Lactose Hydrolysis Research Articles

Lactose hydrolysis in sheep and cow milk with varying fat levels to evaluate enzymatic production of lactulose

AbstractThis study aimed to perform lactose hydrolysis using free and immobilised β-galactosidase in cow and sheep milk with different fat levels, followed by lactulose synthesis through the isomerisation of glucose to fructose using immobilised glucose isomerase. There was no significant difference (P > 0.05) in lactose hydrolysis (>95%) using the free enzyme in the different types of milk. In contrast, significant differences were observed for the hydrolysis of whole cow milk (WCM) and skimmed cow milk (SCM) with the immobilised enzyme (IE). WCM required a longer hydrolysis time (43 h). Thus, the fat content may have affected the lactose hydrolysis of bovine milk with immobilised β-galactosidase. No difference was observed for whole sheep milk and skimmed sheep milk hydrolysed by IE, possibly due to the presence of smaller fat globules, which did not affect the lactose hydrolysis. No significant differences were observed for lactulose synthesis in whole milk from the different species evaluated. The lactulose synthesis in cow and sheep milk by the enzymatic route may be a promising alternative, though further studies are required to optimise the production of this prebiotic in situ.

Recombinant production of Paenibacillus wynnii β-galactosidase with Komagataella phaffii

BackgroundThe β-galactosidase from Paenibacillus wynnii (β-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used β-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, β-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce β-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the β-gal-Pw in K. phaffii either extracellularly or intracellularly.ResultsFirstly, 11 different signal peptides were tested for extracellular production of β-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of β-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular β-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric β-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for β-gal-Pw production compared to the AOX1 promoter. After partial purification, a β-gal-Pw enzyme preparation with a total β-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter).ConclusionThis study showed that the β-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular β-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.

Objectives for the use of raw materials and component composition for the production of low-lactose ice cream from goat milk

A huge number of people all over the world suffer from lactose intolerance. The second problem is an allergy to cow milk protein. People with lactose intolerance, as well as those who suffer from cow milk allergies, are forced to significantly limit their choice of ice cream or give it up completely. The article describes a solution to the problem of eating ice cream by children and adults suffering from lactose intolerance and an allergy to cow milk protein, and presents ways to overcome it.The goal of the research. Objectives for the use of raw materials and component composition for the production of low-lactose ice cream from goat milk. During the research, the quality indicators of the selected raw materials have been studied, the characteristics of the main ingredients for the production of low-lactose ice cream have been substantiated and given. Goat milk, in terms of its quality indicators, is most suitable for the production of ice cream, because it contains more protein, fat, solids, less lactose and does not cause allergies, and therefore it has been chosen as the main raw material for the production of ice cream. A theoretical and practical analysis of the chemical composition and properties of goat milk has been carried out, and compared with cow. To reduce the lactose content, a dual method of lactose hydrolysis has been chosen - the use of probiotic starter cultures and the lactase enzyme. In the process of combined hydrolysis at a temperature of 37-39⁰ C for 4-6 hours, a reduction in lactose content of less than 0.9% is achieved, while the concentration of cells of probiotic microorganisms in active form reaches 108 CFU/cm3. It is proposed to use the “Lactomix KM” stabilization system, which is usually used in the production of fermented milk products.

Unveiling the genetic basis and metabolic rewiring behind the galactose-positive phenotype in a Streptococcus thermophilus mutant

Streptococcus thermophilus (S. thermophilus) is a widely used starter culture in dairy fermentation, but most strains are galactose-negative and only metabolize glucose from lactose hydrolysis. In this study, we aimed to uncover the mechanisms underlying the acquisition of a stable galactose-positive (Gal+) phenotype in a mutant strain of S. thermophilus IMAU10636. By treating the wild-type strain with the mutagenic agent N-methyl-N-nitro-N-nitrosoguanidine, we successfully isolated a Gal+ mutant, S. thermophilus IMAU10636Y. Comparative enzyme activity assays revealed that the mutant exhibited higher β-galactosidase and galactokinase activities, but lower glucokinase and pyruvate kinase activities compared to the wild-type. High-performance liquid chromatography analysis confirmed the mutant’s enhanced ability to utilize lactose and galactose, leading to increased glucose secretion. Integrated genome and transcriptomics analyses provided deeper insights into the underlying genetic and metabolic mechanisms. We found that the metabolism regulatory network of the glycolysis / Leloir pathway was altered in the mutant, possibly due to the upregulation of the gene expression in the galR-galK intergenic region. This likely led to increased RNA polymerase binding and transcription of the gal operon, ultimately promoting the Gal+ phenotype. Additionally, we identified a mutation in the scrR gene, encoding a LacI family transcriptional repressor, which also contributed to the Gal+ phenotype. These findings offer new perspectives on the metabolic rewiring and regulatory mechanisms that enable S. thermophilus to acquire the ability to metabolize galactose. This knowledge can inform strategies for engineering and selecting Gal+ strains with desirable fermentation characteristics for dairy applications.

The Characterization of a Low-Calorie and Lactose-Free Brown Fermented Milk by the Hydrolysis of Different Enzymatic Lactose.

The adoption of brown fermented milk in the normal diet and daily beverages is accompanied by significant sugar intake and a high public health burden. To reduce the sugar content in dairy products while maintaining optimal nutritional properties, a novel low-calorie, lactose-free brown fermented milk was developed through enzymatic hydrolysis and the Maillard reaction. The optimal product was achieved using low-temperature lactase, where the lactose and glucose content were reduced 33-fold and 2.4-fold to 0.06 g/100 g and 13.32 g/L, respectively, meeting the criteria for being lactose-free (<0.5 g/100 g). Meanwhile, hazardous compounds such as 5-hydroxymethylfurfural and 3-deoxyglucosone were reduced by more than 20%. After 28 days of storage, the water-holding capacity and suspension stability remained notably stable, and the protein composition was also more enriched compared to commercial milk. It is expected that this low-calorie dairy product may promote growth in the dairy market.

Analysis of catalytic sites in FeY zeolite prepared by sono-assisted exchange of iron (II) ions

In the refining industry, Y-type faujasite (FAU) plays an irreplaceable role, being widely used to convert crude oil into more valuable products such as gasoline. However, the demand for fuels and chemicals stimulates the search for alternatives, showing the applications of FAU beyond producing value-added chemicals from petroleum-based derivatives. These alternative applications are further enhanced from active sites that emerge in zeolites when iron is incorporated by a sonochemical treatment. Given that ultrasonic irradiation promotes mass transfer, uniform coverage of active sites is expected to produce more efficient catalysts. Likewise, Fe-zeolites catalyze a wide range of reactions in mild conditions, linked to the research of bio-refinery, biomimicry and environmental remediation. This work explores the potential of a Fe-Y zeolite prepared by a sono-assisted ion exchange method in different applications: biomass conversion (lactose hydrolysis), enzyme mimetic materials (partial oxidation of benzene to phenol) and transformation of renewable energy (photodegradation of nitrobenzene and rhodamine B). The modification of FAU was performed at different sonication times 5, 15, 30 and 120 min and characterized by X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), N2 adsorption-desorption isotherms, magnetic and non-magnetic thermogravimetric analysis (MTGA and TGA), zeta potential (ζ), Fourier-transformed Infrared (ATR-FTIR), and ultraviolet–visible diffuse reflectance spectroscopy (UV–Vis DRS). Finding that the modification of zeolite Y with Fe species enhances its activity for the tested applications, owing their catalytic activity primarily to α-Fe (II) sites. Presenting the sono-assisted ion exchange method as an alternative for introducing Fe species and enhancing the catalytic activity of zeolites.

Enhancement of β-galactosidase catalytic activity and stability through covalent immobilization onto alginate/tea waste beads and evaluating its impact on the quality of some dairy products

The current study aimed to evaluate the hydrolysis of whole fat milk (WFM) and sweet whey (SW) using β-galactosidase (β-gal) after covalent immobilization onto activated alginate/tea waste (Alg/TW) beads as a novel carrier. The optimum temperature for free and Alg/TW/β-gal was 40 °C and the ideal pH was 7.0. However, Alg/TW/β-gal displayed better stabilities at high temperatures and a wide pH range. Additionally, the value of Km and Vmax for Alg/TW/β-gal was higher than the free enzyme. The Alg/TW/β-gal showed better residual activity (78.6 %) after 90 storage days at 4 °C. The reusability of Alg/TW/β-gal was very good as it conserved its full activity after 15 consecutive cycles and conserved 93 % of its initial activity after 10 cycles with ONPG (O-nitrophenyl-β-D-galactopyranoside) and lactose as a substrate, respectively. The impact of Alg/TW/β-gal on WFM and SW using HPLC analysis revealed a remarkable decrease in lactose concentration and increase of glucose and galactose concentrations. The SW exhibited higher degree of lactose hydrolysis (97.3 %) compared to WFM (62.4 %). Besides, SW had a prominent increase in total phenolic content (96.8 mg/L) compared to WFM (54.3 mg/L). The antioxidant activity had increased after enzyme treatment in both WFM and SW. The GC–MS analysis for volatile compounds identified twenty-five flavour constituents. Finally, Alg/TW/β-gal has a potential application for obtaining healthy, acceptable, and commercial dairy products of low lactose.

Preparation, characterisation and functional effects of goat milk whey protein isolate with low lactose after heat and non‐heat treatments

This study investigates the physicochemical and functional properties of goat milk whey with heat and non‐heat treatments (pasteurisation, high‐temperature heating, ultrasonic treatment and microwave heating) and lactose hydrolysis. The optimal lactose hydrolysis conditions were obtained through orthogonal test. The results revealed the lactose and glucose in low‐lactose goat milk whey remained largely unchanged through all four treatments, while high‐temperature and microwave treatments induced protein structure alteration. The foaming and emulsifying properties of low‐lactose whey powder were enhanced by heating and ultrasonic treatments, although foam stability decreased. Our findings provide valuable insights into exploiting more nutritional goat milk products.

A Novel β-Galactosidase from Kluyvera intermedia and its Potential for Hydrolyzing Lactose in Milk

ABSTRACT A new β-galactosidase (EC 3.2.1.23) discovered in Kluyvera intermedia (β-gal-Kli) was recombinantly produced in Escherichia coli BL21 (DE3). The bioreactor cultivation resulted in a maximum β-gal-Kli activity of 124.6 ± 25.5 μkat oNPGal,37 °C/Lculture. The β-gal-Kli was purified 4.5fold to a specific activity of 172.3 μkat oNPGal,37 °C/gprotein. The β-gal-Kli showed maximum activity at pH 6.5 and 50°C and was sufficiently active and stable at an industrially relevant temperature of 8°C (half-life of 63 days). The β-gal-Kli was compared to a commercially available β-galactosidase (opti-lactase LX2) for lactose hydrolysis in milk (45.3 ± 0.9 glactose/L) at 8°C. With the activity of 2.7 nkatlactose/mLmilk, “lactose-free” (lactose <0.1 g/L) was realized by β-gal-Kli after 72 h, while 192 h was needed for opti-lactase LX2. Additionally, the galactooligosaccharide synthesis was observed with both these two enzymes. In conclusion, the β-gal-Kli showed its potential for the production of “lactose-free” dairy products.

Immobilization of the β-galactosidase enzyme by encapsulation in polymeric matrices for application in the dairy industry

Lactose intolerance affects approximately 65% of the global adult population, leading to the demand for lactose-free products. The enzyme β-galactosidase (βG) is commonly used in the industry to produce such products, but its recovery after lactose hydrolysis is challenging. In this scenario, the study aims to encapsulate βG within capsules, varying in dimensions and wall materials, to ensure their suitability for efficient industrial recovery. The enzyme βG was encapsulated through ionic gelation using alginate and its blends with pectin, maltodextrin, starch, or whey protein as wall materials. The capsules produced underwent evaluation for encapsulation efficiency, release profiles, activity of the βG enzyme, and the decline in enzyme activity when reused over multiple cycles. Alginate at 5% wt/vol concentrations, alone or combined with polymers such as maltodextrin, starch, or whey protein, achieved encapsulation efficiencies of approximately 98%, 98%, 80%, and 88%, respectively. The corresponding enzyme recovery rates were 34%, 19%, 31%, and 48%. Capsules made with an alginate-pectin blend exhibited no significant hydrolysis and maintained an encapsulation efficiency of 79%. Encapsulation with alginate alone demonstrated on poor retention of enzyme activity, showing a loss of 74% after just 4 cycles of reuse. Conversely, when alginate was mixed with starch or whey protein concentrate, the loss of enzyme activity was less than 40% after 4 reuses. These results highlight the benefits of combining encapsulation materials to improve enzyme recovery and reuse, offering potential economic advantages for the dairy industry.

β-galactosidase immobilization on ceramic ultrafiltration membrane for simultaneous lactose hydrolysis and protein separation

Protein and lactose of cheese whey can be separated using ultrafiltration membranes and lactose can be hydrolyzed by β-galactosidase. This study investigated the integration of ultrafiltration and enzymatic hydrolysis for simultaneous protein concentration and lactose hydrolysis. β-galactosidase was immobilized on ceramic membrane via surface activation with gelatin and subsequent enzyme cross-linking with glutaraldehyde. Membrane geometries, buffer types, gelatin and enzyme concentrations were optimized. The optimal results yielded a 63 % enzyme immobilization efficiency and a 93 % lactose hydrolysis rate by using disc membrane, sodium acetate buffer (pH 4.8), sodium phosphate buffer (pH 7.5), 0.1 g/L gelatin concentration, and 5.0 g/L enzyme concentration. The optimized enzymatic membrane was evaluated with synthetic and real whey. In synthetic whey, 85 % lactose hydrolysis and 89 % protein rejection were achieved. While in real whey, 72 % lactose hydrolysis and 83 % protein recovery were obtained. This approach offers a single-step, efficient, and scalable method for whey valorization with potential for industrial applications.

A cost-effective enzyme kinetics and inhibition model for biochemistry education and research.

Enzyme kinetics and inhibition studies are crucial in biochemistry education and research. Conventional methods often require expensive equipment and reagents, potentially limiting their accessibility in limited resource settings. Our approach sought to develop a cost-effective experimental design for studying enzyme kinetics and inhibition. Lactase was chosen as a protein model and its activity was investigated by measuring glucose production from lactose hydrolysis. In the study, commercially available lactase pills were used as an enzyme source, while milk was used as a substrate. Instead of scientific equipment, glucometers were used to measure lactase activity. Enzyme kinetics were evaluated using Michaelis-Menten and Lineweaver-Burk plots. In the study, the effects of temperature, pH, and inhibitors were also investigated. The results of our study aligned with established enzyme kinetics theories and previous studies. Lactase showed temperature and pH-dependent activity, with decreased activity observed at both low and high extremes. Results also showed that galactose acts as a competitive inhibitor of lactase. The approach presented here offers a cost-effective procedure for studying enzyme kinetics and inhibition. It can act as a valuable tool for educational purposes and for preliminary research in settings with limited resources.

New perspective into the interaction behavior explore of Nano-berberine with alpha-lactalbumin in the presence of beta-lactoglobulin: Multi-spectroscopic and molecular dynamic investigations

The present study was focus on the interaction of alpha lactalbumin (α-LA) and Nano-berberine (Nano-Ber) in the absence and presence of beta lactoglobulin (β-LG) as binary and ternary systems by the exertion of different spectroscopic techniques, conductometry, β-galactosidase activity, and molecular simulation measurements. The outcomes of fluorescence quenching clarified the dominance of static quenching mechanism throughout the interaction of α-LA-Nano-Ber and (α-LA-β-LG) Nano-Ber complexes and also determined the KSV values to be (4.68±0.05) × 104 and (5.18±0.06) × 104 M−1, respectively. The measured thermodynamic parameters exhibited the participation of electrostatic forces in the binding interaction of α-LA and α-LA-β-LG complex with Nano-Ber. Moreover, the negative value of ΔG0 represents the spontaneous mode of this binding process in both systems. Synchronous fluorescence and resonance light scattering data demonstrated the impact of β-LG attendance on altering the interaction behavior of α-LA and Nano-Ber. The changes in the secondary structure of both proteins were affirmed by circular dichroism spectroscopy (CD) technique. According to the displayed β-galactosidase activity in the Michaelis-Menten plot of both binary and ternary systems, a greater Vmax refers to the stronger affinity of lactose and lactose hydrolysis that can be effective on the lactose intolerance regarding dairy products. Additionally, molecular dynamic simulations were employed to simulate the atomic-level interactions and explore the stability and conformational changes of protein-Nano Ber complexes over time. The results of these comprehensive biophysical investigations shed light on the binding characteristics, structural alterations, and dynamic behavior of Nano-Ber upon interaction with α-lactalbumin in the absence and presence of β-lactoglobulin as the binary and ternary systems. This study reveals the Nano-Ber role in the protein–protein interaction behavior and provides valuable insights into the potential applications of α-lactalbumin in the design of functional food formulations and drug delivery systems

Stabilization of β-D-galactosidase in solution containing chitosan-based membrane: Central composite rotatable design

β-D-galactosidase is a hydrolase enzyme capable of hydrolyzing lactose in milk-based foods. Its free form can be inactivated in solution during the production of low-dosage lactose foods. Then, it is important to study strategies for avoiding the free enzyme inactivation with the aim of circumventing this problem. The stabilization of β-D-galactosidase in aqueous solution after interactions with chitosan/eucalyptus sawdust composite membrane proved to be a potential strategy when optimized by central composite rotatable (CCR) design. In this case, the best experimental conditions for β-D-galactosidase partitioning and stability in an aqueous medium containing the chitosan-based composite membrane reinforced with eucalyptus sawdust were i) enzyme/buffer solution ratio of 0.0057, ii) pH 5.6, iii) membrane mass of 50 mg, and iv) temperature lower than 37 °C. Significance was found for the linear enzyme/buffer solution ratio, linear temperature, and quadratic pH (p < 0.05) in the interval between 0 and 60 min of study. In the interval between 60 and 120 min, there was significance (p < 0.12) for linear temperature, the temperature-enzyme/buffer solution ratio interaction and the interaction between linear pH and linear enzyme/buffer solution ratio. The Pareto charts and response surfaces clearly showed all the effects of the experimental variables on the stabilization of β-D-galactosidase in solution after interactions with the chitosan composite membrane. In this case, industrial food reactors covered with chitosan/eucalyptus sawdust composite membrane could be a strategy for the hydrolysis of lactose during milk-producing processes.

The archaeal highly thermostable GH35 family β-galactosidase DaβGal has a unique seven domain protein fold.

The most extensively studied β-d-galactosidases (EC3.2.1.23) belonging to four glycoside hydrolase (GH) families 1, 2, 35, and 42 are widely distributed among Bacteria, Archaea and Eukaryotes. Here, we report a novel GH35 family β-galactosidase from the hyperthermophilic Thermoprotei archaeon Desulfurococcus amylolyticus (DaβGal). Unlike fungal monomeric six-domain β-galactosidases, the DaβGal enzyme is a dimer; it has an extra jelly roll domain D7 and three composite domains (D4, D5, and D6) that are formed by the distantly located polypeptide chain regions. The enzyme possesses a high specificity for β-d-galactopyranosides, and its distinguishing feature is the ability to cleave pNP-β-d-fucopyranoside. DaβGal efficiently catalyzes the hydrolysis of lactose at high temperatures, remains stable and active at 65 °С, and retains activity at 95 °С with a half-life time value equal to 73 min. These properties make archaeal DaβGal a more attractive candidate for biotechnology than the widely used fungal β-galactosidases.

Isolation and characterization of a β-galactosidase from Lactobacillus helveticus for industrial processing

In this study, a thermostable β-galactosidase from Lactobacillus helveticus (Lb. helveticus) OSU-PECh-4A has been isolated through diafiltration and size-exclusion chromatography. The enzyme consists of a heterodimer with a molecular mass of 110 kDa, with a small and large subunit of 36 kDa and 74 kDa, respectively. The Km and Vmax values for lactose and o-nitrophenyl-β-D-galactopyranoside (oNPG) hydrolysis were, respectively, 29.87 ± 1.05 mM, 1.88 ± 0.02 μmol D-glucose released per min per mg of protein, and 0.067 ± 0.003 mM, 1.70 ± 0.05 μmol o-nitrophenol (oNP) released per min per mg of protein. This β-galactosidase is significantly activated by Mg+2 (2–10 mM) and slightly inhibited by D-Glucose. The enzyme can also hydrolyze 57 ± 3% of lactose after 12 h of reaction at 45°C and under high lactose concentration. We propose that this enzyme provides a significant advantage from a practical and consumer point of view due to its origins as a probiotic source and improved features for important industrial applications, such as lactose hydrolysis and the potential to produce galacto-oligosaccharides (GOS).

The covalent immobilization of β-galactosidase from Aspergillus oryzae and alkaline protease from Bacillus licheniformis on amino-functionalized multi-walled carbon nanotubes in milk

This study aimed was to covalently immobilize β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis on amino-functionalized multi-walled carbon nanotubes. In this study, a two-level factorial design was employed to investigate the impact of seven continuous variables (activation pH, glutaraldehyde molarity, activation time (0–8 h), buffer solution pH (8-0), buffer solution molarity, MWCNT-NH2-glutaraldehyde quantity, and stabilization time (0–180 h)) on the immobilization efficiency and enzymatic activity of protease and β-galactosidase. Furthermore, the effect of time on the percentage of enzymatic activity was examined during specific intervals (24, 48, 72, 96, and 120 h) of the immobilization process. The analysis of variance results for protease enzymatic activity revealed a notable influence of the seven variables on immobilization efficiency and enzymatic activity. Additionally, the findings indicate that activation time, buffer pH, MWCNT-NH2-glutaraldehyde quantity, and stabilization time significantly affect the activity of the protease enzyme. The interplay between buffer pH and stabilization time is also significant. Indeed, both activation time and the quantity of MWCNT-NH2-glutaraldehyde exert a reducing effect on enzyme activity. Notably, the influence of MWCNT-NH2-glutaraldehyde quantity is more significant (p < 0.05). In terms of beta-galactosidase enzymatic activity, the study results highlight that among the seven variables considered, only the glutaraldehyde molarity, activation time, and the interplay of activation time and the quantity of MWCNT-NH2-glutaraldehyde can exert a statistically significant positive impact on the enzyme's activity (p < 0.05). The combination of activation time and buffer solution molarity, as well as the interactive effect of buffer pH and MWCNT–NH2–glutaraldehyde, can lead to a significant improvement in the stabilization efficiency of the protease of carbon nanotubes. The analysis of variance results demonstrated that the efficiency of covalently immobilizing β-galactosidase from Aspergillus oryzae on amino-functionalized multi-walled carbon nanotubes is influenced by the molarity of glutaraldehyde, buffer pH, stabilization time, and the interplay of activation time + buffer pH, buffer pH + activation time, activation time + buffer molarity, and glutaraldehyde molarity + MWCNT-NH2-glutaraldehyde (p < 0.05). Through the optimization and selection of optimal formulations, the obtained results indicate enzyme activities and stabilization efficiencies of 64.09 % ± 72.63 % and 65.96 % ± 71.77 % for protease and beta-galactosidase, respectively. Moreover, increasing the enzyme stabilization time resulted in a reduction of enzyme activity. Furthermore, an increase in pH, temperature, and the duration of milk storage passing through the enzyme-immobilized carbon nanotubes led to a decrease in enzyme stabilization efficiency, and lactose hydrolysis declined progressively over 8-h. Hence, the covalent immobilization of β-galactosidase from Aspergillus oryzae and protease from Bacillus licheniformis onto amino-functionalized multi-walled carbon nanotubes is anticipated to be achievable for milk applications.

Kinetics and products of Thermotoga maritima β-glucosidase with lactose and cellobiose

Galacto-oligosaccharides (GOS) are prebiotic compounds that are mainly used in infant formula to mimic bifidogenic effects of mother’s milk. They are synthesized by β-galactosidase enzymes in a trans-glycosylation reaction with lactose. Many β-galactosidase enzymes from different sources have been studied, resulting in varying GOS product compositions and yields. The in vivo role of these enzymes is in lactose hydrolysis. Therefore, the best GOS yields were achieved at high lactose concentrations up to 60%wt, which require a relatively high temperature to dissolve. Some thermostable β-glucosidase enzymes from thermophilic bacteria are also capable of using lactose or para nitrophenyl-galactose as a substrate. Here, we describe the use of the β-glucosidase BglA from Thermotoga maritima for synthesis of oligosaccharides derived from lactose and cellobiose and their detailed structural characterization. Also, the BglA enzyme kinetics and yields were determined, showing highest productivity at higher lactose and cellobiose concentrations. The BglA trans-glycosylation/hydrolysis ratio was higher with 57%wt lactose than with a nearly saturated cellobiose (20%wt) solution. The yield of GOS was very high, reaching 72.1%wt GOS from lactose. Structural elucidation of the products showed mainly β(1 → 3) and β(1 → 6) elongating activity, but also some β(1 → 4) elongation was observed. The β-glucosidase BglA from T. maritima was shown to be a very versatile enzyme, producing high yields of oligosaccharides, particularly GOS from lactose.Key points• β-Glucosidase of Thermotoga maritima synthesizes GOS from lactose at very high yield.• Thermotoga maritima β-glucosidase has high activity and high thermostability.• Thermotoga maritima β-glucosidase GOS contains mainly (β1-3) and (β1-6) linkages.Graphical

Diaminated Cellulose Beads as a Sustainable Support for Industrially Relevant Lipases.

Environmentally persistent polystyrene or polyacrylic beads are used as supports in enzyme large-scale bioprocesses, including conversion glucose isomerization for high-fructose corn syrup production, hydrolysis of lactose, and synthesis of active pharmaceutical ingredients. In this paper, we report the development of a novel sustainable and scalable method to produce diaminated cellulose beads (DAB) as highly efficient alternative supports for industrially relevant lipases. Regenerated cellulose beads were grafted with diaminated aliphatic hydrocarbons via periodate oxidation and reductive amination. The oxidation step indicated that aldehyde content can be easily tuned through the reaction time and concentration of reactants. Reductive amination of dialdehyde cellulose was more efficient as the length of the diaminated hydrocarbon compound increased. Morphological analysis of DAB showed that cellulose chemical grafting enabled the preservation of the bead shape and internal structure upon freeze-drying. Enzymatic degradability studies demonstrated that chemical functionalization did not undermine enzyme cellulose hydrolysis. The addition of aminated moieties on cellulose dramatically increased absorption efficiency for all industrially relevant lipases used, reaching 100% for Thermomyces lanuginosus lipase (TLL). Storage and recyclability experiments demonstrated that enzymes were retained and recyclable for at least nine cycles, although the activity gradually declined after each cycle. Medium chain triacylglycerol hydrolysis in a SpinChem reactor using TLL immobilized on 1,6 DAB exhibited higher activity compared to acrylic beads (588 vs 459 U/g) suggesting that biodegradable cellulose-based materials could be a valid and attractive alternative to plastics carriers.

Engineering the β-galactosidase from Aspergillus oryzae for making lactose-free and no-sugar-added yogurt

Yogurt usually contains 5% to 7% sugar and 3% to 5% lactose. As β-galactosidases can hydrolyze lactose and improve sweetness, they have the potential to produce lactose-free (LF) and no-sugar-added (NSA) yogurt. In this study, the β-galactosidase AoBgal35A from Aspergillus oryzae was engineered by site-saturation mutagenesis. Results of 19 variants of T955 residue showed that the lactose hydrolysis rate of T955R-AoBgal35A was up to 90.7%, which is much higher than the 78.5% of the wild type. Moreover, the optimal pH of T955R-AoBgal35A was shifted from pH 4.5 to pH 5.5, and the optimal temperature decreased from 60°C to 50°C. The mutant T955R-AoBgal35A was successfully expressed in Komagataella pastoris, which produced extracellularly 4,528 U/mL of β-galactosidase activity. The mutant T955R-AoBgal35A was used to produce LF yogurt. The Streptococcus thermophilus count of LF yogurt increased from 7.9 to 9.5 log cfu/g, which is significantly higher than that of the control group (8.9 log cfu/g). The residual lactose content of LF yogurt was 0.13%, meeting the requirements of the national standard in China for the "lactose-free" label (<0.5%). Furthermore, sugar in yogurt was replaced by whey powder to produce LF-NSA yogurt. The optimal addition content of whey powder was 7.5%. The texture, water-holding capacity, and titratable acidity of LF and LF-NSA yogurt achieved good shelf life stability. Therefore, this study provides an insight for technological implications of β-galactosidases in the dairy industry.