The enzyme, named BWbg1, belongs to a class of proteins known as beta-galactosidases, which catalyze the cleavage of lactose, the disaccharide sugar that makes up a large fraction of the solids in milk, into its two component monosaccharides, glucose and galactose. Beta-galactosidases have long occupied a central place in dairy biotechnology because they support two distinct and commercially significant applications. The first is the production of low-lactose and lactose-free milk and dairy goods for the large population of people who cannot properly digest lactose. The second is the synthesis of galacto-oligosaccharides, or GOS, through transgalactosylation reactions in which the enzyme, instead of simply splitting lactose, stitches galactose units together into short chains that humans cannot digest but that beneficial gut bacteria eagerly consume.

What makes BWbg1 stand out in a crowded field of known beta-galactosidases is its thermal profile. The purified enzyme exhibits maximum activity at 80 degrees Celsius and at a neutral pH of 7, a combination that immediately distinguishes it from the mesophilic enzymes traditionally used in dairy processing. It also demonstrates impressive resilience, retaining more than 72 percent of its activity after six hours of incubation at 55 degrees Celsius. For industrial operators, these are not merely laboratory curiosities. Elevated operating temperatures increase the initial productivity of enzymatic reactions, improve the solubility of substrates such as concentrated lactose solutions, and dramatically reduce the risk of contamination by spoilage and pathogenic microorganisms, which struggle to proliferate at temperatures that the enzyme tolerates with ease.

The path to BWbg1 illustrates the power of functional metagenomic screening, a technique that has become one of the most productive strategies in modern bioprospecting. Rather than trying to culture the microorganisms from the As Burgas hot spring, an approach that would capture only a small fraction of the community because most environmental microbes resist laboratory cultivation, the researchers extracted the collective DNA of the entire microbial ecosystem and cloned it into a metagenomic library. They then screened that library for clones exhibiting beta-galactosidase activity, allowing the function of the gene itself, rather than any prior knowledge of the organism that carried it, to guide the discovery. Sequence analysis of the protein encoded by the recovered gene revealed that BWbg1 belongs to glycoside hydrolase family 35, a well-characterized group of enzymes, yet its properties proved distinctive enough to warrant the label of novelty.

The enzyme’s performance in GOS synthesis is arguably its most commercially compelling feature. Working with a concentrated lactose solution of 40 percent weight per volume at 70 degrees Celsius, BWbg1 converted the substrate into a product mixture containing up to 48 percent galacto-oligosaccharides by weight over a four-hour reaction. That yield, achieved at a temperature at which most conventional enzymes would rapidly denature, positions BWbg1 as a serious candidate for industrial prebiotic production. GOS are among the most widely added prebiotics in infant formula and functional foods, valued for their ability to selectively stimulate the growth of beneficial bacteria such as Bifidobacteria in the gut. A thermostable enzyme that can sustain high-yield synthesis at 70 degrees Celsius offers manufacturers both faster throughput and a substantially lower microbial contamination risk during extended production runs.

Perhaps the most surprising finding, however, concerns what happens when the enzyme is exposed to heat before it is asked to work. The researchers observed a remarkable heat activation effect: after two hours of incubation at 65 degrees Celsius, BWbg1’s ability both to hydrolyze lactose and to produce GOS was enhanced rather than diminished. This behavior has direct implications for how the enzyme could be deployed in real dairy plants. Milk destined for the market is routinely pasteurized, and the two dominant methods are HTST, high-temperature short-time pasteurization, and VAT, the older vat pasteurization approach that uses lower temperatures over longer holding periods. The study demonstrated that BWbg1 retains its activity after both HTST and VAT pasteurization of commercial milk, and that the heat activation it experiences during these treatments actually improves its subsequent performance on the lactose in the milk.

This compatibility with pasteurization regimes is a genuinely practical advantage. In current industrial practice, lactose hydrolysis is typically performed as a separate step after pasteurization, requiring additional equipment, time, and careful hygiene controls. An enzyme that can be added to milk, survive the pasteurization step itself, and then go on to work more effectively afterward could allow manufacturers to merge two processing stages into one streamlined operation. The enzyme’s tolerance of the exact thermal conditions used to make milk safe for consumers means that the same heat treatment that protects public health simultaneously primes the biocatalyst for action, a coincidence of process requirements that enzyme engineers usually struggle to achieve by design.

Equally noteworthy is the enzyme’s behavior at the opposite end of the temperature scale. The study reports that BWbg1 remains capable of hydrolyzing lactose from commercial milk and producing GOS even at 8 degrees Celsius, a temperature within the range of standard refrigerated storage. This cold activity opens the door to applications in which milk is treated during chilled logistics or storage, extending the enzyme’s usefulness well beyond the hot processing floor. A single biocatalyst that functions productively from refrigeration temperatures up to 80 degrees Celsius spans a thermal range that few, if any, commercially established beta-galactosidases can match, and it gives process designers an unusual degree of freedom in choosing where and when in the production chain to deploy it.

The broader significance of the work lies in what it says about the untapped enzymatic wealth of extreme environments. Hot springs have long been recognized as reservoirs of thermostable proteins, because the microorganisms adapted to life at elevated temperatures must maintain enzymes that keep their structure and function under conditions that would destroy the proteins of ordinary organisms. Yet the vast majority of these thermophilic communities remain genetically uncharacterized, and functional metagenomics offers a way to access their catalytic repertoire without ever growing a single cell in the laboratory. The As Burgas spring, flowing through a geologically active region of Galicia, proved to be a productive hunting ground, and the researchers suggest that BWbg1’s versatility, spanning lactose hydrolysis, GOS synthesis, pasteurization compatibility, and cold-temperature activity, makes it a strong candidate for industrial application.

For consumers, the practical outcome of such research is tangible. Lactose intolerance affects a substantial share of the global population, and demand for lactose-free dairy continues to climb, while the market for prebiotic ingredients that support gut health grows in parallel. An enzyme that can serve both markets, converting milk into a digestible product for one consumer group and into a GOS-enriched functional food for another, addresses two major trends in food science with a single biocatalyst. As the team’s results demonstrate, the answer to some of the dairy industry’s most persistent processing challenges may have been waiting all along in the steaming waters of a Spanish hot spring, encoded in the genomes of microbes no one has ever cultured.

Subject of Research: A thermostable GH35 beta-galactosidase from a hot spring metagenome for lactose hydrolysis and galacto-oligosaccharide production in milk

Article Title: A novel thermostable beta-galactosidase for low-lactose and galacto-oligosaccharide-rich milk

Article References: A novel thermostable beta-galactosidase for low-lactose and galacto-oligosaccharide-rich milk. (n.d.). https://doi.org/10.1007/s00253-026-14045-z

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14045-z

Keywords: beta-galactosidase, metagenomics, thermostable enzyme, galacto-oligosaccharides, lactose-free milk, hot spring, bioprospecting, dairy biotechnology, prebiotics, pasteurization, GH35, transgalactosylation

The team’s strategy rests on the Maillard reaction, the same non-enzymatic chemistry that browns bread crust and sears steak. In food science, this reaction can be harnessed deliberately: a reducing sugar is covalently grafted onto the free amino groups of a protein, producing a protein-polysaccharide conjugate with improved solubility, antioxidant activity and interfacial behavior. The researchers chose galacto-oligosaccharides, or GOS, as their grafting partner, a prebiotic carbohydrate derived from lactose that is already valued for feeding beneficial gut bacteria. When carefully controlled, wet-heating glycosylation at 90 degrees Celsius for just 15 minutes can attach GOS to protein while avoiding the pitfalls of overdone Maillard chemistry, such as excessive browning, protein crosslinking and the formation of advanced glycation end-products.

The central insight of the study is that the history of the protein before conjugation matters as much as the conjugation step itself. The team compared two extraction routes. The conventional route used alkaline solubilization at pH 9 with stirring, precipitation at the isoelectric point and freeze-drying, yielding around 39.7 milligrams of protein per gram of flour at best. The microwave-assisted route, optimized through a nine-run screening design in a Labotron 12T system, hit its peak at 400 watts for 20 minutes at pH 9, delivering 293.72 milligrams per gram, roughly seven times the conventional yield. The rapid volumetric heating disrupts the cellular matrix and releases protein efficiently, but crucially it also appears to leave the protein in a partially unfolded state with more reactive amino groups exposed.

That conformational legacy showed up clearly in the structural data. Fourier-transform infrared spectroscopy revealed that the microwave-extracted conjugate, dubbed LPIGHM, showed the strongest evidence of carbohydrate incorporation, with a pronounced growth of the carbon-oxygen stretching envelope between 1200 and 1000 per centimeter and a weakening of the ordered amide bands. Differential scanning calorimetry and thermogravimetric analysis painted the same picture: the microwave-derived conjugate displayed the most complex thermal profile and enhanced structural integrity compared with the non-heated mixture and the conventionally prepared conjugate. Secondary-structure analysis showed beta-sheet content falling from 56.7 percent in native lupin protein to 50.0 percent in LPIGHM, with beta-turns and random coil rising in compensation, a shift from rigid order toward flexible disorder that favors interfacial performance.

Perhaps the most dramatic structural change was in particle size. Native lupin protein isolate was heavily aggregated, with a mean hydrodynamic diameter of 1775 nanometers, a figure the authors caution is skewed by a minority population of large clumps. After glycation, the conjugates shrank dramatically: 152 nanometers for the simple mixture, 99 for the conventionally prepared conjugate and 89.7 nanometers for the microwave-derived one, an apparent 19.8-fold reduction. Surface hydrophobicity collapsed in parallel, from 443 arbitrary units for the native protein to just 62.5 for LPIGHM, as hydroxyl-rich GOS chains replaced exposed apolar side chains on the particle surface. Fluorescence spectroscopy confirmed tertiary structural rearrangement, with tryptophan emission quenching and blue-shifting most strongly in the microwave-derived conjugate.

These structural shifts translated directly into functional gains. Foaming capacity of the native protein was a dismal 10 percent with 4 percent stability; the microwave-derived conjugate reached 64 percent capacity and 36 percent stability, increases of 6.4-fold and 9-fold respectively. Emulsifying activity index climbed from 7.2 to 74.2 square meters per gram, a tenfold improvement, while emulsion stability rose from 40.2 to 71.6 minutes. Water-holding capacity more than doubled to 2.9 grams per gram, and oil-holding capacity rose to 14.6 grams per gram. Solubility at neutral pH improved only modestly, from 31.5 to 33 percent, which the authors attribute to measuring well away from the isoelectric region where glycation exerts its largest effect on plant proteins.

Correlation analysis across the four treatment means reinforced the mechanistic story. The apparent degree of grafting, measured by the loss of free amino groups, was the strongest single predictor of performance, correlating at r = 0.99 with foaming capacity and r = 0.98 with emulsifying activity. Surface hydrophobicity correlated negatively with solubility and interfacial function, and particle size and beta-sheet content tracked negatively with emulsion stability. Notably, zeta potential showed no strong association with any interfacial property, supporting the authors’ argument that stabilization in these conjugates is steric and hydration-driven rather than electrostatic: the attached carbohydrate layer displaces the shear plane outward and screens the underlying charges, so a less negative zeta potential can coexist with more bound sugar and better stability.

The biological results were more nuanced and enzyme-specific. Alpha-glucosidase inhibition improved roughly fourfold in all GOS-containing systems, reaching 60 to 66 percent at 100 milligrams per milliliter, an effect present even in the non-heated mixture, suggesting the oligosaccharide itself contributes to binding at the enzyme’s pocket-shaped active site. Alpha-amylase inhibition, by contrast, was not improved and declined at low concentrations, plausibly because the hydrated sugar chains obstruct the enzyme’s extended substrate-binding cleft. ACE inhibition remained modest throughout. Antioxidant activity rose in an assay-dependent fashion: the microwave-derived conjugate excelled at ABTS radical scavenging, reaching 43.9 percent at the lowest tested concentration versus 12.8 percent for native protein, while the conventionally prepared conjugate showed the strongest reducing power and total antioxidant capacity. Against Caco-2 colorectal cancer cells, antiproliferative activity was substantial, though the native protein was the most active at 86.2 percent, and the microwave-derived conjugate partially preserved activity after heating.

The gut microbiota experiments provided the study’s most forward-looking results. Using pooled fecal slurries from six healthy adult donors in an in vitro fermentation model, the team found that the conjugates, particularly the microwave-derived one, produced the most gas, retained more acetate at 24 hours than the free GOS mixture, and fostered the most even microbial community, with a Shannon index of 3.23 versus 1.85 for the untreated control. Beneficial genera such as Bifidobacterium and Lactobacillus were enriched while opportunistic taxa including Escherichia and Klebsiella declined. PICRUSt2 functional prediction pointed to enhanced carbohydrate and energy metabolism pathways. The authors are careful to note that short-chain fatty acid concentrations declined over the incubation rather than accumulating, that the fermentation was run without simulated digestion, and that the predicted pathways reflect metabolic potential rather than measured activity.

The study’s limitations are candidly acknowledged. The design did not include a microwave-extracted protein carried through without conjugation, so the effects attributed to LPIGHM belong to the combined process rather than to microwave extraction alone. The degree of grafting is an apparent value that cannot distinguish sugar attachment from conformational masking of amino groups, and the correlation analysis rests on only four treatment means. Confirming the full picture will require mass-spectrometric mapping of glycation sites, quantification of advanced glycation end-products and available lysine, digestion studies before fermentation, and trials in real food matrices. Even so, the core message stands: how a plant protein is extracted shapes how well it can be engineered afterward. For food formulators wrestling with the functional shortcomings of legume proteins, the microwave is looking less like a shortcut and more like a strategic first move.

Subject of Research: Microwave-assisted extraction of lupin protein isolate to enhance Maillard glycation with galacto-oligosaccharides for improved functionality and gut fermentability

Article Title: Microwave-assisted extraction enhances wet-heating Maillard glycation of lupin protein with galacto-oligosaccharides: structural basis for improved interfacial functionality and gut fermentability

Article References: Ullah, N., Bamigbade, G., Arachchi, M. J. P., Ali, A., Kamal-Eldin, A., Zhou, F., Miao, S., & Ayyash, M. (2026). Microwave-assisted extraction enhances wet-heating Maillard glycation of lupin protein with galacto-oligosaccharides: structural basis for improved interfacial functionality and gut fermentability. Food Chemistry: X, 39, Article 104518. https://doi.org/10.1016/j.fochx.2026.104518

Image Credits: AI Generated

DOI: 10.1016/j.fochx.2026.104518

Keywords: lupin protein isolate, Maillard reaction, galacto-oligosaccharides, microwave-assisted extraction, protein glycation, emulsifying properties, foaming capacity, gut microbiota, short-chain fatty acids, prebiotics, plant protein, Food Chemistry: X