Food Safety Dr. Wahida H. Alqahtani Disclaimer The texts, tables and images contained in this course presentation are not my own, they can be found on: References supplied Atlases or The web sites FSN 522 Food Safety Dr. Wahida H. Alqahtani

Part 10 out of 2 Enzymatic reactions IN FOOD Dr. Wahida H. Alqahtani Disclaimer The texts, tables and images contained in this course presentation are not my own, they can be found on: References supplied Atlases or The web sites FSN 522 Part 10 out of 2 Enzymatic reactions IN FOOD Dr. Wahida H. Alqahtani

Instructors : Dr. Wahida H. Alqahtani Office : Bldg. 9, Room 57T, Tel: 8050458, Email: wahida@ksu.edu.sa Schedule : Wednesday 10 - 12 am Office Hour: Tuesday 10 - 1pm ,Wednesday 12-1 pm and Thursday 10-12pm, if you can’t come on this time you should take an appointment References : https://www.okcareertech.org/educators/cimc/new-products/clusters/science-technology-engineering-and-mathematics-cluster/pdf-files/enzymesandfoodprep.pdf http://pubs.acs.org/doi/abs/10.1021/ba-1977-0160.ch005 https://en.wikipedia.org/wiki/Coenzyme_A The handout of the parts :

Objectives: To describe What are enzymes. To explain enzyme Class Characterizations. To identify examples of enzymatic Reactions. To Explain the function of enzymes as catalysts in chemical reactions. To outline Coagulation of Milk by Rennet Addition. Preparations: You are expected to prepare for each lecture by reading material to be covered before it is to be presented in class. Examinations: Exams will consist of one-hour exam and final exam. Grading: one - hour exams will be 30%, assignments will be 14% , Presentation will be 16% and final exam will be 40 % .

Enzymatic Reactions

introduction

What are enzymes? Enzymes are highly specialized proteins that catalyze specific biochemical reactions Proteins are chains of individual amino acids

Enzymes Food quality can be changed due to the activity of enzymes during storage or processing Enzymes can also be used as analytical indicators to follow those changes Enzyme-catalyzed reactions can either enhance or deteriorate food quality Changes in color, texture, sensory properties

Enzymes They do so through enzyme catalysis Living organisms must be able to carry out chemical reactions which are thermodynamically very unfavorable Break and/or form covalent bonds Alter large structures Effect three dimensional structure changes Regulate gene expression They do so through enzyme catalysis

Enzymes A common biological reaction can take place without enzyme catalysis …but will take 750,000,000 years With an enzyme….it takes ~22 milliseconds Even improvement of a factor of 1,000 would be good Only 750,000 years Living system would be impossible

Enzyme Class Characterizations Oxidoreductase Oxidation/reduction reactions Transferase Transfer of one molecule to another (i.e. functional groups) Hydrolase Catalyze bond breaking using water (ie. protease, lipase) Lyase Catalyze the formation of double bonds, often in dehydration reactions, during bond breaking Isomerase Catalyze intramolecular rearrangement of molecules Ligase Catalyze covalent attachment of two substrate molecules

Oxidation vs Oxidized The removal of an electron is oxidation (redox reactions). When a food system is oxidized, oxygen is added to an active binding site For example, the result of lipid oxidation is that the lipid may become oxidized. In the food industry, we common speak of “oxidizing agents” versus “reducing agents”. Both are used in foods.

Oxidation vs Oxidized Reducing agents are compounds that can donate an electron in the event of an oxidation reaction. L-ascorbic acid is an excellent reducing agent as are most antioxidants Oxidizing agents induce the removal of electrons Benzoyl peroxide is commonly added to “bleached” wheat flour

Effect of Enzymes

Effect of Enzymes Enzymes are highly specialized class of proteins: A bag of sugar can be stored for years with very little conversion to CO2 and H2O. This conversion is basic to life, for energy. When consumed, it is converted to chemical energy very fast. Both enzymatic and non-enzymatic reactions. Enzymes are highly specialized class of proteins: Specialized to perform specific chemical reactions. Specialized to work in specific environments.

The importance of enzymes in the food industry

Why are enzymes important in the food industry? Added or used to cause particular reaction Advantages Natural, Nontoxic Catalyze specific reactions Active under mild conditions Active at low concentrations Can control rate of reaction Can be inactivated

controls the action of enzymes

What controls the action of enzymes? Temperature Water Content pH Chemicals Alteration of Substrates Alteration of Products

Properties of Enzymes

Properties of Enzymes Specificity of enzymes Sensitivity to pH Sensitivity to temperature Kinetic properties of enzymes

Specificity of enzymes Absolute – one enzyme acts only on one substrate (example: urease decomposes only urea; arginase splits only arginine) Relative – one enzyme acts on different substrates which have the same bond type (example: pepsin splits different proteins) Stereospecificity – some enzymes can catalyze the transformation only substrates which are in certain geometrical configuration, cis- or trans-

Sensitivity to pH Each enzyme has maximum activity at a particular pH (optimum pH) For most enzymes the optimum pH is ~7 (there are exceptions)

Sensitivity to temperature -Each enzyme has maximum activity at a particular temperature (optimum temperature) -Enzyme will denature above 45-50oC -Most enzymes have temperature optimum of 37o

Kinetic properties of enzymes Study of the effect of substrate concentration on the rate of reaction 26

Enzymatic Reactions

Chemical Reactions in Foods Enzymatic Enzymes are proteins that occur in every living system Enzymes can have beneficial and detrimental effects Bacterial fermentations in cheese, pickles, yogurt Adverse color, texture, flavor, and odor High degree of specificity (Enzyme – Substrate) Non-enzymatic Those reactions that do not require enzymes Addition, redox, condensation, hydrolysis.

Enzymatic Reactions sucrose glucose + fructose Enzymatic reactions can occur from enzymes naturally present in a food Or as part of food processing, enzymes are added to foods to enable a desired effect Enzymes speed up chemical reactions (good or bad) and must be controlled by monitoring time and temperature. Typically we think of enzymes as “breaking apart” lipids, proteins, or carbs; but there are several enzyme categories sucrose glucose + fructose sucrase “invertase”

Enzymatic Browning Fruits such as apples, pears, peaches, apricots, and bananas, and vegetables such as potatoes quickly turn brown when their tissue is exposed to oxygen. Such oxygen exposure occurs when the food is sliced or bitten into or when it has sustained bruises, cuts or other injury to the peel. This “browning reaction” is related to the work of an enzyme called phenolase (or polyphenoloxidase), a conjugated enzyme in which copper is present.

Prevention

Prevention - Enzymatic browning can be prevented or slowed in several ways. - Immersing the “injured” food (for example, apple slices) in cold water slows the browning process. -The optimum temperature for enzymes to act is 43ºC(109ºF).The lower temperature decreases enzyme activity, and the water limits the enzyme’s access to oxygen. - Refrigeration slows enzyme activity even more, and boiling temperatures destroy (denature) the enzyme. - A long-used method for preventing browning involves lowering of pH to 2.5-2.7 by the addition of acids such as ascorbic acid, malic or citric acid

Common Enzymatic Reactions

Common Enzymatic Reactions some reactions can also occur without enzymes HYDROLYSIS Food molecules split into smaller products, due to the action of enzymes, or other catalysts (heat, acid) in the presence of water OXIDATION / REDUCTION: Reactions that cause changes in a food’s chemical structures through the addition or removal of an electron (hydrogen). Oxidation is the removal of an electron Reduction is the addition of an electron

Common Enzyme Reactions Carbohydrases : making corn syrup from starch Proteases : Meat tenderizers Lipases : Flavor production in chocolate and cheese Pectinases Glucose oxidase Flavor enzymes Lipoxygenase Polyphenol oxidase Rennin (chymosin)

Example of Enzymes

Enzymes catalyze reactions such as these:

Experiments with Food Enzymes Enzymatic Browning of Fruits and Vegetables Coagulation of Milk by Rennet Addition

Coagulation of Milk by Rennet Addition Pipette 10 ml of milk into each of 3 test tubes. To two of the tubes, add ~1.5 ml of a 1% rennet solution. Mix. (The 3rd tube will serve as a control. It contains no rennet). Place one of the two tubes with rennet into water at ~37C Observe the coagulation.

Coagulation of Sample Control

Coagulation of Sample 1% Rennet Solution, No Heat 5 minutes 10 minutes 20 minutes

Coagulation of Sample 1% Rennet Solution, 37C 5 minutes 10 minutes 20 minutes

The Mechanism Rennet An enzyme obtained from fourth stomach of ruminant animals, and from some microorganisms Cleaves particular bond in K-casein of milk to initiate milk coagulation Coagulates milk protein in cheese making Aids in development of flavor and texture in ripened cheese. Mild heat speeds up the enzyme reaction.

Other Example of Enzymes

Other Examples of Enzymes in Foods Milk Lactase Alkaline phosphatase Lipases Plasmin Fresh vs. canned pineapple Bromelain breaks down gelatin in “Jello” Meat tenderizer – uses bromelain, ficin, or papain Blanching of vegetables – catalase and peroxidase Cloudy vs. clear apple juice Mandarin oranges Onions – enzyme alliinase acts on sulfur cmpds.

Coenzymes

Coenzymes Coenzymes act as group-transfer reagents . A (CoA, CoASH, or HSCoA) is a coenzyme, notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle. All genomes sequenced to date encode enzymes that use coenzyme A as a substrate, and around 4% of cellular enzymes use it (or a thioester, such as acetyl-CoA) as a substrate. In humans, CoA biosynthesis requires cysteine, pantothenate, and adenosine triphosphate (ATP).

Coenzyme classification (1) Metabolite coenzymes - synthesized from common metabolites. (2) Vitamin-derived coenzymes - derivatives of vitamins . Vitamins cannot be synthesized by mammals, but must be obtained as nutrients.

Examples of metabolite coenzymes ATP can donate phosphoryl group ATP S-adenosylmethionine donates methyl groups in many biosynthesis reactions S-adenosylmethionine 50

Vitamin-Derived Coenzymes Vitamins are required for coenzyme synthesis and must be obtained from nutrients . Most vitamins must be enzymatically transformed to the coenzyme . Deficit of vitamin and as result correspondent coenzyme results in the disease .