Samenvatting Lipids + enzymes+ phenolic compounds + carbohydrates - Samenvatting Lipids, phenolic - Studeersnel (2024)

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Samenvatting Lipids, phenolic compounds and enzymes (+ carbohydrates)

LIPIDS

Lipids are a broad range of compounds that are soluble inorganic solvents. A fat is in the solid state and an oil is in theliquid state. Lipids are divided in 2 groups:

 Saponifiable fraction: have an ester group Unsaponifiable fraction: have no ester group

During saponification there is the production of soap of lipidsby a reaction with a base. During this reaction the hydrolysis ofthe ester bond takes place.

Saponifiable fraction Unsaponifiable fractionMono-, di- and triglycerides (esters of 1,2 or 3fatty acids and one glycerol)

Aldehydes + ketones (free fatty acids andoxidation products)Waxes (esters of fatty acids and alcohols) SterolsSterol esters (esters of fatty acids and sterols) Prenol lipidsPhospholipids (esters of fatty acids andphosphate group with glycerol)Glycolipids (esters of 2 fatty acids and glycerolwith one or more sugar units)Saccharolipids (esters of fatty acids with asugar backbone)Sphingolipids (sphingosine backbone is anamide linked to a fatty acid, that can be O-linked to a polar head group)Fatty acids:

Fatty acids are important building blocks of lipids. Fatty acids are primarily carboxylic acids with an unbranchedcarbon chain, generally consisting of an even number of C-atoms. There are 2 categories:

• Saturated fatty acids: have no double bonds
• Unsaturated fatty acids: have double bond(s)  Monosaturated  Polysaturated

Saturated fatty acids have a much higher melting point than unsaturated fatty acids and the ratiosaturated/unsaturated fatty acids is of high importance with respect to their physical properties. They can also bedivided on their length: short chain (4-6 C-atoms), medium chain (8-12 C-atoms) and long chain (14 or more C-atoms)

The unsaturated fatty acids that are found in nature almost always occur in cis configuration, although trans fattyacids do occur in milk fats and in hydrogenated fats. Trans fatty acids are less preferred, because they increase thelevel of LDL-cholesterol in the blood and can increase cardiovascular diseases.

Polysaturated fatty acids have more than 1 double bond. Most of these are organized along the carbon chain inrepeated 1,4-pentadiene structural motif (-C=C-C-C=C-). These fatty acids have not conjugated system.

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Fatty acid composition:

Fats and oils can be characterized by determining the fatty acid composition of their triglycerides .They can bedivided into 4 groups:

 Depot fats from animals: these fats mainly contain palmitic acid (C16:0), oleic acid (C18:1) and stearic acid (C18:0). This results in a relatively high amount of saturated fatty acids compared to vegetable oil. Milk fats: they have a large variety in fatty acid composition. There are relatively a lot of short and medium chain saturated acids. They have high amounts of short chain fatty acids. In milk fat a considerable part of the unsaturated bonds are in the trans- configuration. Fish oils: they have also a large variety in fatty acid composition. They have a high content of unsaturated fatty acids and especially with a long chain. The multiple unsaturated fatty acids can lower the LDL-cholesterol, although the fish oils are very sensitive for oxidation reactions due to their great amount of unsaturated fatty acids. Vegetable oil : there are 2 groups. The first group includes oils that primarily contain fatty acids with a chain length of 16-18 C-atoms. A large part of the C18 fatty acids occur as mono or multiple unsaturated fatty acids. The second group comprises of fats that are saturated to a relatively high degree.

Triacylglyceride (TAG) composition:

P Palmitic acid (C16:0)S Stearic acid (C18:0)O Oleic acid (C18:1)L Linoleic acid (C18:2)M Myristic acid (C14:0)Phospholipids:

Phospholipids are esters of glycerol with 2 fatty acids and one phosphoric acid group. Phospholipids are surfaceactive substances (prefer to be at interfaces between oil and water) as they have a lipophilic (hydrophobic) andhydrophilic group. Because of their structure phospholipids are important structural elements in biological cellmembranes.

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unsaturated fatty acids. To accelerate the reaction a higher temperature, very low or high water activity and pro-oxidants (metal ions) are preferred.

Bv:

The reactivity of the formed peroxy radical is relatively low and therefore it very selectively extracts the weakestbound H-atom from the fatty acid molecule. This is the H-atom that is bound at the α-methylene position (-CH 2 nextto a double bond ) in a unsaturated fatty acid. In particular methylene groups located between 2 double bonds inpolysaturated fatty acids.

Photo oxidation:

Fat oxidation can also be influenced by light: photo oxidation. Thereare ‘sensitizers’ and this can be activated by light. There are 2 types ofsensitizers:

o Type I: the activated sensitizer (Sen*) reacts directly with a substrate (fatty acid RH) forming an alkyl radical that subsequently participates in the normal autoxidation reactions.o Type II: here oxygen is activated by the activated sensitizer (Sen*). This results in the formation of singlet O 2. Singlet O 2 is in a higher energy state than normal triplet O 2. Due to the fact that singlet O 2 is more electrophilic than in triplet O 2 and it can react faster with double C- bonds in fatty acid chains. Typical examples of type II sensitizers are in natural pigments and synthetic colorants. Singlet O 2 reacts directly with the double bond, causing a shift of this double bond. Consequently, hydroperoxides are formed that are different from those that are formed during autoxidation. 4

Riboflavin is a strong sensitizer and is present in milk products. It is able to absorb energy from light.

The resistance of oil or fat against fat oxidation is indicated by the time that elapses until an arbitrary quantity ofperoxides is formed: induction time. After this time has passed, the reaction increases considerably. The longer theinduction time, the more stable fat or oil is.

Lipoxygenase

Fat oxidation can also be the result of an enzymatic catalysed reaction. In many plants and certain animal tissues,lipoxygenase enzymes are present, which are able to catalyse the formation of hydroperoxides from certainunsaturated fatty acids. Lipoxygenases only oxidize a 1,4cis-cis- pentadiene system (only polysaturated fatty acids).

Factors that influence fat oxidation:

• fatty acid composition: in particular unsaturated fatty acids, in which a 1,4-cis-cis-pentadiene system structure is present, are sensitive to oxidation reactions.
• Oxygen: in case of an unlimited oxygen supply, the extent of fat oxidation is not dependant on the oxygen pressure. At very low oxygen pressure, the rate of oxidation does depend on the oxygen pressure. The surface area that comes in contact with oxygen is also important for fat oxidation. in the case of a product with a large-surface volume ratio, a smaller effect of oxygen pressure on fat oxidation is to be expected.
• Storing conditions: generally a higher temperature leads to more oxidation reactions, except when the amount of oxygen becomes limiting. Storage at low temperature will decrease the rate of autoxidation. However, some lipoxygenases might still be active at a relatively low temperature. Enzymatic fat oxidation can only be prevented by inactivating the enzyme by blanching. The presence of light can also stimulate fat oxidation. The water activity also plays an important role in fat oxidation. When Aw is low, an extremely fast oxidation can take place. This is caused by:  metal ions are no longer surrounded by a water jacket and are directly available.  Less H-bonds between water and hydroperoxides exist and therefore hydroperoxides are more available.  Oxygen migrates better into the fat.

Fat oxidation is minimal at an Aw of 0,2 -0,4. At higher Aw values the catalyst are more mobile in water.

• Pro-oxidants: some compounds stimulate fat oxidation (presence of metal ions, metal porphyrins and photosensitizers).
• Anti-oxidants: anti-oxidants can slow down fat oxidation, however they can not prevent it. Primary anti- oxidants stop the chain by inactivating the alkyl-, alkoxy-, and peroxy-radicals. They remove radicals by converting into radicals themselves, but they are more stable and will not react further in the chain. In general compounds with a phenolic hydroxy group are active as primary oxidants. The most used synthetic primary anti-oxidant is BHA, BHT, TBHQ. Ascorbic acid is a natural secondary anti-oxidant and used to remove oxygen.

Hydrolytic rancidity

Acylglycerides can be hydrolysed to glycerol and free fatty acids in the presence of water. This is catalysed by alkali, acids and enzymes (lipase). When lipase is present, free fatty acids will be formed easily. An additional effect of the formation of free fatty acids is an acceleration of fat oxidation process, because free fatty acids react faster than acylglycerides. Hydrolysis of acylglycerides that contain a high amount of short chain fatty acids is easily observed by the smell or taste of the free fatty acids, because they are relatively volatile.

Reactions at higher temperatures

Fats change considerably when they are heated for an extensive period of time at high temperature. The reactions: oxidation, isomerization, dimerization, polymerization and hydrolysis occur.

• Isomerization occurs because cis-fatty acids isomerizes to trans-fatty acids and can cyclize subsequently.

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The terms phenolic compounds and phenolics refer to the entire group of compounds that fit the definition above.Another term is also commonly used and this is polyphenols. The prefix poly refers to the multiple hydroxyl groupsthat often occur in the structures of phenolic compounds. The advantage of trivial names is that they are muchshorter and easier to use in text and conversation than the full chemical name. A downside is that these names cannot directly be related to the structure of the corresponding molecule.

General structural features and molecular properties of phenolic compounds

The structural features and the resulting molecular properties of phenolic compounds can strongly affect overallfood properties. There are three important parameters involved with the molecular properties: polarity, reactivityand the size of the conjugated system.

 Polarity is a measure of the number and strength of different polar bonds in the molecule. Polar molecules generally have a high solubility in water (hydrophilic), whereas apolar molecules are poorly soluble in water but highly soluble in oils (hydrophobic). Mostly the phenolics are medium polar, but the exact polarity and water solubility is strongly dependent on their structural features. Reactivity is important because these molecules can quickly react with each other or other food molecules. This leads to modification or degradation of the original structures, thereby changing the molecular and food properties. The size of the conjugated system is also from importance. A conjugated system is a sequence of alternating double and single bonds. The double bonds in aromatic rings and certain substituents like carbonyls (C=O) can also be a part of the conjugated system. The size of the conjugated system influences the colour of food products, because of the conjugated system absorbing visible light. A general rule of thumb is that a conjugated system with >8 conjugated double bonds only absorbs light in UV range, thus appears colourless.

Substituents can influence the polarity, reactivity and size of the conjugated system, the 6 major types are:

o Hydroxylation is substation with an additional OH-group. Each additional OH-group slightly increases the polarity and thus increases water solubility. Hydroxylation also makes phenolics more reactive. Particularly interesting are phenolic compounds that posses at least 2 OH-groups that are in ortho position. This o-dihydroxybenzene/1,2- dihydroxybenzene structure is commonly referred to as o- diphenol and these are important with oxidation.o Methylation mostly occurs as the attachment of methyl- group (-CH 3 ) to a hydroxyl-group. Methylated hydroxyl groups are called methoxyl-groups. Methylation reduces polarity resulting in lower water solubility, it reduces reactivity resulting in reduced oxidation and lower antioxidant activity and it blocks further structural modification.o Glycosylation is substitution of the aromatic ring with a saccharide (usually a mono- or disaccharide). The saccharide is mostly attached to a hydroxyl group. It increases polarity and water solubility of the phenolic compounds, due to high polarity of the saccharide. Reactivity is reduced by glycosylation. They are called glycosides. If the saccharide is removed, the remaining phenolic without any saccharide attached is called aglycon.o Carboxylation is substitution with a carboxylic acid group (-COOH) and this can occur directly on the aromatic ring or at the end of a carbon chain. It increases the polarity and water solubility due to the polar C-O and O- H bonds. If the carboxylic acid group is deprotonated then the charged group increases the polarity even further. 7

o Extension of the conjugated system can occur by substitution with alkenes or by formation of a fused ring system, although this is dependent on the type of fused ring structure. if the conjugated system gets bigger it can result in becoming coloured and there are more possibilities to form resonance structures.

Monomeric phenolic compounds

Simple phenolics arethe smallest simplestclass of phenolicsbased on the corephenol structure.hydroxybenzoic acidsare hydroxylatedderivatives of benzoicacids.

Hydroxycinnamic acids consist of a benzene ring with a propenoic acid group attached to it. Hydroxycinnamylalcohols are closely related compounds in which the carboxylic acid group is reduced to an alcohol group. Thecarboxylic acid group allows them to form esters or amides with molecules that possess alcohol or amine groups.Hydroxycinnamic and hydroxycinnamyl alcohols serve as building blocks formany other types of phenolic compounds.

Stillbenoids consist of 2 aromatic rings linked via an ethylene bridge (-C=C-).Due to this double bond connecting the 2 rings, they have relatively longconjugated system of 7 double bonds. Despite this, they are mostly stillcolourless or only slightly yellow.

Flavonoids, isoflavonoids and chalcones are often referred to as‘flavonoids’. All of these compounds possess 2 aromatic rings that areconnected via a group consisting of 3 C-atoms. There are differentsubclasses of phenolic compounds.

The names of (iso)flavonoids subclasses are built up via a logical way:

• Base of the name ‘flav’
• ‘iso’ indicates that B-ring is connected at position 3
• Suffix ‘an’ indicates absence of a double bond between position 2 and 3
• Suffix ‘ol’ indicates presence of a hydroxyl-group at position 3
• Suffix ‘one’ indicates presence of carbonyl-group at position 4

Flavanols, flavanones and isoflavans possess 2 chiral centres. Therefore they each have 4 stereoisomers.

di-, oligo- and polymeric phenolic compounds

monomeric phenolic compounds can be converted to dimers, oligomers and polymers by oxidative coupling.

Definitions of tannins usually include: the ability to form complexeswith proteins and water extractable. Tannins are subdivided into 2more defined groups: hydrolysable tannins and condensed tannins.

 Hydrolysable tannins have a central glycosidic core of which at least 2 of the hydroxyl-groups are esterified with benzoic acids. Further structural diversity is introduced by introduction of

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phenolic compounds and PPO are not in contact with each other, they are present in different compartments in theplant cells. If the plant tissue is damaged, enzymatic oxidation can take place, because phenolics, PPO and oxygencome into contact with each other. The key reaction is conversion of phenolic compounds to o-quinones, catalysedby PPO. There are 2 types of follow up reactions:

 Reactions between oxidised and non-oxidised phenolics. These reactions involve oxidative coupling of phenolic compounds, which yields di, oligo- and polymers. The coupling products can have implications for food colour because many of these compounds are brown pigments. This reaction sequence is called enzymatic browning. Reactions between oxidised phenolics and other food molecules. These reactions occur with many different types of food molecules including proteins and amino acids. These reactions lead to changes in flavour, colour and appearance.

It is important to remember that only the first step is an enzymatic reaction that is catalysed by PPO.

Hydroxylation of monophenols (cresolase activity)

Many phenolic compounds are monophenols, they only possess oneOH-group on their aromatic ring. These compounds can be directlyconverted into o-quinones. some PPOs can convert monophenols too-diphenols via hydroxylation. In this reaction, PPO utilises ½ mole ofoxygen to perform addition of a hydroxyl-group to the aromatic ring atthe position ortho to the first OH-group (cresolase activity).

Conversion of o-diphenols to o-quinones (catecholase activity )

All PPOs possess catecholase activity which is the main reactioncatalysed by PPOs. In this reaction the PPO utilises ½ mole of oxygen tooxidise an o-diphenol to an o-quinone.

The overall reaction from a monophenol to an o-quinone can be catalysed by PPOs that possess both cresolase andcatecholase activity.

Factors influencing enzymatic formation of o-quinones

1. The overall PPO activity differs between different sources.
2. The characteristics and properties of the PPO play a role as well. Each plant has its own specific PPO and the properties of these PPOs can differ, even though they all catalyse the same type of reaction(s). The optimum pH and temperature can differ between different kinds of PPOs.
3. The structure of the phenolic compound also has a large influence on its conversion by PPO. Generally, o- diphenols are the preferred substrates of PPO but even between o-diphenols the rate at which they are converted can differ. Other substituents on the ring, including fused rings also influence PPO activity.

The o-diphenols are highly preferred over its two positional isomers as substrates for PPO.

4. The conditions in the food process, ingredient or product are of importance. The most important factors are pH, temperature , contact between enzyme and substrate and availability of oxygen. Most PPOs are optimally active in pH range of 5-7.

The rate and extent of formation of PPO-catalysed enzymatic oxidation is the key determinant for the rate andextent of these follow-up reactions.

Protein-phenolic interactions

Phenolic compounds can interact with other food molecules, but notably proteins. There are 2 main types ofinteractions: covalent and non-covalent interactions:

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• Covalent interactions: these types of interactions are generally considered as irreversible and lead to formation of protein-phenolic conjugates. The main mechanism behind this conjugation is reaction of electron-deficient o-quinones with nucleophilic groups. Typical nucleophilic groups present in food like proteins and peptides are amine (-NH 2 ) or thiol (-SH) groups. Amine and thiol groups are found in proteins and peptides in the side chain. Thiols are much stronger nucleophiles than amines and therefore thiols are more reactive. If the nucleophilic thiol or amine is present in the side chain of an amino acid residue of a protein, a covalent bond can be formed between phenolic and protein. These reactions lead to the formation of protein- bound o-diphenols. Upon further oxidation of the protein- bound o-diphenol, protein-bound o-quinones are formed. This can lead to the formation of various types of cross links between proteins. When a protein-bound o-quinone reacts with a non-oxidised phenolic, it can lead to browning of the protein. These interactions strongly affect protein properties (bv solubility or functionality of the protein).
• Non covalent interactions: these interactions are reversible and cause the forming of protein-phenolic complexes. o Hydrogen bonds: proteins contain groups which can serve as H-bond donors or acceptors. o Hydrophobic interactions: the aromatic rings of phenolics are hydrophobic and proteins also contain various hydrophobic groups. o Ionic bonds: proteins and phenolics can both be charged, depending on the pH. Ion bonds can be formed between oppositely charged groups.

The type of interaction that is formed is dependent on many factors:

1. Protein structure and characteristics; this includes primary, secondary etc structures, the types of side chains and the accessibility of reactive amino acid side chains.
2. Phenolic compound structure and characteristics; including hydrophobicity, solubility and molecular size. Monomeric phenolic compounds generally do not form strong protein-phenolic complexes or stable cross links between proteins.
3. pH of the food/ingredient; this determines the charge of the phenolic compounds and the proteins. At low pH COOH-groups are not charged (undissociated), whereas at neutral or alkaline pH COOH-groups are dissociated and thus negatively charged. Phenolic compounds that are not charged are more hydrophobic, which promotes hydrophobic interactions. Protein-phenolic complexation is maximal around the iso-electric point of proteins.

Protein-phenolic interactions can lead to aggregation of proteins and eventually precipitation of proteins togetherwith phenolics. Aggregation and precipitation happens mostly when if one protein molecule interacts with multiplephenolics. The mechanism underlying aggregation and precipitation is depend on the molar ratio between phenolicsand proteins.

 Low molar ratio phenolic/protein: at a low ratio, oligomeric compounds can simultaneously bind to different proteins and thus forming cross links. The cross linked proteins form aggregates. High molar ratio phenolic/protein: at a high ratio, the proteins are coated with phenolic compounds. The protein is surrounded by a hydrophobic layer of phenolics and therefore reduces water solubility. Proteins can now precipitate by themselves or different proteins can form hydrophobic interactions.

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• Addition of sulphite; this is a very powerful inhibitor of enzymatic browning. This is due to the HSO 3 - , which can interact as nucleophile. The sulfo-phenolics can’t react further to brown pigments and can’t be oxidised anymore by PPO. The reactions with sulphite is irreversible. Sulphite can also react with the active site of PPO and this leads to an irreversible modification of the active site that inactivates the enzyme. So sulphite has a dual-action mechanism.
• Addition of thiol compounds; thiols act as nucleophiles and react with o-quinones forming thio-phenolics.
• Removal of phenolic compounds

Colourful anthocyanins

In anthocyanins and anthocyanidins, the presence of colour is a result ofthe conjugated system that connects the A-ring and the B-ring via the C-ring. Now the conjugated system has 8 double bonds. The colour ofanthocyanins is dependent on the pH. Depending on the food pH, theanthocyanins can occur in one of five different forms.

Effect on food flavour:

Bitterness

Flavour = taste (non-volatile) & aroma (volatile)

Grapefruit contains bitter flavanone glycosides. Grapefruit and other citrusfruits contain different glycosides of the aglycon naringenin and hesperetin. 2common saccharides that are bound to these aglycons are rutinose andneohesperidose. Both saccharides consisting of rhamnose and glucose, buthave different linkage positions. The linkage position of the disaccharide isessential for bitter taste.

Astringency

Astringency is caused by non-covalent protein-phenolic complexation. In particular this isthe result of interactions between phenolic compounds and proline-rich proteins fromsaliva (speeksel). Strong complexation and cross linking of proteins leads to precipitation.if phenolics like condensed tannins interact with proline rich salivary proteins in this way,this results in precipitation of the salivary proteins in the mouth  astringency.

Effect of phenolics on aroma

Nucleophilic groups in amino acids side chains can react with o-quinones. Moreover, free amino acids always possess a free amine anda free carboxylic acid group. This makes them even more reactive.When an amino acid reacts with an o-quinone via nucleophilic attack ofthe amine, the reaction can be followed by so-called Streckerdegradation, which leads to formation of an aldehyde from the aminoacid. The amine performs a nucleophilic attack on one of the C=O’s ofthe o-quinone, which results in loss of the carbonyl oxygen as water and the formation of C=N intermediate. Thenthe free carboxylic acid of the amino acid is lost as CO 2 and water participates in the reaction to eventually form endproducts: aminophenol and aldehyde.

Methods for analysis (blz 181):

 Detection of phenolic compounds by visual observation UV-VIS spectrophotometry Total phenolic content by folin-coicaltue assay Antioxidant assay 13

 Advanced methods for analysis of phenolics

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o Bread; for the preparation of a light bread with a good volume, a certain quantity of gas (CO 2 ) is required, that is formed from fermentable sugars (in particular maltose) in the dough. These sugars originate from the degradation of damaged starch granules by endogenous β-amylase. Native starch granules cannot be degraded well by amylases, however due to the fact that during grinding of the flour some starch granules are damaged, a certain number of them become accessible to these enzymes. In order to increase the efficiency of the degradation α-amylase is added. Α-amylase is an endo-enzyme and β-amylase an exo-enzyme, therefore they work together synergistically. Fungal amylases are preferred due to lower thermostability.o Beer; malt contains an excess of enzymes. During brewing important enzymatic processes take place, in which especially α- and β-amylases and protease from malt are involved.o Starch products; the classic preparation of starch syrup and glucose consist of an acid hydrolysis of starch under pressure and high temperature. Bitter and coloured by-products are formed and corrosion of the equipment can occur. Compared to that, an enzymatic process brings a lot of advantages. Enzymes that are important in this process are: theremostable α-amylase, gluco- amylase, pullulanase, iso-amylase and glucose-isomerase.o Glucose; crystalline glucose can serve as sucrose substitute. A thermostable α-amylase is added to a slurry of starch. Steam is added to this slurry to obtain gelatinization, after which liquefaction of starch takes place in an expansion barrel. In a second step the hydrolysis is completed with gluco- amylase, however to make the degradation more efficient iso-amylase or pullulanase is often added to the gluco-amylase as ‘helper’ enzyme.

• Cellulases; cellulose is important in the cell walls of plants. Two other important polymers that surroundcellulose in the cell walls are hemicellulose and pectin. Cellulose is a β-(1->4) glucan and can have differentvalues for DP. Cellulose contains areas that are crystalline and consequently not easily accessible to anenzyme. However also amorphous areas that can actually be reached by enzymes present. For thedegradation of cellulose there are 3 important enzymes: cellobiohydrolase (CBH), endo-glucanase encellobiase and these are glycosidic hydrolases.o Cellobiohydrolase (CBH); this is an exo-enzyme thatreleases cellobiose from a chain end. It is the essentialenzyme for the degradation of crystalline cellulose and itworks synergistically with endoglucanase.o Endoglucanase; this is and endo-enzyme that cuts atrandom. The enzyme has a low activity on crystallinecellulose, but works well on a soluble cellulosederivative, such as carboxymethyl cellulose (CMC).o Cellobiase (=β-glucosidase); the product of cellulosedegradation by CBH and endoglucanase consists of oligosaccharides, with a lot of cellobiose.Cellobiose is a dimer of 2 glucosyl residues β(1->4)linked. A β-glucosidase can further degrade thesesmall oligosaccharides in particular the dimer cellobiose, to glucose.
• Pectinases; pectin could be seen as the cement that ‘glues’ the building blocks together in the plant cell wall.Pectin is a very complex polysaccharide that consists of areas less linear (smooth regions) and areas that arestrongly branched (hairy regions). The smooth region is also called homogalacturonan and this consists of α-D-galacturonans of which the carboxylic acid groups can be esterified with methanol with a high or low DE.Homogalacturonan degrading enzymes are subdivided into pectin-esterases and pectin-depolymerases.o Pectin esterase; this enzyme is also called pectin methylesterase/ PME and is classified as anesterhydrolase or carbohydrate esterase. The enzyme saponifies pectin with a high degree ofesterification by which methanol and pectin with lower DE are formed.o Pectin depolymerase; these have three important groups: Polygalacturonase; this enzyme is also indicated as PG. it is a glycosidic hydrolase. The bestsubstrate is pectic acid, but also pectin with a low DE is easily degraded. The enzymehydrolyses the glycosidic linkages next to a free carboxyl group.

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 Pectate lyase; is abbreviated to PAL and belongs to the lyase group. This enzyme cleaves the glycosidic linkage of pectin with s low DE or pectic acid by β-elimination. In this reaction a double bond is introduced in the newly formed non-reducing chain end galacturonic acid residue. Pectin lyase; also called pectin trans-eliminase is usually abbreviated to PL and is classified as lyase. It cleaves the glycosidic linkage next to a methylated carboxyl acid group from pectin with a high DE. The reaction proceeds with β-elimination, as a result of which a double bond is introduced at the newly formed non-reducing chain end.

Application of pectinases

During ripening of a tomato enzymes such as polygalacturonase and pectin esterases are produced that can causefor example fruit softening. During the processing of tomato it can also be advantage that the polygalacturonaseactivity in the tomato is reduced. When processing the tomato to a homogenous mass thefruit’s endogenous pectin esterase and polygalacturonase work together by which thepectin with a high DE and high molecular weight is degraded. The result is a thin-liquid(cold-break juice) and this can be easily concentrated for tomato sauce or paste. Only ifthe enzymes are immediately activated during the process of tomatoes heat, one canobtain a tomato juice with a smooth consistency. This way of processing is called hot-break.

If a fruit is processed to juice by pressing, a turbid juice will usually be the result. In somecases this is desired, but it can also be undesired. It is assumed that the cloud consists ofcell wall fragments and protein parts that are surrounded by a coating of pectin with ahigh DE. Stability of these stable cloud parts can be lost under the influence of pectindegrading enzymes.

Protein acting enzymes, classification

Proteolytic enzymes belong to the group of hydrolysates and they cleave a peptide linkage in a protein or syntheticsubstrate by introducing an H 2 O molecule. Proteases can be classified on the basis of 2 characters:

 Point of attack; the polymer substrate (bv proteins) can be cleft internally or from the end. Endopeptidases cleave protein molecules into smaller peptide chains. Exopeptidases act from the end of a peptide chain and generally release one amino acid at a time. The endo- and exoproteases often effectively work together on the hydrolysis of a protein, because endopeptidase creates a new chain that the exopeptidase can act on. There can made a sub-division of the exopeptidases based on the fact that in a protein chain an amino- terminus and carboxyl-terminus are present. o Carboxypeptidases work from the side with a free carboxyl group o Aminopeptidases always cleave the peptide from the end with a free amino group

Proteases are rather specific and the percentage of peptide linkages from a protein that can be cleaved bycertain protease is not high.

 The most commonly used classification is based on the active group in the catalytic centre (active side): o serine proteases; these enzymes have serine and histidine in the catalytic centre. The OH-group of serine plays an essential role in the catalytic activity. Therefore they are inhibited by DFP which forms an ester-linkage with this OH-group. o Cysteine proteases; these enzymes have cysteine and histidine in their catalytic centre. The SH-group in the cysteine an essential role plays. They are inhibited by sulphydryl reagents.

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Lipase-lipid hydrolysis

Lipases are responsible for the hydrolytic conversion oftriglycerides into glycerol and free fatty acids. A specificproperty of lipase is the activity at the interface betweenwater and oil. Activation of the enzyme takes place at theinterface between oil and water: interfacial activation.

CARBOHYDRATES

Carbohydrates that are important in relation to foods occur in the form of:

 Monosaccharides; single sugars (bv. Glucose en fructose) Disaccharides; two sugars linked together (bv saccharose) Oligosaccharides; 2 tot 20 suikers Polysaccharides; long chains of sugars which can be present in vegetable raw material as storage carbohydrate or as structural element of the cell wall Glycosides; carbohydrates which are linked to another compound, the aglycon. The aglycon can vary from a simple molecule to a more complex molecule.

Monosaccharides:

Structural properties of monosaccharides:

Aldose or ketose

Pyranose or furanose

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Hexose or pentose

Alpha or beta anomer

Each D-sugar has an L-enantiomer, which is the mirror image of the whole D-molecule. In a D-enantiomer thehydroxyl group on the highest chiral C-atom is positioned on the right side of the Fischer projection and in an L-enantiomer on the left side.

When a monosaccharide is in open form , the carbonyl group (C=O) can be oxidized by for instance Cu2+-ions. Thesugar reduces the ion. Therefore such a sugar is called a reducing sugar. Most reducing sugars have a reducingterminal sugar residue. Since reducing sugars can open the ring structure, they can convert from one anomeric forminto the other anomeric form via the open chain form. This conversion is also called mutarotation and this istemperature dependent.

Uronic acids= monosaccharides of which the primary hydroxyl group at C6 is oxidised to a carboxylic group (COOH).Since they have an acid group, they can have a negative charge depending on the pH.

Disaccharides:

Monosaccharides can be linked to each other via glycosidic linkages and consequently form oligosaccharides andpolysaccharides. When this linkage is made between the hemiacetal OH-groups of 2 monosaccharides, anonreducing disaccharide is formed and when a hemiacetal OH group and an alcoholic OH-group are involved in thelinkage, a reducing disaccharide is formed.

Hydrolysis is the cleavage of the glycosidic bond and this can occurenzymatically or chemically. With hydrolysis you can release reducingsugars.

Oligosaccharides:

20