san xuat enzym trong trai cay

Enzymes in Fruit Juice Production and Fruit Processing 5.1.2.1 Introduction The first application of enzymes in the fruit juice industry was the use of pectinases for apple juice clarification in the 1930s [500]. The fast clarification of juice after breakdown of pectin by pectinases and the decrease in juice viscosity resulted in a shorter process and greatly improved the quality of industrial apple juice. Later, pectinases were applied to depectinization of red-berry juice. The use of pectinases and amylases to degrade apple pectin and starch during the hot clarification stage prevents post-bottling haze formation, and thus concentration of the juice by a factor of six has become possible. This results in a smaller storage volume, cheaper transportation, and better concentrate stability without spoilage. Treatment of apple pulp with pectinases and hemicellulases was introduced later: by lowering the viscosity of the pulp, the press capacity and the yield were significantly improved. Depectinization of juice with pectinases with high arabanase activity improved product quality further by preventing araban haze forma- tion after concentration of the juice. In the production of clear and stable red-berry juice or concentrate, hot maceration with enzymes ensures a higher juice yield and a better 112 5 Industrial Enzymes color extraction [501]. Nowadays, enzyme suppliers provide fruit juice producers with tailor-made enzyme preparations optimally blended on the basis of fruit composition for improved quality and stability of finished products, together with shorter process duration and larger plant capacity. In association with new equipment and processing technologies, industrial enzymes allow processors to add value to raw material for food and feed and to reduce waste quantity. Industrial processes are very diverse and numerous. Here the most common are described, but differences still exist depending on the company and plant. 5.1.2.2 Biochemistry of Fruit Cell Walls The pulp of fruits and vegetables is composed of cells. They are surrounded by a cell wall, which resists internal pressure and external shock (Fig. 34). Polysaccharides constitute 90–100 % of the structural polymers of walls of growing plant cells, known as primary cell walls. Secondary cell walls develop from primary cell walls during cell growth. Cell wall composition depends on fruit species and evolves in dependence on agronomic and climatic conditions, fruit ripeness, the type and duration of storage of the fruit (cell growth and cell senescence). The composition of plant cell walls has been widely studied, and numerous models of the three-dimensional structure have been proposed [502–504]. Three major independent domains are distinguished: the xylo- glucan network, the pectin matrix, and the structural proteins. The cellulose–xyloglu- can network is embedded in the pectin matrix. Pectin is the major structural polysaccharide component of fruit lamellas and cell walls. Three pectic polysaccharides are present in all primary cell walls: homogalacturonan and rhamnogalacturonans I and II [505] (Fig. 35). Recent models divide pectin into so-called smooth regions of Fig. 34 Fruit cells (fc) and middle lamellae (ml) connecting the cell walls Fig. 35 Model of pectin smooth regions (SR) and hairy regions (HR) model [506]. Ara ¼ arabinose, Gal ¼ galactose, GalA ¼ galacturonic acid, Rha ¼ rhamnose, Xyl ¼ xylose 5.1 Enzymes in Food Applications 113 unbranched homogalacturonan (60–90 %) and hairy regions of highly branched rhamnogalacturonan I (10–40 %) [507], [508]. Homogalacturonan is a homopolymer of (1!4)-a-D-galactosyluronic acid residues, capable of forming gels. Carboxyl groups of the galactosyluronic acid residues of primary cell wall homogalacturonan can be methyl-esterified at the C-6 position and acetyl-esterified at the C-2 or C-3 position. Helical chains of homogalacturonan that are less than 50 % methyl-esterified can form a gel-like structure and condense by cross-linking with calcium ions, which are present in primary cell walls. Degree of methylation, molecular weight, and pectin content are specific to fruit species (Table 10). Table 10. Fruit pectin composition Fruit Apple Blackcurrant Grape Orange peel Pear Pineapple Strawberry Pectin, wt % 0.5–1.6 1.0–1.2 0.1–0.4 3.5–5.5 0.7–0.9 0.04–0.1 0.5–0.7 Methylation wt % 80–92 50–80 50–65 65 50–70 22–40 20–60 114 5 Industrial Enzymes These characteristics must be taken into consideration in choosing the right pectinase balance for best fruit processing. During ripening, the protopectin in primary cell wall is slowly transformed into soluble pectin by the action of endogenous pectinases present in the fruit. The decrease in molecular weight is due to pectin depolymerases, and the decrease in degree esterification mainly to pectin methylester- ase. However, these activities are very low. Homogalacturonan contains chains of up to about 200 galacturonic acid units, 100 nm long, with some rhamnose. A xylogalactur- onan subunit (XGalA), substituted with xylose at the C-3 position of the galacturonic acid residue, has been identified as part of the galacturonan backbone. It can be methyl- esterified. Rhamnogalacturonan I (RGI) has a backbone of up to 100 repeats of the disaccharide rhamnose-galactose. The side chain can vary in size from a single glycosyl residue to 50 or more glycosyl residues [505]. In general, about half of the rhamnosyl units of RGI have sidechains, but this can vary with cell type and physiological state. [509]. Arabinans are mostly 5-linked arabinofuranosyl units forming helical chains, but arabinosyl units can be interconnected at each free O-2, O-3, and O-5 position to form a diverse group of branched arabinans [510]. The RGI of primary cell walls is branched with (1!5)-a-L-arabinan, (1!3)- or (1!2)-a-L-arabinosyl residues, and arabinogalac- tan (AG) type II with a (1!3),(1!6)-b-D-galactan backbone, with (1!3)-a-L-arabinosyl residues [512, 511]. Other macromolecules such as cellulose, xyloglucan, and arabi- nogalactan proteins (AGP) are associated with the plasma membrane [513]. Rhamno- galacturonan II (RGII) is a low molecular weight (ca. 4.8 kdalton) complex polysaccharide with a backbone of nine (1!4)-a-D-galactosyluronic acid residues and four side chains attached to O-2 or O-3 of the backbone. The side chains are composed of twelve different sugars [514] — apiose, 2-O-methyl- l-fucose, 2-O-methyl-D-xylose, aceric acid (3-C-carboxy-5-deoxy-l-xylose), Kdo (3-deoxy- D-manno-octulosonic acid), Dha (3-deoxy-D-lyxo-heptulosaric acid) — bound in more than 20 different linkages. The two main hemicelluloses of all primary cell walls are xyloglucan and arabinoxylan. Hemicelluloses bind tightly via hydrogen bonds to the surface of cellulose linking or cross-linking microfibrils to create a cellulose–hemi- cellulose network. Interconnections with the pectic polysaccharides are of primary importance for the integrity of the pectin network. Cellulose is a (1!4)-b-D-glucan which accounts for about 20–30 % of the dry matter of most primary cell walls and is particularly abundant in secondary cell walls. In 1993-1994, VINCKEN and VORAGEN demonstrated that xyloglucan was a key structure of apple cell walls for the degradation of cell-wall-embedded cellulose (around 57 % of the apple cell-wall matrix) [515]. The cellulose–xyloglucan network determines the strength of the cell wall and is embedded in an independent pectin matrix, hemicelluloses, and proteins. In apples the xyloglucan fraction makes up about 24 % of the total amount of sugar. About 5 wt % of some primary cell walls is made up of the hydroxyprolin-rich structural glycoprotein extensin, which binds some polysaccharides together. Fractions of the different macromolecules in fruit are given in Table 11. Starch is present in unripe apples in amyloplasts. This is the largest biological molecule, with a molecular weight of 10 5 –10 9 dalton. Starch is composed of two components: amylose and amylopectin [516]. Amylose, the minor component, is a Table 11. Fruit cell wall composition in g/kg fresh matter 5.1 Enzymes in Food Applications 115 Fruit EIR Pectin Hemicellulose Cellulose Lignin Protein Total Apple Pear Mango 20 15 25 272 281 408 169 148 91 349 267 236 2 69 27 76 82 127 868 847 889 Pineapple 13 Strawberry 12 Raspberry 20 163 411 168 267 66 89 210 232 177 85 11 73 94 255 277 819 975 784 Cherry Papaya 13 26 396 364 49 165 130 124 169 4 244 127 988 784 *Ethanol-insoluble residue. (1!4)-a-D-glucan and has a linear structure. Various degrees of polymerization have been ascribed to this fraction, with chain lengths in the range of 100–1000 glucose units. Amylopectin contains a-(1!6) and a-(1!4) glucose linkages. Amylopectin exhibits branching at the 1!6 position, and its degree of polymerization is far higher than that of amylose. The ratio amylose/amylopectin can vary in natural starches in the general range 1/3 to 1/4. 5.1.2.3 Cell-Wall-Degrading Enzymes Pectinases Progress in enzymology has been so fast that certain activities are not yet described in the International Enzyme Classification (E.C. no.). Many microorganisms produce enzymes that degrade fruit cell walls. Commercial pectinases for the fruit juice industry come from selected strains of Aspergillus sp. Enzymes are produced during fungal growth, purified, and concentrated. Pectinases are defined and classified on the basis of their action toward pectin (Fig. 36). Pectin lyase (PL, E.C. 4.2.2.10) is a pectin Fig. 36 Fruit pectin and pectin-degrading enzymes. Ara ¼ arabinose, Gal ¼ galactose, GalA ¼ galacturonic acid, OMe ¼ methyl ester, OAc ¼ ethyl ester, Rha ¼ rhamnose, Xyl ¼ xylose. 116 5 Industrial Enzymes depolymerase of the endo type which has a great affinity for long, highly methylated chains and acts by b-elimination of methylated a-1,4 homogalacturonan with the formation of C4-C5 unsaturated oligo-uronides [517]. Pectin methylesterase (PME, E.C. 3.1.1.11) removes methoxyl groups from pectin, and at the same time decreases the affinity of PL for this substrate. This results in the formation of methanol and less highly methylated pectin. PME from Aspergillus has a strong affinity for highly meth- oxylated pectin such as apple pectin and acts according to a multichain mechanism [518]. Demethylation with PME generates free carboxylic acid groups and the pectin becomes negatively charged. Polygalacturonase (PG, E.C. 3.2.1.15) exists in two forms: endo-PG and exo-PG. Both types act only on pectin with a degree of esterification of less than 50–60 %. Endo-PG acts randomly on the a-1,4-polygalacturonic backbone and results in a pronounced decrease in viscosity. Exo-PG acts at the nonreducing end of the chain. Exo- PG releases small fragments from the chain and does not significantly reduce the viscosity. Seven endo-PGs, two exo-PGs, and seven PL isoenzymes from Aspergillus niger have been described [519], [520]. Different enzymes acting on rhamnogalacturonan I were identified and purified from Aspergillus sp. [521]. RGase A was identified as a hydrolase that splits the a-D-GalAp-(1!2)-a-L-Rhap linkage of RGI, while RGase B appeared to be a lyase that splits the a-l-Rhap-(1!4)-a-D-GalAp linkage by b-elimina- tion. Two novel enzymes were also identified: a rhamnogalacturonan rhamnohydrolase and a rhamnogalacturonan galacturonohydrolase. As an accessory enzyme for the RGases, rhamnogalacturonan acetyl esterase (RGAE) was also described. Although the structure of RGII substrate has been described, enzymes able to hydrolyze it are still unknown and have not been described yet (2003). Arabanases are pectinases, since they remove arabinose covalently bound to the homogalacturonan backbone. Three enzymes have been described: an endo-arabinanase (a-1!5; ABFA) and two arabinofuranosidases, namely, exo-arabinofuranosidase A (a-1!2;a-1!3) (ABFA) and exo-arabinofuranosidase B (a-1!3;a-1!5; ABFB; E.C. 3.2.1.55). All three are produced by Aspergillus niger [522]. High activities are required for apple and pear processing. In general, pectinase activity and ratio of the different pectinases can vary in different commercial preparations. Hemicellulases Hemicellulases are enzymes that hydrolyze arabinogalactans, galac- tans, xyloglucans, and xylans. Arabinanases are classified as pectinases when they act on pectin side arabinans. They are also classified as hemicellulases when acting on arabinogalactans or arabinoxylans. Aspergillus sp. produces enzymes that hydrolyse arabino-(1!4)-b-D-galactans type I and arabino-(1!3)-(1!6)-b-D-galactans type II [512], [523], [524]. The exo-(1!3)-b-D-galactanase is able to release galactose and (1!6)-b-D-galacto- biose. Aspergillus enzyme is able to bypass a branch point in a b-(1!3) backbone. The action of this enzyme is enhanced by the presence of ABFB. Xylanases hydrolyse the (1!4)-b-D-xylans in synergy with ABFs [525]. Amylases In fruit juice industry, fungal acid amylase and amyloglucosidase are used to process fruits which contain starch. This is the case for unripe apples at harvest time. 5.1 Enzymes in Food Applications 117 Aspergillus niger produces acid a-amylase and amyloglucosidase [526]. These exogenous enzymes are applied after starch gelatinization (occurring above 75 8C) for preventing post-bottling haze formation (starch retrogradation). Acid endoamylase (AA) acts on amylose and amylopectin. It produces dextrins, which are substrates for glucoamylase or amyloglucosidase (AG) a-1!4,1!6 exo-hydrolase, which release glucose from the non-reducing end of the chain. 5.1.2.4 Apple Processing After oranges, apples are the most important raw material worldwide for the production of clear juice and clear concentrate. In 2004/2005 1.3 10 6 t of apple juice was produced [528]. The major producers of apple juice are China, Poland, and Argentina [527]. 5.1.2.4.1 Apple Pulp Maceration The current trend is to process apple juice from table varieties having defects or not sold for table consumption (Golden Delicious, Granny Smith, Jonagold, Red Delicious). Apples processed in the crop period are easily pressed with a relatively high yield. They are stored at low temperature in controlled atmosphere for several months and processed according to market demand. During storage, the insoluble protopectin is slowly transformed into soluble pectin by endogenous apple pectinases (protopecti- nase type), and starch is slowly degraded by endogenous apple amylases into glucose and consumed during post-harvest metabolism. Soluble pectin content can increase from 0.5 up to 5 g per kilogram of over-ripe apple [529]. Apples become difficult to press unless macerated with pectinases (Fig. 37). In the traditional process, enzymes are used at two different stages (Fig. 38). Application of commercial pectinases from Aspergillus sp. in apple mash is necessary because activity of endogenous enzymes is too low to cause an immediate noticeable effect. Because apple pectin is highly methylated, commercial enzyme preparations must contain a high concentration of pectinlyase or pectin methylesterase in association with polygalacturonase and arabanase, together with side activities such as rhamnogalacturonase and xylogalacturonase. Rapidase Press (DSM), Rapidase Smart (DSM), Pectinex UltraSP (Novozymes), and Rohapect MA+ (AB Enzymes) pectinases sold for apple pulp maceration contain enzymes necessary to obtain a good pressability and high yield throughout the entire season. Fig. 37 Effect of pectinases on percentage apple juice yield during the processing season 118 5 Industrial Enzymes Fig. 38 Production of apple concentrate Commercial products contain more or less the enzyme activities described below, but with different ratios from non-GM and GM organisms. Pulp enzyming results in a fast decrease in pulp viscosity, a large volume of free-run juice, and fast pressing. The yield is over 90 % with hydraulic press and pomace leaching, compared to 75–80 % maximum without enzyme treatment. 5.1.2.4.2 Apple Juice Depectinization Pectin is the main cause of juice turbidity. One liter of juice with 13 % dry matter can contain 2–5 g of pectin after pressing, depending on the ripeness of the fruit. Pectinase dosage is determined in the laboratory by performing the alcohol test. Acidified ethanol (2 volumes of ethanol containing 0.5 % HCl þ 1 volume of apple juice) is used to precipitate residual pectin with a molecular weight of up to 3000 dalton. It is important to use acidified ethanol; otherwise, precipitation of organic acids or calcium pectate occurs because of the pH change after mixing ethanol with the juice, and thus a a false positive result is obtained. Pectinlyase or pectin methylesterase plus polygalacturonase and arabanase are the most important enzymes. Arabinose represents 55 % of neutral sugars in hairy regions of apple pectin. Arabanase activity prevents haze formation after juice concentration. The juice clarification is carried out after enzymatic depectiniza- tion [530]. The first stage consists of destabilization of the cloud by PL, which has no Fig. 39 Apple juice depectinization 5.1 Enzymes in Food Applications 119 visible effect but results in a strong decrease in the viscosity of the juice (Fig. 39). PL cuts pectin at random, and cutting 1–2 % of the linkages is enough to reduce apple juice viscosity by 50 %. PG becomes active only after the action of PME. Because of its high molecular weight, PG cannot hydrolyze pectin with a degree of methylation greater than 50–60 % due to steric hindrance. At this stage, PL is no longer active and pectin hydrolysis is due to the system PME/PG. The second stage is cloud flocculation. The sedimentation necessary for the clarification of apple juice occurs only after enzymatic degradation of pectin and starch. The cloud is composed of proteins that are positively charged at the pH of the juice (3.5–4.0) since their isoelectric point lies between pH 4.0 and 5.0. These proteins are bound to hemicelluloses that are surrounded by pectin as a negatively charged protective colloid layer. Pectinases such as Rapidase C80 Max (DSM), Pectinex C80 Max (Novozymes), and Rohapect DAL (AB Enzymes) partially hydrolyze the pectin gel, and thus results in the electrostatic aggregation of oppositely charged particles (positive proteins and negative tannins and pectin), flocculation of the cloud, and then clarification of the juice [530]. The optimal pH for this mechanism is 3.6. The alcohol test shows whether juice depectinization is complete. At the beginning of the processing season, unripe apples contain 5–7 g of starch per liter of juice. At this point, the iodine test gives a dark blue color and starch can be precipitated with iodine. Starch is present as granules 2–13 mm in size, composed of 30 % amylose and 70 % amylopectin chains. The latter can fix 20 wt % iodine. Apples contain endogenous amylases, but the process is too short and activities are too low to degrade the starch. When the juice is heated to 75–80 8C, starch changes from an insoluble form to a soluble form (gelatinization with water). If no amylase is added, these molecules can subsequently undergo retrogradation and form large aggregates that are difficult to hydrolyze and clog filters. The addition of fungal amylase and amyloglucosidase results in the hydrolysis of soluble starch into glucose monomers. Retrogradation and post-bottling haze formation are prevented, and clarification and filterability are improved. Prepara- tions such as Hazyme DCL (DSM) and AMG (Novozymes) contain a-amylase and amyloglucosidase from Aspergillus niger. The dosage is determined by the iodine test. The third stage is clarification with finings and sheet filtration, which is being replaced by micro- or ultrafiltration (UF). Inorganic membranes have cut-offs from 20 000 to 500 000 dalton. Insoluble particles and soluble undegraded molecules such as parts of RGI, RGII, proteins including enzymes, and polyphenols, remain to a greater or lesser extent in the retentate, depending on the cut-off of the filtration membrane. Recently, it 120 5 Industrial Enzymes was found that undegraded RGI, RGII, and dextrins can foul UF membranes. Rapidase UF (DSM) and Novoferm 43 (Novozymes) contain rhamnogalacturonases and other side activities to improve ultrafiltration flow rate. In conclusion, apple juice is easily produced after pulp maceration and juice depecti- nization with enzymes, with possible concentration of up to 708 brix (percentage by weight of soluble solids in a syrup at 68 8F), without risk of gel or haze formation. A few producers make ‘‘natural’’ or cloudy apple juice, e.g., in Germany and the USA. Until now, they did not add enzymes, because the existing commercial pectinases induce fast clarification of the juice. DSM sells a purified pectin methylesterase without pectin depolymerase activity. It can be used in the cloudy-juice process at the maceration stage to improve the yield, and also in the French cider process with calcium for defecation with flotation techniques. 5.1.2.5 Red-Berry Processing The production of clear juice and concentrate from blackcurrant, raspberry, or strawberry requires enzymatic maceration and depectinization [531], [532]. Clarifica- tion, filtration, and concentration are difficult because these juices have high pectin content (typical content of residual low molecular weight pectin of 7 g L 1 compared to 0.5 g L 1 in apple juice). It is assumed that pectin hairy regions remain as soluble colloid in the juice and hemicelluloses tend to bind to phenolics and proteins during processing and storage. The result is the formation of irreversibly linked brown complexes that enzymes can no longer break down. An additional problem is related to the frequent contamination of red berries, mainly strawberry and raspberry, with Botrytis cinerea. This parasitic fungus, growing on rotten berries, secretes a b-1,3-1,6- linked glucan into the berries with a molecular weight of ca. 10 6 dalton [533]. This gum reduces the filterability and the clarity of the juice. It is possible to hydrolyze this glucan with b-glucanases with Filtrase (DSM)Glucanex (Novozymes). Single-Stage Red-Berries Process The single-stage process consists of simultaneous blackcurrant or blackberry maceration and depectinization (Fig. 40). Pectinases are used to improve juice and color extraction while retaining the organoleptic properties of the fruit. However, the extracted color is sometimes partly destabilized by anthocya- nases (side activities of pectinases) or by oxidation. Oxidation can be chemical or enzymatic, due to the endogenous polyphenol oxidase (PPO) of the fruit, and is catalyzed by metal ions. It is therefore recommended that the pulp be heated to 90 8C to inhibit fruit oxidases prior to maceration with enzymes. Some red berries are very acidid (pH 2.6–2.8) and have high contents of phenolics and anthocyanins, which are inhibitors of pectinases. Hence, red-berry processing requires commercial pecti- nases that are especially stable under these conditions. This is the case for Klerzyme 150 (DSM), Klerzyme Intense (DSM), and Pectinex BEXXL (Novozymes). Two-Stage Red-Berries Process The two-stage process consists of enzymatic macera- tion of fruit pulp, followed by a second addition of enzyme for juice depectinization at low temperature. Raspberries or gooseberries are heated to 90 8C for at least two minutes to increase color extraction and to destroy fruit polyphenol oxidase. The pulp is then cooled to 20–25 8C for enzymatic maceration. After pressing, the extracted juice is [...]... is not permitted to use enzymes in the production of premium orange juice, they can be used in other applications Enzymes can increase the yield of solids recovery during pulp washing, facilitate the production of highly concentrated citrus base, improve recovery of essential oil from peel, debitter juice, and clarify lemon juice [528] An example of citrus processing using enzymes is fruit peeling... Citrus 12XL (DSM), which degrades the pectin part of the cloud Enzymes are added to the juice in amounts of 10 g hL at 8–10 8C to avoid juice oxidation The insoluble solids are removed by filtration or ultrafiltration The brilliant lemon juice can be concentrated at 658 brix 1 5.1 Enzymes in Food Applications 5.1.2.7 Conclusion 123 Nowadays, enzyme producers provide a wide range of pectinases for processing... (equipment, process stages, type of enzymes .) In Europe, a Code of Practice has been developed to maintain quality and authenticity standards [534] Members of Association of the Industry of Juices and Nectars from fruits and vegetables (AIJN) in parallel with the Association of Microbial Food Enzyme Producers (AMFEP) have established references for fruit juice composition and enzyme specifications, in line... and processes including enzymes allow processors to obtain higher juice yield (productivity) together with higher quality of finished products The use of specific pectinases adapted to the fruit process improves the shelf life of juices and concentrates (stability of color and freedom from turbidity) Apart from juice processing, the wide range and the high specificity of commercial enzymes open the way to... pure´e, cloudy or clear juice from apricot, peach, kiwi, mango, guava, papaya, and banana are often processed without enzymes The main problem is viscosity, which can be decreased with pectinases [528] Pectinases and amylases can be later used for clear juice production 121 122 5 Industrial Enzymes Fig 41 Citrus processing Citrus Fruits The development of frozen concentrated orange juice started in 1940...5.1 Enzymes in Food Applications Fig 40 Production of blackcurrant concentrate depectinized with pectinases The low processing temperature after heating prevents aroma losses, and high-quality juices and concentrates . maceration with enzymes ensures a higher juice yield and a better 112 5 Industrial Enzymes color extraction [501]. Nowadays, enzyme suppliers provide fruit juice producers with tailor-made enzyme preparations. Cell-Wall-Degrading Enzymes Pectinases Progress in enzymology has been so fast that certain activities are not yet described in the International Enzyme Classification (E.C. no.). Many microorganisms produce enzymes. Industrial Enzymes Fig. 38 Production of apple concentrate Commercial products contain more or less the enzyme activities described below, but with different ratios from non-GM and GM organisms. Pulp enzyming