VOLUME V

INDUSTRIAL PRODUCTION WITH IMMOBILIZED ENZYMES: ANTIBIOTICS, FOOD ADDITIVES AND DAIRY PRODUCTS

(Flowsheets of the processes and other drawings are not shown on this site. Please contact the author, if needed)

INDUSTRIAL PRODUCTION WITH IMMOBILIZED ENZYMES: ANTIBIOTICS, FOOD ADDITIVES AND DAIRY PRODUCTS

476. A specific D-hydantoinase from calf liver was chosen by Snamprogetti as the principal catalyst for immobilization and scale-up of the process. The enzyme preparation has a specific activity of about 3 U/mg protein that is entrapped in cellulose triacetate fibers (paragraph 408). 70 U are entrapped per gram of polymer and about 80% of this amount is found active in fibers (Marconi, 1978).

3.     Technological characteristics of the process

477. A stirred tank reactor for the hydrolysis of 3 kg of 5-phenylhydantoin per day (the reaction actually takes place over 5 hr) has been described (Marconi & Morisi, 1979). The operation conditions are 30°C, pH 8.5; the half-life of the immobilized enzyme is 20 days. A similar approach is used also for the preparation of another intermediate for semisynthetic antibiotics, i.e. D(-)-p-hydroxyphenylglycine. As Snamprogetti reported (Process for the Preparation of D-Phenylglycine, 1977), they developed the method using an ion exchange resin that removes the D-amino acid product from the reaction mixture, thus preventing the oxidation of the amino group of the amino acid by the nitrous acid that is used for the oxidation reactions.

4.     Scale of the process

478. In 1977 Snamprogetti planned to install a plant with a capacity of 200 kg of D-phenylglycine per day (60 tons/year). Production was to be batchwise over a 24-hour period (Process for the Preparation of D-Phenylglycine, 1977); one synthesis of phenyl hydantoin, one enzymic hydrolysis to the carbamoyl derivative and eight oxidation reactions could be performed per day. At present presumably less than 50 tons of D-phenylglycine is produced per year, with an estimated enzyme-derived amount less than 1 ton/year (Poulsen, 1985).

479. The following equipment items were necessary (Process for the Preparation of D-Phenylglycine, 1977): one autoclave for hydantoin synthesis with a nominal capacity of 5 m³; four stirred, thermostatted vessels for enzymic hydrolysis with a nominal capacity of 6.5 m³ each; one filter press with a filter area of 5.0 m²; three column reactors for chemical oxidation of the carbamoyl derivative with a nominal capacity of 3 m³ each; three centrifugal pumps for the reactors with a capacity of 10 m³/hr; one vacuum evaporator with a capacity of 500 kg water/hr and a maximum temperature of 50°C; one centrifugal separator with a basket capacity of 50 kg; one vacuum dryer with a maximum temperature of 50°C.

480. More recent data about this process indicated (Samejima et al., 1980) that D(-)-p-hydroxyphenylglycine (an intermediate in the synthesis of semisynthetic penicillins and cephalosporins, especially amoxicillin) from its hydantoin precursor by hydantoinase was going to be produced industrially by Kanegafuchi Chemical Industry Co. in Japan. In 1982 there was a report on the setting-up of an industrial plant in Singapore to produce this compound using immobilized Bacillus brevis cells containing the enzyme dihydropyrimidinase, on the basis of the process patented by Kanegafuchi (Tramper, 1985). This plant has a production capacity of less than 50 tons per annum. A yield of 300 tons in 1983 and a rapid further increase to 700 tons per year was reported (Tramper, 1985).

B.     PRODUCTION OF L-ASPARTIC ACID BY IMMOBILIZED MICROBIAL CELLS CONTAINING ASPARTASE

481. L-Aspartic acid is widely used for medicines and food additives. It has been produced industrially by fermentation and by enzymatic methods from ammonium fumarate using aspartase as a catalyst. Rhis reaction had previously been carried out on an industrial scale in a batch process using soluble enzyme. However, there were some disadvantages, as was indicated in paragraph 70 for the case of the soluble amino acylase process. The results of extensive studies of continuous production of L-aspartic acid using immobilized Escherichia coli cells containing aspartase led Tanabe Seiyaku Co. to industrialize the method in 1973. In 1976 this process was also scaled up by Kyowa Hakko Kogyo Co., using immobilized aspartase on a polymeric carrier.

1.     Background

482. The enzyme aspartase catalyzes a one-step stereospecific addition of ammonia to the double bond of fumaric acid. Fumaric acid is dissolved in a 25% ammonia solution and the ammonium fumarate is passed through a reactor containing the enzyme catalyst. The reactor must be capable of removing the heat produced since the reaction is exothermic.

483. It is necessary to extract aspartase from microbial cells before immobilization since it is an intracellular enzyme. Extracted intracellular enzymes are generally unstable, and most of the immobilization methods that Tanabe Seiyaku tried resulted in low activity and poor yield (Chibata, 1980). They indicated that although entrapment of the soluble aspartase to a polyacrylamide gel lattice gave relatively active immobilized enzyme, its stability under operating conditions was not sufficient (see paragraph 485). Therefore, Tanabe Seiyaku considered this immobilized aspartase as unsatisfactory for the industrial production of L-aspartic acid. As a result of further studies, the company chose the direct immobilization of whole microbial cells of E. coli by entrapping them in polyacrylamide gel. This system has been operating industrially for the automatic and continuous production of L-aspartic acid since 1973. This is considered to be the first industrial application of immobilized microbial cells (Chibata, 1980).

2.     Immobilized enzymes (cells) preparations

484. Tanabe Seiyaku uses the enzyme aspartase from E. coli. After entrapment the cells are incubated in 1 mM Mg²⁺, which is thought to cause autolysis of the entrapped cells and increases the effective activity greatly (Hultin, 1983). When the immobilized E. coli cells are suspended at 37°C for 24-48 hours in substrate solution, their activity increases by a factor of 10 (Chibata, 1978a). The increase of enzyme activity is observed even in the presence of chloramphenicol, an inhibitor of protein synthesis. Therefore, this activation is not considered to be the result of protein synthesis but rather to increased permeability for substrate and/or product caused by autolysis of E. coli cells in the gel lattice. This was confirmed by electron micrographs of immobilized E. coli cells. Once lysis occurs, the substrate, ammonium fumarate, and the product, L-aspartate, passes easily through the gel; however, aspartase does not leak out of the gel lattice (Chibata, 1978a).

485. The immobilized catalyst is prepared as follows (Chibata, 1980). Escherichia coli cells (100 kg, wet weight) collected from culture broth are suspended in 400 liters of physiological saline. 75 kg of acrylamide monomer, 4 kg of N,N'-methylene-bis-acrylamide, 50 liters of 5% a-dimethylaminopropio-nitrile, and 50 liters of 1% potassium persulfate are added to this suspension. The mixture is allowed to stand at below 40°C for 10-15 min, and the resulting stiff gel is made into round 2-3 mm granules. The half-life for this preparation at 37°C is estimated to be 120 days. In comparison, the half-life for the gel entrapped extracellular aspartase being equal to 30 days and for the intact E. coli cells, 10 days.

486. As Tanabe Seiyaki indicated, immobilization of enzymes and microbial cells in polyacrylamide gel has some limitations. Aspartase, for example, is partially inactivated during the immobilization procedure by the action of acrylamide monomer, beta-dimethylaminopropionitrile or by potassium persulfate. As a further improvement in the productivities of immobilized microbial cell systems, the company has found that kappa-carrageenan, a polysaccharide from seaweed, is one of the most suitable matrices for immobilization of enzymes and microbial cells (Chibata, 1980). Apparently, kappa-carrageenan easily becomes a gel under mild conditions and causes little inactivation of enzymes in the course of their immobilization. Thus, E. coli immobilized with the carrageenan and then treated with glutaraldehyde and hexamethylenediamine shows the highest aspartase productivity. When the productivity of the immobilized preparation with polyacrylamide is taken as 100, that of immobilized cells with carrageenan hardened with glutaraldehyde and hexamethylenediamine is 1500 - around 15 times greater. The last preparation is also very stable, with a half-life of 680 days (almost 2 years). As a result, in 1978 Tanabe Seiyaku Co. switched from the conventional polyacrylamide method to this new carrageenan method for industrial production of L-aspartic acid.

487. After examining several adsorbents the Kyowa Hakko Kogyo Co. selected Duolite A7, a phenol-formaldehyde resin, for adsorbing aspartase used in their continuous production of L-aspartic acid with a packed bed reactor (Konechy, 1984).

488. A technique using polyurethane prepolymers for the immobilization of E. coli cells containing aspartase activity was reported by W.R. Grace & Co. (Maryland). The resultant E. coli "foam" is ground to an average particle size of 0.5 cm in a Cumberland mill (Fusee, 1987).

3.     Technological characteristics of the processes

489. Ammonium fumarate serves as the principal substrate for the preparation of L-aspartic acid using immobilized aspartase (see also paragraph 482). The flow sheet of the process is essentially the same as that for the optical resolution of amino acid racemates (para. 461 in Vol. IV). That is, a 1 M solution of ammonium fumarate containing 0.001 M MgCl₂ is passed through the column packed with the immobilized E. coli cells at 37°C and pH 8.5. The flow velocity is equal to 0.6 volume/hr. In a typical run the 2400 liters of effluent is adjusted to pH 2.8 with 60% sulfuric acid at 90°C, then cooled to 15°C and kept for 2 hr. Crystalline L-aspartic acid is collected by centrifugation and washed. By this simple procedure pure L-aspartic acid can be obtained without recrystallization, and the yield is usually over 95% (Chibata, 1980). The industrial process is fully automatic and continuous.

490. Japan's Mitsubishi Petrochemical Co. announced in 1986 (Genet. Engin. Biotechnol. Mon., 1986) that they would begin to make L-aspartic acid from fumaric acid and ammonia using the microorganism Brevibacterium flavum containing aspartase. The company indicated that E. coli, which is usually used for that purpose, has a weak cell structure that must be protected by immobilizing the bacterium if the microorganism is to be recycled. B. flavum, on the other hand, has a hard cell wall that can withstand repeated centrifugations without need of immobilization, thereby reducing the cost for L-aspartic acid production.

491. W.R. Grace & Co. (Maryland) reported on a bench-scale system that they claimed would be "an ideal representation of an industrial-scale system" (Fusee, 1987). The residence time of a column reactor was 32 min, with a linear flow rate of 5 cm/min (the latter was chosen to approximate rates that will be used for industrial-scale reactors). After the effluent was collected, adjusted to the isoelectric point of L-aspartic acid at pH 2.8 with H₂SO₄ at 90°C, and cooled, a white, crystalline material was collected by filtration and washed with water. The crystalline product was determined to be >99% L-aspartic acid, and the yield (227 g) was 85% of theoretical.

4.     Economic estimations

492. A comparison of the cost for production of L-aspartic acid by the conventional batch process using intact cells and the continuous process using immobilized cells is shown in Figure 1. The major savings are in the cost of the enzyme which is reduced by a factor of approximately 9 because immobilized cells are substantially more stable than intact cells. There is also a 30% reduction in labor cost since the immobilized process is automated. As a result, the overall production costs of the immobilized cell system are about 60% that of the conventional batch process using intact cells. Furthermore, the procedure employing immobilized cells is advantageous from the standpoint of waste treatment (Chibata, 1980).

5.     Scale of the processes

493. Tanabe Seiyaku Co. uses a 1000-liter column with immobilized cells and produces 1700 kg/day (89% of the theoretical yield) or 51 ton/month of L-aspartic acid (Chibata, 1980). A similar industrial process has been operating at Kyowa Hakko Kogyo Co. since 1976.

494. In 1986 Mitsubishi Petrochemical Co. was planning to operate a plant producing L-aspartic acid using B. flavum aspartase (see paragraph 490) with a capacity of 1,000 tons per year (Genet. Engin. Biotechnol. Mon., 1986).

495. Purification Engineering, Inc. (Baltimore, MD) has recently claimed that it can make some 2,000 tons of L-aspartic acid a year - almost as much as is required for all of G.D. Searle's (Skokie, IL) $500 million aspartame production - in a 208-liter immobilized-cell column. The $4 million plant producing L-aspartic acid and L-phenylalanine has been on-line since December 1984 (McCormick, 1985).

C.     PRODUCTION OF L-MALIC ACID BY IMMOBILIZED MICROBIAL CELLS CONTAINING FUMARASE

496. L-Malic acid is mainly used in the pharmaceutical field as an antidote for hyper-ammoniemia and a component of amino acid infusion, and is becoming of greater market interest as a food acidulant in competition with citric acid, for example, in confectionery products. Malic acid, which is found naturally in most fruits, is a weaker acid than citric or tartaric. It possesses a tart flavor that builds up slowly and then gradually diminishes, blending well with essences and other flavors. The racemic mixture of DL-malic acid is produced by a cheap chemical synthesis from malic anhydride and water, whereas the natural L-form of malic acid produced by fermentation in batch process using microbial broth is rather expensive for the acidulant market.

497. The continuous industrial production of L-malic acid by means of immobilized microbial cells Brevibacterium ammoniagenes and then B. flavum containing the enzyme fumarase has been carried out by Tanabe Seiyaku Co. since 1974 (Chibata, 1978a). Some time later a process for the enzymic hydration of fumaric acid to L-malic acid by fiber-entrapped fumarase was recommended for industrial operation (Marconi & Morisi, 1979). The fumarase was extracted from microbial cells and purified prior to its entrapment in fibers. In 1976, Kyowa Hakko industrialized a similar process with fumarase adsorbed on an anion exchange resin (Konechy, 1984).

1.     Background

498. L-Malic acid is produced as a result of hydration of fumaric acid by fumarase, either intracellularly or extracted from a microbial producer. The reaction reaches an equilibrium when about 80% of the fumaric acid is converted to L-malic acid.

499. According to Tanabe Seiyaku (Chibata, 1978b), immobilized cells of B. ammoniagenes form succinic acid as a by-product of L-malic acid production, and separation of these two products is very difficult. Therefore, the critical point of success for industrial production of pure L-malic acid is the prevention of succinic acid formation during the enzyme-catalyzed reaction. The treatment of the immobilized cells with detergents, particularly with bile extract, which is readily available, is considered by Tanabe as the most suitable for industrial purposes. The detergent suppresses succinic acid formation and greatly enhances the formation of L-malic acid by the immobilized cells. The most effective conditions are incubation of the immobilized cells in 1 M sodium fumarate (pH 7.5) containing 0.3% bile extract at 37°C for 20 hr (Chibata, 1978b).

2.     Immobilized cells and enzymes

500. Tanabe Seiyaku used the fumarase-containing cells B. ammoniagenes immobilized in polyacrylamide gel for the first three years (1974-1977) of the industrial production of L-malic acid (Chibata, 1978a, 1980). This led to better stability of the enzymic catalyst as compared with the intact cells. Thus, the activity of intact cells under operating conditions (37°C) rapidly decreased and its half-life was 6 days. On the other hand, immobilized cells treated with bile extract had a half-life of 55 days at the same temperature.

501. Tanabe Seiyaku, however, has indicated that immobilization of microbial cells in polyacrylamide gel had some limitations. That is, some enzymes are inactivated during the immobilization procedure by the action of acrylamide monomer, beta-dimethylaminopropionitrile, potassium persulfate or heat of the polymerization reaction (see also paragraph 486). Thus, in order to find a more general immobilization technique and to improve the productivities of the elements of the immobilized microbial cell system the company switched over to kappa-carrageenan as a gel matrix (par. 486). They also found that B. flavum has a higher fumarase activity and stability after immobilization with carrageenan as compared with the former B. ammoniagenes (Chibata, 1980).

502. When the productivity of immobilized B. ammoniagenes with polyacrylamide is taken as 100, that of immobilized B. flavum with the same carrier is 273 and for B. flavum with carrageenan is 897. The half-life at 37°C for the immobilized microbial systems was as follows:

B. ammoniagenes
         Polyacrylamide         53 days
         Carrageenan             75 days

B. flavum
         Polyacrylamide         94 days
         Carrageenan           160 days

503. For immobilization, both cell suspension and carrageenan dissolved in physiological saline are warmed to 37-60°C. Both are mixed, and the mixture is cooled and/or contacted with aqueous solution containing K⁺, NH₄⁺, Ca²⁺ (or other) ions as a gel-inducing agent. After treatment, the gel is granulated to a suitable particle size. If the operational stability of immobilized cells is not satisfactory, they can be treated with hardening bifunctional agents such as glutaraldehyde and hexamethylenediamine to obtain stable immobilized cell preparations (Chibata, 1980).

504. In order to obtain even more stable preparations and to improve the productivity of immobilized fumarase cells, during the late 1970's Tanabe Seiyaku Co. started to add polycationic polymers to the immobilization medium. The stabilization effect is particularly evident with polyethyleneimine (Tosa et al., 1982). The heat stability of the immobilized preparation increased so that the column could be operated at relatively high temperatures of 50-55°C for long times (half-lives of 128 days at 50°C, 74 days at 55°C and 243 days at 37°C). The productivity of B. flavum immobilized with kappa-carrageenan and polyethyleneimine increased to 21 times that of B. ammoniagenes immobilized with polyacrylamide (2100 at 45°C and 1600 at 37°C as compared with the data in paragraph 502). In 1980 the industrial production system of L-malic acid was changed to incorporate this immobilization method.

505. According to the Snamprogetti procedure (Marconi & Morisi, 1979), fumarase is extracted and partially purified from microbial cells by mechanical disruption, removal of nucleic acids, and precipitation with ammonium sulfate. The purified enzyme solution is entrapped in cellulose triacetate fibers that display about 40% of the entrapped activity. The characteristics of the immobilized enzyme have not been disclosed, and according to Marconi and Morisi (1979), the entrapped fumarase showed "good" stability both under storage and operating conditions at pH values of 7.0-8.0, as compared with the free enzyme which was "rather unstable", especially when dissolved in water at a low protein concentration.

3.     Technological characteristics of the processes

506. Tanabe Seiyaku uses a column packed with immobilized cells. The inlet solution of 1 M sodium fumarate at pH 7.0 is passed through the column at 37°C at a flow rate of 0.2 hr^-1; the reaction at these conditions reaches an equilibrium with about 80% conversion of fumaric acid to L-malic acid. From the effluent of the column, L-malic acid is obtained by ordinary methods, that is fumaric acid is separated by acidifying the effluent; the yield is 70% of the starting material, fumaric acid (Chibata, 1978b).

507. In the Snamprogetti process, a solution of 0.5 M sodium fumarate at pH 7.0 and 25°C is continuously pumped through a column packed with fiber-entrapped fumarase. 50% of the fumarate is converted to L-malate, and the activity decreases to 25% of the initial value after 100 days of operation. L-Malic acid is separated from the reaction mixture by precipitation as the calcium salt from which pure L-malic acid is obtained; the unchanged fumarate is recycled (Marconi & Morisi, 1979).

508. In China, L-malic acid is produced with 15% polyacrylamide gel immobilized Candida rugosa cells from fumarate in 82-85% yield in a continuous column operation (Zhang, 1982). The author reports, "pilot plant experiments and industrial production [of L-malic acid] have been accomplished".

4.     Economic estimation and scale of the processes

509. Economic estimates and data on the scales of the processes of L-malic acid industrial production by means of immobilized enzymes or cells are generally lacking in the literature. Tanabe Seiyaku reported only that, "both the economic efficiency and the quality of the product have so far been satisfactory" (Chibata, 1980). According to Snamprogetti, a comparison between the production cost of citric acid by fermentation and that of L-malic acid by fiber-entrapped fumarase "was done and gave comparable production costs for the two organic acids" (Marconi & Morisi, 1979).

510. According to Samejima and coworkers (1980), L-malic acid is now produced industrially with immobilized microbial cells and enzyme by Tanabe Pharmaceutical Co. and Kyowa Hakko Kogyo Co. Recent data indicate that by using 1000-liter columns packed with the immobilized B. flavum cells, 42.2 kg of L-malic acid/hr can be produced continuously for 6 months (Chibata et al., 1987).


D.     DEACYLATION OF PENICILLIN AND CEPHALOSPORIN DERIVATIVES BY IMMOBILIZED PENICILLIN AMIDASE

511. Beta-lactam antibiotics make up a large group of physiologically active compounds including natural and semisynthetic penicillins, cephalosporins, and cephamycins. Many of them are characterized by unique antimicrobial properties, bacteriocidal action, and low toxicity, and are widely used for practical applications in medicine. Because of their practical effectiveness, the synthesis and modification of ß-lactam antibiotics is an important problem of enzyme engineering (Svedas et al., 1980). In the mid-1980s the world market value of antibiotics for pharmaceutical use was estimated annually at nearly 5 billion US $, and ß-lactams accounted for 53.5% of it in 1980 and was expected to increase to 63.5% by 1990 (Kieslich, 1985). In 1989, the best-selling single enantiomer drugs - "the optically active top ten" - between them had sales of over $10 billion. Those drugs included several penicillin- and cephalosporin-based antibiotics (Sheldon, 1992; Hodgson, 1992). The world's largest producer of antibiotics in general is China, with an annual output of over 10,000 tons at a value of $300 million (Han Ying-Shan, 1991).

512. Market research indicates that chiral molecules in general (penicillins and cephalosporins constitute a large fraction of them) are likely to take an increasing share of the drug market; Frost and Sullivan's 1991 report "The European Market for Pharmaceutical Intermediates" (cit. in Hodgson, 1992) estimates that sales of enantiomeric intermediates in Western Europe will increase from $91 million in 1991 to $136 million in 1996; their share of the pharmaceutical intermediate market will increase from 16 to 20 percent over that period.

513. Synthesis of new ß-lactam antibiotics usually proceeds in two major steps. For the first step a specific natural ß-lactam compound (e.g. penicillin or cephalosporin) is hydrolyzed by penicillin amidase forming a semiproduct such as 6-aminopenicillanic acid (6-APA) or 7-aminodeacetoxy-cephalosporanic acid (7-ADCA). For the second step the semiproducts form new "semisynthetic" antibiotics as a result of chemical or enzymatic conversion.

514. Thus, 6-APA and 7-ADCA are important intermediates in the production of semisynthetic penicillins and cephalosporins in the pharmaceutical industry. Penicillins are well-known medical preparations and have been widely used since the 1930's. Cephalosporins, on the other hand, have demonstrated some considerable advantage over penicillins in broad spectrum antimicrobial activity, low toxicity, and also outstanding efficacy against various penicillin resistant strains (Hyun et al., 1993). 6-APA and 7-ADCA have been produced commercially for some years by the deacylation of benzylpenicillin (penicillin G) and a cephalosporin derivative, respectively, using immobilized penicillin amidase (also known as penicillin acylase) (Ishimura & Suga, 1992; Stambolieva et al., 1992). The first industrial process for 6-APA production was realized in the early 1970's by three manufacturers simultaneously, Squibb (USA), Astra (Sweden), and Riga Biochemical Plant (USSR). 7-ADCA production using immobilized penicillin amidase was industrialized by Riga Biochemical Plant (USSR) in 1980.

1.     Background

515. Penicillin amidase catalyzes the deacylation of penicillin with formation of 6-APA and the side chain acid. Several types of penicillinamidases are known that are distinguishable by their substrate specificity. The enzyme of one group, which is usually of fungal or of actinomycete origin preferentially hydrolyzes phenoxymethylpenicillin (penicillin V), whereas that of the other group, generally of bacterial origin, preferentially hydrolyzes benzylpenicillin (penicillin G). In the latter case the hydrolysis products are 6-APA and phenylacetic acid.

516. In contrast to most other industrial processes using immobilized cells, the deacylation of benzylpenicillin results in the formation of ionogenic compounds which at the normal operating pH are charged (at least one of them). To maintain the reaction pH in the desired range it is therefore necessary to add alkali or use large amounts of a buffering salt. This in turn affects the design and operation of immobilized penicillin amidase reactors.

517. The production of 6-APA by fermentation is uneconomical as the yields are poor and is always accompanied by production of a mixture of penicillins. The isolation of 6-APA, therefore, requires a complex process (Borkar et al., 1978). The chemical method of 6-APA production from its respective penicillin usually depends upon the selective deacylation of penicillins. The ß-thiazolidine ring has to be protected from cleavage. In addition, the method requires subzero temperatures, and the problems of corrosion are severe because of aggressive solvents.

518. Before immobilized penicillin amidase was introduced commercially, 6-APA had been industrially produced batchwise using intact bacterial cells containing the enzyme. Cells were used only once and the wet microbial mass was usually discarded. Immobilization of penicillin amidase allowed the process to run continuously. The enzymatic method for deacylation of penicillins has advantages over others as the hydrolysis proceeds to completion under mild conditions such as at temperatures slightly higher than ambient and at pH values near neutrality. Under these conditions, both penicillin and 6-APA are stable and the product isolated is free from contamination by degraded compounds. Besides the evident economic advantages that immobilized systems offer on account of their recycling potential, in the present context the need to exclude macromolecular allergenic impurities is another overriding reason for the exclusive use of the immobilized catalyst. Presently, the substantial part of 6-APA manufactured in Sweden and Italy and all 6-APA produced in the USSR and in Japan is obtained using immobilized penicillin amidase.

519. In the overall process penicillin G is separated from the fermented broth by solvent extraction; the end product of the extraction is generally a crude concentrated solution of penicillin G,K from which the penicillin is crystallized by azeotropic distillation with butanol. The dried crystals of penicillin G are dissolved in water and upon hydrolysis yield 6-APA and phenylacetic acid. If the 6-APA plant were located adjacent to the penicillin fermentation plant, it would be possible to hydrolyze directly the crude solution of penicillin G obtained from the solvent extraction, by-passing the crystallization stage. The phenylacetic acid product can be used as a precursor in penicillin fermentation (Giacobbe et al., 1978).

2.     Commercial preparations of immobilized penicillin amidase

520. Penicillin amidase from the E. coli strain, ATCC 11105, from Bacillus megaterium strain, ATCC 14945, and from mutants of these two strains are the most commonly used in industry (Robas et al., 1993). Recently, following a mutagenesis program with E. coli ATCC 11105, a mutant designated as E. coli G133 was selected by the Rhone-Poulenc Rorer Company. In this mutant, penicillin amidase production is less sensitive to glucose repression (Robas et al., 1993).

521. A number of immobilized systems, patented for commercial production of penicillin amidase, vary significantly (see table below). The enzyme covalently coupled to Sephadex G 200 activated by cyanogen bromide is described in a patent, assigned to the Swedish firm, Astra. Bayer A.G. patented a series of hydrophilic supports covering polyacrylamide maleic acid copolymer prepared by heating at 80°C and also patented maleic anhydride-polyol(meth)acrylate copolymer derivatives for the immobilization of penicillin amidase (Ger. Offen. 2,157,972; 2,215,509; 2,215,512; 2,215,687). Bayer A.G. also patented the covalent attachment of the enzyme to soluble macromolecules like dextran or starch followed by encapsulation of the complex in a polymer. Novo Laboratory holds patents on the enzyme immobilization by intermolecular cross-linking with bifunctional reagents like glutaraldehyde either in the presence of an inert filler like cellulose or by later admixture with the filler (Ger. Offen 2,345,185; 2,345,186).

522. Cross-linking of the enzyme in a nonionic methacrylate resin support with glutaraldehyde added in the presence of a water soluble diamine has been described in a patent assigned to the Beecham group (Ger. Offen. 2,334,900; 2,336,829). Adsorption of the enzyme by ion exchangers like DEAE-cellulose and CM-cellulose and subsequent cross-linking with glutaraldehyde has also been patented by Beecham. At Snamprogetti a partially purified penicillin amidase from E. coli, entrapped in spun fibers of cellulose triacetate has been successfully utilized on a laboratory scale since the early 1970's (Marconi et al., 1973) and then industrialized. Tanabe Seiyaku Co. holds a patent according to which whole cells of E. coli and other organisms containing the enzyme have been entrapped in polyacrylamide. Several patents describe immobilized purified penicillin amidase from E. coli attached to cellulose derivatives such as DEAE cellulose, amino-chloro-S-triazyl DEAE cellulose, dichloro-S-triazyl DEAE cellulose and filter cloth (S. African Pat. 6,804,164; 6,804,009; British Pat. 1,183,260). Toyo Jozo company has developed a process for the production of 7-ADCA in which the extracellular penicillinamidase adsorbed on celite or covalently coupled to activated porous fibers is used as a catalyst (Genet. Engin. Biotechnol. Mon., 1986). Spofa, United Pharmaceutical Works (Czechoslovakia) has been using covalently cross-linked disrupted microbial cells with a "high content" of penicillin amylase for commercial production of 6-APA (Czechoslov. Pat. 203,607).

523. In a Chinese industrial process (Zhang, 1982) an E. coli strain having "high penicillin acylase activity" was immobilized in agar gel. The microbial cells are mixed with an equal volume of 8% agar gel and poured into an organic solvent with stirring. After washing, the resultant gel beads are crosslinked with 1% glutaraldehyde solution and washed. Another immobilized preparation is obtained by entrapment of the microbial cells in gelatin and crosslinked with glutaraldehyde, then layered and cut into 2 mm gel film particles. Both immobilized preparations have been successfully used in the pharmaceutical industry since 1978 to produce 6-APA, and later, 7-ADCA (Zhang, 1982; Wang et al., 1982).

524. As an alternative to the use of preformed polymers, Boehringer Mannheim has developed a method for immobilization of penicillin amidase in which the enzyme is derivatized with epoxides derived from a vinyl group, the soluble protein derivative being subsequently copolymerized with materials like acrylamide as carriers. The size of the particles and their physical properties have a bearing not only on agitation in the reactor but also on the separation of the catalyst by sedimentation or filtration (Konechy, 1984).

525. Immobilization of penicillin amidase by the Snamprogetti method is as follows (Giacobbe et al., 1978). Ten kg of a solution of the enzyme containing glycerol is buffered with phosphate to pH 8, is added to a solution of 5 kg of cellulose triacetate in 71.4 liters of methylene chloride at 4°C with stirring. The emulsion that is formed is extruded through a spinneret into a coagulation bath containing toluene. The fibers formed are then dried to remove all organic solvents. Penicillin amidase as well as the other proteins present in the enzyme preparation are physically entrapped within the microcavities of the porous fibers to yield 20-30% of the initial enzyme activity. One kg of dry fibers produces about 240 kg of 6-APA for a period of more than 4 months (6-APA Process. Plant Description and Chemical Consumption, Snamprogetti, 1977).

526. Genin, a Mexican company, announced in 1982 that it was going to make immobilized penicillin amidase by entrapment in kappa-carrageenan gel of genetically engineered E. coli that overproduced the enzyme. The method avoided disrupting the E. coli cells to extract the amidase. A leader of the research project announced that the E. coli strain was the subject of an international patent application (McGraw Hill's Biotechnology Newswatch, 1982a).

527. Rohm GmbH (F.R.G.) has developed "EUPERGIT - Penicillin-amidase", penicillin amidase from E. coli covalently bonded to EUPERGIT-C, which is a macroporous beaded polymer composed of methacrylamide, N-methylen-bis- methacrylamide and allylglycidyl ether (Kramer & Lehmann, 1984). The beaded shape of the catalyst and its mechanical rigidity allows easy filtration: emptying times of about 15-45 min were reported for 1500 liters from a batch reactor (Kramer & Lehmann, 1984).

3.     Technological Characteristics of Processes

528. Toyo Jozo used immobilized penicillin amidase in a column reactor that is composed of six layers or packs of enzyme bed. After about 30 batch reactions, a spent pack is removed from the inlet side of the reactor column and a new pack is added to the outlet side. In this way the reaction can be carried out by batchwise recycling of the penicillin G solution through the reaction column at a temperature of 30-36°C. Constant pH is maintained by automatic addition of 4 N NaOH solution. After batchwise reaction, the reaction mixture is transferred to a crystallization vessel and methanol (about half the volume of total reaction mixture) is added. 6-APA is precipitated from the solution by adjusting pH with 6 N hydrochloric acid. The precipitate is filtered, washed with methanol and dried in vacuo. These steps give a purity of more than 98% for 6-APA, and a total yield of 86% of theoretical.

529. In the Toyo Jozo process, the conditions are as follows. One batch of reaction gives 125 kg of 6-APA per 3-3.5 hr recycling period. Two batches of reaction are carried out per day. The main equipment of the plant includes: a jacketed 5,000-liter vessel as a reservoir; a 200-liter tank of 4 N NaOH connected to an automatic valve for pH regulation; a 198-liter reactor column that is composed of 6 layers of the enzyme-bed; a recycling pump with a capacity of 100,000 liters/hr; two 7,000-liter jacketed vessels as crystallizers; a vacuum evaporator and a filter press.

530. Snamprogetti performs the hydrolytic reaction batchwise, recycling through a column packed with the fiber-entrapped penicillin amidase (strings made of these fibers are assembled parallel to the axis of a tube), a water solution of penicillin or cephalosporin (6-12%, w/v) at a temperature of 37°C and a pH value of about 8.0 that is kept constant by continuous automatic addition of an alkaline solution. Once a degree of hydrolysis of 98% is reached, the reaction mixture is conveyed to the 6-APA or 7-ADCA recovery tank, and a fresh antibiotic solution is fed into the reactor. The product is precipitated by adjusting the pH of the reaction mixture to the isoelectric point (pH 4.2) in the presence of an organic solvent miscible with water; the precipitate is collected by filtration, washed, and finally dried under vacuum. The purity of the resulting 6-APA is usually higher than 96-98%, and the overall yield is between 85 and 90% (Marconi & Morisi, 1979). Snamprogetti indicated that the company's plant operates for 15 hr daily and processes 4 batches lasting 3 hr each. Half an hour is needed after each batch to discharge the hydrolyzed penicillin solution and start a new batch (6-APA Process. Plant Description and Chemical Consumption, Snamprogetti, 1977).

531. Snamprogetti indicated that the life of penicillin amidase immobilized in fibers under operating conditions is strictly dependent on the purity of cephalosporin G when 7-ADCA is the product of enzymatic conversion. Provided that the purity of cephalosporin is higher than 95%, it was estimated than one kg of enzyme fibers (dry basis) produces about 150 kg of 7-ADCA. The overall yield of 7-ADCA is 85-90% (7-ADCA Process. Plant Description and Chemical Consumption, Snamprogetti, 1977).

532. The equipment for the 12 ton/year Snamprogetti plant includes: a 40-liter tank for a soda solution; two stainless steel stirred jacketed 300-liter vessels; an enzymic stainless steel 100-liter reactor containing penicillin amidase fibers; a recycling centrifugal pump with a capacity of 15,000 liters/hr; a 100-liter tank for HCl solution; one NaOH solution pump with a capacity of 12 l/hr; a stirred stainless 2,000-liter vessel; one centrifugal separator with a basket capacity of 70 kg; and a vacuum dryer operating at a maximum temperature of 40°C (6-APA Process. Plant Description and Chemical Consumption, Snamprogetti, 1977).

533. In the Tanabe Seiyaku Co. process, a solution of 0.05 M penicillin G in 0.01 M borate-phosphate buffer (pH 8.5) is passed through the column packed with E. coli cells immobilized in polyacrylamide gel at 30°C and a space velocity of 0.12. From the column effluent, 6-APA is obtained in about 80% yield based on starting penicillin G (Chibata, 1978b). The half-life of the immobilized catalyst under the operating conditions is 42 days at 30°C and 17 days at 40°C.

534. In the Chinese industrial process (see par. 523) after 285 cycles of 6-APA production over 7.5 months, the hydrolytic rate was unchanged (Wang et al., 1982). According to another source (Zhang, 1982), the immobilized cells column for 7-ADCA production was used 23 times over 30 days without a decrease in activity; a similar column was operated on an industrial scale 72 times over 3 months.

535. An industrial plant for 6-APA production with immobilized disrupted bacterial cells containing penicillin amidase has been in use since 1979 in Slovenska Lupca (Czechoslovakia). The half-life of the crosslinked catalyst (see par. 522) is a minimum of 500 2-hr cycles in a batch reactor at 37°C, pH 7.8.

536. According to a Genin company representative (par. 526), genetically engineered E. coli cells entrapped in carrageenan give a 6-APA yield of 90% or higher at 6-APA concentrations up to 70 g/l. In their process penicillin G is pumped down the column of a packed bed reactor. In Genin's scaled-up pilot plant the enzymatic reaction has been carried out with 3,000 liters of working volume, and the specific activity of the cells is comparable to that obtained in an 8-liter reactor (McGraw-Hill's Biotechnology Newswatch, 1982a).

537. Hindustan Antibiotics Ltd. described their pilot plant producing 6-APA with penicillin amidase covalently bound to modified cellulose (Borkar et al., 1978). The enzymatic reaction proceeds in a continuous stirred 100-liter reactor, where the penicillin G solution is added to a 50 g/l enzyme suspension at a ratio of 2:1 (v/v), and water is added to the mixture at a ratio of 10:3 (v/v). The reaction mixture was kept stirred at 40°C during the course of the reaction and pH was maintained between 7.5 to 7.8 by addition of alkali. The quantity of bound enzyme used for conversion of 4.6 kg of penicillin G was 3.6 kg (containing 371 g of protein).

538. A fully automated computer-controlled batch reactor for 6-APA production is described (Dunn et al., 1985), where penicillin amidase immobilized on Eupergit C, made by Rohm Pharma, acts as a catalyst (see par. 527). The reactor performed more than 400 reaction cycles with a single batch of catalyst, and the productivity of the enzyme was reported as a minimum of 20 tons of 6-APA, isolated in 99% purity, per 100 kg of catalyst. One cycle runs for 80-120 min reaction time with 8% penicillin G (potassium salt) and 8% wet catalyst, with a conversion rate of 98-99% and a product yield of 50% based on the weight of penicillin G (potassium salt). With an average reaction time of 100 min, a filling time of 20 min and an emptying time of 15-45 min (see par. 527), a complete cycle takes about 3 hrs. Thus, in a 24-hr operation, 8 batch cycles can be carried out (Kramer & Lehmann, 1984).

539. The industrial process in Russia presently operates with immobilized penicillin amidase (par. 522) that performs about 700 reaction cycles over its useful life, as compared to 300 in the late 1970's and to 100 in the early 1970's. The total yield of 6-APA is 86-88% (Bartoshevich et al., 1986).

4.     Economic Estimates

540. Economic estimates from Snamprogetti (Italy) for the production of 6-APA integrated with the manufacture of penicillin G itself (Marconi & Morisi, 1979) indicate that a higher overall yield of 6-APA is obtained by omitting the crystallization of penicillin G. Shown below is a comparison of the conventional process (penicillin amidase immobilized in fibers) and the chemical method with this integrated method. The cost of the integrated process is lowest because the cost of utilities, chemicals, and labor encountered in the crystallization of penicillin G is absent. The calculations are based on a plant capacity of 40 tons/year and a penicillin G price of $14.35/BU.

541. Economic estimates showed that the application of immobilized penicillin amidase on an industrial scale in the USSR saved 90 million rubles (US $130 million that time) for the period from 1978-1985 (Bartoshevich et al., 1986).

5.     Scale of the Processes

542. 6-APA, an important intermediate in the synthesis of semisynthetic penicillin, is currently produced by Astra in Sweden, Bayer in West Germany, Beecham in England, Minmedbioprom in the U.S.S.R., Novo in Denmark, Spofa in Czechoslovakia, Squibb in the U.S.A., Toyo Jozo in Japan, by an industrial plant in China, and possibly, some others.

543. According to published data, Toyo Jozo in Japan produces 5 tons of 6-APA per month; Spofa, produces 30 tons of 6-APA per year in its "Biotika" plant in Slovenska Lupca. Pilot plants have also been running for a long time at Snamprogetti in Italy, Tanabe Seiyaku Co. in Japan, Hindustan Antibiotics Ltd. in India, Genin in Mexico. All the 6-APA produced in the Soviet Union and Japan is made with immobilized penicillin amidase. According to Poulsen (1985), approximately 4500 ton/year of 6-APA is produced worldwide with an estimated enzyme consumption of 4-5 ton/year.

E.     USE OF IMMOBILIZED LACTASE IN DAIRY PROCESSING

544. The use of lactase (ß-galactosidase) in the dairy industry is one of the most promising commercial uses of immobilized enzymes. Lactase hydrolyzes lactose, the principal carbohydrate of milk, a sugar with poor solubility properties and a relatively low degree of sweetness, to galactose. The relative sweetness of lactose is 20-40 under various conditions, whereas those of glucose and common sugar (sucrose) are 70-75 and 100, respectively. The hydrolysis of lactose offers some advantages in a dairy product. First, it improves the digestibility of lactose. It is well known that some infants, adults, and some ethnic groups are unable to digest lactose probably because of the lack of lactase in the mucosa of the small intestine (Marconi & Morisi, 1979). Consuming milk products causes abdominal pain, cramps, diarrhea, and general intestinal upset. This problem is circumvented if lactose in the product is hydrolyzed by lactase to the readily utilizable sugars, glucose and galactose (Porro et al., 1992; Bakken et al., 1992). Moreover, the low solubility of lactose can result in its crystallization in dairy food with concomitant appearance of a sandy or gritty texture, deposit formation and protein destabilization. Products that suffer most from such crystallization are condensed milk, sweetened condensed milk, condensed and dried wheys, ice cream and frozen milks. Using lactase to process such products could reduce lactose concentrations to acceptable values (Finocchiaro et al., 1980). Another advantage is the possibility of preparing or improving new food and dairy products with higher solubility characteristics and a higher degree of sweetness.

545. Moreover, lactose, a major byproduct of cheese manufacturing, causes many pollution problems, and its utilization becomes more and more interesting because of the need for environmental protection. Modern dairy processing plants sometimes produce over 1 million liters of whey daily, and traditional means of whey disposal are no longer acceptable. Because whey and ultrafiltration permeate from the manufacture of cheese and whey protein have very high biochemical oxygen demands of about 50 gm O₂/l, they are hazardous wastes that cannot be discharged without expensive treatment (Prenosil et al., 1984). The upgrading of whey and ultrafiltration permeate can be accomplished by enzymatic hydrolysis of lactose by lactase.

546. By-production of whey in the U.S.A. alone exceeds 15 million tons/yr (Greenberg & Mahoney, 1981), of which only one-half is further processed. France produces 7 million tons/yr of whey, of which protein constitutes 45-50 thousand tons, and lactose, 330-350 thousand tons (Lenoir, 1981). The British Food Association reported that only 24% of 103 surveyed food companies dealt with the utilization of dairy wastes (Fullbrook, 1983). The economic potential of hydrolyzed lactose syrup in Britain has been calculated at approximately $35 million per year (Greenberg & Mahoney, 1981). This estimate is based on the relative sweetness of the sugars, a 70% conversion of lactose to monosaccharides and a value of $0.22/kg for sucrose. Clearly as the price of sucrose increases, the economic potential of hydrolyzed lactose increases.

1.     Background

547. Cheese whey, the major byproduct of the dairy industry, contains about 6.5% solids of which 70% (4-5% of dry mass) is lactose. Lactose can be hydrolyzed with ion exchange resins, strong mineral acids or enzymes. Ion exchange resins and strong acids tend to destroy the whey protein by irreversible denaturation. The use of hydrolyzing enzymes lowers the lactose content without adversely affecting the proteins and other components of whey. Through immobilization, enzymes can be repeatedly used for lower operating and separation costs. Milk treated by enzymatic methods retains its original nutritional value because glucose and galactose, the products of lactose hydrolysis, are not removed.

548. Lactase hydrolyzes the lactose in whey or milk to equal quantities of glucose and galactose. Compared with the sweetness of sucrose, which has a standard reference value of 100, the glucose-galactose syrup has a value of about 70, which is similar to that of glucose and considerably sweeter than lactose itself (see par. 399 and 544). Hydrolyzed lactose solutions can be evaporated to concentrated syrups, since glucose and galactose, unlike lactose, are both very soluble. Syrups of 70-80% sugar content are fluid and have a good shelf life.

549. Only 25% of the lactose must be hydrolyzed to prevent crystallization during prolonged storage, because small amounts of glucose and galactose inhibit the formation of lactose crystals. This avoids a sandy texture in ice cream and frozen deserts due to crystallized sugar. The need for stabilizers is thereby eliminated.

2.     Commercial Preparations of Immobilized Lactase

550. Lactases occur rather widely in nature and have been isolated from animals, plants, and microorganisms. Enzymes of plant and animal origin are of little commercial value but several microbial lactases are technologically important. The major enzymes of commercial interest are those from the yeasts Kluyveromyces or Saccharomyces lactis, Kluyveromyces or Saccharomyces fragilis, and the fungi Aspergillus niger and A. oryzae.

551. Lactases are rather expensive. Typical prices for the partially purified enzymes are: K. lactis $1.02 per mkatal, A. niger $7.56 per mkatal and E. coli $660 per mkatal, assuming that 1 mkatal hydrolyzes 1 mmole or 0.23 g of lactose per second under optimum conditions. These prices make it essential that the enzymes be immobilized if they are to be used in large-scale manufacturing processes (Greenberg & Mahoney, 1981).

552. The fungal lactases (from Aspergillus) with acid pH optima (2.5-4.5) are especially suitable for hydrolysis of lactose in acid whey, but they are more sensitive than others to product inhibition by galactose. The yeast enzymes from Kluyveromyces or Saccharomyces sp. have neutral pH optima (6-7) making them suitable for the hydrolysis of lactose in milk or sweet whey, and they are also less inhibited by galactose. However, they are less thermostable than the fungal enzymes. Moreover, the use of the yeast lactases is limited by their requirement for activating ions to reach full activity. Of the bacterial lactases only that from E. coli (pH optima 6.5-7.5) is available commercially, in both crude and pure form. It is not used, however, in food processing because of its cost and the fact that it toxicity is a problem with crude extracts of coliforms.

553. Not all lactase sources are acceptable or have GRAS (generally recognized as safe) status when the enzyme is going to be used in food systems. Lactase preparations from A. niger, A. oryzae and from Saccharomyces sp. (lactis or fragilis) are considered safe because those sources already have a history of safe use and have been subjected to numerous tests (Gekas & Lopez-Leiva, 1985). Currently GRAS status is valid for A. niger, A. oryzae, K. fragilis and K. lactis (Gekas & Lopez-Leiva, 1985; Food Technol., 1985b) for use in the production of lactase-treated milk and lactose-reduced milk.

554. Because of the rather high price of lactases as compared to the low value of the waste product whey, the direct addition of lactase to the substrate is economically prohibitive. This problem is overcome by immobilization of the enzyme with any of a variety of techniques and support carriers. However, from a list of lactase immobilized commercial preparations shown below a preference for some specific methods and immobilizing agents is quite obvious: adsorption and crosslinking with glutaraldehyde, and covalent coupling with a solid carrier.

555. Glutaraldehyde as a bifunctional crosslinking agent is usually accepted in the food industry and moreover displays beneficial disinfectant properties (Gekas & Lopez-Leiva, 1985). Phenol-formaldehyde resin is often the carrier in this method. It is used in scaled-up systems in the form of fluidized bed reactors. Another common carrier related to the covalent coupling method is porous silica in the form of beads. Coupling of the activated beads to the enzyme is commonly achieved again with glutaraldehyde. A similar technique is used with other inorganic carriers like porous alumina and ferrites.

556. Rohm GmbH (Darmstadt, FRG) has used a macroporous, bead-shaped carrier with a porosity of 3-4 mg/g for A. oryzae lactase immobilization. The enzyme was bound covalently by oxirane groups. Because of their rigid structure the 0.1-0.3 mm diameter beads are pressure stable and show "good flow properties" (Plainer & Sprossler, 1982). The immobilized lactase has the brand name Plexazym LA. The authors claim (Sprossler & Plainer, 1983) that the immobilization method produces a much higher immobilization yield than standard immobilization techniques (adsorption, gel entrapment, microencapsulation). The beads are hard and resistant to compression, similar to an ion-exchange resin. According to Rohm GmbH, 1 kg of this immobilized lactase can produce 80% hydrolysis of 960 liters of whole whey within 24 hr (Sprossler & Plainer, 1983).

557. Immobilized A. oryzae lactase was produced recently at the Technisch-Chemisches Laboratorium, RTH Zentrum in Zurich, Switzerland. The enzyme is adsorbed on Duolite S-761, ion-exchange resin, and crosslinked by glutaraldehyde. The price per unit activity of immobilized enzyme, including the carrier price and enzyme deactivation, is optimal at a level of 20 mg enzyme/g Duolite (Prenosil et al., 1984). The half-life for the immobilized enzyme is 31 days under operating conditions (40°C, pilot plant, deionized whey).

558. Snamprogetti used K. lactis lactase as the purified commercial preparation Maxilact from Gist Brocades, Delft, Holland. To prepare the fiber-entrapped lactase, the enzyme powder is suspended in pH 7.2 buffer containing 1 mM EDTA and 2 mM MgSO₄, and allowed to stand for 24 hr at 4°C with gentle stirring. Insoluble material is removed by centrifugation and a 30% (w/w) solution of clarified extract in glycerol is prepared. The solution is entrapped in cellulose triacetate according to the standard procedure of Snamprogetti (see par. 406).

559. Lehigh Valley Dairy (Allentown, Pennsylvania) in their pilot plant has used an immobilized lactase-on-alumina catalyst (Coughlin et al., 1978). The catalyst comprised A. niger lactase immobilized on 150-micron diameter porous alumina particles with a pore diameter of about 4,000 A and a surface area of about 4 m²/g and showed "good fluidization characteristics and long term mechanical, biological, and chemical stability during operation" (Coughlin et al., 1978). Less then 0.5% of the catalyst was lost by attrition and elutriation from a fluidized reactor over a 2-wk period.

560. Corning Glass Works of Corning, NY recently patented lactase immobilized on microporous ceramic beads that showed "improved immobilized enzyme stability ... by a factor of about 300" (U.S. Pat. 4,409,247). Earlier preparations of the immobilized enzyme comprised A. niger lactase chemically bound to silane functional groups on the treated porous glass bead surface. Such immobilized systems having up to 50 mg of enzyme bound to one gram of porous glass generally retained 30-80% the activity of the free enzyme. Each gram of lactase-coated glass could hydrolyze about 6 grams of lactose per hour at 35°C (Canad. Chem. Process., 1977).

561. Sumitomo Chemical and Shin Nippon Chemical Industries have developed "high-purity immobilized lactase for use in hydrolysis of milk sugar" (Genet. Engin. Biotechnol. Mon., 1984a).

3.     Technological Characteristics of the Processes

       Production of low lactose milk

562. The enzymatic hydrolysis of lactose can be achieved either with soluble enzymes, usually in batch fermentation processes, or with immobilized enzymes (Bakken et al., 1992; Porro et al., 1992). Although the subject of this review is immobilized enzyme systems, the following table represents some data on soluble lactase systems that have been used to diminish lactose content in milk. High residence times are usually necessary for such systems (several hours to several weeks) as compared with those for immobilized enzymes (usually several minutes).

563. Currently two companies use large-scale processing of milk with soluble lactase (Gekas & Lopez-Leiva, 1985). The Valio Laboratory's system uses K. lactis lactase ("Maxilact" 40,000) in a ratio of 0.4 kg/m³ to treat 40,000 of UHT milk per day. Tetra Pak company uses a soluble lactase technology in which milk produced and bottled aseptically is hydrolyzed by "Lactozym" during storage at room temperature; 70-80% hydrolysis occurs in one month with only 0.01-0.03 ml of enzyme per liter of milk.

564. The only industrial process for the treatment of milk with an immobilized enzyme system has been operating at Centrale del Latte of Milan, Italy since 1977, utilizing the Snamprogetti technology. The immobilized enzyme is lactase from yeast (a purified commercial preparation Maxilact, from Gist-Brocades, Delft, Holland) entrapped in triacetate cellulose fibers (see par. 554, 558), and the reaction is performed batchwise at low temperature. The processed milk, after having reached the desired degree of hydrolysis of lactose, is separated from the enzyme fibers, sterilized and finally distributed.

565. The flow chart for the Snamprogetti pilot plant that preceded the industrial plant is shown in Figure 2. This plant has been used for the hydrolysis of milk lactose since 1974. It comprises essentially a 300-l stainless steel receiving tank, a continuous sterilizer (Cherry Burrel, Pilot, Cherry Co., Cedar Rapids, Iowa, USA), a 20-liter jacketed glass column (internal diameter, 15 cm; height, 56 cm) containing the entrapped lactase (4 kg of wet fibers), a peristaltic pump, and a 500-liter aseptic jacketed tank. The glass column and the aseptic tank are thermostatted at 4-7°C. The lactase fibers are placed parallel to the column axis and fixed at the two extremities. Raw skimmed milk, processed in the sterilizer at a sterilization temperature of 142°C for 3 sec, is rapidly cooled to 4 to 7°C, and pumped to an aseptic tank through the lactase column at a flow rate of 7 l/min. Recirculation is continued until the desired lactose conversion is obtained. No contamination of treated milk occurs, e.g. from the leakage of enzyme from fibers (Marconi, 1978). Loss of activity from a 0.5-kg sample of enzyme fiber that processed about 10,000 liters of milk in 50 batches is less than 10% (Marconi & Morisi, 1979).

566. Because microbial contamination in this process may cause serious problems, care is taken to assemble the pilot plant so that all piping and related plant equipment, except the enzyme reactor, can be steam-sterilized. This kept the bacterial count in the product within the limits prescribed for pasteurized milk (in Italy less than 30,000 per ml). In fact, the total bacterial count at 32°C in the product was always less than 10,000 per milliliter (Marconi, 1978; Marconi & Morisi, 1979). As regards the quality of the product obtained, the tests performed demonstrated that "the organoleptic characteristics remain unaltered except for a very slight increase in sweetness" (Marconi, 1978). As regards the shelf-life of the product, it "can be stored at least 3-4 months if kept in the refrigerator at 4°C" (Marconi, 1978).

567. Rohm GmbH developed a pilot plant for processing whole milk (3.5% fat, 4.6% lactose) with Plexazym LA (see par. 459). Because of the optimal heat stability of the immobilized enzyme at neutral pH, the packed bed reactor is operated at 55°C. At a space velocity of 20 hr^-1 a productivity of 13 tons of milk/kg Plexazym is reached in 30 days. The packed bed is not plugged by droplets of the fatty emulsion or by the casein micelles of milk because of the morphological properties of the support (Plainer & Sprossler, 1982).

       Processing of whey

568. Before 1980 processes using immobilized lactase were developed mainly on a pilot plant scale. Probably the first pilot plant was that of Valio Cooperative Dairies Association in Finland, who began in 1976 operating their 5 ton/day continuous column bioreactor for cheese whey lactose hydrolysis with phenol-formaldehyde resin-bound Aspergillus niger lactase (Linko, 1985). This technology was developed by Valio in cooperation with the Technical Research Centre of Finland. Since 1978 whey has been processed on an industrial scale both to improve the digestibility of pig feed and to obtain hydrolyzed lactose products for various food applications (Linko, 1985).

569. One of the first pilot plants was developed jointly by Lehigh University (Bethlehem, Pennsylvania), the University of Connecticut (Storrs, Connecticut), and Lehigh Valley Dairy (Allentown, Pennsylvania). The pilot plant hydrolysis unit comprises two 2 m fluidized-bed reactors of 7.6 cm I.D., each containing about 4 kg of catalyst (A. niger lactase adsorbed on a porous alumina and crosslinked with glutaraldehyde) and having a maximum daily throughput capacity of about 160 liters of raw (unfiltered) acidic cheese whey. Both columns are equipped with conical inlet sections above quick-acting ball valves that could start or stop liquid flow without loss of catalyst from the bottom of a column. Such conical inlets provide good liquid distribution and minimize channelling during operation. Moreover, ultrafiltration and ion exchange capabilities are included in the pilot plant in a scheme that permits any sequence of one or more of the three operations. The flow diagram is shown in Figure 3 (Coughlin et al., 1978).

570. With reference to Figure 3, the whey is first heated to the desired hydrolysis temperature (45 to 60°C) in a stainless steel shell and tube heat exchanger (American Standard SSCP-D3014) from which it is pumped through the fluidized-bed immobilized enzyme hydrolyzers at a flow rate of 0.5 l/min and at pH 4.9. Hydrolyzed whey flows through a particle trap to remove any catalyst that might elutriate, and further through an ultrafiltration and/or ion exchange unit if post-hydrolysis treatment is desired. Very little of the catalyst is eluted; a liquid fluidized bed is characterized by very gentle agitation of the catalyst particles and a very sharp liquid catalyst interface at the top of the bed. If pre-hydrolysis treatment is desired, raw whey can be ultrafiltered and/or demineralized. Ultrafiltration is achieved by a Romicon (1115-45-XM50) filter, which has a molecular weight cutoff of 50,000 and an average flux of about 600 l/m²/day and permits recovery of valuable whey protein. Ion exchange is used for demineralization. The pilot plant demineralization consists of an anion exchange column containing Duolite ES-340 resin and a cation column containing Duolite C-20 resin (Coughlin et al., 1978).

571. When raw, unfiltered whey is fed directly to the hydrolyzer, some difficulty is experienced in maintaining constant temperature during these experiments: there is a slow but discernable decrease in conversion that could not be completely explained by the temperature variations experienced. Sanitization with Iosan involved washing the bed in the expanded state for approximately 1 hr; an increase in activity (and hence enhanced conversion) is observed directly after sanitization, which is, however, lost soon after the feed of raw whey is started again, and the slow decrease in conversion continues. Based on the pilot plant experiments a plant producing 50 tons of whey/day is projected (Coughlin et al., 1978), taking into account the following aspects of the process:
       -lactose conversion 70%
       -catalyst half-life 60 days
       -total catalyst required 710 kg
       -reactor diameter 51 cm
       -reactor height 6.9 m
Economic estimates of plant production are given in paragraphs 593-595.

572. Snamprogetti used its pilot plant with yeast lactase entrapped in cellulose triacetate fibers (see par. 558) for the hydrolysis of lactose in sweet whey (Marconi & Morisi, 1979). Proteins are separated from the whey by ultrafiltration; the permeate, containing the mineral salts and lactose, is continuously fed to the enzyme reactor at 60°C, pH 4.5. The effluent from the reactor is demineralized by ion-exchange treatment and concentrated to 80% total solid. The following parameters for the 100 liters of whey/day reactor have been estimated:
       -lactose conversion 95%
       -catalyst average life 90 days
       -total catalyst required 1 kg (dry mass)
       -reactor diameter 8 cm
       -reactor height 1 m
       -temperature 60°C
       -flow rate 16 l/hr

573. Snamprogetti also developed a 1000-liter reactor for the hydrolysis of lactose in raw, untreated sweet whey. The stainless steel reactor, 100 cm in height and 120 cm in diameter, and containing 5 kg (dry weight) of entrapped yeast lactase, operated at 25°C, pH 6.5-7.0 for 3.5 hr to reach 95% conversion of lactose. Average life for the catalyst is 90 days.

574. According to Corning Glass Works' data (Ford & Pitcher, 1975), two important variables that affect the observed half-life of immobilized lactase under operating conditions are feed composition and temperature. From the table shown below it is seen that the higher the feed purity, the longer the half-life. The normal salt content of the whey feed is detrimental to enzyme half-life although it affects activity insignificantly. Removal of 90 to 95% of the salt in deproteinized acid whey results in dramatically improved enzyme stability.

575. In fact, a half-life of 62 days at 50°C is determined experimentally for the hydrolysis of lactose in deproteinized, de-ashed acid whey in the pilot plant's reactor (Fig. 4), a vertical cylindrical column of 10 cm in diameter and 70 cm in enzyme bed height (Ford & Pitcher, 1975). The column operated in downflow mode. Figure 5 indicates the general principle of operation of the modified hydrolysis unit (Dohan et al., 1980). The process is run at constant throughput and constant conversion, and the temperature is slightly raised to compensate for the thermal de-activation of the composite. Cleaning is achieved by backflushing the bed with dilute acetic acid. The estimated life of such a system is around two years under laboratory conditions (Dohan et al., 1980).

576. Rohm GmbH used a pilot plant with a packed bed reactor containing Plexazym LA (see par. 554) to hydrolyze lactose in filtered acid whey at pH 4.5 with a space velocity of 50 hr^-1 (Plainer & Sprossler, 1982). The rate of hydrolysis decreases from an initial value of 100% to about 90% over 60 days of operation. After 100 days of operation, the degree of hydrolysis is still 80%. Seventy tons of whey/kg Plexazym LA is reached in 60 days. The pilot reactor by Rohm GmbH contains 1 kg of immobilized lactase. A day's run consists of 20 hr of hydrolysis (960 liters of whey is typically hydrolyzed) and 4 hr of cleaning with a 0.1% solution of a quaternary ammonium salt (Sprossler & Plainer, 1983).

577. A fully automated pilot plant "Lactohyd" for the enzymatic hydrolysis of lactose in sweet whey is under operation at the Technisch-Chemisches Laboratorium, EHT Zentrum, Zurich (Switzerland), and its simplified flow diagram is shown in Figure 6. The plant contains fixed bed reactors with a total capacity of 800-1000 liters of whey/day (Prenosil et al., 1984). A. niger (then later A. oryzae) lactase immobilized on the ion exchange resin, Duolite S-761, by adsorption and crosslinking with glutaraldehyde (see par. 557) is used as the enzyme catalyst. "Quickfit" glass is used for easy plant construction and visual inspection under operating conditions. Four fixed bed reactors are installed so that every combination of connections is possible. Three of them are used for hydrolysis with A. niger lactase, with the fourth on standby, waiting to be exchanged with any reactor in need of in situ regeneration. This reactor in turn is put on standby after its regeneration. In fact, only one reactor - instead of three used with enzyme from A. niger - is required to achieve 90-95% lactose conversion with A. oryzae lactase, which is more active (Prenosil et al., 1984).

578. The pilot plant "Lactohyd" has been operated with catalyst regeneration and re-immobilization performed in situ. During the plant's operation, microbial contamination "was of no problem" (Prenosil et al., 1984). The basic features of the pilot plant are listed below:

579. In 1978 at the Milk Marketing Board, England and Wales Technical Division (Crudgington, Telford Salop, U.K.) facility a semi-industrial plant for the enzymatic hydrolysis of lactose in cheddar sweet whey was put in operation (Dohan et al., 1980). Analysis of production of the plant was used to develop the application of hydrolyzed lactose syrups. A. niger lactase is immobilized according to Corning Glass Works' method, that is by covalent binding to a controlled-pore silica carrier with the silane glutaraldehyde technique (see par. 554). The plant operated continuously five days a week and processed about 30,000 liters of whey a week, resulting in about 1.7 tons of syrup (1200 kg of solids).

580. The whey pathway in the plant is as follows. Raw whey is pasteurized and sent to an ultrafiltration plant. The permeate fraction is then de-ashed by passage over an ion-exchange demineralization unit, and held in a standard dairy tank for storage. The hydrolysis plant operates at 360 l/hr and achieves 80% hydrolysis. The hydrolysis plant is entirely automatic and operates 16 to 20 hours per day. It is monitored by a series of alarms and automatic shut-down procedures. When no more feed is available at the end of a run, the plant terminates production automatically and prepares for the cleaning cycle. While the system is cleaned by standard dairy cleaners, the lactase bed is fluidized every day for half an hour with dilute acetic acid. The temperature varies from 32 to 39°C during half a year operation, and the enzymatic system is very stable (Dohan et al., 1980).

581. A similar pilot plant ran concurrently at Union Laitierre Normande in Conde/Vire, France, with the main goal to examine other feeds and operating conditions (Dohan et al., 1980). The pilot facility installed there has a capacity of 500 l/hr at 80% conversion. The plant operates four days a week for 6 to 8 hours per day. Daily cleaning takes 2 to 3 hours. During these runs different kinds of whey feed are used (casein and whey, sweet whey, etc.), and various models of whey pretreatment are studied, electrodialysis and partial demineralization in particular (Dohan et al., 1980).

582. The Nutrisearch Co., a joint venture of the Kroger Company and Corning Glass Works, which was begun in 1982 (see par. 600) built a new $15 million plant in Winchester (Kentucky, USA) that uses the immobilized enzyme technology of Corning and continuous fermentation technology of Kroger (Mans, 1984). The plant operates in three stages, that is protein removal from cottage cheese whey in an ultrafiltration system, hydrolysis of lactose in the permeate with immobilized lactase, and fermentation of the resultant simpler sugars to produce baker's yeast. In each 20 hour operating day the Nutrisearch plant converts 400 tons of raw cottage cheese whey into protein concentrate and baker's yeast. The plant is located next to Kroger's Winchester Farms Dairy, from which raw whey is pumped through an underground pipe and stored in 120 m³ raw whey tanks. Whey is also trucked in from other Kroger dairies in the midwest. Reverse osmosis systems have been installed at these dairies to remove one-half to two-thirds of the water from the whey to reduce shipping costs. When whey is received, it is centrifuged and sterilized in a typical dairy type high temperature-short time plate pasteurizer. The pasteurized whey is stored at 55°C in 160 m³ tanks (Mans, 1984).

583. Before the enzymatic hydrolysis step, protein is removed from the whey in a four-stage ultrafiltration system. The permeate, containing 95% of the liquid and lactose, is pumped to three 200 m³ tanks for in-process storage. From these, the stream, which contains about 5% lactose by weight, is split, and pumped to the two hydrolysis columns. The retentate from ultrafiltration, which contains most of the whey protein, leaves the unit at approximately 20% total solids. About two-thirds of these solids are protein, 30% is lactose and the reminder is minerals (Mans, 1984).

584. The hydrolysis columns are 1 m diameter, 5 m high stainless steel pressure vessels filled with immobilized enzyme (30/45 mesh particles) that operate in the downflow mode. Temperature in the columns is controlled at about 20°C. Product (glucose-galactose syrup) from the hydrolysis columns is pumped into 80 m³ storage tanks and then directed to the fermentation stage (Mans, 1984).

585. Summed up below are data available on technological applications of immobilized lactase.

4.     Economic Estimates

586. Two main methods of lactose hydrolysis exist: the acid (catalytic) and enzymatic methods. The first method is characterized by very severe pH and temperature conditions (pH 1-2, temperature 100-150°C), while the second, as shown in the preceding paragraphs, is carried out under considerably milder conditions (pH 3.5-8, temperature 5-60°C). The literature contains a comparison of the economics of these two principal whey hydrolysis systems, with the Corning Glass immobilized enzyme process and the hot acid resin process developed by Permutit (Gekas & Lopez-Leiva, 1985) as examples. The study shows that great difficulties arise in attempting a straight comparison between the two systems. However, both systems appear to be viable if the sucrose price is established at US $353/ton (1980).

587. A more recent comparison of the two methods, acid and enzymatic (immobilized), has shown a processing cost of 0.51 Hfl per kg of syrup produced by acid hydrolysis, and 0.73 Hfl per kg of syrup produced by the enzymatic hydrolysis (Gekas & Lopez-Leiva, 1985). A selling price of 0.92 Hfl can be expected. This analysis has been made based on the following data:

The enzymatic product had 77.5% dry matter, while the acid hydrolyzed product had 62.5% dry matter (Gekas & Lopez-Leiva, 1985).

588. The experiences of the Milk Marketing Board (U.K.) and Union Laitierre Normande (France) jointly with Corning Glass Works (USA) in the semi-industrial enzymatic hydrolysis of lactose in various kinds of whey, particularly cheddar sweet whey and casein acid whey (see par. 579-581), led the companies to the following conclusions (Dohan et al., 1980). Under conditions typical in Western Europe, an 80% lactose hydrolysate of permeate can be achieved at a total cost close to 0.5 French Francs per kg of lactose, or 0.02 FF/liter of permeate at 40 g/liter. This cost, based on a typical 200 m³/day plant, includes capital depreciation, labor, chemicals, utilities, enzymes, etc. De-ashing of whey is estimated as 0.25 to 0.80 FF/kg lactose (varies according to level and technology used; 50% by electrodialysis would be at 0.25 FF while total de-ashing by ion-exchange would be at 0.80 FF), and concentration is estimated as 0.5 FF/kg lactose. Taking these fractions together, total cost "at the gate of the 200,000 liter/day plant" of an hydrolyzed lactose syrup is 1.25-1.80 FF/kg lactose, which is "quite attractive" (Dohan et al., 1980).

589. Shown below is the equipment cost estimate for Corning's pilot plant designed to process 4.5 tons of deproteinized acid whey per day. Processing costs include labor and supply costs (as shown below in par. 593), capital or equipment costs taken at 20% annually (including depreciation, maintenance, taxes, etc.) and cost of the immobilized lactase. The total cost, though, does not include the cost of de-ashing or concentration and assumes no cost for deproteinized whey (Ford & Pitcher, 1975).

590. Processing costs in Corning's pilot plant were estimated in the range $22-88 per ton of lactose, depending on plant size, cost for immobilized enzyme, and percent hydrolysis. One particular example is shown below.

591. Reducing the salt level by ion exchange or electrodialysis results in projected costs of $44-110 per ton, including capital costs comparable to those for the hydrolysis system. Similar additional costs would be encountered for concentrating the product from 5% solids to the 50% or higher solids level necessary for sweetener substitution. Overall cost of the sugar product is estimated, according to Ford & Pitcher (1975) to be $180-220 per ton. That price looked attractive, according to Corning Glass Works (Ford & Pitcher, 1975), as compared to the 1974 price for corn syrups ($370/ton, dry basis).

592. For the commercial-scale plant that was announced by Corning Glass Works in 1979 and where deproteinized whey (optimal demineralization) has been continuously treated in a packed-bed reactor followed by vacuum concentration of the hydrolyzed syrup (the product throughput is 200 m³/day), the estimated costs expressed as ¢/kg of hydrolyzed lactose are 9-13¢ for demineralization by ion exchange, 9-11¢ for 80% hydrolysis of lactose, and 7¢ for concentration of syrup to 50-60% solids. A zero value is placed on the deproteinized whey which may be the permeate from an ultrafiltration system (Finocchiaro et al., 1980).

593. Shown below are data by Lehigh/Connecticut Universities and Lehigh Valley Dairy on process economics based on a projected throughput of 45 tons of whey/day (for the main aspects of this basic case study, see par. 571).

594. The next table gives operating and fixed costs for the above 45 tons of whey/day hydrolysis plant; here, depreciation and interest are taken as 15% of the capital investment, which is equivalent to a straight line depreciation over a 20 year lifetime, zero salvage value, and a borrowing cost of 10% simple interest per year (Coughlin et al., 1978).

595. From the table above it can be seen that a major component of hydrolysis cost is the price and lifetime of the immobilized lactase, which have been assumed to be $220/kg and 2 months (Coughlin et al., 1978). As the projected cost for the immobilized lactase is estimated to be $23/kg for production of the enzyme in large quantity (Coughlin et al., 1978), the process will be more profitable in the near future. Besides, even with the current high cost of the enzyme, the overall cost of hydrolysis can be significantly reduced by extending the lifetime of the catalyst. Operating costs and investments for other related unit processes for whey hydrolysis, as well as the daily values that may be realized from whey are summarized in the following table:

596. In this table waste treatment savings means the cost of treating the whey as a waste, a cost that will be saved by utilizing the whey. Then, the hydrolysis of 70% of the lactose in 45 ton/day of whey produces an increase in sweetness equivalent to 897 kg/day of sucrose, if sucrose is valued at $ 0.472/kg, this translates to an added value of $423 as shown in the table (Coughlin et al., 1978).

597. Shown below are economic estimates available in the literature for hydrolysis of lactose in whey on pilot and industrial scales. Although a straight comparison of the different systems is difficult, especially for pilot plants, because of the different bases used for the calculations, different plant sizes, etc, nevertheless, a rough comparison of economic estimates is useful.

598. From the above table it can be concluded that since the cost of corn syrup in Western Europe in the early 1980's was 44-56 cents per kg, hydrolyzed lactose syrup provides a cheaper alternative. In the U.S. the comparison is not as favorable since corn syrup is less costly (22-27¢/kg); however, there are other considerations that may make the process economically viable in the U.S. For example, it may be worthwhile to recover the more valuable whey protein and convert the lactose to hydrolyzed lactose syrup than to pay whey disposal charges. The continuous success of the Nutrisearch Company, now Nutrisearch International Corporation (see par. 560), is an appropriate example in this respect.

5.     Scale of the Processes

599. In the early 1980's, the concept of industrial processes for lactose hydrolysis in dairy products with immobilized enzymes evolved into reality. In paragraph 585 the current state of the technological application of immobilized lactase preparations on pilot and industrial scale is shown. The development of immobilized lactase research and applications is typical of the development of enzyme engineering and reflects generally the basic trend of industrial biotechnology, that is formation of joint venture companies, especially in the United States.

600. In 1982 Corning Glass Works formed two joint venture companies - in the USA and Western Europe - to make and market hydrolyzed whey sugars and protein. Jointly with Britain's Milk Marketing Board, which operates 22 creameries producing half of all the whey in the U.K. - 680 million liters a year (McGraw Hill's Biotechnology Newswatch, 1982b) - Corning Glass Works formed a firm named Specialist Dairy Products, which built and operated a small unit for market development at the Ashton Creamery (McGraw Hill's Biotechnology Newswatch, 1982b). Jointly with the Kroger Company (Cincinnati, USA), Corning has created Nutrisearch Co., which in 1984 started up a $15 million plant in Winchester, Kentucky for processing cottage cheese whey with immobilized lactase in particular (see par. 582-584). The Nutrisearch plant, which is the biggest in the world in the area of enzyme engineering for the dairy industry, converts 400 tons of raw cottage cheese whey per day, or about 130,000 tons/year (Mans, 1984).

601. In March, 1986 Eastman Kodak Company (Rochester, New York) joined the Nutrisearch Co. The new company is being incorporated as Nutrisearch International Corporation with Corning, Kroger, and Kodak as equal partners (Food Technol., 1986c). Currently, Nutrisearch has plants in Winchester (Kentucky), in Maelor (France), and in Wales (U.K.) and additional plant locations will be selected to meet future growth needs. Among products currently made by Nutrisearch are baker's yeast, "cream" yeast for ethanol applications, whey protein concentrates used in egg and milk replacement applications, sweet protein concentrates to replace sweetened condensed milk, and many specialty blends (Food Technol., 1986c).

602. As for the hydrolysis of lactose in milk, low-lactose milk is being produced in Italy on a small scale and finds a market, particularly among those who are somewhat lactose-intolerant. At the end of the 1970's, Centrale del Latte in Milan produced 10 tons of low-lactose milk daily (Marconi & Morisi, 1979; Marconi, 1980). The process relies on the use of sterilized milk, and as a result, the product has a cooked flavor that may not be acceptable in some countries in North America or Europe where consumers are accustomed to the taste of milk given a milder heat treatment. On the other hand, the product is also sweeter and may thereby appeal to some population groups, such as children (Greenberg & Mahoney, 1981).

603. For pilot plants, there are three other processes designed to handle milk: Gist Brocades, Rohm GmbH, and Sumitomo (see par. 585). These are continuous processes with short residence times (Gekas & Lopez-Leiva, 1985).

604. The major industrial application for processing of deproteinized whey is likely to be in the production of hydrolyzed lactose syrups. Such sweet syrups have been used by a number of firms as a source of sugar and, in some cases, of protein in bakery products, confectionery, ice cream, feedstuffs for cattle instead of molasses, fruit drinks and so forth (see par. 544, 548, 549).

605. Hydrolyzed demineralized lactose syrup is produced by Valio Process in Finland. It contains: glucose, 10%; galactose, 20%; lactose, 10%; and protein plus salts, 10%. It is used at 12.3% by weight in an ice cream mix in order to substitute a part of normal sucrose and milk. Various mix formulations have been tested during ice cream manufacture, and the optimum formula is: 11% fat, 10% milk (80% hydrolyzed), 16% sugars (a portion of sucrose has been replaced by corn syrup sweeteners) (Gekas & Lopez-Leiva, 1985).

606. In several cases low-lactose milk, obtained by means of immobilized lactase, has been used in order to accelerate the ripening of cheddar cheese with significant cost savings. 80% hydrolyzed milk was also used in the manufacture of blue cheese. The product showed improved proteolytic activity. An orange flavor beverage prepared from hydrolyzed and deproteinized cheese whey was recommended as a shelf-stable drink for athletes (Gekas & Lopez-Leiva, 1985).

607. According to Poulsen (1985), less than 10,000 tons of d.s. lactose hydrolysates were produced in 1984 (which obviously does not include the Nutrisearch process) with an estimated amount of immobilized enzyme of less than 5 tons/year.