BIOTECHNOLOGY - V. H. Herasymenko - 2006

Part II. Special Biotechnologies

CHAPTER 10. APPLICATION OF IMMOBILIZED ENZYMES IN BIOTECHNOLOGY

10.5. APPLICATION OF BIOTECHNOLOGIES WITH IMMOBILIZED ENZYMES IN THE DAIRY INDUSTRY

Currently, in the dairy industry, biotechnologies utilizing immobilized Enzymes can be employed for removing lactose from milk and its processed products, in cheese-making processes, and for extending the shelf life of milk.

During milk Processing for cheese production, a large amount of whey is generated, which contains small quantities of protein, various salts, and lactose (about 5%). For instance, The production of 1 ton of cheese yields 9 tons of whey. Each ton of whey contains about 5 kg of high-quality protein, B-group Vitamins, a complex of free Amino Acids, and all essential mineral elements, including Calcium and phosphorus. However, the most valuable component of the whey is lactose, with nearly 50 kg present in every ton.

Lactose, a disaccharide or milk sugar, is a valuable raw material for the food and microbiological industries. Due to its low solubility and mild sweetness, it cannot be used in the food industry without prior Treatment. When acted upon by the enzyme lactase or β-galactosidase, it is hydrolyzed into two Monosaccharides—glucose and galactose—which are 1.5 times sweeter than sugar. They are utilized in the food industry, for example, in the production of ice cream with an extended shelf life (up to 4 months) and as a replacement for molasses in obtaining saccharide products.

Whey can be used in the microbiological industry as a substrate for cultivating fodder Yeast strains that exhibit lactase activity (Saccharomyces fragilis, Zygosaccharomyces lactis, Candida pseudotropicalis, and others).

Such processing of dairy whey reduces environmental pollution, as these milk processing byproducts are largely underutilized. Chemical Hydrolysis of lactose is economically unfeasible due to the high cost of purifying the resulting products.

Removal of lactose from dairy whey. There are currently no industrial-scale plants for processing dairy whey, although the availability of cheap raw Materials and the valuable products formed during hydrolysis generate considerable interest. Pilot plants are operational in several countries. For example, in the USA, Corning Glass has established the processing of deproteinized and demineralized whey. Operating at 50 ^oC, the plant uses lactose immobilized by covalent bonding to porous silica gel via glutaraldehyde. The enzyme half-life depends primarily on the COMPOSITION OF THE initial raw material, reaching up to 62 days when processing demineralized deproteinized whey. The production cost of 1 ton of sugars obtained via Enzymatic hydrolysis of whey lactose at this facility is approximately 175 dollars.

In 1978, England initiated the processing of dairy whey to remove lactose using a semi-industrial plant (Fig. 10.3). Currently, it employs lactose immobilized on porous silica gel through covalent bonding with glutaraldehyde using the Corning Glass method. Pre-pasteurized, deproteinized, and demineralized whey is fed into the Reactor. Over 5 days of continuous operation, the plant processes about 30 tons of whey, yielding 1.7 tons of sugar syrup or 1.2 tons of dry sugars. The lactose conversion rate is 80%. A similar plant involving the same partners was commissioned in 1978 in France, with a capacity of 500 liters/hour of whey. At an industrial plant with a daily output of 200 tons of sugar syrup at an 80% lactose conversion rate, the cost of producing 1 kg of the resulting sugars ranges from 1.25 to 1.80 French francs.

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Fig. 10.3. Scheme of the semi-industrial plant for lactose hydrolysis in dairy whey

(according to Klesov A. A., 1982)

To extract lactose from dairy whey, the biocatalyst (immobilized lactase) is placed horizontally in the reactor as a fixed bed.

Removal of lactose from milk. The necessity of removing lactose from whole milk stems from the fact that some individuals, particularly in Asian and African countries, cannot digest lactose due to a lack of lactase in their bodies. Consuming milk leads to diarrhea, flatulence, and abdominal pain in these individuals. Eliminating lactose from milk can solve a vital issue of improving Nutrition for a significant portion of the population, especially children in economically disadvantaged countries.

However, numerous challenges arise due to the specific PHYSICOCHEMICAL PROPERTIES OF this product. Because milk is a colloidal solution, using a fixed-bed reactor carries a potential risk of Column clogging. Furthermore, to maintain high quality and prevent microbial contamination, the technology requires milk processing at low operating temperatures. Under these conditions, The activity of the immobilized enzyme decreases, as the optimum operating Temperature for lactase is 37 ^oC. This reduction in lactase activity necessitates an increased holding time of the milk in the reactor. In turn, this may lead to milk contamination with the enzyme and the carrier, the removal of which from milk is costly.

The successful overcoming of many of these problems has enabled the Structure/175.html">Implementation of the first commercial project for producing lactose-free milk. Since 1975, an industrial pilot plant utilizing lactase has been operating in Italy (Fig. 10.4).

Fig. 10.4. Scheme of the pilot plant for producing lactose-free dietary milk using immobilized

lactase

(according to Klesov A. A., 1982)

The sources for commercial soluble lactase preparations are Fungi (whose lactase is more suitable for processing acidic whey) and Yeasts (whose lactase is better suited for milk treatment).

To prevent reactor clogging, the flow rate of milk through the column was significantly increased. Yeast lactase was immobilized by entrapping droplets of the enzyme within hollow Cellulose triacetate fibers, which are wound into coils, arranged very loosely parallel to the reactor axis, and anchored at its top and bottom. This design simultaneously ensures low resistance to liquid flow and a very large enzyme surface area. The durability of cellulose triacetate prevents the carrier from entering the final product. The enzyme immobilized by this method exhibits high stability: 0.5 kg of enzyme-bearing cellulose triacetate fibers lost only 10% of their initial enzymatic activity after processing 500,000 liters of milk at 4-7 ^oC.

To eliminate microbial contamination, the columns are periodically flushed with bactericidal solutions.

Due to the high cost of lactase (yeast β-galactosidase), economic feasibility can be achieved through the high operational Stability of the enzyme and high lactose conversion. This challenge was addressed by employing a packed-bed reactor with recirculation (as packed-bed systems require less enzyme for an equivalent product yield). The setup features a 20-meter reactor containing 4 kg of lactase immobilized by entrapment in cellulose triacetate fibers. The enzyme exhibits remarkable stability, losing only 20% of its activity over 50 days of operation. The unit has a capacity of at least 8,000 liters of milk per day. Skimmed milk is processed by sterilization for 3 s at 142 ^oС, followed by rapid cooling to 4-7 ^oС, and is then repeatedly pumped through the reaction column at a rate of 7 L/min until the required conversion degree (75%) is attained. Lactose-free milk can be stored for 3-4 months at 7 ^oС.

Commercial soluble lactase preparations are primarily derived from fungi (whose lactase is better suited for acid whey processing) and yeasts (whose lactase is more appropriate for milk treatment).

Cheese production. The USE OF IMMOBILIZED enzymes holds considerable promise for milk processing in cheesemaking. Rennet is the primary milk-clotting enzyme utilized in cheese production. Its action on whole milk releases the protein casein, which upon coagulation forms the structural foundation of rennet-curd cheesemaking. Milk clotting (coagulation) occurs in two stages. The first, enzymatic stage involves structural alterations in casein, rendering it less stable, while the second, non-enzymatic stage leads to precipitation in the presence of Calcium Ions. Furthermore, rennet significantly influences cheese quality and, consequently, The Economics of production.

Natural rennet is obtained from the abomasums of young calves fed exclusively on a milk diet. Virtually all countries with developed dairy industries face a shortage of calf abomasums and, consequently, of rennet. In the former Soviet Union, annual cheesemaking requirements demanded 200–250 tons of rennet, extractable from 8–10 million calf abomasums (Artamonov, 1989). It has been estimated that the total global demand for rennet amounts to approximately USD 100 million.

To replace young calf stomachs in cheese production, processors turned to abomasums from older calves. Although these contain less rennet, they feature elevated levels of Pepsin and undesirable ballast substances, which adversely affect the quality of the resulting cheeses. This has driven intensive research into alternative microbial sources of rennet.

Microorganisms produce A wide variety of enzymes. Since the late 19th century, it has been known that certain microorganisms synthesize milk-coagulating enzymes, including Bac. subtilis, Bacterium prodigiosum, Aspergillus oryzae, and others. Microbial milk-clotting enzymes are produced on an industrial scale in Japan, the USA, Denmark, and other countries. Enzyme producers utilized in Japan include Mucor parasiticus, in the USA the fungus Endothia parasitica, and in Denmark the mold Mucor miehei. Cooney and Emerson manufacture an enzyme known as rennilase, France produces formase, and the former Soviet Union produced mesenterin GR and mucorin. Microbial enzymes can be used individually or combined with natural rennet, thereby cutting the required amount of the latter in half.

Unfortunately, microbial rennet has certain limitations. It remains active over extended periods and exhibits a broader Specificity compared to animal-derived rennet. Consequently, microbial rennet tends to degrade the natural flavor profile of cheeses, negatively impacts conservation and ripening processes, and impairs organoleptic properties. Therefore, Introduction/32.html">Genetic Engineering techniques have recently been adopted to obtain higher-quality rennet.

The application of immobilized rennet or alternative proteases offers significant promise, presenting opportunities for enzyme reactivation, prolonged reuse, and process automation, all of which ensure substantial economic benefits.

Another promising avenue is The Use of immobilized enzymes for milk stabilization. Trypsin treatment causes milk to sour more slowly and retain its original sensory qualities for extended periods. Treatment with immobilized trypsin extends the shelf life of milk by 2–3 weeks. The use of immobilized enzymes is more efficient than conventional milk processing Methods such as sterilization or high-frequency current treatment.