Anita Kruger

1. Anita Kruger time@mfm.sun.ac.za 072 5454 959

2. FOOD CHEMISTRY 3FCHE30 FACULTY OF SCIENCE Department of Horticulture & Food Technology SEMESTER 2 MODULE 4 Other Carbohydrate Gels

3. CARBOHYDRATES • Monosaccharides • Polysaccardies & Oligosaccharides • Storage Carbohydrate – Animals • Glycogen (from Glucose) • Storage Carbohydrate – Plants • Starch • Structural Polysaccharide – Plants • Cellulose • Gums

4. Pectin Substances-Plant Gums • Heteropolysaccharides • “Hydrocolloid” – water-binder • Hydrophillic colloid: imbinding large quatities of water • Fill intercellular spaces, middel lamella of plant tissue • Wide channels in young fruit tissue

5. Pectin Substances-Plant Gums • Plant cell wall showing Pectin

6. Pectin Substances-Plant Gums

7. Pectin Substances-Plant Gums • Role of Pectin in Plant Tissue (fruits) • In outer cell walls, closely associated with cellulose – precursor of pectin: Protopectin • Absorb water and transfer it among cells • Responsible for firmness, texture (fruits & veggies) • Softening during ripening • Breakdown of colloidal stability in fruit juices

8. Pectin Substances-Plant Gums • Change in pectin substances during ripening • Protopectin in middle lamella between cell walls soluble pectin • Reduce cell wall thickness • Softening & Ripening • Decrease the degree of esterification of carboxyl groups with methyl alcohol

9. Pectin Substances-Plant Gums Pectin is a polymer of α-Galacturonic acid with a variable number of methyl ester groups. • Methylated ester of Polygalacturonic acid • Chains of 300 to 1000 glalacturonic acid units • Joined with 1α→4 linkages • This structure shown here is three methyl ester forms (-COOCH3) for every two carboxyl groups (-COOH) • hence it is has a 60% degree of esterification, normally called a DE-60 pectin

10. Pectin Substances-Plant Gums • Transformation of Protopectin to Pectin • Pectin-rich Plant Materials (Pomace, Citrus) • Heated with acidified water - Hydrolyzed • Protopectin (cellulose) form water soluble pectin (Same transformation during ripe fruit) • Precipitated form aqueos solutions by alcohol/acetone as jelly-like coagulum, which dissolve in water (water soluble) • Pectin = Negatively charged colloid/electrolite • Commercial pectin contains a number of impurities: hemicelluloses, pentosans, galactoses • Can be purified by repeated precipitation and redissolution (more later…)

11. Pectin Substances-Plant Gums • General Structure • Long, “unbranced” chains of polygalacturonic acid, with carboxyl groups partially esterfied with methyl alcohol - Heteropolysaccharides • High molecular masses (20 000 – over 400 000) • Joined by a-1,4 glycosidic bonds • Gums have acid groups as well as hydroxyl groups and long-chain structures = Good emulsifier • Acidic goups act as strongly hydrophilic portion of molecule • Rest of molecule in the chain act as less hydrophilic portion (almost hydrophobic)

12. Pectin Substances-Plant Gums • Guar Gum / Locust Bean Gum • Polysaccharide: Galactomannans • Structure: Galactose-phosphate and galacomannan groups • Guar gum (legume) • is the ground endosperm of seed from the guar plant (similar to soybean) • Molecular mass: 220 000 • Not able to bond easily with proteins/other polysaccharides • Form gels at 2 – 3% concentration • Five to eight times the thickening of starch • Many uses in pharmaceutical industry • food stabilizer/source of dietary fiber

13. Pectin Substances-Plant Gums • Approximately 85% of guar gum is guaran • water soluble polysaccharide consisting of linear chains of mannose with 1β→4 linkages to which galactose units are attached with 1α→6 linkages • The ratio of galactose to mannose is 1:2 Guaran is the principal polysaccharide in guar gum.

14. Pectin Substances-Plant Gums • Locust Bean • Derived from the seed endosperm of carob trees • Form pliable gums • Both consist mostly of galactomannans • Cold water soluble • USES • Salad dressings • Ice-cream • Baked goods (stabilizing and water retention)

15. Pectin Substances-Plant Gums • Agar / Carrageenans • Agar • extracted form seaweed • polymer of agarobiose, a disaccharide composed of D-galactose and 3,6-anhydro-L-galactose. • Consists mainly of galactose • Uses: Solid medium in microbiological applications, culturing bacteria, cellular tissue and DNA fingerprinting • Potent gel former • Forms gel at about 0.04%

16. Agarobiose is the repeating disaccharide unit in agar Pectin Substances-Plant Gums

17. Pectin Substances-Plant Gums • Carrageenan • Extracted from seaweed • Differs from agar in that they have sulfate groups • (-OSO3-) in place of some hydroxyl groups • Complex sulphated galactan • Negatively charged on sulphate group • Form gels of varying viscosity based on Ca++ or K+ • Can interact with protein due to ionic bonding • Uses: Stabilizers, thickener in instant puddings and desserts

18. Pectin Substances-Plant Gums • Changes in structure during hydolysis • Undergo hydrolysis by acid or alkali or suitable enzymes (alkaline hydrolysis) • First step: Removal of number of methoxyl groups • Leaving ultimately polygalacturonic acid • Called pectic acid – completely free of methoxyl groups

19. Pectin Substances-Plant Gums • Extraction of Pectin • Mainly form apple and citrus peels • Soak in warm water • Remove colloidel material, carbohydrates, acid and other water soluble inpurities • Rest of material are dried • Until moisture content of ca 10%

20. Pectin Substances-Plant Gums • Extraction • Water:moist peel – 3:1 • Water:dry peel – 30:1 • Water:peel acidified until pH 1,3 and pH1,4 • 1 hour at 90°C - 100°C (longer at 60°C) • Increase pH to pH 4,5 using Na2CO3 • Press the mixture • Extraxt is filtered with EtOH/acetone/AlCl3 • Pectin is floccultaed out of mixture • Neutralize mixture with Na2CO3 or NH4OH

21. Pectin Substances-Plant Gums • Extraction… • Metal salts are removed from the presipitate later through acidified EtOH • Pectin is further purified by dissolving it in water and presipitating with EtOH until the product is pure

22. Pectin Substances-Plant Gums • Marketing of Commercial Pectin • Available in the pure state • Characterized according to • 1. Jellying power (grade) • Increases with increasing molecular weight • 2. Degree of methoxylation • High methoxyl or low methoxyl pectins • Determines the mechanism of gel formation • 3. Rate of solidification of the jellies • Rapid/medium/slow set pectins • Rate and temperature of setting is also governed by the extend of esterification

23. Pectin Substances-Plant Gums • Rapid pectins: higher degree of methoxylation • At equal degrees of esterification, pectins with a higher degree of polymerization require shorter setting times.

24. Pectin Substances-Plant Gums • 1. Jellying Power – Pectin GRADES • Number of parts of sugar required to gel one part of pectin to acceptable firmness OR • Number of parts of sugar that can be gelled by 1 part of pectin under standard conditions • Usual conditions (Standard conditions) • pH 3.2 – 3.5 • Sugar 65 – 70% • Pectin 0.2 – 1.5% • Commercial grades: vary from 100 – 500 • Gels of different firmnesses can be formed by playing around with the pectin:sugar:acid ratio

25. 1. Jellying Power – Pectin GRADES… • Example: If you require a minimum of 0.1 g of pectin to 65% sugar and at a pH of 3.2 the pectin grade for that would be 650 • 0.1 x 10 = 1 = 1 part of pectin • 65 x 10 = 650 = pectin grade • Factors contributing to the gelling properties of pectin • Chain length • Degree of methoxylation • pH • Sugar content

26. Pectin Substances-Plant Gums • 2. Degree of Methoxylation

27. Pectin Substances-Plant Gums • Pectin as extracted normally has more than 50% of the acid units esterified, and is classified as "high methyl ester (HM) pectin". HM pectin formula

28. Pectin Substances-Plant Gums • Modification of the extraction process, or continued acid treatment, will yield a "low methyl ester LM) pectin" with less than 50% methyl ester groups. LM pectin formula

29. Pectin Substances-Plant Gums • 3. Rate of solidification of the jellies • Rapid-set pectin • Degree of methoxylation over 70% • Forms gels with sugar and acid at optimum pH 3.0 – 3.4 • Begins at 85ºC • Gel strenghts depend on molecular weight • The higher the molecular weight, the firmer the gel • Gel strength is not influenced by degree of methoxylation • Used in manufacturing of preserves in order to prevent whole fruit or chunks settling to the bottom or rising to the top of the jar, instead of being evenly distributed throughout the jam

30. Pectin Substances-Plant Gums • Slow-set pectin • Degree of methoxylation 50 – 70% • Forms gels with sugar and acid at an optimum pH 2.8 – 3.2 and at lower temperatures than rapid-set pectin • Forms jelly below 55ºC

31. Pectin Substances-Plant Gums • Uses of Pectin in Food Manufacturing • Jams, jellies, marmalades and preserves • Dehydrating agent must be present for pectin to form a gel • Typical dehydrating agents used for precipitation of pectin are alcohol or acetone • In jams and jellies it is the sugar that plays the dehydrating role • In forming a good jelly, suitable pectin-acid-sugar ratio should be maintained

32. Pectin Substances-Plant Gums • Syneresis – “weeping” • If the pH drop lower than 3, you will find that the gel becomes firmer and may even exhibit syneresis. • Water is expelled from the gel • Common in Fruit Jellies • See syrup forms on the jelly after a couple of months in storage • Factors contributing to Syneresis • Low pH (too much citric acid added) leads to degrading of the pectin netwok – water cannot hold gel structure efficiently anymore • Excess water (low TSS) • Not enough pectin

33. Pectin Substances-Plant Gums • Pectolytic Enzymes • Pectin enzymes are capable of degrading pectic substances • Commercially important for the treatment of fruit juices/beverages • To aid in filtration/clarification/increasing yields • Pectolytic Enzymes have to be added during production of most fruit juices and veggie soups • Also used for the production of galacturonic acids • Presence of pectic enzymes in fruit/veggies can result in excessive softening • Pectins may also cause “cloud” separation in tomato and fruit juices

34. Pectin Substances-Plant Gums • Commercial Uses • Clarification of fruit juices/wines • Aiding the disintegration of fruit pulps • By reducing the large pectin molecules into smaller units, and eventually into galacturonic acid – compound become water soluble – loses their suspending power • Viscosity is reduced • Insoluble pulp particles rapidly settle out

35. Pectin Substances-Plant Gums • Pectic enzymes • Most microorganisms produce at least one pectic enzyme • Almost all fungi and many bacteria produce these enzymes • Which readily degrade the pectin layers holding plant cells together • Leads to separation and degradation of the cells – plant tissue becomes soft • Bacterial degradation of pectin in plant tissues is responsible for the spoilage known as “soft rot” in fruits/veggies

36. Pectin Substances-Plant Gums • Pectinesterase (PE) • (or Pectin Pectyl-Hydrolase) • Found in bacteria, fungi, higher plants • Large amounts in citrus fruits & tomatoes • PE catalyzes the hydrolytic removal of the methoxyl groups (saponification) from the pectin molecule (methylgalacturonic acid) • Requires the presence of a free carboxyl group next to the methoxyl group to be saponified • Optimum activity at pH 7.5 • Methoxyl group may be released as methanol • Could be considered a toxic by-product • Reaction is catalyzed by pectin esterase • PE is specific for galacturonide esters, will not attack nongalacturonide methyl esters to any large extent

37. Pectin Substances-Plant Gums • HTST (high temperature short time) pasteurization is used to deactivate pectolytic enzymes and maintain cloud stability in fruit juices • Pectin is a protective colloid that helps keep insoluble particles in suspension • Cloudiness is required in commercial products – provide desirable appearance • Destruction of high levels of pectinesterase during production of tomato juice/puree is of vital importance. • Pectinesterase will act quite rapidly once the tomato is broken • HOT-BREAK method: the tomatoes are broken up at high temperature so that the pectic enzymes are destroyed instantaneously

38. Pectin Substances-Plant Gums • Polygalacturonase (PG) • Also known as pectinase • Catalyses the Hydrolysis the glycosidic linkages (a-1,4 bonds) in individual units in pectin chains • Catalyses the glycosidic hydrolysis of the bond between galacturonic acid units

39. Pectin Substances-Plant Gums • PG can be divided into • Endoenzymes: act within molecule on a-1,4 linkages – Endopolygalacturonases • Cause rapid decrease in viscosity of pectin solutions without considerable increase in reducing groups • Attack the molecule at random, breaking it to shorter chains – “liquefying” enzymes • Fruits/filamentous fungi, NOT in yeasts or bacteria • Exoenzymes: catalyze the stepwise hydrolysis (split) of galacturonic acid molecules, from nonreducing end of chain - Exopolygalacturonases • Plants (carrot/peaches)/fungi/bacteria

40. Pectin Substances-Plant Gums • Some PG act principally on methylated substrates (pectins) – Polymethyl galacturonases • Other PG act on substrates with free carboxylic acid groups (pectic acids) – Polygalacturonases • Split glycosidic bonds between two unesterified units

41. Pectin Substances-Plant Gums • Pectin Transeliminase (PT) • Catalyses a dehydration reaction that breaks the alpha-1,4 glycosidic bond • Splits the glycosidic bonds of a glucuronide chain by trans elimination of hydrogen from the 4- and 5-position of the glycuronide moiety • Attacks only glycosidic bonds between methoxylated units • Glycosidic bonds in pectin are highly susceptible to this reaction • PT are of the endotype and are obtained exclusively from filamentous fungi, Aspergillus niger • The purified enzyme has an optimum pH 5.1 – 5.2

42. Pectin Substances-Plant Gums • Amount of enzyme required for clarification of juice • Depends on type of enzyme • Amount of bentonite/glucose powder • Bentonite is an activated clay used to form a heavy complex with colloidal material and to remove the presipitate through filtration. • Type and pH of Juice • Time and temperature of method used

43. Pectin Substances-Plant Gums • Enzyme treatment is usually followed by a pasteurisation process • To destroy yeasts • Inactivate Pectolitic and oxidising enzymes • Denaturise proteins

44. Pectin Substances-Plant Gums • Role of Pectin in Cloud Formation • When juices are produced from fruit, the initial extraction process produces a liquor (or serum) as well as a particulate component • Cloudy juices consist of a suspension of very fine particles in a clear medium (serum) • Some particulate will form a sediment – settle at the bottom of the tank • Rest remains as cloud in juice • Some juices require this cloud to give the juice its specific consumer appeal and mouthfeel • Tomato juice/guava juice/orange juice/lemon juice

45. Pectin Substances-Plant Gums • Cloud Structure • Differs from one juice to another, although the basic structure is the same • In orange juice the cloud consists of micelles (colloidal particles) that contain a positively charged protein core coated by a layer of negatively charged pectin • Neg. charged due to the free carboxyl ( –COO-)groups on pectin • These neg. charges cause electrostatic repulsion between micelles (electrostatic charge of the suspended particles) • Prevents them from settling down • Maintenance of the cloud • Interference with the cloud structure will very likely precipitate it

46. Pectin Substances-Plant Gums • Clarification of Cloudy Juices • Removal of the particulate matter • Negative charges (surface of particles) will create insoluble complexes with other soluble polyelectrolytes of opposite charge • Will then precipitate easily • WINE: polyethylenamine is used for the clarification of cloudy wines • The precipitate thus formed will settle, carrying with it all suspended matters as well as tannins • Another method for attaining similar effects consists of treating cloudy liquids with commercial preparations of pectolytic enzymes, “Pectinol” • Usually obtained from molds and contain PE and endo-PG

47. Pectin Substances-Plant Gums • Clarification of Cloudy Juices • Can also be done by filtration of the juice of wine • Adsorbing the cloud onto a stationary phase • Requires you to run juice through a column or over a bed of adsorbent (ex. Polyethyleneamine) • Positive charges on the resin attracts negatively charged cloud and binds it • Bentonite is used in the wine industry • Adding a flocculant to the juice • Adding aluminium sulphate: the Al++ precipitates the cloud due to electrostatic attraction. • Using pectolytic enzymes • PE and PG can disrupt pectin and therefore cloud structure – leading to precipitation

48. Pectin Substances-Plant Gums • Industrial Manufacture of Pectins • Commercial Pectins are manufactured in two main forms • “Liquid pectins”: concentrated solutions of pectins extracted from waste plant materials (pomace/citrus peels) • “dry-pectin powders”: • Commercial practice the resulting products are not pure substances • Their degree of purity depends largely on • The methods of manufacture • Molecular size of pectin substances • Degree of esterification • Amount of accompanying ballast material present

49. Pectin Substances-Plant Gums • Manufacturing procedures comprise the following main steps • (1) Removal of material • (2) Removal of ballast • (3) Acid hydrolysis of protopectin and dissolution of pectin • (4) Precipitation • By alcohols or acetone • Or mineral salts such as aluminium hydroxide with opposite electric charge to negatively charged pectinic acids • (5) Purification and drying

50. Pectin Substances-Plant Gums • The use of HM-pectin in fruit jellies/jams • Dissolving dry pectin • Pectin must be in a complete gel form to be used to maximum capacity • A standard pectin solution: • Dry pectin with correct amount of water • Mix quickly with Mechanical mixer • Until clear viscous solution • Measured part is added to jam mixture • Or dry pectin can be mixed with sugar before adding to jam – this will prevent lumps • At high temperature pectin are degraded (Grade of pectin) • Ideally pectin will be added in the final stage of the evaporation process when the jam mixture are boiling