Enzymatic synthesis of food ingredients in low-water media

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Fed, Regist. (1993)58, 2229-2300 Fed. Regist. (1993)58, 2206-2228 led. Regist. (1991)56, 60880-60891 Fed.Regist. (1993)58, 2302-2426 led. Regist. (1993)58, 2448-2456 Fed Regist. (1993)58, 2944-2949 DraftProposal for a Council Directive on the Use of Claims Concerning Foodstuffs (SPA/62/ORIG-Fr/Rev.2, 23/11/92)(1992) Commissionof

the EuropeanCommunities 16 Fed. Regist. (1993) 58, 2431-2447 17 Code of Federal Regulations (1992) [21 C.F.R.§130], US Government Printing Office 18 Fed. Regist. (1992) 57, 23989-24004

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Review Current and potential applications of enzymes in low-water media to the synthesis of food ingredients/additives are described. Examples given include biotransformations of fats and oils, and the synthesis of emulsifiers, flavours, peptides and oligosaccharides. Advantages associated with biotechnological methods of production are briefly discussed.

Enzymes have been applied to the production of food and food ingredients for centuries on purely empirical grounds, with little knowledge of the underlying principles involved. The first enzyme systems dedicated to food processing were developed only in the second half of this century as a result of a more comprehensive understanding of the structure and function of biological macromolecules at the molecular level. However, close examination of today's food industry shows that enzymes are employed mainly for the degradation (hydrolysis) of food biopolymers rather than for synthetic purposes. This situation arose from the belief that enzymes are very delicate catalysts, easily inactivated by extreme conditions such as high temperatures or the presence of organic solvents; furthermore, a large number of commercially interesting food additives have relatively poor solubilities in water at ambient temperatures. Recent recognition of the fact that enzymes can function perfectly well in near-anhydrous conditions, displaying highly enhanced stability '-3, has significantly widened the scope of application of biocatalysts for food additive/ingredient manufacture. There are several important advantages associated with the use of enzymes in low-water environments, hi vivo the synthesis and hydrolysis of the same molecules are catalysed by different enzymes. The elimination of water from the reaction media in vitro enables the 'reversal' of

Enzymatic synthesis of food ingredients in low-water media EvgenyN. Vulfson hydrolytic enzymes and therefore avoids the need for expensive co-factors or activated substrates that are required for their 'synthetic' counterparls. It is also worth mentioning that water itself is not an ideal medium for synthetic purposes, since it often participates in side reactions and may complicate product recovery. However, the most striking features of anhydrous enzymatic catalysis are the greatly enhanced operational stability of enzymes, and solvent-dependent alterations to their catalytic properties. For example, porcine pancreatic iipase (EC 3. l. 1.3) remains active in anhydrous octane at 100°C (Ref. 4), while subtilisin (EC 3.4.4.16), whose natural function is protein hydrolysis, readily catalyses the acylation of sugars in polar organic solvents 5. The main purpose of this paper is to demonstrate the scope for the application of enzymes in 'non-conventional media' in the food industry as well as to review their current and potential uses in the manufacture of food additives.

Biotransformations of fats and oils

The scope for the application of lipases (EC 3.1.1.3) in the oleochemical industry is enormous. Fats and oils are produced worldwide at a level of - 6 x l07 t/year and a very substantial part of this (>2 x l06 t/year) is utilized in hydrolysis, alcoholysis and glycerolysis reactions Evgeny N. Vulfson is al the Department of Biotechnology & Enzymology, (Fig. I, Steps 1-3). The products of all three reactions AFRC Institute of Food Research, Reading Laboratory, Earley Gate, are used extensively in the food industry, either directly or as intermediates for the production of emulsifiers, Whileknights Road, Reading, UK RG6 2EF. Trends in Food Science & Technology July 1993 IVol. 41

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209

flavours, etc. The conditions for steam fat-splitting and the conventional glycerolysis of oils involve temperatures of 240-260°C and high pressures (alcoholysis is typically performed under slightly milder conditions) 6. The resulting products (fatty acids and their esters, mono- and diglycerides) are often unusable in foods as obtained and hence require redistillation to remove impurities and products of degradation. In addition, highly unsaturated heat, sensitive oils cannot be used directly without prior hydrogenation. Conservation of energy and minimization of thermal degradation are probably the major attractions in replacing the current chemical technologies with biological o n e s f'7. There have already been several reports of enzymatic fat-splitting processes. For example, Miyoshi Oil & Fat Co., Japan, have reported the commercial use of Candida cylindracea lipase in the manufacture of fatty acids 8. The company claim that the enzymatic method yields a higher-quality product that is cheaper overall than the conventional Colgate-Emery process. The fact that industry is seriously considering exploring enzymatic alternatives is illustrated by the rapidly growing number of patents and publications in this field. The underlying trend in the latest developments, as exemplified by Refs 9-1 l, is to move away from using organic solvents and added emulsifiers. The three major reactions (hydroly:~is% alcoholysis ") and glycerolysis ~l) have been directly performed in a mixture of substrates, thus providing very high productivity. In all cases,

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Fig. 1

Biotransformations of fats and oils. R,,.R,~:fatty acid residues. ROH: typically methanol. 2.10

simplicity, superior quality of the final product, and very high yields were claimed. It should be stressed that fatty acids and their alkyl esters and partial glycerides are essentially cheap, bulk products, and the cost of enzymes for their synthesis often remains too high for their wide commercial use. This is less critical, however, for higher added-value products such as structured triglycerides. Synthesis of structured triglycerides Obviously the properties and, therefore, the commercial value of fats depend on their fatty acid composition. Traditionally an upgrading of low-quality fats has been achieved either by blending in natural fats and oils with different triglyceride compositions or by chemical (trans)esterification in the presence of an alkaline catalyst. However, chemical catalysts randomize fatty acids in triglyceride mixtures, which often prevents the tbrmat|on of products with the required physicochemical characteristics. A typical example of a high-value, asymmetric triglyceride mixture is cocoa butter. It consists of predominantly 1,3-disaturated-2-oleoyl glycerol, where pahnitic, stearic and oleic acids account for more than 95% of the total fatty acids. The most important features of cocoa butter, which are vital for its use and responsible for its unique sensory characteristics in chocolate, are the crystal structure and a very sharp melting profile between 25°C and 35°C. Thus, it is a brittle solid at room temperature but melts completely just below body temperature, leaving no 'greasy' sensation in the mouth. The potential of 1,3-specific lipases tbr the manufacture of cocoa butter substitutes was clearly recognized more than a decade ago when UnileveP-' and Fuji Oil ~~ filed their patent applications. In both cases the process relied on lipase-catalysed transesterification (Fig. I, Step 4) or acidolysis (Fig. I, Step 5) of a cheap oil such as palm mid fraction, which contains a significant amount of 1,3-dipalmitoyl-2-oleoyl glycerol, with tristearin or stearic acid, respectively. The reactions can be conducted continuously or batchwise with or without organic solvents. Pilot-scale trials have been per|brmed successfully on a tonne scale, but Unilever had to postpone commercialization due to a sharp drop in the price of cocoa butter on the world market. The opposite decision has been taken by Fuji Oil; the company is currently manufacturing an enzymaticaily produced cocoa butter substitute. Comprehensive reviews of this technology, including.analysis of the product composition, are available (see, for example, Ref. 14). In principle, the same approach is applicable to the synthesis of many other structured triglycerides possessing valuable dietetic or nutritional qualities. For example, milk fat substitutes have been prepared as a partial replacement for the milk fat in baby foods, in contrast to plant oils, triglycerides of human milk contain palm|tic acid almost exclusively in the 2-position, with 1,3-dioleoyl-2-paimitoyi glycerol being the major individual component. This triglyceride and functionally similar fats are readily obtainable by acidolysis of palm

Trends in Food Science & Technology July 1993 IVol. 41

oil top fraction, which generally contains up to 80% tripalmitin with oleic acid ~-~.The large-scale manufacture of these products as baby food supplements will be initiated by the end of this year. Acidolysis, catalysed by 1,3-specific lipases (Fig. 1, Step 5) is likely to become the key to manufacturing yet another group of nutritionally important products: medium-chain triglycerides of octanoic and decanoic acids. These are used in common medical practice to provide a dense form of calories to patients with pancreatic insufficiency and other forms of malabsorption, as these substrates are easier to hydrolyse by pancreatic esterases than long-chain fats, and consequently the resulting monoglycerides are absorbed more efficiently. However, medium-chain fats do not provide essential fatty acids; this may lead to the development of deficiency syndromes in the patients. Recently it has been shown that this problem can be overcome by using structured triglycerides containing octanoic acid in the 1- and 3-positions and an essential fatty acid in the 2-position 16. Much attention has also been paid to the production of high-value polyunsaturated fatty acids such as arachidonic, eicosapentaenoic and docosahexaenoic acids. Many of the n-3 polyunsaturated fatty acids have been reported to have beneficial therapeutic and nutritional effects (see Ref. 17 and references cited therein). The commercial interest mainly centres around mild methods of concentrating and recovering the fatty acids in a form suitable tbr incorporation into specific dietary formulations. Substantial enrichment of polyunsaturated fatty acids in the monoglyceride fraction has been achieved by alcoholysis catalysed by a 1,3-specific lipase. This approach has already found a commercial application ~.

Enzymatic synthesisof emulsifiers This is another emerging area where the application of enzymes in non-aqueous media is likely to result in a number of new, high-quality products ~'~. Carbohydrate fatty acid esters are widely used as emulsifiers in a great variety of food formulations (low-fat spreads, sauces, ice creams, mayonnaises, etc.). Current chemical manufacturing methods are typically based on high-temperature (trans)esterification in the presence of an alkaline catalyst2°. The main drawback of this conventional m e t h d is the formation of undesirable side products, the safety of which causes increasing concern from regulatory authorities. For example, recent toxicological studies of the emulsifier Polysorbate 80 (polyoxyethylene oleate) suggest that it causes inflammation and squamous hyperplasia of the forestomach in male and female mice and ulcers of the forestomach in female mice -'~. These findings are hardly surprising since polysorbates are produced by oxyethylation of sorbitan esters under basic conditions and elevated temperatures; this promotes ester exchange, resulting in more or less random addition of ethylene oxide to the hydroxyl groups. The starting material for this synthesis, food-grade sorbitan esters, is also a rather complex mixture of isomers. A Trends in Food Science & Technology July 1993 [Vol. 4]

typical preparation of this emulsifier contains more than 60 individual compounds, many of which have been identified by gas chromatography - mass spectrometry as various isomers of sorbitan, isosorbide (up to 15), and their mono-, di- and tri-esters -'2. Additionally there is growing environmental concern regarding the biodegradability of conventional detergents used for cleaning purposes in all industries, including food manufacturing. The enzymatic synthesis of sugar fatty acid esters has been successfully performed in anhydrous organic solvents z~. However, the low productivity of this approach, the low operational stability of enzymes in solvents such as pyridine and dimethylsulphoxide and difficulties associated with the use of these solvents in a large-scale manufacture prevented commercialization of this approach. An alternative method that relied upon prior 'hydrophobization' of the sugar followed by solvent-free enzyme-catalysed esterification appeared to be more attractive from the industrial standpoint. Adelhorst et ai. 24 have performed solvent-free esterification of simple alkyi glycosides using molten fatty acid and immobilized Candida antarctica lipase. The reaction was carded out at moderate temperatures (60-80°C) with excellent regioselectivity (Fig. 2a). A range of 6-O-acylglucopyranosides has been prepared in up to 90% yield from an equimolar mixture of substrates, and Novo-Nordisk has taken this process to pilot-scale trials 2s. The synthesis is claimed to be economical, and the products, intended for cleaning purposes, are claimed to be nontoxic and rapidly biodegradable. Mono- and diesters of monosaccharides have been prepared in high yields in a similar solvent-free process (Fig. 2b) using sugar acetals as the starting materials 2~'. As in the process described above, prior 'hydrophobization' of the sugars was required to carry out the reaction in the absence of added solvents. This methodology can be readily extended to the preparation of disaccharide esters. The synthesis has been performed on a multigram scale using crude, commercial fatty acid mixtures yielding up to 88% product with a monoester content of up to 94% and no appreciable amount of side products. Although acetalization and subsequent hydrolysis do not seem to present any technological difficulties, the overall production process may be complicated. Hence, it remains to be seen whether these additional steps can be justified by resultant improvements in quality and superior emulsifying properties of the final product; this is currently being investigated in detail. (Poly)glycerol-based surfactants can also be produced enzymatically at ambient temperatures and, ultimately, in solvent-free processes. Mono- and diglycerides have been prepared by the direct coupling of glycerol and free fatty acids in membrane bioreactors 27 or in a batch mode 28. Batchwise preparation is also applicable to the production of polyglyceroi fatty acid esters. Lysophospholipids constitute another class of food emulsifiers currently prepared on a large scale using phospholipase-mediated hydrolysis. This batch reaction

211

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Enzymaticpreparationof flavours

The world market for flavouring compounds is currently valued at I CHs O about US$3 billion, amounting to II ~ RCOOH approximately a quarter of the value CHs~ C ~ CH~ of the total food additives market. Due to the complexity of many natural aroH~O~ ~ mas, in terms of both the composition and structure of individual compoR-COzOH= nents, the application of biotechnoDeacetalization H(OH) < logical methods, particularly enzymes, to their production has attracted keen attention over the past decade. In o. addition, biotechnologically produced 0 flavours are often categorized by II regulatory authorities as 'natural' or CHs~ C ~ CHs 'nature identical', and are consequently preferred by consumers. Many Fig.2 flavours are comparatively highLipase-catalysedsynthesesof sugar-basedsurfactants.R0:ethyl groupor higheralkyl chain. added-value products, which means that the cost of biocatalysts plays a is typically performed in a -30% (w/w) phospholipid less significant role in the overall economics of producemulsion with aqueous buffer in the presence of phos- tion. A survey of this extensive and expanding area is pholipase A2 (EC 3. I. 1.4)-~'. However, this process suf- beyond the scope of this review. A few selected examples fers from several complications, one of which is the are given below; comprehensive reviews of the subject necessity to inactivate phospholipase A.~ alter com- are available elsewhere .~2'~.~. Esters of carboxylic acids are important components pletion of the reaction, since it is practically impossible to recover and reuse the enzyme from the heterogeneous of natural aromas, contributing to the flavour in most reaction mixture. Irreversible inactivation of phospho- fruits and in many other tbods. The enzymatic synthesis iipase A, is achieved either by a combination of alkaliz- of more than 50 flavouring esters has been described to ation and heat treatment or by digestion with protease(s) date "u, and in principle the reaction can be carried out in followed by temperature-induced denaturation of the a mixture of alcohol and carboxylic acid without the need protease 2~'. Thus, the conventional production of lyso- for added solvents, resulting in very high productivity phospholipids presents a rather unusual case, in which and almost quantitative yields (see, for example, enzyme stability is a major drawback in the manu- Ref. 34). Although chemical synthesis of the majority of facturing process. these compounds is relatively straightforward, the appliIdeally one would like to run such a process in a cation of enzymes seems to offer some advantages homogeneous reaction mixture and, if possible, continu- where the esterification of heat/acid-labile alcohols such ously. This would avoid complications arising from the as terpenes is concerned 35, or when optically pure isonecessity to inactivate the phospholipase and result in mers are required. more cost-efficient use of the enzyme. However, phosMany compounds used for flavouring exist as optical pholipase A., displays poor activity in primary alcohols isomers, each isomer possessing different flavour or other organic solvents that would be a natural choice characteristics. For example, the D- and L-isomers of for phospholipid biotransformations. Another problem carvone have herbal and spearmint flavours, respectively, associated with the use of phospholipase A, in such a while only the L-isomer of menthol has the desired system would be the requirements for the presence of taste. The resolution of racemic mixtures is often diffiCa :+. Fungal lipases, on the other hand, are known to cult to achieve chemically, whereas the required comfunction perfectly well in nearly anhydrous solvents, ponent can usually be obtained enzymatically in one step. and have no requirements for co-factors; some of them Figure 3a illustrates the hydrolysis of menthyl esters accept phospholipids as substrates (see Ref. 30 for a catalysed by a stereospecific esterase (EC 3. I. I. l) in an recent review and references cited therein). Thus, Mucor aqueous-organic two-phase system, which can be used o.

OH•

212

Trends in FoodScience& TechnologyJuly 1993 IVol. 41

(a) CHa

to prepare optically pure L-menthol 36. This important flavouring and other terpenes have also been optically resolved via (trans)esterification in organic solvents 37.3~. Intramolecular (trans)esterification (e.g. lactonization) is another reaction of potential interest to the food industry (Fig. 3b). Various lactones are currently used as flavours and fragrances; however, their chemical syntheses present certain difficulties. Unfortunately, the enzymatic route also suffers from several limitations. The reaction is rather slow and lactonization is often accompanied by oligomerization~-4°; therefore, the practical value of this approach remains to be established. Currently, enzymatic resolution of racemic lactones 4' seems to be a more appropriate route from a practical standpoint. Long-chain aliphatic, aromatic and terpenoid aldehydes exhibit distinct organoleptic qualities that are highly valued in the food industry. Although the majority can be produced chemically, in some cases the food industry has to rely on agrochemical and biotechnological sources. In vivo, aldehydes are mainly synthesized as intermediates in the formation of their corresponding alcohols. Therefore, the alcohols are usually obtainable in much higher quantities. The oxidation of alcohols is readily catalysed by alcohol dehydrogenase (EC l.l.l.2) 4-', as illustrated in Fig. 3c. Coenzyme regeneration required by this reaction can be achieved by several means, for example by simultaneous reduction of 'sacrificial' aldehyde catalysed by the same enzyme. Enzymatic peptide synthesis Short peptides are yet another group of compounds that are important in the sensory appreciation of foods. A large number of specific oligopeptide structures possessing sweet (e.g. aspartame and alitame), salty (e.g. ornithine-containing dipeptides), sour and umami (mainly acidic di- and tripeptides), bitter (peptides rich in hydrophobic amino acids) and astringent (7-glutamyi dipeptides) flavour profiles have been described and evaluated 4.~.~4.Short peptides not only provide a particular taste and flavour in a variety of products; they also play an important role as "food hormones' (see, for example, Ref. 45), controlling many physiological functions, even appetite. The identification and isolation of biologically active and flavour peptides is ongoing, but feasible approaches to their production have already been assessed. The application of conventional, chemical methods of peptide synthesis is rather limited by the food acceptability of the solvents and reagents used and by expenses associated with the activation/protection of amino acids. Biotechnology offers three alternatives: the purification of oligopeptides from protein hydrolysates, microbiological production by fermentation, and direct enzymatic coupling of amino acids. Although the preferential route of synthesis is, obviously, dependent on the particular structure, some general conclusions can easily be drawn. Enzymatic hydrolysis could be difficult to control, while separatioa of a ~eptide from the mixture produced Trends in Food Science& TechnologyJuly 1993 IVol. 41

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CHa

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CH(CH3)2

CH(CH3)2

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(b)

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O

CH(CHa)2 D.menthol ester

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H,O

+

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Fig. 3 Potential usesof enzymesfor the production of flavours: (a), resolutionof racemates;(b), lactonization;(c), oxidation-reduction.

may be very expensive if a high-purity final product is required. Product recovery is likely to be much cheaper from a fermentation broth, but the expression of relatively short oligopeptides is rather problematic at present. In contrast, the synthesis of relatively short oligopeptides using proteinases in low-water media is a rapidly developing area 46"47, and the commercial feasibility of this approach has been unambiguously proven in the manufacture of aspartame ~8"4H,which is currently used in more than 500 different products 44. It should be mentioned that until recently there has been some doubt regarding the practical applicability of the enzymatic approach to the preparation of structures more complex than di- and tripeptides. This is because the majority of early syntheses (for reviews, see Refs 46 and 47) included conventional protection/deprotection steps for the elongation of the oligopeptide chain. However, the enzymatic assembly of an oligopeptide can be substantially simplified if N-unprotected amino acid esters are used as substrates. This strategy has been successfully implemented for the three-step preparation of enkephalin precursors4% biologically active pentapeptides present in milk hydrolysates 45. This process has also been run continuously for over 1000 hours in a threestage bioreactor yielding, under steady-state conditions, up to 0.7 g of pentapeptides per day ~°. Furthermore, short peptides can be synthesized enzymatically in good yields in heterogeneous, equimolar mixtures of substrates in the total absence or in the presence of small 213

amounts of added solvents, thus providing productivity approaching the theoretical maximum 5'.

Enzymaticoligosaccharidesynthesisand sugarmodification Carbohydrate-converting enzymes, especially hydrolases, have found a wide range of applications in the food i n d u s , . They are used for the hydrolysis and modification of starches as well as in the brewing, baking, fruit and wine processing, confectionery and dairy industries, with their sale accounting for a third of the worldwide enzyme markeP 2. All these applications are essentially based on the hydrolytic activity of the enzyme, but glycosidases (as any other hydrolases) can also be forced to synthesize oligosaccharides under appropriate reaction conditions. Due to the peculiarity of the glycosidase's catalytic mechanism (Fig. 4), there are generally two options for the utilization of their synthetic potential: reversal of hydrolysis and transglycosylation. Reversal of hydrolysis relies on the establishment of a thermodynamic equilibrium in the reaction mixture, while transglycosylation proceeds under kinetically controlled conditions, whereby the enzyme-bound glycon (glycosyl moiety of substrate) can be discharged by water or transferred to an alternative accepter, typically a sugar or another alcohol (see Ref. 53 for a recent review). In both cases it is preferable to perform the reaction in a low-water medium, either to shift the equilibrium towards synthesis or to minimize undesirable hydrolysis of the activated glycon donor. The main advantage of using transglycosylation rather than reverse hydrolysis is the high activity of the enzyme, which facilitates obtaining good yields in a relatively short period of time, Both reactions have been extensively studied as routes for the production of short oligosaccharides with desirable properties for use in |'oods "~4,and the first products are currently being manufactured in Japan on a multi-tonne scale. Meiji Seika uses transglycosylation

catalysed by a fungal fructosyltransferase (EC 2.4.1.10) to produce non-reducing oligosaccharides from sucrose, which serves as a donor and accepter of glycon. The product (which has the trade name 'Neosugar') contains mainly a mixture of di-, tri- and tetrasaccharides, with fructosyl units linked through ~l(2~l) glycosidic bonds 54. Another Japanese company, Yakuit Honsha, uses [l-galactosidase (EC 3.2.1.23) to catalyse the transglycosylation of lactose to produce 6-galactosyl lactose, one of the biologically active oligosaccharides present in human milk. The oligosaccharide mixture obtained is claimed to possess a range of physiological activities and is used as a supplement in a number of products such as fermented milk drinks and infant food formulae (see Ref. 55 and references cited therein). Both products are claimed to be less calorific and cariogenic than usual sugars, and in both cases the reactions are carried out at nearly saturating concentrations of the sugars. Carrying out enzymatic reactions in low-water media allows the reversal of hydrolytic reactions, the manipulation of enzyme specificity and facilitation of product recovery. Furthermore, the addition of organic solvents to the medium can have an important impact on the equilibrium of non-hydrolytic reactions. For example, the isomerization of glucose catalysed by glucose isomerase (EC 5.3.1.5), which is a key process in the production of high-fructose syrup, typically results in a final concentration of fructose that is insufficient for some commercial applications. The required 55% concentration of fructose is simply unobtainable in water at ambient temperatures. Consequently, in industrial processes, the enzymatic isomerization is usually followed by chromatographic enrichment. This relatively expensive step can be avoided if the same reaction is carried out in ethanol-water mixtures. Thus, the shift in equilibrium that is achieved is sufficient to obtain the required fructose content in one step ~.

Conclusions

Hopefully, the variety of examples given above adequately demonstrates the potential of low-water enzymology in the production of food ingredients and additives. In many instances biotechnological methods offer attractive economical alternatives to the conventional chemical approaches, while the introduction of highly ,< "specific enzymatic syntheses should relieve public con~ O R cern over the adverse effects of contaminants or by+ROH products in chemically produced food additives. The ® development of novel systems will undoubtedly provide routes for manufacturing other complex natural com+AOH ~ ~ - ~ O A ponents of foods, Today, however, the cost of some Hydrolysis enzymes still remains too high for the manufacture of Transgly©osylation many low-added-value products. This probably represents the major obstacle for a wider use of enzymatic ReverseSynthesis methods in the food industry. At the same time, the impressive progress in genetics and in process techFig.4 nology enables the enzyme industry to offer biocatalysts Mechanismsof enzymatictransglycosylationand reversalof hydrolysis.AOH is a with greatly improved properties and often at reduced glycon accepter,typicallya mono- or disaccharide,and the oxonium ion in costs 57. In future this should shift the economic balance brackets is a postulatedenzyme-boundintermediate. in favour of biotechnological methods.

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References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1.5 16 17 18 19 20

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Forthcoming articles Fuzzy logic and neural network applications in food science and technology, by T. Eerik~inen, S. Linko, P. Linko, T. Slimes and Y-H. Zhu Lipid encapsulation technology - techniques and applications to food, by Ryuchi Matsuno and Shuji Adachi Applications of membrane technology in food processing, by F. Petrus Cuperus and Herry H. Nijhuis Plastics packaging of food products: the environmental dimension, by David Brown

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