applications of proteases in the food industry

8/7/2019 Applications of proteases in the food industry

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Applications of proteases in the food

industry

Certain proteases have been used in food processing for centuries andany record of the discovery of their activity has been lost in the mists oftime. Rennet (mainly chymosin), obtained from the fourth stomach(abomasum) of unweaned calves has been used traditionally in theproduction of cheese. Similarly, papain from the leaves and unripe fruitof the pawpaw (Carica papaya) has been used to tenderise meats.

These ancient discoveries have led to the development of various foodapplications for a wide range of available proteases from many sources,usually microbial. Proteases may be used at various pH values, andthey may be highly specific in their choice of cleavable peptide links orquite non-specific. Proteolysis generally increases the solubility ofproteins at their isoelectric points.

The action of rennet in cheese making is an example of the hydrolysisof a specific peptide linkage, between phenylalanine and methionineresidues (-Phe105-Met106-) in the -casein protein present in milk (see

reaction scheme [1.3]). The -casein acts by stabilising the colloidalnature of the milk, its hydrophobic N-terminal region associating with thelipophilic regions of the otherwise insoluble - and -caseinmolecules, whilst its negatively charged C-terminal region associateswith the water and prevents the casein micelles from growing too large.Hydrolysis of the labile peptide linkage between these two domains,resulting in the release of a hydrophilic glycosylated and phosphorylatedoligopeptide (caseino macropeptide) and the hydrophobic para- -casein, removes this protective effect, allowing coagulation of the milk toform curds, which are then compressed and turned into cheese (Figure4.1). The coagulation process depends upon the presence of Ca2+ andis very temperature dependent (Q10 = 11) and so can be controlledeasily. Calf rennet, consisting of mainly chymosin with a small butvariable proportion of pepsin, is a relatively expensive enzyme andvarious attempts have been made to find cheaper alternatives frommicrobial sources These have ultimately proved to be successful and
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microbial rennets are used for about 70% of US cheese and 33% ofcheese production world-wide.

Figure 4.1. Outline method for the preparation of cheese.

The major problem that had to be overcome in the development of themicrobial rennets was temperature lability. Chymosin is a relativelyunstable enzyme and once it has done its major job, little activityremains. However, the enzyme from Mucor mieheiretains activityduring the maturation stages of cheese-making and produces bitter off-flavours. Treatment of the enzyme with oxidising agents (e.g. H2O2,peracids), which convert methionine residues to their sulfoxides,reduces its thermostability by about 10C and renders it morecomparable with calf rennet. This is a rare example of enzymetechnology being used to destabilise an enzyme Attempts have beenmade to clone chymosin into Escherichia coliand Saccharomyces


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cerevisiae but, so far, the enzyme has been secreted in an active formonly from the latter.

The development of unwanted bitterness in ripening cheese is anexample of the role of proteases in flavour production in foodstuffs. The

action of endogenous proteases in meat after slaughter is complex but'hanging' meat allows flavour to develop, in addition to tenderising it. Ithas been found that peptides with terminal acidic amino acid residuesgive meaty, appetising flavours akin to that of monosodium glutamate.Non-terminal hydrophobic amino acid residues in medium-sizedoligopeptides give bitter flavours, the bitterness being less intense withsmaller peptides and disappearing altogether with larger peptides.Application of this knowledge allows the tailoring of the flavour of proteinhydrolysates. The presence of proteases during the ripening of cheesesis not totally undesirable and a protease from Bacillusamyloliquefaciens may be used to promote flavour production incheddar cheese. Lipases from Mucor mieheiorAspergillus nigeraresometimes used to give stronger flavours in Italian cheeses by a modestlipolysis, increasing the amount of free butyric acid. They are added tothe milk (30 U l-1) before the addition of the rennet.

When proteases are used to depolymerise proteins, usually non-specifically, the extent of hydrolysis (degree of hydrolysis) is describedin DH units where:

(4.1)

Commercially, using enzymes such as subtilisin, DH values of up to 30are produced using protein preparations of 8-12% (w/w). The enzymesare formulated so that the value of the enzyme : substrate ratio used is2-4% (w/w). At the high pH needed for effective use of subtilisin, protonsare released during the proteolysis and must be neutralised:

subtilisin (pH 8.5)H2N-aa-aa-aa-aa-aa-COO- H2N-aa-aa-aa-COO- + H2N-aa-aa-COO- + H+ [4.1]

where aa is an amino acid residue.


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Correctly applied proteolysis of inexpensive materials such as soyaprotein can increase the range and value of their usage, as indeedoccurs naturally in the production of soy sauce. Partial hydrolysis ofsoya protein, to around 3.5 DH greatly increases its 'whippingexpansion', further hydrolysis, to around 6 DH improves its emulsifyingcapacity. If their flavours are correct, soya protein hydrolysates may beadded to cured meats. Hydrolysed proteins may develop properties thatcontribute to the elusive, but valuable, phenomenon of 'mouth feel' insoft drinks.

Proteases are used to recover protein from parts of animals (and fish)would otherwise go to waste after butchering. About 5% of the meat canbe removed mechanically from bone. To recover this, bones aremashed incubated at 60C with neutral or alkaline proteases for up to4 h. The meat slurry produced is used in canned meats and soups.

Large quantities of blood are available but, except in products suchblack puddings, they are not generally acceptable in foodstuffs becauseof their colour. The protein is of a high quality nutritionally and is de-haemed using subtilisin. Red cells are collected and haemolysed inwater. Subtilisin is added and hydrolysis is allowed to proceedbatchwise, with neutralisation of the released protons, to around 18 DH,when the hydrophobic haem molecules precipitate. Excessivedegradation is avoided to prevent the formation of bitter peptides. Theenzyme is inactivated by a brief heat treatment at 85C and theproduct is centrifuged; no residual activity allowed into meat products.The haem-containing precipitate is recycled and the light-brownsupernatant is processed through activated carbon beads to removeany residual haem. The purified hydrolysate, obtained in 60% yield, maybe spray-dried and is used in cured meats, sausages and luncheonmeats.

Meat tenderisation by the endogenous proteases in the muscle afterslaughter is a complex process which varies with the nutritional,

physiological and even psychological (i.e. frightened or not) state of theanimal at the time of slaughter. Meat of older animals remains tough butcan be tenderised by injecting inactive papain into the jugular vein of thelive animals shortly before slaughter. Injection of the active enzymewould rapidly kill the animal in an unacceptably painful manner so theinactive oxidised disulfide form of the enzyme is used. On slaughter, theresultant reducing conditions cause free thiols to accumulate in the


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muscle, activating the papain and so tenderising the meat. This is a veryeffective process as only 2 - 5 ppm of the inactive enzyme needs to beinjected. Recently, however, it has found disfavour as it destroys theanimals heart, liver and kidneys that otherwise could be sold and, beingreasonably heat stable, its action is difficult to control and persists intothe cooking process.

Proteases are also used in the baking industry. Where appropriate,dough may be prepared more quickly if its gluten is partially hydrolysed.A heat-labile fungal protease is used so that it is inactivated early in thesubsequent baking. Weak-gluten flour is required for biscuits in orderthat the dough can be spread thinly and retain decorative impressions.In the past this has been obtained from European domestic wheat butthis is being replaced by high-gluten varieties of wheat. The gluten inthe flour derived from these must be extensively degraded if such flouris to be used efficiently for making biscuits or for preventing shrinkage ofcommercial pie pastry away from their aluminium dishes.

The use of proteases in the leatherand wool industries

The leather industry consumes a significant proportion of the world'senzyme production. Alkaline proteases are used to remove hair fromhides. This process is far safer and more pleasant than the traditionalmethods involving sodium sulfide. Relatively large amounts of enzymeare required (0.1-1.0 % (w/w)) and the process must be closelycontrolled to avoid reducing the quality of the leather. After dehairing,hides which are to be used for producing soft leather clothing and goodsare bated, a process, often involving pancreatic enzymes, thatincreases their suppleness and improves the softness of theirappearance.

Proteases have been used, in the past, to 'shrinkproof' wool. Wool fibresare covered in overlapping scales pointing towards the fibre tip. Thesegive the fibres highly directional frictional properties, movement in thedirection away from the tip being favoured relative to movement towardsit. This propensity for movement solely in the one direction may lead toshrinkage and many methods have been used in attempts to eliminate


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the problem (e.g. chemical oxidation or coating the fibres in polymer). Asuccessful method involved the partial hydrolysis of the scale tips withthe protease papain. This method also gave the wool a silky lustre andadded to its value. The method was abandoned some years ago,primarily for economic reasons. It is not unreasonable to expect its useto be re-established now that cheaper enzyme sources are available.

The use of enzymes in starchhydrolysis

Starch is the commonest storage carbohydrate in plants. It is used by

the plants themselves, by microbes and by higher organisms so there isa great diversity of enzymes able to catalyse its hydrolysis. Starch fromall plant sources occurs in the form of granules which differ markedly insize and physical characteristics from species to species. Chemicaldifferences are less marked. The major difference is the ratio ofamylose to amylopectin; e.g. corn starch from waxy maize contains only2% amylose but that from amylomaize is about 80% amylose. Somestarches, for instance from potato, contain covalently bound phosphatein small amounts (0.2% approximately), which has significant effects onthe physical properties of the starch but does not interfere with itshydrolysis. Acid hydrolysis of starch has had widespread use in thepast. It is now largely replaced by enzymic processes, as it required theuse of corrosion resistant materials, gave rise to high colour and saltashcontent (after neutralisation), needed more energy for heating and wasrelatively difficult to control.


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Figure 4.2. The use of enzymes in processing starch. Typical conditionsare given.

Of the two components of starch, amylopectin presents the greatchallenge to hydrolytic enzyme systems. This is due to the residuesinvolved in -1,6-glycosidic branch points which constitute about 4 -6% of the glucose present. Most hydrolytic enzymes are specific for -1,4-glucosidic links yet the -1,6-glucosidic links must also be cleavedfor complete hydrolysis of amylopectin to glucose. Some of the mostimpressive recent exercises in the development of new enzymes have

concerned debranching enzymes.

It is necessary to hydrolyse starch in a wide variety of processes whichm be condensed into two basic classes:

1. processes in which the starch hydrolysate is to be used bymicrobes or man, and


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2. processes in which it is necessary to eliminate starch.

In the former processes, such as glucose syrup production, starch isusually the major component of reaction mixtures, whereas in the latterprocesses, such as the processing of sugar cane juice, small amounts

of starch which contaminate non-starchy materials are removed.Enzymes of various types are used in these processes. Althoughstarches from diverse plants may be utilised, corn is the world's mostabundant source and provides most of the substrate used in thepreparation of starch hydrolysates.

There are three stages in the conversion of starch (Figure 4.2):

1. gelatinisation, involving the dissolution of the nanogram-sizedstarch granules to form a viscous suspension;

2. liquefaction, involving the partial hydrolysis of the starch, withconcomitant loss in viscosity; and

3. saccharification, involving the production of glucose and maltoseby further hydrolysis.

Gelatinisation is achieved by heating starch with water, and occursnecessarily and naturally when starchy foods are cooked. Gelatinisedstarch is readily liquefied by partial hydrolysis with enzymes or acidsand saccharified by further acidic or enzymic hydrolysis.

The starch and glucose syrup industry uses the expression dextroseequivalent or DE, similar in definition to the DH units of proteolysis, todescribe its products, where:

(4 .2)

In practice, this is usually determined analytically by use of the closelyrelated, but not identical, expression:

(4 .3)

Thus, DE represents the percentage hydrolysis of the glycosidiclinkages present. Pure glucose has a DE of 100, pure maltose has a DEof about 50 (depending upon the analytical methods used; see equation
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(4.3)) and starch has a DE of effectively zero. During starch hydrolysis,DE indicates the extent to which the starch has been cleaved. Acidhydrolysis of starch has long been used to produce 'glucose syrups' andeven crystalline glucose (dextrose monohydrate). Very considerableamounts of 42 DE syrups are produced using acid and are used inmany applications in confectionery. Further hydrolysis using acid is notsatisfactory because of undesirably coloured and flavoured breakdownproducts. Acid hydrolysis appears to be a totally random process whichis not influenced by the presence of -1,6-glucosidic linkages.

Table 4.2 Enzymes used in starch hydrolysis

EnzymeEC

numberSource Action

-Amylase 3.2.1.1

Bacillus amyloliquefaciens

Only -1,4-oligosaccharide linksare cleaved to give -dextrinsand predominantly maltose (G2),G3, G6 and G7 oligosaccharides

B. licheniformis

Only -1,4-oligosaccharide linksare cleaved to give -dextrinsand predominantly maltose, G3,G4 and G5 oligosaccharides

Aspergillus oryzae,A. niger

Only -1,4 oligosaccharide links

are cleaved to give -dextrinsand predominantly maltose andG3 oligosaccharides

Saccharifying-amylase

3.2.1.1B.

subtilis(amylosacchariticus)

Only -1,4-oligosaccharide linksare cleaved to give -dextrinswith maltose, G3, G4 and up to50% (w/w) glucose

-Amylase 3.2.1.2 Malted barleyOnly -1,4-links are cleaved,from non-reducing ends, to givelimit dextrins and -maltose

Glucoamylase 3.2.1.3 A. niger -1,4 and -1,6-links arecleaved, from the nonreducingends, to give -glucose

Pullulanase 3.2.1.41 B. acidopullulyticus Only -1,6-links are cleaved togive straight-chain maltodextrins
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The nomenclature of the enzymes used commercially for starchhydrolysis is somewhat confusing and the EC numbers sometimes lumptogether enzymes with subtly different activities (Table 4.2). Forexample, -amylase may be subclassified as liquefying orsaccharifying amylases but even this classification is inadequate toencompass all the enzymes that are used in commercial starchhydrolysis. One reason for the confusion in the nomenclature is the useof the anomeric form of the released reducing group in the productrather than that of the bond being hydrolysed; the products of bacterialand fungal -amylases are in the -configuration and the productsof -amylases are in the -configuration, although all these enzymescleave between -1,4-linked glucose residues.

The -amylases (1,4- -D-glucan glucanohydrolases) are

endohydrolases which cleave 1,4- -D-glucosidic bonds and canbypass but cannot hydrolyse 1,6- -D-glucosidic branchpoints.Commercial enzymes used for the industrial hydrolysis of starch areproduced by Bacillus amyloliquefaciens (supplied by variousmanufacturers) and by B. licheniformis (supplied by Novo Industri A/Sas Termamyl). They differ principally in their tolerance of hightemperatures, Termamyl retaining more activity at up to 110C, in thepresence of starch, than the B. amyloliquefaciens -amylase. Themaximum DE obtainable using bacterial -amylases is around 40 butprolonged treatment leads to the formation of maltulose (4- -D-glucopyranosyl-D-fructose), which is resistant to hydrolysis byglucoamylase and -amylases. DE values of 8-12 are used in mostcommercial processes where further saccharification is to occur. Theprincipal requirement for liquefaction to this extent is to reduce theviscosity of the gelatinised starch to ease subsequent processing.

Various manufacturers use different approaches to starch liquefactionusing -amylases but the principles are the same. Granular starch isslurried at 30-40% (w/w) with cold water, at pH 6.0-6.5, containing 20-80

ppm Ca

2+

(which stabilises and activates the enzyme) and the enzymeis added (via a metering pump). The -amylase is usually supplied athigh activities so that the enzyme dose is 0.5-0.6 kg tonne-1 (about 1500U kg-1dry matter) of starch. When Termamyl is used, the slurry of starchplus enzyme is pumped continuously through a jet cooker, which isheated to 105C using live steam. Gelatinisation occurs very rapidlyand the enzymic activity, combined with the significant shear forces,
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begins the hydrolysis. The residence time in the jet cooker is very brief.The partly gelatinised starch is passed into a series of holding tubesmaintained at 100-105C and held for 5 min to complete thegelatinisation process. Hydrolysis to the required DE is completed inholding tanks at 90-100C for 1 to 2 h. These tanks contain baffles todiscourage backmixing. Similar processes may be used with B.amyloliquefaciens -amylase but the maximum temperature of 95Cmust not be exceeded. This has the drawback that a final 'cooking'stage must be introduced when the required DE has been attained inorder to gelatinise the recalcitrant starch grains present in some types ofstarch which would otherwise cause cloudiness in solutions of the finalproduct.

The liquefied starch is usually saccharified but comparatively small

amounts are spray-dried for sale as 'maltodextrins' to the food industrymainly for use as bulking agents and in baby food. In this case, residualenzymic activity may be destroyed by lowering the pH towards the endof the heating period.

Fungal -amylase also finds use in the baking industry. It often needsto be added to bread-making flours to promote adequate gas productionand starch modification during fermentation. This has becomenecessary since the introduction of combine harvesters. They reducethe time between cutting and threshing of the wheat, which previously

was sufficient to allow a limited sprouting so increasing the amounts ofendogenous enzymes. The fungal enzymes are used rather than thosefrom bacteria as their action is easier to control due to their relative heatlability, denaturing rapidly during baking.

Production of glucose syrup

The liquefied starch at 8 -12 DE is suitable for saccharification toproduce syrups with DE values of from 45 to 98 or more. The greatestquantities produced are the syrups with DE values of about 97. Atpresent these are produced using the exoamylase, glucan 1,4- -glucosidase (1,4- -D-glucan glucohydrolase, commonly calledglucoamylase but also called amyloglucosidase and -amylase), whichreleases -D-glucose from 1,4- -, 1,6- - and 1,3- -linked glucans.


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In theory, carefully liquefied starch at 8 -12 DE can be hydrolysedcompletely to produce a final glucoamylase reaction mixture with DE of100 but, in practice, this can be achieved only at comparatively lowsubstrate concentrations. The cost of concentrating the product byevaporation decrees that a substrate concentration of 30% is used. Itfollows that the maximum DE attainable is 96 - 98 with syrupcomposition 95 - 97% glucose, 1 - 2% maltose and 0.5 - 2% (w/w)isomaltose ( -D-glucopyranosyl-(1,6)-D-glucose). This material is usedafter concentration, directly for the production of high-fructose syrups orfor the production of crystalline glucose.

Whereas liquefaction is usually a continuous process, saccharification ismost often conducted as a batch process. The glucoamylase most oftenused is produced by Aspergillus nigerstrains. This has a pH optimum of4.0 - 4.5 and operates most effectively at 60C, so liquefied starchmust be cooled and its pH adjusted before addition of the glucoamylase.The cooling must b rapid, to avoid retrogradation (the formation ofintractable insoluble aggregates of amylose; the process that gives riseto the skin on custard). Any remaining bacterial -amylase will beinactivated when the pH is lowered; however, this may be replaced laterby some acid-stable -amylase which is normally present in theglucoamylase preparations. When conditions are correct theglucoamylase is added, usually at the dosage of 0.65 - 0.80 litreenzyme preparation.tonne-1 starch (200 U kg-1). Saccharification is

normally conducted in vast stirred tanks, which may take several hoursto fill (and empty), so time will be wasted if the enzyme is added onlywhen the reactors are full. The alternatives are to meter the enzyme at afixed ratio or to add the whole dose of enzyme at the commencement ofthe filling stage. The latter should give the most economical use of theenzyme.


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Figure 4.3. The % glucose formed from 30% (w/w) 12 DE maltodextrin,at 60C and pH 4.3, using various enzyme solutions.200 U kg-1Aspergillus nigerglucoamylase; -----------400 U kg-1A.nigerglucoamylase; 200 U kg-1A.nigerglucoamylase plus 200 U kg-1Bacillusacidopullulyticus pullulanase. The relative improvement on the addition

of pullulanase is even greater at higher substrate concentrations.

The saccharification process takes about 72 h to complete but may, ofcourse, be speeded up by the use of more enzyme. Continuoussaccharification is possible and practicable if at least six tanks are usedin series. It is necessary to stop the reaction, by heating to 85C for 5min, when a maximum DE has been attained. Further incubation willresult in a fall in the DE, to about 90 DE eventually, caused by theformation of isomaltose as accumulated glucose re-polymerises with theapproach of thermodynamic equilibrium (Figure 4.3).

The saccharified syrup is filtered to remove fat and denatured proteinreleased from the starch granules and may then be purified by passagethrough activated charcoal and ion-exchange resins. It should beremembered that the dry substance concentration increases by about
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11 % during saccharification, because one molecule of water is takenup for each glycosidic bond hydrolysed (molecule of glucose produced).

Although glucoamylase catalyses the hydrolysis of 1,6- -linkages, theirbreakdown is slow compared with that of 1,4- -linkages (e.g. the ratesof hydrolysing the 1,4- , 1,6- and 1,3- -links in tetrasaccharides arein the proportions 300 : 6 : 1). It is clear that the use of a debranchingenzyme would speed the overall saccharification process but, forindustrial use such an enzyme must be compatible with glucoamylase.Two types of debranching enzymes are available: pullulanase, whichacts as an exo hydrolase on starch dextrins; and isoamylase(EC.3.2.1.68), which is a true endohydrolase. Novo Industri A/S haverecently introduced a suitable pullulanase, produced by a strainofBacillus acidopullulyticus. The pullulanase from Klebsiella

aerogenes which has been available commercially to some time isunstable at temperatures over 45C but the B.acidopullulyticus enzymes can be used under the same conditions asthe Aspergillus glucoamylase (60C, pH 4.0-4.5). The practicaladvantage of using pullulanase together with glucoamylase is that lessglucoamylase need be used This does not in itself give any costadvantage but because less glucoamylase is used and fewer branchedoligosaccharides accumulate toward the end of the saccharification, thepoint at which isomaltose production becomes significant occurs athigher DE (Figure 4.3). It follows that higher DE values and glucose

contents can be achieved when pullulanase is use (98 - 99 DE and 95 -97% (w/w) glucose, rather than 97 - 98 DE) and higher substrateconcentrations (30 - 40% dry solids rather than 25 - 30%) may betreated. The extra cost of using pullulanase is recouped by savings inevaporation and glucoamylase costs. In addition, when the product is tobe used to manufacture high-fructose syrups, there is a saving in thecost of further processing.

The development of the B. acidopullulyticus pullulanase is an excellentexample of what can be done if sufficient commercial pull exists for a

new enzyme. The development of a suitable -D-glucosidase, in orderto reduce the reversion, would be an equally useful step for industrialglucose production. Screening of new strains of bacteria for a novelenzyme of this type is a major undertaking. It is not surprising that moredetails of the screening procedures used are not readily available.


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Production of syrups containingmaltose

Traditionally, syrups containing maltose as a major component havebeen produced by treating barley starch with barley -amylase. -Amylases (1,4- -D-glucan maltohydrolases) are exohydrolases whichrelease maltose from 1,4- -linked glucans but neither bypass norhydrolyse 1,6- -linkages. High-maltose syrups (40 - 50 DE, 45-60%(w/w) maltose, 2 - 7% (w/w) glucose) tend not to crystallise, even below0C and are relatively non-hygroscopic. They are used for theproduction of hard candy and frozen deserts. High conversion syrups

(60 - 70 DE, 30 - 37% maltose, 35 - 43% glucose, 10% maltotriose,15% other oligosaccharides, all by weight) resist crystallisation above4C and are sweeter (Table 4.3). They are used for soft candy and inthe baking, brewing and soft drinks industries. It might be expectedthat -amylase would be used to produce maltose-rich syrups fromcorn starch, especially as the combined action of -amylase andpullulanase give almost quantitative yields of maltose. This is not doneon a significant scale nowadays because presently available -amylases are relatively expensive, not sufficiently temperature stable

(although some thermostable -amylases from speciesofClostridium have recently been reported) and are easily inhibited bycopper and other heavy metal ions. Instead fungal -amylases,characterised by their ability to hydrolyse maltotriose (G3) rather thanmaltose (G2) are employed often in combination with glucoamylase.Presently available enzymes, however, are not totally compatible;fungal -amylases requiring a pH of not less than 5.0 and a reactiontemperature not exceeding 55C.

High-maltose syrups (see Figure 4.2) are produced from liquefied starch

of around 11 DE at a concentration of 35% dry solids using fungal -amylase alone. Saccharification occurs over 48 h, by which time thefungal -amylase has lost its activity. Now that a good pullulanase isavailable, it is possible to use this in combination with fungal -amylases to produce syrups with even higher maltose contents.
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High-conversion syrups are produced using combinations of fungal -amylase and glucoamylase. These may be tailored to customers'specifications by adjusting the activities of the two enzymes used butinevitably, as glucoamylase is employed, the glucose content of the finalproduct will be higher than that of high-maltose syrups. The stability ofglucoamylase necessitates stopping the reaction, by heating, when therequired composition is reached. It is now possible to produce starchhydrolysates with any DE between 1 and 100 and with virtually anycomposition using combinations of bacterial -amylases, fungal -amylases, glucoamylase and pullulanase.

Table 4.3 The relative sweetness of food ingredients

Food ingredient Relative sweetness (byweight, solids)

Sucrose 1.0

Glucose 0.7

Fructose 1.3

Galactose 0.7

Maltose 0.3

Lactose 0.2

Raffinose 0.2

Hydrolysed sucrose 1.1Hydrolysed lactose 0.7

Glucose syrup 11 DE


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The sucrose industry is a comparatively minor user of enzymes butprovides few historically significant and instructive examples of enzymetechnology The hydrolysis ('inversion') of sucrose, completely or

partially, to glucose and fructose provides sweet syrups that are morestable (i.e. less likely crystallise) than pure sucrose syrups. The mostfamiliar 'Golden Syrup' produced by acid hydrolysis of one of the lesspure streams from the cane sugar refinery but other types of syrup areproduced using yeast (Saccharomyces cerevisiae) invertase. Althoughthis enzyme is unusual in that it suffers from substrate inhibition at highsucrose levels ( > 20% (w/w)), this does not prevent its commercial useat even higher concentrations:

[4.2]

Traditionally, invertase was produced on site by autolysing yeast cells.The autolysate was added to the syrup (70% sucrose (w/w)) to beinverted together with small amounts of xylene to prevent microbialgrowth. Inversion was complete in 48 - 72 h at 50C and pH 4.5. Theenzyme and xylene were removed during the subsequent refining andevaporation. Partially inverted syrups were (and still are) produced byblending totally inverted syrups with sucrose syrups. Now, commerciallyproduced invertase concentrates are employed.

The production of hydrolysates of a low molecular weight compound inessentially pure solution seems an obvious opportunity for the use of animmobilised enzyme, yet this is not done on a significant scale, probablybecause of the extreme simplicity of using the enzyme in solution andthe basic conservatism of the sugar industry.

Invertase finds another use in the production of confectionery with liquidor soft centres. These centres are formulated using crystalline sucroseand tiny (about 100 U kg-1, 0.3 ppm (w/w)) amounts of invertase. At thislevel of enzyme, inversion of sucrose is very slow so the centre remains


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solid long enough for enrobing with chocolate to be completed. Then,over a period of days or weeks, sucrose hydrolysis occurs and theincrease in solubility causes the centres to become soft or liquid,depending on the water content of the centre preparation.

Other enzymes are used as aids to sugar production and refining byremoving materials which inhibit crystallisation or cause high viscosity.In some parts of the world, sugar cane contains significant amounts ofstarch, which becomes viscous, thus slowing filtration processes andmaking the solution hazy when the sucrose is dissolved. This problemcan be overcome by using the most thermostable -amylases (e.g.Termamyl at about 5 U kg-1) which are entirely compatible with the hightemperatures and pH values that prevail during the initial vacuumevaporation stage of sugar production.

Other problems involving dextran and raffinose required thedevelopment of new industrial enzymes. A dextran is produced by theaction of dextransucrase (EC 2.4.1.5) from Leuconostocmesenteroides on sucrose and found as a slime on damaged cane andbeet tissue, especially when processing has been delayed in hot andhumid climates. Raffinose, which consists of sucrose with -galactoseattached through its C-1 atom to the 6 position on the glucose residue,is produced at low temperatures in sugar beet. Both dextran andraffinose have the sucrose molecule as part of their structure and both

inhibit sucrose crystal growth. This produces plate-like or needle-likecrystals which are not readily harvested by equipment designed for theapproximately cubic crystals otherwise obtained. Dextran can produceextreme viscosity it process streams and even bring plant to a stop.Extreme dextran problems arc frequently solved by the use of fungaldextranases produced from Penicillium species. These are used (e.g.10 U kg-1 raw juice, 55C, pH 5.5, 1 h) only in times of crisis as theyare not sufficiently resistant to thermal denaturation for long-term useand are inactive at high sucrose concentrations. Because only smallquantities are produced for use, this enzyme is relatively expensive. An

enzyme sufficiently stable for prophylactic use would be required inorder to benefit from economies of scale. Raffinose may be hydrolysedto galactose and sucrose by a fungal raffinase (see Chapter 5).

Glucose from cellulose
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There is very much more cellulose available, as a potential source ofglucose, than starch, yet cellulose is not a significant source of pureglucose. The reasons for this are many, some technical, some

commercial. The fundamental reason is that starch is produced inrelatively pure forms by plants for use as an easily biodegradableenergy and carbon store. Cellulose is structural and is purposefullycombined and associated with lignin and pentosans, so as to resistbiodegradation; dead trees take several years to decay even in tropicalrainforests. A typical waste cellulolytic material contains less than halfcellulose, most of the remainder consisting of roughly equal quantities oflignin and pentosans. A combination of enzymes is needed to degradethis mixture. These enzymes are comparatively unstable of low activityagainst native lignocellulose and subject to both substrate and productinhibition. Consequently, although many cellulolytic enzymes exist and itis possible to convert pure cellulose to glucose using only enzymes, thecost of this conversion is excessive. The enzymes might be improved bystrain selection from the wild or by mutation but problems caused by thephysical nature of cellulose are not so amenable to solution. Granularstarch is readily stirred in slurries containing 40% (w/v) solids and iseasily solubilised but, even when pure, fibrous cellulose formsimmovable cakes at 10% solids and remains insoluble in all but themost exotic (and enzyme denaturing) solvents. Impure cellulose often

contains almost an equal mass of lignin, which is of little or no value asa by-product and is difficult an expensive to remove.

Commercial cellulase preparations from Trichoderma reeseiconsist ofmixtures of the synergistic enzymes:

a. cellulase (EC 3.2.1.4), an endo-1,4-D -glucanase;b. glucan 1,4- -glucosidase (EC 3.2.1.74), and exo-1,4- -

glucosidase; andc. cellulose 1,4- -cellobiosidase (EC 3.2.1.91), an exo-

cellobiohydrolase (see Figure 4.4).They are used for the removal of relatively small concentrations ofcellulose complexes which have been found to interfere in theprocessing of plant material in, for example, the brewing and fruit juiceindustries.
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Lactose is present at concentrations of about 4.7% (w/v) in milk and thewhey (supernatant) left after the coagulation stage of cheese-making.Its presence in milk makes it unsuitable for the majority of the world's

adult population, particularly in those areas which have traditionally nothad a dairy industry. Real lactose tolerance is confined mainly topeoples whose origins lie in Northern Europe or the Indian subcontinentand is due to 'lactase persistence'; the young of all mammals clearly areable to digest milk but in most cases this ability reduces after weaning.Of the Thai, Chinese and Black American populations, 97%, 90% and73% respectively, are reported to be lactose intolerant, whereas 84%and 96% of the US White and Swedish populations, respectively, aretolerant. Additionally, and only very rarely some individuals suffer frominborn metabolic lactose intolerance or lactase deficiency, both of whichmay be noticed at birth. The need for low-lactose milk is particularlyimportant in food-aid programmes as severe tissue dehydration,diarrhoea and even death may result from feeding lactose containingmilk to lactose-intolerant children and adults suffering from protein-calorie malnutrition. In all these cases, hydrolysis of the lactose toglucose and galactose would prevent the (severe) digestive problems.

Another problem presented by lactose is its low solubility resulting incrystal formation at concentrations above 11 % (w/v) (4C). Thisprevents the use of concentrated whey syrups in many food processesas they have a unpleasant sandy texture and are readily prone tomicrobiological spoilage. Adding to this problem, the disposal of suchwaste whey is expensive (often punitively so) due to its high biologicaloxygen demand. These problems may be overcome by hydrolysis of thelactose in whey; the product being about four times as sweet (see Table4.3), much more soluble and capable of forming concentrated,microbiologically secure, syrups (70% (w/v)).

Lactose may be hydrolysed by lactase, a -galactosidase.
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[4.3]

Commercially, it may be prepared from the dairy yeast Kluyveromycesfragilis (K. marxianus var. marxianus), with a pH optimum (pH 6.5-7.0)suitable for the treatment of milk, or from the fungi Aspergillusoryzae orA. niger, with pH optima (pH 4.5-6.0 and 3.0-4.0, respectively)more suited to whey hydrolysis. These enzymes are subject to varyingdegrees of product inhibition by galactose. In addition, at high lactose

and galactose concentrations, lactase shows significant transferaseability and produces -1,6-linked galactosyl oligosaccharides.

Lactases are now used in the production of ice cream and sweetenedflavoured and condensed milks. When added to milk or liquid whey(2000 U kg-1) and left for about a day at 5C about 50% of the lactoseis hydrolysed, giving a sweeter product which will not crystallise ifcondensed or frozen. This method enables otherwise-wasted whey toreplace some or all of the skim milk powder used in traditional ice creamrecipes. It also improves the 'scoopability' and creaminess of the

product. Smaller amounts of lactase may be added to long-life sterilisedmilk to produce a relatively inexpensive lactose-reduced product (e.g.20 U kg-1, 20C, 1 month of storage). Generally, however, lactaseusage has not reached its full potential, as present enzymes arerelatively expensive and can only be used at low temperatures.

Enzymes in the fruit juice, wine,brewing and distilling industries

One of the major problems in the preparation of fruit juices and wine iscloudiness due primarily to the presence of pectins. These consistprimarily of -1,4-anhydrogalacturonic acid polymers, with varyingdegrees of methyl esterification. They are associated with other plant


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polymers and, after homogenisation, with the cell debris. The cloudinessthat they cause is difficult to remove except by enzymic hydrolysis. Suchtreatment also has the additional benefits of reducing the solutionviscosity, increasing the volume of juice produced (e.g. the yield of juicefrom white grapes can be raised by 15%), subtle but generally beneficialchanges in the flavour and, in the case of wine-making, shorterfermentation times. Insoluble plant material is easily removed byfiltration, or settling and decantation, once the stabilising effect of thepectins on the colloidal haze has been removed.

Commercial pectolytic enzyme preparations are producedfrom Aspergillus nigerand consist of a synergistic mixture of enzymes:

a. polygalacturonase (EC 3.2.1.15), responsible for the randomhydrolysis of 1,4- -D-galactosiduronic linkages;

b. pectinesterase (EC 3.2.1.11), which releases methanol from thepectyl methyl esters, a necessary stage before thepolygalacturonase can act fully (the increase in the methanolcontent of such treated juice is generally less than the naturalconcentrations and poses no health risk);

c. pectin lyase (EC 4.2.2.10), which cleaves the pectin, by anelimination reaction releasing oligosaccharides with non-reducingterminal 4-deoxymethyl- -D-galact-4-enuronosyl residues,without the necessity of pectin methyl esterase action; and

d. hemicellulase (a mixture of hydrolytic enzymes including: xylanendo-1,3- -xylosidase, EC 3.2.1.32; xylan 1,4- -xylosidase, EC3.2.1.37; and -L-arabinofuranosidase, EC 3.2.1.55), strictly nota pectinase but its adventitious presence is encouraged in orderto reduce hemicellulose levels.

The optimal activity of these enzymes is at a pH between 4 and 5 andgenerally below 50C. They are suitable for direct addition to the fruitpulps at levels around 20 U l-1 (net activity). Enzymes with improvedcharacteristics of greater heat stability and lower pH optimum are

currently being sought.

In brewing, barley malt supplies the major proportion of the enzymeneeded for saccharification prior to fermentation. Often other starchcontaining material (adjuncts) are used to increase the fermentablesugar and reduce the relative costs of the fermentation. Although maltenzyme may also be used to hydrolyse these adjuncts, for maximum


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economic return extra enzymes are added to achieve their rapidsaccharification. It not necessary nor desirable to saccharify the starchtotally, as non-fermentable dextrins are needed to give the drink 'body'and stabilise its foam 'head'. For this reason the saccharificationprocess is stopped, by boiling the 'wort', after about 75% of the starchhas been converted into fermentable sugar.

The enzymes used in brewing are needed for saccharification of starch(bacterial and fungal -amylases), breakdown of barley -1,4- and -1,3- linked glucan ( -glucanase) and hydrolysis of protein (neutralprotease) to increase the (later) fermentation rate, particularly in theproduction of high-gravity beer, where extra protein is added. Cellulasesare also occasionally used, particularly where wheat is used as adjunctbut also to help breakdown the barley -glucans. Due to the extreme

heat stability of the B. amyloliquefaciens -amylase, where this is usedthe wort must be boiled for a much longer period (e.g. 30 min) toinactivate it prior to fermentation. Papain is used in the later post-fermentation stages of beer-making to prevent the occurrence ofprotein- and tannin-containing 'chill-haze' otherwise formed on coolingthe beer.

Recently, 'light' beers, of lower calorific content, have become morepopular. These require a higher degree of saccharification at lowerstarch concentrations to reduce the alcohol and total solids contents of

the beer. This may be achieved by the use of glucoamylase and/orfungal -amylase during the fermentation.

A great variety of carbohydrate sources are used world wide to producedistilled alcoholic drinks. Many of these contain sufficient quantities offermentable sugar (e.g. rum from molasses and brandy from grapes),others contain mainly starch and must be saccharified before use (e.g.whiskey from barley malt, corn or rye). In the distilling industry,saccharification continues throughout the fermentation period. In somecases (e.g. Scotch malt whisky manufacture uses barley malt

exclusively) the enzymes are naturally present but in others (e.g. grainspirits production) the more heat-stable bacterial -amylases may beused in the saccharification.


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Glucose oxidase and catalase in thefood industry

Glucose oxidase is a highly specific enzyme (for D-glucose, butsee Chapter 8), from the fungi Aspergillus nigerand Penicillium, whichcatalyses the oxidation of -glucose to glucono-1,5-lactone (whichspontaneously hydrolyses non-enzymically to gluconic acid) usingmolecular oxygen and releasing hydrogen peroxide (see reactionscheme [1.1]). It finds uses in the removal of either glucose or oxygenfrom foodstuffs in order to improve their storage capability. Hydrogenperoxide is an effective bacteriocide and may be removed, after use, by

treatment with catalase (derived from the same fungal fermentations asthe glucose oxidase) which converts it to water and molecular oxygen:

catalase2H2O2 2H2O + O2 [4.4]

For most large-scale applications the two enzymic activities are notseparated. Glucose oxidase and catalase may be used together whennet hydrogen peroxide production is to be avoided.

A major application of the glucose oxidase/catalase system is in theremoval of glucose from egg-white before drying for use in the bakingindustry. A mixture of the enzymes is used (165 U kg-1) together withadditional hydrogen peroxide (about 0.1 % (w/w)) to ensure thatsufficient molecular oxygen is available, by catalase action, to oxidisethe glucose. Other uses are in the removal of oxygen from the head-space above bottled and canned drinks and reducing non-enzymicbrowning in wines and mayonnaises.

Medical applications of enzymes

Development of medical applications for enzymes have been at least asextensive as those for industrial applications, reflecting the magnitude ofthe potential rewards: for example, pancreatic enzymes have been in
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use since the nineteenth century for the treatment of digestivedisorders. The variety of enzymes and their potential therapeuticapplications are considerable. A selection of those enzymes which haverealised this potential to become important therapeutic agents is shownin Table 4.4. At present, the most successful applications areextracellular: purely topical uses, the removal c toxic substances andthe treatment of life-threatening disorders within the blood circulation.

Table 4.4 Some important therapeutic enzymes

EnzymeEC

numberReaction Use

Asparaginase 3.5.1.1 L-Asparagine H2O L-aspartate + NH3 Leukaemia

Collagenase 3.4.24.3 Collagen hydrolysis Skin ulcersGlutaminase 3.5.1.2 L-Glutamine H2O L-glutamate + NH3 Leukaemia

Hyaluronidasea 3.2.1.35 Hyaluronate hydrolysis Heart attack

Lysozyme 3.2.1.17 Bacterial cell wall hydrolysis Antibiotic

Rhodanaseb 2.8.1.1 S2O32- + CN- SO32- + SCN-Cyanide

poisoning

Ribonuclease 3.1.26.4 RNA hydrolysis Antiviral

-Lactamase 3.5.2.6 Penicillin penicilloatePenicillinallergy

Streptokinase

c

3.4.22.10 Plasminogen plasmin Blood clotsTrypsin 3.4.21.4 Protein hydrolysis Inflammation

Uricased 1.7.3.3 Urate + O2 allantoin Gout

Urokinasee 3.4.21.31 Plasminogen plasmin Blood clots

a Hyaluronoglucosaminidaseb thiosulphate sulfurtransferasec streptococcal cysteine proteinased urate oxidasee plasminogen activator

As enzymes are specific biological catalysts, they should make the mostdesirable therapeutic agents for the treatment of metabolic diseases.Unfortunately a number of factors severely reduces this potential utility:
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preparations with only biocompatible buffering salts and mannitol diluentadded. The costs of such enzymes may be quite high but stillcomparable to those of competing therapeutic agents or treatments. Asan example, urokinase (a serine protease, see Table 4.4) is preparedfrom human urine (some genetically engineered preparations are beingdeveloped) and used to dissolve blood clots. The cost of the enzyme isabout100 mg-1, with the cost of treatment in a case of lung embolismbeing about10000 for the enzyme alone. In spite of this, the marketfor the enzyme is worth about70M year-1.

A major potential therapeutic application of enzymes is in the treatmentof cancer. Asparaginase has proved to be particularly promising for thetreatment of acute lymphocytic leukaemia. Its action depends upon thefact that tumour cells are deficient in aspartate-ammonia ligase activity,which restricts their ability to synthesise the normally non-essentialamino acid L-asparagine. Therefore, they are forced to extract it frombody fluids. The action of the asparaginase does not affect thefunctioning of normal cells which are able to synthesise enough for theirown requirements, but reduce the free exogenous concentration and soinduces a state of fatal starvation in the susceptible tumour cells. A 60%incidence of complete remission has been reported in a study of almost6000 cases of acute lymphocytic leukaemia. The enzyme isadministered intravenously. It is only effective in reducing asparaginelevels within the bloodstream, showing a half-life of about a day (in a

dog). This half-life may be increased 20-fold by use of polyethyleneglycol-modified asparaginase.

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Cc ng dng ca protease trong ngnh cng nghip thc phm

proteases Mt s c s dng trong ch bin thc phm trongnhiu th k v bt k h s v s pht hin ca cc hot ng ca h b mt trong sng m ca thi gian. Rennet (ch yu l chymosin),thu c t d dy th t (d mi kh) ca b unweaned c sdng truyn thng trong sn xut pho mt.Tng t nh vy, papain tl v qu cha chn ca pawpaw (Carica u ) c s dng tenderise tht. Nhng khm ph ny c xa dn n s pht trinca cc ng dng thc phm khc nhau cho mt lot cc protease csn t nhiu ngun, thng l vi khun. Protease c th c s dngti cc gi tr pH khc nhau, v h c th c nh gi cao c thtrong s la chn ca h v cc lin kt peptide cleavable hoc rt
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khng c th. S phn gii protein thng lm tng ha tan caprotein ti cc im isoelectric ca h.-casein trong sa (xem phn ng n [1,3]). Cc hnh ng ca ddy trong vic a ra pho mt l mt v d ca s thy phn ca mtlin kt peptide c th, gia phenylalanine v d lng methionine (-Phe105-Met106-) trong cc protein -casein phn t, trong khi in tchm kt khu vc ca mnh C-thit b u cui vi cc nc v nukhng th khng ha tan - v -casein bng cch n nh cc tnh chtkeo ca sa, k nc khu vc N-u cui ca n gn vi cc vnglipophilic ca Cc hnh vi ngn nga cc mixen casein t tngtrng qu ln. -casein, loi b hiu ng ny bo v, cho php ng tca sa to thnh sa ng, trong Thy phn ca cc lin ktpeptide khng n nh gia hai lnh vc, dn n s ra i ca mtoligopeptide glycosyl ha v phosphoryl ha a nc (caseino

macropeptide) v para cc k- sau c nn v bin thnh ph mt(hnh 4.1).Qu trnh ng mu ph thuc vo s c mt ca Ca2 + vrt nhit ph thuc (Q10 = 11) v nh vy c th c kim sot ddng. B-d dy, bao gm ch yu l chymosin vi mt t l nh nhngbin ca pepsin, l mt enzyme tng i t tin v nhiu n lc c thc hin tm gii php thay th r hn t cc ngun vi sinhvt ny cui cng c chng minh l thnh cng v rennets vi sinhvt c s dng cho khong 70% M pho mt v 33% sn xut phomt trn ton th gii.________________________________________

Hnh 4.1. Phc tho phng php cho vic chun b ca pho mt.________________________________________Cc vn ln c khc phc trong s pht trin ca vi sinh vtc nhit rennets lability. Chymosin l mt enzyme tng i nnh v mt khi n lm cng vic chnh ca n, t hot ng vncn. Tuy nhin, cc enzyme t Mucor miehei gi li hot ng trongcc giai on trng thnh ca vic ra pho mt v sn xut ngoihng v cay ng. iu tr cc enzyme vi oxy ha cht (v d nhH2O2, peracids), m chuyn i d lng methionine sulfoxides ca

h, lm gim thermostability ca n vo khong 10C v lm cho nnhiu hn so vi b-d dy. y l mt v d him hoi ca cng nghenzym c s dng gy mt n nh mt n lc enzyme cthc hin sao lu chymosin vo Escherichia coli v nm menSaccharomyces cerevisiae, nhng, cho n nay, cc enzym ny c tit ra trong mt hnh thc hot ng ch t th hai.S pht trin ca cay ng khng mong mun trong qu trnh chn pho


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mt l mt v d v vai tr ca protease trong sn xut hng v trongthc phm. Cc hnh ng ca protease ni sinh trong tht sau khi gitm rt phc tp nhng "treo" hng v tht cho php pht trin, ngoitenderising n. N c tm thy rng peptide c d lng axitamino acid thit b u cui cho tht, ngon ming hng v tng t nhca bt ngt. Thit b u cui khng k d lng acid amin trongoligopeptides c trung bnh cho hng v ng, cay ng c t mnhm vi chui axit amin nh hn v bin mt hon ton vi peptide lnhn. p dng cc kin thc ny cho php may ca cc hng v cabt m thy phn. S hin din ca protease trong qu trnh chn capho mt l khng hon ton khng mong mun v mt protease tBacillus amyloliquefaciens c th c s dng thc y sn xutpho mt cheddar hng v . Lipases t Mucor miehei hoc Aspergillusniger i khi c s dng to hng v mnh hn trong pho mt bi mt lipolysis khim tn, lm tng lng axit butyric min ph. Hc thm vo sa (30 U l-1) trc khi b sung cc men dch v ny.Khi protease c s dng depolymerise protein, thng khng cbit, mc thy phn (mc thy phn) c m t trong cc nv DH hp:

(4.1)Thng mi, cc enzym s dng nh subtilisin, DH gi tr ln n 30c sn xut bng cch s dng ch phm protein ca 8-12% (w /w). Cc enzyme c xy dng sao cho gi tr ca enzyme: cht nn tl s dng l 2-4% (w / w). Ti pH cao cn thit s dng hiu qu

subtilisin, proton c gii phng trong s phn gii protein v phic v hiu ha:subtilisin (pH 8,5)H2N-aa-aa-aa-aa-aa-COO-H2N-aa-aa-aa-COO-+ H2N-aa-aa-COO-+ H+ [4,1]ni aa l mt d lng acid amin.p dng ng s phn gii protein ca cc vt liu r tin nh lprotein u nnh c th tng phm vi v gi tr s dng ca h, l thcs xut hin t nhin trong sn xut nc tng. Mt phn thy phnprotein u nnh, khong 3,5 DH thy phn lm tng ng k hn

na 'm rng nh' ca n,, n khong 6 DH ci thin nng lc can to nh tng. Nu mi v ca h l chnh xc, u nnh thy phnprotein c th c thm vo tht cha khi. Thy phn protein c thpht trin ti sn ng gp vo s kh nm bt, nhng c gi tr,hin tng "ming cm thy trong nc ngt.Protease c s dng phc hi protein t cc b phn ca ngvt (c) nu khng s i n cht thi sau khi git m.Khong 5% ca


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tht c th c loi b c hc t xng. phc hi ny, xng cnghin 60C vi protease trung tnh hoc kim cho n 4 h. Cc bntht sn xut c s dng trong cc loi tht ng hp v xp.s lng ln ca mu c sn nhng, ngoi tr trong cc sn phmbnh trng ming nh mu en, h thng khng c chp nhntrong thc phm v mu sc ca h. Protein ny l ca mt cht lngcao dinh dng v l subtilisin s dng de-haemed. t bo c thuthp v haemolysed trong nc.Subtilisin c thm vo v thy phnc php tin hnh batchwise, vi trung ha ca cc proton phthnh, khong 18 DH, khi cc phn t haem k nc kt ta. xungcp qu mc l trnh ngn nga s hnh thnh cc chui axit aminng.enzyme ny l bt hot bi mt x l nhit ngn ti 85C v snphm c ly tm, khng hot ng d c php thnh cc snphm tht. Cc kt ta haem cha c ti ch v gn nu nh sngc x l qua than hot tnh ht loi b bt k haem cn li. Ccthy phn tinh khit, thu c nng sut 60%, c th c phun kh vc s dng trong cc loi tht c cha khi, xc xch v tht ntra.Tht tenderisation ca protease ni sinh trong c bp sau khi git m lmt qu trnh phc tp khc nhau vi cc nh dinh dng, sinh l vthm ch tm l (c ngha l s hi hay khng) ca ng vt ti thiim git m. Tht ca ng vt ln tui vn cn kh khn nhng c thc tenderised bng cch tim papain khng hot ng vo tnh mchhuyt mch xut pht ca cc ng vt sng lu trc khi git

m. Injection ca enzyme hot ng nhanh chng s git cc con vtmt cch khng th chp nhn au n nn cc hnh thc disulfuakhng hot ng ca enzyme oxy ha c s dng. Trn c s gitm, vic gim iu kin kt qu gy thiol t do tch t trong c, kchhot cc papain v tenderising tht. y l mt qu trnh rt hiu quv ch c 2-5 trang / pht khng hot ng ca enzyme cn phi ctim. Gn y, tuy nhin, n c tm thy khng a v n ph hytri tim ng vt, gan v thn m nu khng c th c bn v, chp l nhit n nh, hnh ng ca n l kh kim sot v vn cn tnti vo qu trnh nu.

Protease cng c s dng trong ngnh cng nghip nngbnh. Khi thch hp bt, c th c chun b nhanh hn nu glutenca n l mt phn thy phn. Mt nhit khng n nh nm proteasec s dng n khng hot ng sm trong nng tip theo. btyu-gluten l cn thit cho bnh bch-quy c bt c th ly lan mngv gi hin th trang tr. Trong qu kh, iu ny c ly t la mtrong nc chu u nhng iu ny ang c thay th bng cc


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ging cao gluten la m. Cc gluten trong bt c ngun gc t nhngphi c rng ri xung cp nu nh bt c s dng hiu qu lm bnh quy hoc ngn nga co rt ca bnh pie thng mi rakhi cc mn n bng nhm ca h.Vic s dng protease trong ngnh cng nghip da v lng cu

Ngnh cng nghip da chim mt t trng ln trong sn xut enzymetrn th gii. protease kim c s dng loi b lng t da. Qutrnh ny n nay an ton hn v d chu hn so vi phng phptruyn thng lin quan n sulfua natri. Lng tng i ln enzymec yu cu (0,1-1,0% (w / w)) v qu trnh phi c kim sot chtch trnh lm gim cht lng ca da ny. Sau khi co lng, da s c s dng sn xut qun o bng da mm mi v hng hac bated, quy trnh, thng lin quan n men ty, lm tng b sungv ci thin s mm mi ca s xut hin ca h.Protease c s dng, trong qu kh, len 'shrinkproof. si lenc cp trong chng cho quy m ch n u si. Nhng cungcp cho cc si tnh nh hng cao tnh ma st, chuyn ng theohng i t u c a thch so vi phong tro i vi n. iu nyxu hng di chuyn duy nht trong mt hng c th dn n co rt vnhiu phng php c s dng trong cc n lc loi b ccvn (v d nh qu trnh oxy ha ha hc hoc ph cc si trongpolymer). Mt phng php tham gia thnh cng trnh thy phn mtphn ca cc th thut quy m vi papain protease. Phng php ny

cng cho lng mt v nh vo gi tr ca n. Phng php b bqua mt s nm trc, ch yu v l do kinh t. N khng phi l khnghp l mong i s dng ca n s c ti lp ngay by gi rngcc ngun enzyme r hn c sn.Vic s dng cc enzym thy phn tinh bt trong

Tinh bt l carbohydrate lu tr ph bin trong cc nh my. N cs dng bi cc nh my mnh, bi vi khun v cc sinh vt cao hn c mt s a dng tuyt vi ca cc enzym c kh nng xc tcthy phn ca n. Tinh bt t cc ngun thc vt xy ra dng ht c

kch thc khc nhau r rt v c tnh vt l t loi ny sang loi. Hacht khc bit l t c nh du. S khc bit chnh l t s caamylose amylopectin, v d nh tinh bt t ng bp cha sp ch c2 amylose% nhng t amylomaize l khong 80% amylose. Mt s tinhbt, v d t khoai ty, c cha phosphat covalently rng buc vi slng nh (khong 0,2%), trong c tc ng ng k n cc tnhcht vt l ca tinh bt, nhng khng can thip vi thy phn ca


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n.Acid thy phn tinh bt c s dng rng ri trong qu kh. l bygi phn ln c thay th bng quy trnh enzym, v n yu cu sdng vt liu chng n mn, dn n mu sc cao v ni dungsaltash (sau khi trung ha), cn nhiu nng lng hn si m vtng i kh kim sot.________________________________________

Hnh 4.2. Vic s dng enzyme trong ch bin tinh bt. iu kin tiubiu c a ra.________________________________________Trong hai thnh phn ca tinh bt, amylopectin trnh by cc thch thcln cho cc h thng enzym thy phn. -1,6-glycosidic m to thnhkhong 4 - 6% ca hin ti glucose. iu ny l do d lng tham giavo ngnh im cng phi c ct thy phn hon ton ca

amylop -1,4-glucosidic nhng cc -1,6-glucosidic lin kt Hu htcc enzym thy phn c c th cho cc lin kt ectin glucose. Mt s bi tp gn y n tng nht trong s pht trin cacc enzym mi c lin quan debranching enzyme.N l cn thit hydrolyse tinh bt trong mt lot cc quy trnh m mc c c thnh hai lp c bn:1. cc qu trnh trong cc thy phn tinh bt c s dng bi vikhun hoc ngi n ng, v2. cc qu trnh trong n l cn thit loi b tinh bt.Trong cc quy trnh c, chng hn nh sn xut xi-r ng, tinh bt

thng l thnh phn chnh ca hn hp phn ng, trong khi cc qutrnh sau ny, chng hn nh ch bin nc ma, mt lng nh tinhbt gy nhim khng tinh bt nguyn liu c loi b. Enzym cc loic s dng trong cc qu trnh ny. Mc d tinh bt t cc nh mya dng c th c s dng, ng l ngun phong ph nht th gii vcung cp hu ht cc b mt c s dng trong vic chun b thyphn tinh bt.C ba giai on trong vic chuyn i ca tinh bt (hnh 4.2):1. gelatinisation, lin quan n vic gii th ca tinh bt nanogram ckch thc ht to thnh mt h thng treo nht;

2. ha lng, lin quan n thy phn mt phn ca tinh bt, vi s mtmt ng thi nht; v3. ng ha, lin quan n vic sn xut glucose v maltose do thyphn na.Gelatinisation t c bng cch nung nng tinh bt vi nc, v xy

ra nht thit v t nhin khi loi thc phm giu tinh bt c nuchn. tinh bt Gelatinised l do thy phn d ha lng mt phn vi cc


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enzym hoc axit v saccharified bi enzym thy phn axt hoc hnna.Cc tinh bt v ngnh cng nghip xi-r ng s dng cc biu thctng ng hoc DE dextrose, tng t nh trong nh ngha cho ccn v DH ca s phn gii protein, m t sn phm ca mnh, trong:

(4 .2)Trong thc t, iu ny thng c xc nh phn tch bng cch sdng cc biu thc lin quan cht ch, nhng khng ging nhau,:

(4 .3)Nh vy, DE i din cho thy phn t l phn trm ca cc mi linkt glycosidic hin nay. Pure glucose c mt DE 100, tinh khit maltosec mt DE trong khong 50 (ph thuc vo phng php phn tch sdng; phng trnh xem (4.3)) v tinh bt c mt DE ca hiu qukhng. Trong qu trnh thy phn tinh bt, DE cho thy mc m tinhbt c ct. Acid thy phn tinh bt t lu c s dng snxut 'xir glucose "v thm ch c ng kt tinh (dextrosemonohydrat). Rt nhiu ng k ca 42 xi-r DE c sn xut bngcch s dng acid v c s dng trong nhiu ng dng trong bnhko. Hn na thy phn bng acid l khng tha ng v mu sc vhng v undesirably sn phm phn hy. -1,6-glucosidic. Acid thydng nh l mt qu trnh hon ton ngu nhin m khng b nhhng bi s hin din ca mi lin kt________________________________________

Bng 4.2 Cc enzyme thy phn tinh bt c s dng trongEnzyme EC Ngun s hnh ng-dextrin v ch yu l maltose (G2), G3, G6 v G7 oligosaccharides-1,4-oligosaccharide lin kt c ct cung cp cho -Amylase3.2.1.1 Bacillus amyloliquefaciens Ch-dextrin v ch yu l maltose, G3, G4 v G5 oligosaccharides -1,4-oligosaccharide lin kt c ct cung cp cho B. licheniformisChAspergillus oryzae, A. -dextrin v oligosaccharides ch yu l maltosev G3 -1,4 lin kt oligosaccharide c ct cung cp cho Chniger-dextrin vi maltose, G3, G4 v ln n 50% (w / w) glucose -1,4-oligosaccharide lin kt c ct cung cp cho -amylase 3.2.1.1B. subtilis (amylosacchariticus) Ch Saccharifying -1,4-lin kt c ct, t khng lm gim kt thc, cung cp chodextrin gii hn v maltose -Amylase 3.2.1.2 mch nha la mch Ch


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-glucose -1,4 -1,6-lin kt c ct, t cc u nonreducing, cungcp cho Enzym glucoamylase 3.2.1.3 A. niger v-1,6-lin kt c ct cung cp cho chui thng

maltodextrins Pullulanase 3.2.1.41 acidopullulyticus B. Ch________________________________________Cc thut ng ca cc enzym c s dng thng mi thy phntinh bt l hi kh hiu v nhng con s EC i khi mt ln cng vicc hot ng tinh t enzyme khc nhau (Bng 4.2). -amylase c thsubclassified nh ha lng hoc saccharifying amylases nhng thmch phn loi ny l khng bao gm tt c cc enzyme c sdng trong thy phn tinh bt thng mi. V d, v cc snphm -amylases l trong cu hnh- Mt l do cho s nhm ln trongdanh php l vic s dng cc hnh thc anomeric ca nhm gim pht

hnh trong sn phm hn l ca tri phiu ang c thy phn, ccsn phm ca vi khun v nm -1,4 d lng glucose lin kt. , mcd tt c cc enzyme tch gia -amylases l trong cu hnh, ca-D-glucosidic. -D-glucosidic v c th b qua nhng khng thhydrolyse 1,6 - branchpoints -D-glucan glucanohydrolases) lendohydrolases m tch 1,4 - tri phiu -amylases (1,4 - CcThng mi enzyme c s dng cho cng nghip thy phn tinh

bt c sn xut bi amyloliquefaciens Bacillus (c cung cp binh sn xut khc nhau) v bi licheniformis B. (c cung cp bi

Novo Industri A / S l Termamyl). -amylase. Chng khc nhau ch yuv s khoan dung ca h v nhit cao, Termamyl gi li hot ngnhiu hn ln n 110C, trong s hin din ca tinh bt, so vi B. ccamyloliquefaciens . -D-glucopyranosyl-D-fructose), m l kh nngchu thy phn bng enzym glucoamylase v amylases DE-amylasesvi khun l khong 40, nhng ko di iu tr dn n s hnh thnhca maltulose (4 - Ti a c th t c bng cch s dng gi trDE ca 8-12 c s dng trong hu ht cc qu trnh thng mi ning ha hn na l xy ra. Yu cu ch yu i vi ha lng nmc ny l gim nht ca tinh bt gelatinised d dng x

l tip theo.-amylases nhng cc nguyn tc u ging nhau. Nhiu nh sn xuts dng phng php tip cn khc nhau ha lng bng cch sdng tinh bt tinh bt mn l slurried mc 30-40% (w / w) vi nclnh, pH 6,0-6,5, c cha 20-80 ppm Ca2 + (m n nh v kch hotcc enzyme) v enzyme c thm vo (thng qua mt my bm nhlng). -amylase thng c cung cp ti cc hot ng cao liu


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enzyme l 0,5-0,6 kg / tn-1 (khong 1500 kg U-1dry vt cht) ca tinhbt. Cc Khi Termamyl c s dng, cht lng ca tinh bt cng vimen c bm lin tc thng qua mt l phn lc, trong c unnng n 105C bng cch s dng hi nc trc tip.Gelatinisationxy ra rt nhanh chng v hot ng ca enzym, kt hp vi cc lclng ct ng k, bt u s thy phn. Thi gian c tr ti cc niphn lc l rt ngn. Cc tinh bt mt phn gelatinised c a vomt lot cc t chc ng duy tr 100-105C v c t chc trong 5pht hon tt qu trnh gelatinisation. Thy phn cho DE yu cuc hon thnh trong xe tng ang nm gi ti 90-100C 1 n 2h. Nhng b cha cc vch ngn ngn backmixing. -amylase nhngnhit ti a ca 95C khng c vt qu. quy trnh tng t cth c s dng vi B. amyloliquefaciens Ny c nhc im l snkhu cui cng "nu n phi c a ra khi cc DE yu cu c

t c gelatinise cc ht tinh bt ngoan c tn ti trong mt sloi tinh bt m nu khng s gy ra tnh trng c trong cc gii phpca sn phm cui cng.Cc tinh bt ho lng thng saccharified nhng tng i s lngnh c phun kh bn nh 'maltodextrins' cho ngnh cng nghipthc phm ch yu l s dng lm i l bulking v trong thc n trem. Trong trng hp ny, hin tng hot ng enzym c th b phhy bng cch gim pH cho n cui giai on lm nng.-amylase cng thy s dng trong ngnh cng nghip nngbnh. Nm N thng cn phi c b sung vo bt lm bnh m

thc y sn xut kh y v sa i trong qu trnh ln men tinhbt. iu ny tr nn cn thit k t khi gii thiu cc kt hp thuhoch. H gim thi gian gia ct v p ca la m, m trc cho php mt gii hn ny mm lm tng s ca cc enzymni sinh. Cc enzym nm c s dng thay v nhng t vi khun nhl hnh ng ca h c d dng hn kim sot do lability nhittng i ca h, bin tnh nhanh chng trong qu trnh nng.Sn xut xi-r ng

Cc tinh bt ho lng ti 8 -12 DE l ph hp vi ng ha sn

xut xi-r vi cc gi tr DE ca 45-98 hoc nhiu hn. S lng snxut ln nht l xi-r vi DE gi tr trong khong 97.-D-glucose t ),trong pht hnh -D-glucan glucohydrolase, thng c gi lenzym glucoamylase m cn amyloglucosidase gi v amylase- -glucosidase (1,4 - Hin nay chng c sn xut bng cch s dngexoamylase, glucan 1,4 - -glucans lin kt. - v 1,3 - -, 1,6 - 1,4- V l thuyt, k ho lng tinh bt mc 8 -12 DE c th c thy


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phn hon ton sn xut mt hn hp phn ng enzymglucoamylase cui cng vi DE ca 100 nhng, trong thc t, iu nyc th t c ch nng b mt tng i thp. Cc chi ph tptrung cc sn phm ca ngh nh rng nng bc hi b mt ca30% c s dng. -D-glucopyranosyl-(1,6)-D N sau cc DE tia t c l 96-98 vi 95 thnh phn xi-r - ng 97%, 1 - maltose2% v 0,5 - 2% (w / w) isomaltose ( -glucose). vt liu ny c sdng sau khi tp trung, trc tip cho sn xut xi-r fructose cao hoc sn xut ng tinh th.Trong khi ha lng thng l mt qu trnh lin tc, ng ha thngc thc hin nh mt qu trnh hng lot. enzym glucoamylasethng nht c s dng l sn phm ca chng Aspergillusniger. iu ny c pH ti u 4,0-4,5 v hot ng hiu qu nht ti 60C,do , tinh bt ho lng phi c lm lnh v iu chnh pH ca n

trc khi b sung cc enzym glucoamylase ny. Vic lm mt phi bnhanh chng, trnh s thoi ha (s hnh thnh cc tp hp khcha khng ha tan ca amylose; cc qu trnh cho php tng ti datrn trng). l bnh thng hin din trong cc ch phm enzymglucoamylase. -amylase c bt hot khi pH c h xung, tuynhin, iu ny c th c thay th sau bi mt s amylase acid-n nh- Bt k vi khun cn li Khi iu kin l chnh xc enzymglucoamylase c thm vo, thng liu 0,65-0,80 lt enzymepreparation.tonne-1 tinh bt (200 kg U-1). ng ha thng cthc hin ti rng ln khuy ng xe tng, m c th mt vi gi lm

(v c sn phm no), v vy thi gian s b lng ph nu enzyme cthm vo ch khi cc l phn ng c y .Cc la chn thay th l o cc enzyme mt t l c nh hoc thm ton b liuenzyme s bt u ca giai on y. Loi th hai nn cho vic sdng kinh t nht ca enzyme.________________________________________

Hnh 4.3. Cc ng% hnh thnh t 30% (w / w) 12 DE maltodextrin,ti 60C v pH 4.3, s dng nhiu gii php enzyme.U 200 kg-1Aspergillus niger enzym glucoamylase; ----------- U 400 kg-1 A.

nigerglucoamylase, 200 kg U-1 A. niger enzym glucoamylase cng vi200 kg U-1 Bacillus acidopullulyticus pullulanase. S ci thin tngi v vic b sung cc pullulanase cn ln nng b mt cao hn.________________________________________Qu trnh ng ha c khong 72 h hon thnh nhng c th, ttnhin, c y mnh bi vic s dng cc enzyme nhiu hnna. ng ha lin tc c th v thc t nu t nht su xe tng c


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s dng trong series. N l cn thit ngn chn cc phn ng, bngcch nung nng n 85C trong 5 pht, khi mt DE ti a c tc. Hn na bnh s dn n s gim v DE, khong 90 DEcui cng, gy ra bi s hnh thnh ca isomaltose nh glucose tchly-polymerises li vi cch tip cn ca trng thi cn bng nhit nglc hc (Hnh 4.3).Cc xi-r saccharified c lc loi b cht bo v protein bin tnhra t cc ht tinh bt v sau c th c tinh ch bng cch i quathan hot tnh v nha trao i ion. Nn nh rng vic tng nng cht kh khong 11% trong ng ha, bi v mt phn t nc cly ln cho mi tri phiu glycosidic thy phn (phn t glucose snxut).v 1, , 1,6 - (v d nh t l thy phn vi 1,4 - , phn tch ca

h l chm so vi 1,4 --mi lin kt Mc d enzym glucoamylase xc

tc thy phn 1,6 --mi lin kt -lin kt trong tetrasaccharides ctheo t l 300: 6: 1). 3 - R rng l vic s dng mt loi enzymedebranching s tng tc qu trnh ng ha tng th, nhng, sdng cng nghip nh mt loi enzyme phi ph hp vi enzymglucoamylase. Hai loi enzyme debranching c sn: pullulanase, hotng nh mt hydrolase exo vo dextrin tinh bt; v isoamylase(EC.3.2.1.68), m l mt endohydrolase ng s tht. Novo Industri A /S mi y gii thiu mt pullulanase ph hp, c sn xut bimt dng acidopullulyticus Bacillus. Cc pullulanase t aerogenesKlebsiella b sn v mt thng mi cho mt s thi gian l khng

n nh nhit trn 45C nhng cc enzym acidopullulyticus B. cth c s dng theo cc iu kin tng t nh cc enzymglucoamylase Aspergillus (60C, pH 4,0-4,5). Li th thc t ca vic sdng pullulanase cng vi enzym glucoamylase l enzym glucoamylaset cn c s dng ny khng t n a ra bt c li th chi phnhng v t c s dng enzym glucoamylase v t oligosaccharidesnhnh tch ly n cui ng ha ny, thi im m isomaltose snxut tr nn quan trng xy ra ti DE cao hn (Hnh 4.3). N sau gitr DE cao hn v ni dung glucose c th t c khi pullulanasec s dng (98-99 DE v 95 - 97% (w / w) glucose, ch khng phi

97-98 DE) v cao hn nng cht nn (30 - 40% kh cht rn hn l25 - 30%) c th c iu tr. Chi ph ph thm ca vic s dngpullulanase c thu li bng cch tit kim trong chi ph bc hi venzym glucoamylase. Ngoi ra, khi sn phm c s dng snxut xi-r cao-fructose, c mt tit kim trong chi ph x l thm.S pht trin ca cc pullulanase acidopullulyticus B. l mt v d tuytvi v nhng g c th c thc hin nu ko thng mi tn ti


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cho mt enzyme mi. -D, lm gim s o ngc, s l mt bcu hu ch cho sn xut ng cng nghip. S pht trin ca mtglucosidase ph hp- Chiu chng mi ca vi khun i vi mtenzyme mi ca loi ny l mt cng vic ln. N khng phi l ngcnhin khi bit thm chi tit v th tc kim tra c s dng l khngc sn.Sn xut xi-r c cha maltose

. Theo truyn thng, xi-r c cha maltose nh l mt thnh phn chyu c sn xut bng cch x l tinh bt la mch vi la mchamylase- -lin kt. lin kt nhng khng cng khng b qua 1,6hydrolyse - -D-glucan maltohydrolases) l exohydrolases m phthnh maltose t 1,4 - glucans -Amylases (1,4 - xir maltose cao(40-50 DE, 45-60% (w / w) maltose, 2 - 7% (w / w) glucose) c xu

hng khng kt tinh, thm ch di y 0C v l tng i khng htm. Chng c s dng sn xut ko cng v sa mc nglnh. Xi-r cao chuyn i (60-70 DE, 30 - 37% maltose, 35 - 43%ng, 10% maltotriose, 15 oligosaccharides% khc, tt c theo trnglng) chng li s kt tinh trn 4C v ngt ngo (Bng 4.3). Chngc s dng cho ko mm v trong sn xut bia, bnh, nc gii khtcc ngnh cng nghip. v pullulanase cho nng sut gn nh nhlng ca maltose. -amylase s c s dng sn xut xir giumaltose t tinh bt ng, c bit l cc hnh ng kt hp caamylase, N c th c d kin thermostable-amylases t loi

Clostridium gn y c bo co) v c th d dng b c ch bing v cc ion kim loi nng khc -amylases l tng i t tin,khng nhit n nh (mc d mt s iu ny khng c thchin trn quy m ln hin nay bi v hin nay c sn . -amylases, ctrng bi kh nng hydrolyse maltotriose (G3) hn l maltose (G2)c s dng thng xuyn kt hp vi enzym glucoamylase. Thayvo nm -amylases yu cu pH ca khng t hn 5.0 v nhit phn ng khng qu 55C. Hin nay cc enzyme sn c, tuy nhin,khng hon ton tng thch; nm

-amylase mt mnh. xir maltose cao (xem hnh 4.2) c sn xut ttinh bt ho lng ca khong 11 DE nng 35% cht rn kh bngcch s dng nm -amylase nm b mt hot ng ca n. ngha xy ra hn 48 h, do thi gian nm-amylases sn xut xi-rvi ni dung maltose thm ch cao hn. By gi m mt pullulanasett l c sn, ta c th s dng kt hp viv enzym glucoamylase. Chuyn i cao c sn xut bng cch


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s dng loi xi r kt hp ca amylase nm- y c th l ph hp vithng s k thut ca khch hng bng cch iu chnh cc hot ngca hai enzyme c s dng nhng chc chn, l enzymglucoamylase c s dng, hm lng ng ca sn phm cuicng s c cao hn so vi xi-r cao maltose. S n nh ca enzymglucoamylase i dng cc phn ng, bng cch nung nng, khi ccthnh phn cn t c. amylases, enzym glucoamylase vpullulanase. -amylases vi khun, nm By gi c th sn xutthy phn tinh bt vi bt k DE t 1 n 100 v vi thnh phn hunh bt k bng cch s dng cc kt hp ca________________________________________Bng 4.3 Cc v ngt tng i ca cc thnh phn thc phmThc phm ngt thnh phn tng i (tnh theo trng lng, chtrn)

Sucrose 1,0Glucose 0,7Fructose 1,3Galactose 0,7Maltose 0,3Lactose 0,2Raffinose 0,2Thy phn sucrose 1,1Thy phn lactose 0,7Glucose-mt 11 DE


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trong nhng nguyn cht t sui t cc nh my ng ma nhng ccloi xi-r c sn xut bng cch s dng nm men (Saccharomycescerevisiae) invertase. Mc d loi enzyme ny l khng bnh thng ch n b c ch c cht cc cp sucrose cao (%> 20 (w / w)),iu ny khng ngn chn s dng thng mi ca n nng caohn:

[4,2]Theo truyn thng, invertase c sn xut trn trang web caautolysing t bo nm men. autolysate c thm vo cc xi-r(sucrose% 70 (w / w)) c o ngc vi nhau vi s lng nhca xylen ngn chn s pht trin ca vi khun. Inversion chon thnh trong 48 - h 72 ti 50C v pH 4,5. Cc enzyme v xylen bloi b trong qu trnh tinh luyn tip theo v bay hi. xir mt phn ongc c (v vn ang) c sn xut bng cch pha trn honton o ngc vi xir xir sucrose. By gi, thng mi sn xut tptrung invertase c tuyn dng.Vic sn xut thy phn ca mt hp cht trng lng phn t thptrong dung dch cht tinh khit c v nh mt c hi r rng cho vic sdng ca mt loi enzyme c nh, nhng iu ny khng c thchin trn quy m ln, c l v cc k n gin ca vic s dng ccenzyme trong dung dch v c bn bo th ca ngnh cng nghipng.Invertase thy khc s dng trong sn xut bnh ko vi cc trung tmcht lng hoc mm. Cc trung tm ny c xy dng bng cch s

dng sucrose tinh th v nh (khong 100 U kg-1, 0,3 trang / pht (w /w)) S tin ca invertase. cp ny ca enzyme, o ca sucrosel rt chm v vy trung tm ny vn cn vng chc lu enrobingvi s c la c hon tt. Sau , trong khong thi gian ca ngyhoc tun, sucrose thy phn xy ra v s gia tng ha tan lm chocc trung tm tr thnh mm, lng, ty thuc vo lng nc trongvic chun b trung tm.enzyme khc c s dng vin tr cho sn xut ng v tinh chbng cch loi b cc vt liu kt tinh, gy c ch nht cao. mts ni trn th gii, ng ma cha mt lng ln tinh bt, m tr

nn nht, do lm chm qu trnh lc v lm cho m gii php khisucrose c gii th. -amylases (v d nh Termamyl khong 5 Ukg-1) m hon ton tng thch vi nhit cao v gi tr pH l u tinp dng trong giai on bc hi chn khng ban u ca sn xutng. Vn ny c th c khc phc bng cch s dngthermostable nhtCc vn khc lin quan n dextran v raffinose yu cu pht trin


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chuyn i ny l qu nhiu. Cc enzyme c th c ci thin bngcch la chn ging t t nhin hoc bng cch t bin, nhng vn gy ra bi bn cht vt l ca cellulose l khng tun theo giiphp. Ht tinh bt l sn sng khuy ng trong slurries c cha 40%(w / v) cht rn v c th d dng solubilised nhng, ngay c khinguyn cht, hnh thc bnh si cellulose bt ng sn mc 10% vvn cn cc cht rn ha tan trong tt c, nhng k l nht (v enzymebin tnh) dung mi . Khng tinh khit c cha cellulose thng gnnh mt khi lng bng nhau ca lignin, c gi tr t hoc khng cnh l mt sn phm ph v l kh khn tn km loi b.cc ch phm cellulase Trichoderma reesei thng mi t bao gmhn hp ca cc enzyme hip ng:a. endo-1 ,4-D-glucanase; cellulase (EC 3.2.1.4), mtb. -glucosidase; v -glucosidase (EC 3.2.1.74), v exo-1, 4 - glucan

1,4 -c. -cellobiosidase (EC 3.2.1.91), mt exo-cellobiohydrolase (xem hnh4.4). cellulose 1,4 -Chng c s dng cho vic loi b cc nng tng i nh cakhu phc hp cellulose c tm thy can thip vo vic x lnguyn liu thc vt, v d, cc ngnh cng nghip sn xut bia vnc tri cy.________________________________________

Hnh 4.4. cng ca mi quan h gia cc hot ng enzyme trong

thy phn cellulose. | | i din cho hiu ng c ch. l hot ngkim sot tc v c th bao gm mt hn hp cc enzym hot ngtrn cellulose ca cc mc khc nhau ca tinh th. Endo-1, 4 -glucanase- -glucosidase v cellobiohydrolase-exo. N hot ng vic hai synergistically exo-1, 4 - l mt sn phm-c chenzyme. Exo-1, 4 - glucosidase, Exo-cellobiohydrolase l sn phmc ch v thm vo dng nh l bt hot trn rng buc vi bmt ca tinh th cellulose.________________________________________Thch hp phn tch kinh t cho thy rng cc ngun gi r cacellulose chng minh l thng t hn cc ngun glucose hn so vitinh bt dng nh t hn. Tng i tinh khit cellulose c gi trtheo ng ngha ca n, nh l mt bt giy v Vn nguyn liu, hinlnh vi gi hn gp i so vi tinh bt ng. Vi tnh trng thiu btgiy th gii ngy cng tng ca n khng th c nhn thy thc tl mt ngun thay th ng trong tng lai gn. Kin thc v hthng enzyme c kh nng lignocellulose xung cp l tin nhanh


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chng nhng c phn chc l s thay th lignocellulose tinh bt nhmt ngun xir glucose s dng thc phm. l, tuy nhin, rt cth l n c th c s dng, trong mt qu trnh lin quan n vics dng ng thi c hai enzym v nm men ln men, sn xutethanol, vic s dng glucose ca nm men loi b hiu ng c chca n trn cc enzym. glucosidase (EC 3.2.1.21, cn c gi l qutrnh cellobiase cho hiu qu ti a (Hnh 4.4). Cn lu rngcellobiose l mt ng khng ln men v phi c thy phn bngcch b sung,Vic s dng lactases trong ngnh cng nghip sa

Lactose l hin nay nng khong 4,7% (w / v) trong sa v sacc (gn) cn li sau khi giai on ng mu ca pho mt. s hin dinca n trong sa lm cho n khng thch hp cho phn ln dn s

ngi ln ca th gii, c bit l trong cc lnh vc c truyn thngkhng c mt ngnh cng nghip sa. Bt dung np lactose l hn chch yu ngi dn c ngun gc nm Bc u hay tiu lc a n v l do "s kin tr lactase ; ngi tr ca tt c cc ng vt cv r rng c th tiu ha sa, nhng trong nhiu trng hp kh nngny gim sau khi cai sa. Ca Thi Lan, Trung Quc v ngi M daen dn s, 97%, 90% v 73% tng ng, c bo co l khngdung np lactose, trong khi 84% v 96% ca M v Thy in trngqun, tng ng, c khoan dung. Thm vo , v ch c rt t khimt s c nhn b bm sinh khng dung np lactose chuyn ha hoc

thiu lactase, c hai u c th c nhn thy khi sinh. S cn thitcho sa t lactose l c bit quan trng trong cc chng trnh vin trlng thc nghim trng nh mt nc m, tiu chy v thm ch tvong c th do n sa cho tr em c cha lactose sa ti v ngiln b suy dinh dng protein-calo. Trong tt c cc trng hp, thyphn lactose glucose v galactose s ngn chn s (nghim trng)cc vn tiu ha.Mt vn khc c trnh by bi lactose l ha tan thp dn nhnh thnh tinh th nng trn 11% (w / v) (4C). iu ny ngn cnvic s dng cc loi xi r sa tp trung nhiu thc phm qu trnh

khi h c mt kt cu ct kh chu v c th d dng d b h hng visinh. Thm vo vn ny, vic x l cht thi sa ny th tn km(thng punitively nh vy) do nhu cu oxy sinh hc cao can. Nhng vn ny c th c khc phc bng cch thy phn calactose trong sa, cc sn phm c khong bn ln nh ngt (xemBng 4.3), nhiu hn na ha tan, c kh nng hnh thnh tp trung,microbiologically an ton, xi-r (70% (w / v)).


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-galactosidase. Lactose c th b thy phn bi lactase, mt[4,3]

Thng mi, n c th c lm t sa nm men Kluyveromycesfragilis (K. marxianus var. Marxianus), vi pH ti u (pH 6,5-7,0)thch hp cho vic iu tr ca sa, hoc t nm Aspergillus oryzae hayA. niger, vi pH Optima (pH 4,5-6,0 v 3,0-4,0, tng ng) ph hphn sa thy phn. Cc men ny c th mc khc nhau ca sc ch sn phm ca galactose. -1,6-oligosaccharidesgalactosyl. Ngoi ra, nng cao lactose v galactose, lactasetransferase ng k cho thy kh nng v sn xut lin ktLactases ang c s dng trong sn xut kem v ng hng liuv sa c c. Khi thm vo sa hay sa lng (2000 U kg-1) v cn li khong mt ngy ti 5C khong 50% lactose c thy phn, to ramt sn phm ngt ngo m s khng kt tinh khi c c, ng

lnh. Phng php ny cho php nu khng-lng ph sa thay thmt s hoc tt c cc sa bt tch kem c s dng trong cng thcnu n kem truyn thng bng. N cng ci thin ca scoopability vbo ca sn phm. S tin nh hn ca lactase c th c thm vosa tit trng di cuc sng sn xut mt sn phm lactose-gimtng i r tin (v d: 20 U kg-1, 20C, 1 thng dung lng lutr). Ni chung, tuy nhin, lactase s dng khng t c timnng ca mnh, nh cc enzym hin nay l tng i t tin v ch cth c s dng nhit thp.Enzym trong nc tri cy, bia, ru v cc ngnh cng nghip chng

ct

Mt trong nhng vn quan trng trong vic chun b cc loi ncp tri cy v ru l my ch yu l do s hin din ca pectins. -1,4-anhydrogalacturonic acid, vi mc khc nhau ca methyl esteha. Nhng bao gm ch yu l cc polyme Chng c kt hp vicc polyme cy trng khc v, sau khi homogenisation, vi cc mnhv t bo. Cc my m h gy ra l kh khn loi b ngoi trenzym thy phn. iu tr nh vy cng c nhng li ch b sung cavic gim nht dung dch, tng khi lng nc sn xut (v d nh

sn lng ca nc p t nho trng c th c nng ln 15%),nhng thay i tinh t nhng nhn chung c li trong hng v v, trongtrng hp lm ru, thi gian ln men ngn hn. Khng ha tannguyn liu thc vt c th d dng loi b bng cch lc, hoc giiquyt v gn, mt khi nh hng n nh ca cc pectins trn my mkeo c g b.Cc ch phm thng mi pectolytic enzyme c sn xut t


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Aspergillus niger v bao gm mt hn hp hip ng ca cc enzym:a. -D-galactosiduronic mi lin kt; polygalacturonaza (EC 3.2.1.15),chu trch nhim v s thy phn ngu nhin ca 1,4 -b. pectinesterase (EC 3.2.1.11), cc phin bn methanol t methyl estepectyl, mt giai on cn thit trc khi polygalacturonaza vic c thhot ng y (s gia tng hm lng methanol trong nc ciu tr nh thng t hn so vi nng t nhin v khng gy ranguy c sc khe) ;c. -D-galact-4-enuronosyl, m khng c s cn thit phi hnh ngpectin esteraza methyl; v pectin lyase (EC 4.2.2.10), m s tch ccpectin, bi mt phn ng loi b pht hnh oligosaccharides vi thit bu cui khng lm gim d lng 4-deoxymethyl-d. -L arabinofuranosidase-, EC 3.2.1.55 -xylosidase, EC 3.2.1.37; v -xylosidase, EC 3.2.1.32; xylan 1,4 - hemixenlulaza (mt hn hp

cc enzym thy phn bao gm: endo xylan-1, 3 - ), ng khng phi lmt pectinase nhng s hin din ngu nhin ca n c khuynkhch gim mc hemixenluloza.Cc hot ng ti u ca cc enzym ny mt pH gia 4 v 5 vthng di 50C. H rt thch hp b sung trc tip cho bt giy nqu cc cp khong 20 U l-1 (hot ng mng).Enzym c tnhcht ci thin s n nh nhit ln hn v pH thp hn ti u angc tm kim.Trong sn xut bia, mch nha la mch cung cp cc t l ch yu cacc enzyme cn thit cho ng ha trc khi ln men.Thng th vt

liu khc c cha tinh bt (adjuncts) c s dng lm tng lngng ln men v gim chi ph tng i ca qu trnh ln men. Mcd men mch nha cng c th c s dng hydrolyse adjunctsny, cho ti a enzyme tr li kinh t ph c thm vo t cng ha nhanh chng ca h.N khng cn thit cng khng mongmun ho a ng tinh bt hon ton, nh dextrin khng ln men cnthit cung cp cho c th "thc ung v n nh 'u' bt ca n. Vl do ny qu trnh ng ha c dng li, bng cch un si cadch nha ', sau khong 75% tinh bt c chuyn i thnh ngln men.

-glucanase) v thy phn protein (protease trung tnh -1,3 - glucanlin kt ( -1,4 - v -amylases), phn tch v la mch Ccenzyme c s dng trong sn xut bia l cn thit cho ng hatinh bt (vi khun v nm ) tng t l ln men (sau ny), c bittrong vic sn xut bia trng lc cao, ni protein thm vo. la mch-glucans. Xenlulaza cng thnh thong c s dng, c bit ni lam c s dng nh thuc h tr m cn gip phn hy cc -


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amylase, ni ny c s dng dch nha phi c un si trong mtthi gian di hn nhiu (v d 30 pht) v hiu ha n trc khi lnmen. Do s n nh nhit cc oan ca B. amyloliquefaciensPapain c s dng trong cc giai on sau qu trnh ln men bia

sau ny lm ngn chn s xut hin ca protein v tannin c cha'lnh, my m "nu khng hnh thnh trn lm lnh bia.Gn y, "nh sng" bia, ni dung nhit thp hn, tr nn ph binhn. Nhng i hi mt mc cao hn cc nng ng ha tinhbt thp hn gim ru v tng cht rn ni dung ca bia. -amylasetrong sut qu trnh ln men. iu ny c th t c bng vic sdng ca enzym glucoamylase v / hoc nmMt lot cc ngun carbohydrate c s dng trn ton th gii sn xut ung c cn c chng ct. Nhiu ngi trong s ny c s lng ng ln men (nh ru rum t mt ma v ru t

nho), nhng ngi khc c cha ch yu l tinh bt v phi csaccharified trc khi s dng (v d nh ru t ng, la mch, mchnha hoc la mch en). Trong ngnh cng nghip chng ct, ngha tip tc trong sut thi gian ln men. -amylases vi khun c thc s dng trong ng ha ny. Trong mt s trng hp (v dnh Scotch whisky sn xut mch nha mch nha la mch s dng cquyn) cc enzym t nhin hin nay, nhng nhng ngi khc (v dnh ht tinh thn sn xut) th cng n nh nhitGlucose oxidase v catalase trong ngnh cng nghip thc phm

glucono-1 ,5-lacton (m mt cch t nhin khng enzymicallyhydrolyses Glucose oxidase l enzyme rt c th (i vi glucose-

applications of proteases in the food industry

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