention is a critical factor in protein functionality because it affects the texture, color, and sensory properties of products. o Water binding depends on the composition and conformation of the protein molecules. o Water interacts with proteins in a number of ways, and significant amounts of water bounded by proteins are retained by hydrogen bonding. Structural water is held H bonding between polypeptide groups of the proteins. Binding of water to proteins is related to the polar hydrophilic groups, such as amino, carboxyl, hydroxyl, carbonyl, and sulfhydryl groups o The binding of water is due to the dipolar character of water. Proteins that contain numerous charged amino acids will tend to bind large amounts of water. Foam Formation and Foam Stabilization o Proteins function as foam-forming and foam-stabilizing components in many foods, e.g., baked goods, sweets, desserts, and beer. The most widely used protein foaming agents are egg white, gelatins, casein, other milk proteins, soy proteins, and gluten. Serum albumin foams very well, while egg albumin does not. o Foams consist of an aqueous continuous phase and a gaseous (air) dispersed phase. Foam can be defined as a two-phase system consisting of air cells separated by a thin continuous liquid layer called the lamellar phase. To perform this function proteins must be amphiphilic. This is achieved when part of the protein structure contains predominantly amino acids with hydrophobic side chains and another part contains mostly hydrophilic side chains. o To be effective surfactants, proteins need to have flexible polypeptide chains, so that they are able to orient at the interface. Only proteins that have little secondary structure and are able to unfold at the interface are effective emulsifiers. Gel Formation o A gel is an intermediate phase between a solid and a liquid. Gel is a substantially diluted system that exhibits no steady-state flow. o Gelation is a basic process in the processing of various foods, milk gels, comminuted meat and fish products, other meat products, fruit jellies, bread doughs, pie and cake fillings, coagulated egg white, and others. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 32 o Protein gels may be utilized to simulate the textural properties and mouthfeel of lipids. Gels are formed when partially unfolded proteins develop uncoiled polypeptide segments that interact at specific points to form a three dimensional cross-linked network. Emulsifying Effect o Emulsions are dispersed systems of one or more immiscible liquids. They are stabilized by emulsifiers – compounds which form interface films and thus prevent the disperse phases from flowing together. The adsorption of a protein at the interface of an oil droplet is thermodynamically favored because the hydrophobic amino acid residues can then escape the H bridge network of the surrounding water molecules. o Contact of the protein with the oil droplet results in the displacement of water molecules from the hydrophobic regions of the oil–water boundary layer. o The suitability of a protein as an emulsifier depends on the rate at which it diffuses into the interface and on the deformability of its conformation under the influence of interfacial tension (surface denaturation). The diffusion rate depends on the temperature and the molecular weight, which in turn can be influenced by the pH and the ionic strength. o A protein with ideal qualities as an emulsifier for an oil-in-water emulsion would have a relatively low molecular weight; a balanced amino acid composition in terms of charged, polar, and nonpolar residues; good water solubility; well-developed surface hydrophobicity; and a relatively stable conformation. Table 5. Functional properties of food proteins Source: Cheung.,et al. (2015. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 33 Table 6 Functional properties of proteins in food systems Source: deMan, et al., (2018). 2.2.8 Animal and Plant Proteins The main difference between animal and plant protein is that animal protein is a complete protein, containing all essential amino acids, whereas plant proteins are incomplete proteins, providing only several essential amino acids to the diet. Furthermore, animal proteins are 90% absorbable while plant proteins are 60-70% absorbable. Importantly, there are certain nutrients which are only included in animal protein. They are heme-iron, vitamin B12, vitamin D, DHA, and zinc. Heme-iron is in red meat and is readily absorbable by the body in contrast to the non-heme-iron present in plant protein. Vitamin B12 is in fish, meat, poultry, and dairy products. Vitamin D occurs in oily fish, eggs, and dairy products. Moreover, DHA is an essential omega-3 fatty acid, which mainly occurs in oily fish. Zinc mainly occurs in beef, pork, and lamb. The differences in both proteins include molecular structure, amino acid profile, digestibility, and technical functionality in food, i.e. the ability to gel, emulsify, bind water etc. These inherent differences influence their bioavailability from a human nutrition perspective, as well as the sensory quality of foods containing animal or plant proteins. These fundamental differences mean that designing plant-based foods to mimic animal foods requires much more than simple substitution of one ingredient with another (LiDay, 2022). Plant-based foods provide less complete protein nutrition because of lower digestibility and source-specific deficiencies in essential amino acids, compared with animal proteins. Plant proteins can be subjected to various processes to bring their functionality closer to that of animal proteins (e.g. hydrolysis to improve solubility), some processes that improve functionality diminish amino acid bioaccessibility or bioactivity, creating negative nutritional This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 34 consequences. Much more research and innovation are required to enhance the potential of plant proteins. Nutritional and functional synergies between plant and animal proteins may offer a path to creating nutritious and attractive foods (LiDay, 2022).. 2.2.9 Test Methods - Determination of Overall Protein Concentration Kjeldahl method- involves the digestion of food with a strong acid so that nitrogen is released, which is then quantified using a titration technique. Protein quantity is then calculated from the nitrogen concentration of the food using a conversion factor (usually 6.25 which is equivalent to 0.16 g nitrogen per gram of protein). This is considered the standard method for protein measurement but has its disadvantages. A conversion factor of 5.6 is recommended for shrimp and fish, 5.4 for cereal products and 4.59 for red seaweed (Hayes, 2020) Dumas method-is fast and does not use chemicals, but is costly to set up and is not very accurate as it does not measure true protein. Direct measurement methods using UV-spectroscopy and refractive index measurement UV spectrophotometric methods, including the Biuret, Bradford and Lowry methods are easy to use, not costly and can quantify small amounts of protein. However, they can give false positive protein readings depending on the sample preparation method used and solubility of the test sample (Hayes, 2020). HPLC - Direct amino acid analysis involves hydrolysis of the protein with HCL and subsequent quantification of the amino acids using HPLC. Table 7 Different analytical methods for the determination of protein. Source: Hayes, 2020 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC759795 This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 35 Methods Based on Different Solubility Characteristics o Salting out- proteins are precipitated from aqueous solutions when the salt concentration exceeds a critical level, which is known as salting-out, because all the water is "bound" to the salts, and is therefore not available to hydrate the proteins. Ammonium sulfate [(NH4)2SO4] is commonly used because it has a high water-solubility, although other neutral salts may also be used, e.g., NaCl or KCl. Generally a two-step procedure is used to maximize the separation efficiency. o Isoelectric Precipitation- The isoelectric point (pI) of a protein is the pH where the net charge on the protein is zero. Proteins tend to aggregate and precipitate at their pI because there is no electrostatic repulsion keeping them apart. Proteins have different isoelectric points because of their different amino acid sequences (i.e., relative numbers of anionic and cationic groups), and thus they can be separated by adjusting the pH of a solution. When the pH is adjusted to the pI of a particular protein it precipitates leaving the other proteins in solution. o Solvent Fractionation- the solubility of a protein depends on the dielectric constant of the solution that surrounds it because this alters the magnitude of the electrostatic interactions between charged groups. As the dielectric constant of a solution decreases the magnitude of the electrostatic interactions between charged species increases. The optimum quantity of organic solvent required to precipitate a protein varies from about 5 to 60%. Solvent fractionation is usually performed at 0oC or below to prevent protein denaturation caused by temperature increases that occur when organic solvents are mixed with water. o Denaturation of Contaminating Proteins-many proteins are denatured and precipitate from solution when heated above a certain temperature or by adjusting a solution to highly acid or basic pHs. Proteins that are stable at high temperature or at extremes of pH are most easily separated by this technique because contaminating proteins can be precipitated while the protein of interest remains in solution. Separation due to Different Adsorption Characteristics o Adsorption chromatography involves the separation of compounds by selective adsorption-desorption at a solid matrix that is contained within a column through which the mixture passes. Separation is based on the different affinities of different proteins for the solid matrix. Affinity and ion-exchange chromatography are the two major types of adsorption chromatography commonly used for the separation of proteins. Separation can be carried out using either an open column or high-pressure liquid chromatography. o Ion Exchange Chromatography relies on the reversible adsorption-desorption of ions in solution to a charged solid matrix or polymer network. This technique is the most commonly used chromatographic technique for protein separation. The buffer conditions (pH and ionic strength) are adjusted to favor maximum binding of the protein of interest to the ion-exchange column. Contaminating proteins bind less strongly and therefore pass more rapidly through the column. The protein of interest is then eluted using another buffer solution which favors its desorption from the column (e.g., different pH or ionic strength). This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 36 o Affinity Chromatography uses a stationary phase that consists of a ligand covalently bound to a solid support. The ligand is a molecule that has a highly specific and unique reversible affinity for a particular protein. The sample to be analyzed is passed thru the column and the protein of interest binds to the ligand, whereas the contaminating proteins pass directly through. The protein of interest is then eluted using a buffer solution which favors its desorption from the column. This technique is the most efficient means of separating an individual protein from a mixture of proteins, but it is the most expensive, because of the need to have columns with specific ligands bound to them. Separation Due to Size Differences o Dialysis is used to separate molecules in solution by use of semipermeable membranes that permit the passage of molecules smaller than a certain size through, but prevent the passing of larger molecules. A protein solution is placed in dialysis tubing which is sealed and placed into a large volume of water or buffer which is slowly stirred. Low molecular weight solutes flow through the bag, but the large molecular weight protein molecules remain in the bag. Dialysis is a relatively slow method, taking up to 12 hours to be completed. It is therefore most frequently used in the laboratory. Dialysis is often used to remove salt from protein solutions after they have been separated by salting-out, and to change buffers. o Ultrafiltration- a solution of protein is placed in a cell containing a semipermeable membrane, and pressure is applied. Smaller molecules pass through the membrane, whereas the larger molecules remain in the solution. The separation principle of this technique is therefore similar to dialysis, but because pressure is applied separation is much quicker. That portion of the solution which is retained by the cell (large molecules) is called the retentate, whilst that part which passes through the membrane (small molecules) forms part of the ultrafiltrate. Ultrafiltration can be used to concentrate a protein solution, remove salts, exchange buffers or fractionate proteins on the basis of their size. o Size Exclusion Chromatography -this technique, sometimes known as gel filtration, also separates proteins according to their size. A protein solution is poured into a column which is packed with porous beads made of a cross-linked polymeric material (such as dextran or agarose). Molecules larger than the pores in the beads are excluded, and move quickly through the column, whereas the movement of molecules which enter the pores is retarded. Thus molecules are eluted off the column in order of decreasing size. Molecular weights of unknown proteins can be determined by comparing their elution volumes Vo, with those determined using proteins of known molecular weight: a plot of elution volume versus log(molecular weight) should give a straight line. Separation by Electrophoresis- electrophoresis relies on differences in the migration of charged molecules in a solution when an electrical field is applied across it. It can be used to separate proteins on the basis of their size, shape or charge. Non-denaturing Electrophoresis a buffered solution of native proteins is poured onto a porous gel (usually polyacrylamide, starch or agarose) and a voltage is applied across the gel. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 37 Denaturing Electrophoresis- proteins are separated primarily on their molecular weight. Proteins are denatured prior to analysis by mixing them with mercaptoethanol, which breaks down disulfide bonds, and sodium dodecyl sulfate (SDS), which is an anionic surfactant that hydrophobically binds to protein molecules and causes them to unfold because of the repulsion between negatively charged surfactant head-groups. This type of electrophoresis is commonly called sodium dodecyl sulfate -polyacrylamide gel electrophoresis, or SDS-PAGE. To determine how far proteins have moved a tracking dye is added to the protein solution, e.g., bromophenol blue. This dye is a small charged molecule that migrates ahead of the proteins. After the electrophoresis is completed the proteins are made visible by treating the gel with a protein dye such as Coomassie Brilliant Blue or silver stain. The relative mobility of each protein band is calculated: Electrophoresis is used to determine the protein composition of food products. The protein is extracted from the food into solution, which is then separated using electrophoresis. SDS- PAGE is used to determine the molecular weight of a protein by measuring Rm, and then comparing it with a calibration curve produced using proteins of known molecular weight: a plot of log (molecular weight) against relative mobility is usually linear. Denaturing electrophoresis is more useful for determining molecular weights than non-denaturing electrophoresis, because the friction to movement does not depend on the shape or original charge of the protein molecules. Isoelectric Focusing Electrophoresis- this technique is a modification of electrophoresis, in which proteins are separated by charge on a gel matrix which has a pH gradient across it. Proteins migrate to the location where the pH equals their isoelectric point and then stop moving because they are no longer charged. This methods has one of the highest resolutions of all techniques used to separate proteins. Amino Acid Analysis is used to determine the amino acid composition of proteins. A protein sample is first hydrolyzed (e.g. using a strong acid) to release the amino acids, which are then separated using chromatography, e.g., ion exchange, affinity or absorption chromatography. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 38 PROGRESS CHECK Select the what is required in the questions and explain your choice. 1. About protein denaturation. Which is the wrong statement? a. The primary structure of protein does not change. b. Globular proteins are converted into fibrous proteins. c. Fibrous proteins are converted into globular proteins. d. The biological property of proteins is destroyed.. 2. About proteins. Which is the wrong statement? a. Proteins occur in living cells. b. Proteins generally contains N, C and H c. Hydrolysis of proteins in acidic solutions forms amino acids (AAs). d. Their solubilities in water don’t reach minimum value at isoelectric points. 3. About enzymes. Which is the wrong statement? a. All enzymes found in the cells are invariably proteins which catalyze biological reactions. b. Enzymes act efficiently at moderate temperatures and pH c. Coenzymes increase activities of enzymes. d. Enzymes are not specific in their actions and substarates. 4. About AAs, Which is the wrong statement? a. In nature, about 20 amino acids in proteins b. The human body can synthesize the 20 amino acids in proteins. c. The simplest amino acid is glycine. d. There are 10 essential amino acids. 6. Which of the statement is correct? a. All AAs except lysine are optically active. b. All AAs are optically active. c. All AAs except glycine are optically active. d. All AAs except glutamic acids are optically active. 7. Which of the following AAs is neutral? a. Aspartic Acid b. Glycine c. Lysine d. Arginine 8. About Carbohydrates. Choose the best answer and explain. a. Hydrates of Carbon b. Polyhydroxy aldehydes and ketones c. Polyhydroxy acid compounds d. None of these 9. The difference between α- D glucose and β- D glucose is a. number of OH groups. b. size of hemiacetal rings c. conformation d. Configuration This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 39 10. Kjeldahl's method can be for Nitrogen determination on ? a. Aniline b. Methyl amine c. Urea d. none of the above 11. Choose the answer based on these statements. Statement. Dumas method is more applicable to N containing organic compounds than Kjeldahl's method. Reason: Kjeldahl's method does not give satisfactory results for compounds in which N is linked to O atom. a. Both statements are correct. b. Both statements are correct but the reason is not the correct explanation for the statement c. Statement is correct but reason is not. d. Both the statement and the reason in incorrect. 12. What compound gives a positive reaction in Tollens test but negative iodoform test. a. b. c. 13. Chromatographic technique is used in: a. for testing the purification of drinking water. b. for detecting chiral compounds c. in the food industry d. all of these 14. Which is used to separate the analytes of a mixture in food raw materials or plants for more advanced use? a. preparative HPLC b. analytical HPLC c. both a and b d. none 15. Chromatographic techniques in food analysis is based on : a. physical adsorption b. chemical adsorption c. hydrogen bond d. absorption 16. Denaturing of proteins cause na lose in? a. primary structure b. sequence of amino acids c. three dimensional structure d. peptide bond 17. Why is lactose less sweeter than fructose? 18. Why is High Fructose Corn Syrup less expensive that sucrose? 19. Why can sugars may be used to control freezing point and crystallization in ice cream, or boiling point in candy making? 20. Show can sugars in jams and jellies prevent the growth of microorganisms,? This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 40 REFERENCES/SOURCES Cheung, Peter C.K. ed-in-Chief, Mehta, Bhavbhuti M. ed. (2015). Handbook of Food Chemistry https://earthwormexpress.com/wp-content/uploads/2021/10/. deMan, John M., Finley, John W., Hurst, Jeffrey W., Lee, Chang Yong (2018). Principles of Food Chemistry, 4th ed. http://www.springer.com/series/5999 Engelking, Larry R. (2015). Textbook of Veterinary Physiological Chemistry (3rd ed.). Elodie Melro, Alexandra Filipe,b., Dora Sousa,c., Medronho, Bruno and Romano, Anabela ( 2021). Revisiting lignin: a tour through its structural features, characterization methods and applications, New Journal of Chemistry. https://pubs.rsc.org/en/content/articlelanding/2021/nj/d0nj06234k/unauth#!divCitation Hayes M. (2020). Measuring Protein Content in Food: An Overview of Methods. Foods. doi: 10.3390/foods9101340. PMID: 32977393; PMCID: PMC7597951. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7597951/table/foods-09-01340- t001/?report=objectonly Hasa (2019). What is the Difference Between Animal and Plant Protein. https://pediaa.com/ Herrero, Miguel, Alejandro Cifuentes, Ibáñez, Elena; del Castillo, Maria Dolores., Advanced Analysis of Carbohydrates in Foods. https://digital.csic.es/bitstream1/1094f Kumar, Krishan & Kumar, Rajesh & Kumar, Vinod & Sagwal, Sony. (2016). Role of Chemistry in Food Processing and preservation. Li Day, Julie A. Cakebread, Simon M. Loveday (2022). Food proteins from animals and plants: Differences in the nutritional and functional properties, Trends in Food Science & Technology. ISSN 0924-2244, https://doi.org/10.1016/j.tifs.2021.12.020. (https://www.sciencedirect.com/science/article/pii/S0924224421006774) Mauricio, Diego; Osorno, Sanchez and Castro, Cristina (2018). Cellulose Application in Food Industry: A Review, Emergent Research on Polymeric and Composite Materials DOI: 10.4018/978-1-5225-3023-7.ch002 Mahu (2018). Difference Between Enzymatic and Nonenzymatic Browning https://www.differenceenzymatic-and-nonenzymatic-browning/ Naviglio D, Gallo M. (2020) Application of Analytical Chemistry to Foods and Food Technology. Foods. doi: 10.3390/foods9091296. PMID: 32942539; PMCID: PMC7554738. Oliveira, Fabíola & Coimbra, Jane & De Oliveira, Eduardo Basilio & Giraldo-Zuniga, Abraham & Rojas, Edwin. (2016). Food Protein-Polysaccharide Conjugates obtained via the Maillard Reaction: A Review. Critical reviews in food science and nutrition. 56. 10.1080/10408398.2012.755669. This module is a property of Technological University of the Philippines Visayas and intended for EDUCATIONAL PURPOSES ONLY and is NOT FOR SALE NOR FOR REPRODUCTION. 41 Rocco, Anna, Aturki, Zeineb, Fanali, Salvatore (2013). Chiral separations in food analysis, TrAC Trends in Analytical Chemistry, , https://doi.org/10.1016/j.trac.2013.05.022. (https://www.sciencedirect.com/science/article/pii/S016599361300191X) Storz, Henning. (2014). Bio-based plastics: status, challenges and trends. Landbauforschung Volkenrode. 63. 321-332. 10.3220/LBF_2013_321-332. Shukla Pooja, Meghani Sonali, Patel Charmi, Shah Dhrumi, Rathod Zalak R* and Saraf Meenu S. (2022). A Review on Qualitative and Quantitative Analysis of Carbohydrates Extracted from Bacteria. Acta Scientific Pharmaceutical Sciences (ISSN: 2581-3226) https://actascientific.com/ASPS/pdf/ASPS-06-0858.pdf Tangboriboon, Nuchnapa & Phudkrachang, Prapaphan & Mulsow, La-Orngdow & Kunchornsup, Wissawin & Sirivat, Anuvat. (2013). Removal of water extractable proteins from concentrated natural rubber latex by eggshells. Journal of Elastomers and Plastics. 45. 253-269. 10.1177/0095244312452273. Tomasik, Piotr and Horton, Derek (2012) Enzymatic conversions of starch, Advances in Carbohydrate Chemistry and Biochemistry. ISSN 0065-2318, ISBN 9780123965233, https://doi.org/10.1016/B978-0-12-396523-3.00001-4. (https://www.sciencedirect.com/science/article/pii/B9780123965233000014) van der Maarel, Marc & Veen, Bart & Uitdehaag, Joost & Leemhuis, Hans & Dijkhuizen, Lubbert. (2002). Properties and applications of starch-converting enzymes of the ??- amylase family. Journal of biotechnology. 94. 137-55. 10.1016/S0168-1656(01)00407-2. Importance of Food Chemistry(2012). https://discover.hubpages.com/education/Importance-of-Food- Chemistry britannica.com linkspringer.com https://food.ku.dk/english/research_at_food/research_fields/food-chemistry/. https://www.foodscience-avenue.com/2015/02/production-of-high-f https://study.com/academy/lesson/glycogen-definition-storage-breakdown.html http://epgp

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