Abstract
The growing need in the current market for innovative solutions to obtain lactose-free (L-F) milk is caused by the annual increase in the prevalence of lactose intolerance inside as well as the newborn, children, and adults. Various configurations of enzymes can yield two distinct L-F products: sweet (β-galactosidase) and unsweet (β-galactosidase and glucose oxidase) L-F milk. In addition, the reduction of sweetness through glucose decomposition should be performed in a one-pot mode with catalase to eliminate product inhibition caused by H2O2. Both L-F products enjoy popularity among a rapidly expanding group of consumers. Although enzyme immobilization techniques are well known in industrial processes, new carriers and economic strategies are still being searched. Polymeric carriers, due to the variety of functional groups and non-toxicity, are attractive propositions for individual and co-immobilization of food enzymes. In the presented work, two strategies (with free and immobilized enzymes; β-galactosidase NOLA, glucose oxidase from Aspergillus niger, and catalase from Serratia sp.) for obtaining sweet and unsweet L-F milk under low-temperature conditions were proposed. For free enzymes, achieving the critical assumption, lactose hydrolysis and glucose decomposition occurred after 1 and 4.3 h, respectively. The tested catalytic membranes were created on regenerated cellulose and polyamide. In both cases, the time required for lactose and glucose bioconversion was extended compared to free enzymes. However, these preparations could be reused for up to five (β-galactosidase) and ten cycles (glucose oxidase with catalase).
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Introduction
Lactose intolerance is a prevalent ailment affecting a significant portion of the global population. According to epidemiological data, up to 70% of the world’s population may be affected by this dysfunction [1]. Lactose intolerance is manifested by inadequate enzyme β-galactosidase (β-gal) required for lactose hydrolysis in the small intestine. Consequently, consumption of lactose-containing foods (both dairy and non-dairy products) by individuals with β-galactosidase deficiency results in gastrointestinal discomfort [2, 3]. One of the easiest ways to deal with lactose intolerance is its elimination from the daily diet by replacing traditional dairy with lactose-free (L-F) products. Nowadays, L-F-labeled dairy products are perceived as functional foods, substitutes for whole milk, and a low-cost dietary source of calcium with a wide range of availability [4, 5].
In recent years, there has been growing interest in finding technologies that provide nutritional value and functional, sensory, and quality properties of L-F dairy products [4]. Among the existing methods for removing lactose from food are membrane separation, fermentation, and enzymatic hydrolysis. The last one has gained high popularity in industrial technologies [4, 6]. It is the result of high specificity and relatively low cost of bioconversion (especially in the case of immobilized preparations) [7]. In the enzymatic approach, two strategies are proposed: batch, in which enzyme is added before pasteurization, and aseptic, in which the sterile enzyme is added to UHT milk before packaging [6].
Enzymatic lactose hydrolysis allows the production of two kinds of L-F milk: sweet and unsweet, Fig. 1. To get the first, the one-enzyme pathway with β-galactosidase is necessary. The higher sweetness of L-F milk is caused by the products of lactose hydrolysis, glucose, and galactose, both of which have a higher sweetness index than lactose. Therefore, L-F milk is gaining popularity in producing low-calorie dairy desserts without supplementary sweeteners [8]. Receiving unsweet L-F milk is more complicated and involves three enzymatic pathways [9]. Because the noticeable sweetness of L-F milk is mainly the result of glucose [10], its decomposition by glucose oxidase (GOX) is desirable [11]. Although catalase (CAT) does not directly participate in the decomposition of glucose, its presence, mainly in the one-pot mode, is necessary to maintain the high enzymatic activity of GOX inhibited by hydrogen peroxide. Unsweet L-F milk is dedicated to an increasingly more extensive group of consumers who negatively perceive the sweetness of L-F milk [12]. Furthermore, unsweet L-F milk, due to its similar taste to traditional milk, can be applied in savory dishes and compete with non-dairy milk alternatives [13].
Enzymatic pathways to obtain sweet and unsweet L-F milk. The red lines represent the step with product inhibition caused by H2O2, and the green lines show benefits related to the presence of catalase in the reaction mixture (decomposition of H2O2 and generation of O2, which acts as a substrate for GOX) (colour figure online)
Industrial biocatalysis is closely related to enzyme immobilization, hel** to overcome the limitation of free enzymes [14, 49, 50], but others offer a poorer immobilization efficiency by covalent bonding on the PDA coating membrane [51]. The satisfactory results of the covalent immobilization of catalase on polyamide have also been confirmed by literature reports [52, 53].
The proposed covalent immobilization of GOX and CAT onto RC membrane via hydroxyl groups exhibited lower efficiency compared to PA membrane. It is worth noting that the GOX bond involving glutaraldehyde, as highlighted in literature reports, is superior. This bond suggests the conversion of originally occurring hydroxyl groups of PMMA microspheres to aldehyde groups, a crucial step for efficient GOX immobilization [54]. Other references indicated the function of RC membrane solely as a separating layer for free GOX and CAT, allowing enzyme retention in the reactor space [55, 56].
It is essential to recognize the versatility of the two distinct unit processes—lactose hydrolysis for producing sweet L-F milk and glucose decomposition for obtaining L-F milk with a traditional taste. This versatility is a testament to the potential of using two different carriers for immobilization (RC membrane for NOLA and PA membrane for GOX and CAT), which should not be seen as an obstacle to future industrial processes.
The presented results indicate the high stability of catalytic membranes with co-immobilized enzymes onto polyamide. These results are notably intriguing since a polyamide membrane has not been widely discussed in the literature as a preferred carrier for the co-immobilization of GOX and CAT. Instead, the proposition of an ultrafiltration membrane composed of an acrylonitrile copolymer [57] or an ultrafiltration membrane made of polypropylene with a skin layer of RC can be found [58]. Polyamide materials, available in membranes, gels, and non-wovens, are widely acknowledged as popular carriers for enzyme immobilization. These materials gained popularity due to their porous structure, chemical and thermal resistance, resistance to biodegradation, and mechanical properties that enable them to withstand increased pressure in membrane flow bioreactors [59]. Some polyamide carriers require partial hydrolysis of the membrane surface to generate more reactive amine groups necessary for enzyme immobilization [60]. Examples of immobilized preparations created on polyamide (PA) and tailored for industrial applications include laccase bonded on functionalized polyamide 6,6 with an immobilization yield of 2% [60], β-xylosidase bonded on a microfiltration polyamide membrane with an immobilization yield of 20% [61], and laccase bonded on polyamide fabric hydrolyzed by bromelain with an immobilization yield of 68% [62].
The literature review focusing on carriers dedicated to GOX and CAT co-immobilization highlights the potential of the one-step strategy of co-immobilization onto polyamide membranes, which was presented in this work. Furthermore, the results suggest achieving a higher value of the bonded mass of enzymes in co-immobilized preparations, exceeding 0.83 mg to accomplish a bioconversion time close to free enzymes. This goal, whether for one-enzyme or two-enzyme immobilization, can be achieved, e.g., by increasing the surface area of the carrier.
The proposed strategy of immobilizing NOLA, GOX, and CAT via covalent binding on microfiltration membranes (RC and PA) was less effective than encapsulated preparations previously described by the authors [9, 27]. The lower activity of the catalytic membranes may be attributed to enzyme denaturation upon immobilization, changes in active-site conformation, and crowding of enzyme molecules on the carrier [45]. Despite the higher enzymatic activity of encapsulated preparations, their utilization in continuous processes, such as in packed bead reactors, may pose difficulties in maintaining a constant porosity of the deposit (alginate beads). Furthermore, the comparison of enzymes reused in subsequent cycles for encapsulated preparations and catalytic membranes with NOLA, GOX, and CAT [9, 27] indicated a decrease in enzymatic activity in each successive cycle, regardless of the form of the immobilized preparation, suggesting enzymes’ instability under process conditions.
Conclusion
The market potential for L-F products is still growing. Thanks to the enzymatic approach and immobilization technique, lactose hydrolysis can be recognized as an efficient and economical method for obtaining two types of L-F milk (sweet and unsweet) at low temperatures, similar to those used in storing and transporting raw milk. Catalytic membranes featuring individually immobilized enzymes and co-immobilized preparations are a prime illustration of multidisciplinary technology that aligns with the increasing demand for highly efficient biocatalysis in the dairy industry. However, creating catalytic membranes without the time and cost-consuming modification of membrane surface chemistry remains challenging. Fundamental limitations are associated with the low affinity of enzyme molecules to functional groups of the membrane and the risk of excessive stiffening of the spatial structure of the enzyme. Nevertheless, the stable connection of the enzyme with the carrier via covalent interactions enabled the catalytic membranes to be reused multiple times. In addition, the high mechanical stability of catalytic membranes allows their application in continuous industrial processes, offering significant opportunities for optimization at various stages. Nonetheless, the development of novel, cost-effective immobilization strategies that ensure high catalyst loading, elevated activity, and long-term stability of catalytic membranes remains a persistent challenge highlighting the need for ongoing research and innovation in this field.
Data availability
All data generated or analyzed during this study are included in this published article (and its supplementary information files).
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KC: conceptualization, methodology, software, formal analysis, investigation, resources, writing—original draft preparation, visualization. AT: conceptualization, methodology, validation, data curation, writing—review and editing, supervision, project administration, funding acquisition.
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Czyżewska, K., Trusek, A. A catalytic membrane approach as a way to obtain sweet and unsweet lactose-free milk. Bioprocess Biosyst Eng 47, 919–929 (2024). https://doi.org/10.1007/s00449-024-03018-z
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DOI: https://doi.org/10.1007/s00449-024-03018-z