Applications of β- D -galactosidase

Non-immobilized β-D-galactosidase

β-D-galactosidase is widely used in the food industry for lactose hydrolysis of different dairy products such as cheese and whey. A rate of about 70% of the world population suffer from a condition called lactose intolerance that manifests through the digestive system discomfort from eating lactose-containing products, such as milk, milk products or products derived from it. This condition is caused by the absence or low concentration in the body of the β-D-galactosidase enzyme. The food industry uses technology based on this enzyme, for hydrolysis of lactose from milk composition of the consumers, making it available for a new group on the market, namely for the people who suffer from lactose intolerance (Shukla, 1975).

Another way to use the enzyme represents the hydrolysis of lactose from whey (milk serum), a byproduct of dairy technology. Lactose from whey by hydrolysis, it becomes sweet syrup that can be used in other branches of the food industry, as well as pastries or the manufacture of soft drinks. So by using the hydrolyzed whey, it can exploit one of the by-products of the cheese industry, where whey is obtained in large quantities that pose serious environmental problems when it comes to removing it. Conversion of lactose from whey in fermentable monosaccharides, glucose and galactose, it makes the hydrolyzed whey a natural and cheep fermentation medium, which is able to develop microbial cultures (Gekas and Lopez-Leiva, 1985).

Milk treated with β-D-galactosidase and used in the cheese industry has led to a more rapid maturing of cheese than if using milk which has not been subject to hydrolysis. The explanation of this is that lactose crystallizes easily, which limits its use in certain process in dairy industry. Treatment of milk and milk-based products with this enzyme for subtraction of lactose content solves some problems as insolubility and low sweetening ability of this disaccharide (Tweedie et al., 1978; Pivarnik et al., 1995).

The enzyme is also used successfully to obtain galacto-oligosaccharides (GOS) by lactose transglycosylation. Galacto-oligosaccharides are recognized for their probiotic effect, namely they are fermented in colon by beneficial bacterial cultures as they can not be absorbed in the intestine because of their insolubility. Other sources of origin for these galacto-oligosaccharides may be cow’s milk, honey and various fruits and vegetables (Maischberger et al., 2008).

Immobilized β-D-galactosidase

Immobilized β-D-galactosidase can be used for both hydrolysis of lactose in milk or whey and synthesis of oligosaccharides. The technological choice depends on the nature of the substrate, enzyme characteristics, economic and production matters. The primary feature determining the choice and applicability of an enzyme is pH. Fungal enzymes with maximum activity situated in more acidic pH interval are suitable for processing acidic whey, while enzymes from yeasts and bacteria exhibiting high activity in neutral pH domain are suitable for processing milk and sweet whey. Hydrolyzed milk lactose is used in manufacturing of milk flavourings, dairy, cheese and yoghurts. Hydrolysis of lactose in milk for food processing prevents crystallization of lactose in frozen or condensed milk products. Moreover, utilization of hydrolyzed milk in preparation of yoghurts and cheese speeds up the acidification process, as lactose hydrolysis is a rate-limiting step in the development of structure and flavour of cheese. The quality of frozen milk and ice cream was relevantly enhanced by adding Lactozyme that prevents crystallization of lactose by its hydrolysis and reduces its granular characteristics, as well. Hydrolyzed whey concentrates can be used as sweeteners in the preparation of fruit syrups or soft drinks. A variety of immobilization carriers were used to improve the properties of β-D-galactosidase from various sources (Table 1.6, Panesar et al., 2010).

Table 1.6. Carriers for β-D-galactosidase immobilization and their efficiency in hydrolysis of lactose (Panesar et al., 2010)

Enzyme source Immobilization carrier

Lactose hydrolysis yield [%]

Time of hydrolysis [h]

Fungi (Miles Chemie) Polyvinyl alcohol 75 5-6

Escherichia coli Polyacrylamide gel 47 6 Kluyveromyces lactis Thiosulfinate 85-90 2.5 Kluyveromyces fragilis Cellulose granules > 90 5

Kluyveromyces lactis Cotton > 95 2

Kluyveromyces marxianus Calcium alginate 84.8 2.5 Bacillus

stearothermophilus Chitosan > 80 2

Immobilization of β-D-galactosidase in polyvinyl alcohol gel led to a more thermally stable preparation compared to the native enzyme, retaining 70% of activity after 24 hours at 50°C. In the hydrolysis reaction of lactose, 70% hydrolysis yield has been achieved after 5-6 hours. Conversion has dropped to 50% after 30 uses (Panesar et al., 2010).

β-D-galactosidase from Bacillus circulans immobilized on Doulite ES-762 presented a higher hydrolysis yield of 70% in continuous system. The addition of Mg²⁺ and Mn²⁺ in hydrolysis reaction enhanced the activity of β-D-galactosidase enzyme from Kluyveromyces lactis in the hydrolysis reaction of ONPG and lactose (Kim, 2006).

The use of β-D-galactosidase from Kluyveromyces lactis immobilized on silica-CPC led to a 90%

lactose hydrolysis yield, realized in a batch minireactor (Giacomini et al., 1998).

β-D-Galactosidase entrapped in a copolymer of N-isopropylacrylamide and acrylamide was effective in the reaction of hydrolysis of lactose at 5°C in order to obtain milk with low lactose content. It was observed that in the course of lactose hydrolysis the stability of casein from milk has been affected (Miezeliene et al., 2000).

Kinetic model for lactose hydrolysis was established using immobilized β-D-galactosidase from Kluyveromyces fragilis. The immobilized enzyme preparation presented activity at 5°C, thus having applicability in preparation of frozen products, avoiding lactose crystallization (Ladero et al., 2000).

Sephadex G-75 and chitosan granules were tested as supports for immobilization of β-D-galactosidase from Pisum sativum. The enzyme immobilized on chitosan exhibited higher hydrolysis rate of lactose in milk and whey at room temperature and at 4°C than the preparation obtained by immobilization on Sephadex G-75. Therefore, it was considered more suitable for industrial applications, considering its pH stability, optimal temperature, high temperature stability and reuse efficiency, as well (Dwevedi and Kayastha, 2009).

Cross-linked enzyme aggregates (CLEAs) or crystals (CLECs), obtained as carrier-free macroparticles, can display high enzyme activity, enhanced stability and low production costs. In hydrolysis reaction of lactose, CLEA of β-D-galactosidase yielded 78% monosaccharides in 12 h, compared to 3.9% of the free form, under similar operational conditions (Graur et al., 2006).

Confinement of Kluyveromyces lactis β-D-galactosidase within an ultra-filtration membrane has been used for continuous production of galacto-oligosaccharides from lactose, with up to 80%

lactose conversion, at an initial substrate concentration of 250 g/L. The reaction was carried out in a perfectly mixed reactor and the enzyme was recovered in a 10 kDa nominal molecular weight cut-off. This method allows the enzyme action in a fully fluid environment, without significant loss of catalytic activity. However, the membrane still presents a boundary for overall mass transfer of substrate and/or products, and enzyme molecules are disposed to interact with the membrane material (Chockchaisawasdee et al., 2005). Using the same methodology, with a hollow-fiber module containing β-D-galactosidase, lactose hydrolysis has been carried out in continuous operation. A conversion rate close to 95% in skim milk was observed for an initial substrate concentration close to 40 g/L (Novalin et al., 2005).