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Cold-adapted microorganisms possess cold-active enzymes with potential applications in different industries and research areas. In this study, two genes encoding β-d-galactosidases belonging to Glycoside Hydrolase families 2 and 42 from the psychrotolerant Arctic bacterium Arthrobacter sp. S3* were cloned, expressed in Escherichia coli and Komagataella phaffii, purified and characterized. The GH2 β-d-galactosidase is a tetramer with a molecular weight of 450 kDa, while the GH42 β-d-galactosidase is a 233 kDa trimer. The Bgal2 was optimally active at pH 7.5 and 22 °C and maintained 57% of maximum activity at 10 °C, whereas the Bgal42 was optimally active at pH 7.0 and 40 °C and exhibited 44% of maximum activity at 10 °C. Both enzymes hydrolyzed lactose and showed transglycosylation activity. We also found that 2 U/mL of the Bgal2 hydrolyzed 85% of lactose in milk within 10 h at 10 °C. The enzyme synthesized galactooligosaccharides, heterooligosaccharides, alkyl galactopyranosides and glycosylated salicin. The Bgal42 synthesized galactooligosaccharides and 20 U/mL of the enzyme hydrolyzed 72% of milk lactose within 24 h at 10 °C. The properties of Arthrobacter sp. S3* Bgal2 make it a candidate for lactose hydrolysis in the dairy industry and a promising tool for the glycosylation of various acceptors in the biomedical sector. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract Background The 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 K M 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. Results Firstly, 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 µkat oNPGal/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 µkat oNPGal/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 µkat oNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter). Conclusion This 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.

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.

Background: The 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. Results: Firstly, 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). Conclusion: This 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. [ABSTRACT FROM AUTHOR]

The object of this study was the process of milk sugar hydrolysis in liquid whey concentrates. The problem solved was the intensification of the lactose hydrolysis reaction in liquid whey concentrates through the use of a combination of leavening and enzyme preparations. Patterns in the lactose hydrolysis process in liquid concentrates of demineralized whey were studied. It was established that the use of an enzyme preparation for liquid whey concentrates does not make it possible to achieve a degree of lactose hydrolysis higher than 75–77 % within 10 h. The simultaneous use of enzyme and leavening preparations for 6 hours ensures the conversion of more than 95 % of lactose for concentrates with a mass fraction of dry substances of 10–30 % and more than 90 % for a 40 % concentrate. The rational duration of lactose hydrolysis in demineralized whey concentrates for the combination of preparations is 6 hours for 10 and 20 % concentrates, and 8 h for 30 and 40 % concentrates, which enables lactose hydrolysis at the level of 96.8–100 %. The rheological properties of concentrates with a solids content of 30 and 40 % indicate that these systems have a high ability to restore the structure. The dynamics of monosaccharide formation during hydrolysis are similar for 10 and 20 % concentrates, in which galactose slightly predominates the systems. Data on the increase in glucose content in fermented 30 and 40 % concentrates contradict the known data regarding the consumption of glucose by the acidophilic bacillus and its conversion to galactose. This may indicate suppression of the activity of the acidophilic bacillus under conditions of increased osmotic pressure in the concentrates. The results of the work could be used in the technology of whey ice cream, as well as dairy products that require adjustment of the chemical composition, primarily in terms of protein and lactose content. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 μkatoNPGal,37 °C/Lculture. The β-gal-Kli was purified 4.5fold to a specific activity of 172.3 μkatoNPGal,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. [ABSTRACT FROM AUTHOR]

Commercial β-galactosidases exhibit undesirable kinetic properties regarding substrate affinity (Michaelis-Menten constant [ K M ] for lactose) and product inhibition (inhibitor constant [ K i ] for galactose). An in silico screening of gene sequences was done and identified a putative β-galactosidase (Paenibacillus wynnii β-galactosidase, BgaPw) from the psychrophilic bacterium Paenibacillus wynnii. The cultivation of the wild-type P. wynnii strain resulted in very low β-galactosidase activities of a maximum of 150 nkat per liter of medium with o -nitrophenyl-β- d -galactopyranoside (o NPGal) as substrate. The recombinant production of BgaPw in Escherichia coli BL21(DE3) increased the yield ∼9,000-fold. Here, a volumetric activity of 1,350.18 ± 11.82 μkat o NPGal /L culture was achieved in a bioreactor cultivation. The partly purified BgaPw showed a pH optimum at 7.0, a temperature maximum at 40°C, and an excellent stability at 8°C with a half-life of 77 d. Kinetic studies with BgaPw were done in milk or in milk-imitating synthetic buffer (Novo buffer), respectively. Remarkably, the K M value of BgaPw with lactose was as low as 0.63 ± 0.045 m M in milk. It was found that the resulting products of lactose hydrolysis, namely galactose and glucose, did not inhibit the β-galactosidase activity of BgaPw, but instead showed a striking activating effect in both cases (up to 144%). In a comparison study in milk, lactose was completely hydrolyzed by BgaPw in 72 h at 8°C, whereas 2 other known β-galactosidases were less powerful and converted only about 90% of lactose in the same time. Finally, the formation of galactooligosaccharides (GOS) was demonstrated with the new BgaPw, starting with pharma-lactose (400 g/L). A GOS production of about 144 g/L was achieved after 24 h (36.0% yield). [ABSTRACT FROM AUTHOR]

The development of hydrolyzed lactose powder products involves technological, sensory, and nutritional challenges (adhesion in the drying chamber, browning of the powder, and formation of Maillard reaction products). Recent studies have analyzed the use of transglutaminase (TGase) to reduce product adhesion in dryers and the intensity of the Maillard reaction. This study aimed to evaluate the application of TGase to powdered dairy products with and without hydrolyzed lactose. Skimmed milk powder was dried under four different treatments with three replicates resulting in 12 products (Treatment 1 skimmed milk powder; Treatment 2 skimmed milk powder with lactase; Treatment 3 skimmed milk powder with TGase; Treatment 4 skimmed milk powder with lactase and TGase). The product retention capacity in the equipment; compositional, physicochemical, and morphological attributes; particle size; and Maillard reaction markers were analyzed. The use of TGase did not affect drying conditions or powder processing. Transglutaminase increased the particle size of the products without altering their bimodal conformation. The color of the product was not modified regardless of whether TGase was added. In the hydrolyzed product with added TGase, the HMF value increased (6–21 times). The use of TGase did not affect the hydrolysis of lactose, and a decrease in the intensity of the casein bands (glutamine-lysine crosslinking) was observed by electrophoresis. This study led to new hypotheses and possibilities for future studies to elucidate the influence of TGase addition to powdered dairy products. [ABSTRACT FROM AUTHOR]

Research has focused on biomass as a sustainable energy source, driving biomass valorization to convert carbohydrates. The price of lactose has dropped below the production cost, urging exploration for diverse applications, such as conversion into value-added compounds. Zeolites, especially Y zeolite, enhance hexose conversion when modified with metals. Fe-modified zeolites show promise, with controlled Si/Al ratios impacting acidity and selectivity. The recent challenges remain in active site comprehension, catalyst tunability, and reaction optimization. Therefore, this study explores the selective hydrolysis of lactose, limiting undesired side-product formation catalyzed through a sono-assisted modified zeolite Y with Fe. The obtained series of Fe-modified zeolite Y were evaluated for the hydrolysis of lactose in a screening experiment. The samples with the higher potential were further used to optimize reaction conditions by a response surface methodology (RSM). This revealed that the samples prepared at high pH generated more impurities and that higher temperatures and lactose concentrations favored hydrolysis. Despite their apparent similarities, the selected Fe-modified zeolite Y samples (Fe5Y and FeYII) highlight distinct catalytic behaviors and active sites. Employing UV-Vis spectroscopy, FTIR spectroscopy, and magnetic thermal gravimetric analysis (MTGA), various Fe active sites were detected, including α-Fe(II), FeOx nanoparticles, and iron oxides. These active sites give insight into the mechanisms involved with temperature and time, which play critical roles—evidencing the potential of sono-assisted iron modification of zeolite Y to achieve selective lactose hydrolysis. These findings contribute to understanding metal-zeolite composites as bifunctional catalysts and their application in sustainable processes. Furthermore, this research highlights the complexity of catalytic reactions of modified zeolites and their potential for fine-tuning active sites for various applications, particularly lactose hydrolysis. Such advancements hold the promise of propelling the field of sustainable energy and catalysis towards a more efficient and environmentally friendly future.

Abstractβ-Galactosidases are crucial carbohydrate-active enzymes that naturally catalyze the hydrolysis of galactoside bonds in oligo- and disaccharides. These enzymes are commonly used to degrade lactose and produce low-lactose and lactose-free dairy products that are beneficial for lactose-intolerant people. β-galactosidases exhibit transgalactosylation activity, and they have been employed in the synthesis of galactose-containing compounds such as galactooligosaccharides. However, most β-galactosidases have intrinsic limitations, such as low transglycosylation efficiency, significant product inhibition effects, weak thermal stability, and a narrow substrate spectrum, which greatly hinder their applications. Enzyme engineering offers a solution for optimizing their catalytic performance. The study of the enzyme’s structure paves the way toward explaining catalytic mechanisms and increasing the efficiency of enzyme engineering. In this review, the structure features of β-galactosidases from different glycosyl hydrolase families and the catalytic mechanisms are summarized in detail to offer guidance for protein engineering. The properties and applications of β-galactosidases are discussed. Additionally, the latest progress in β-galactosidase engineering and the strategies employed are highlighted. Based on the combined analysis of structure information and catalytic mechanisms, the ultimate goal of this review is to furnish a thorough direction for β-galactosidases engineering and promote their application in the food and dairy industries. [ABSTRACT FROM AUTHOR]

Eggshell membrane (ESM) is used in immobilization studies due to its large surface area, non-toxicity and biodegradability. In this study, β-galactosidase was immobilized on ESM using the adsorption and cross-linking method. The interactions between enzyme and ESM after immobilization were determined by ATR-FTIR (attenuated total reflection fourier transform infrared spectroscopy) and SEM (scanning electron microscope). The optimum temperature of the free enzyme was found to be 35 °C, and this value was 45 °C for the immobilized enzyme. Immobilized enzyme managed to retain more than 50% of its activity after 8 reuses. In the lactose removal experiment from milk, the highest reaction efficiency was found as 55.8% under specified optimization conditions for β-galactosidase immobilized ESM after 3 h. Due to the microfiber protein structure of the ESM, it has improved enzyme stability properties, as a result of the multi protein–protein interactions formed after the immobilization between the proteins in the ESM and the enzyme molecules. [ABSTRACT FROM AUTHOR]

The lactose-free dairy market has significantly grown in the past years in relation to the large population who experience lactose intolerance. Lactose-free dairy products are nutritionally more attractive than alternative lactose-free non-dairy products because of the superior amino acid profile. Of the methods to produce lactose-free dairy products, lactose hydrolysis is the most economical and sustainable since there is no by-product formed in the process and offers the possibility of generating value-added products, like galactooligosaccharides. This review covers the advantages and disadvantages of techniques for hydrolyzing lactose in milk and pure lactose. Mainly, it focuses on enzymatic hydrolysis methods. Additionally, it analyzes scale-up problems with specific techniques and what future research should be conducted to generate novel products of commercial utility. Currently, pre-process hydrolysis using soluble β-galactosidase is the most common way to produce lactose hydrolyzed milk industrially while academic research focuses on immobilized packed bed reactors to recycle enzymes and produce higher yields of value-added products. Further experimental research should be conducted on developing a commercial scale immobilized enzyme bioreactor to produce more economically and environmentally attractive lactose hydrolyzed milk. [ABSTRACT FROM AUTHOR]

Enzymatic hydrolysis of lactose is a crucial step to improve the efficiency and selectivity of membrane-based separations toward the recovery of milk oligosaccharides free from simple sugars. Response surface methodology was used to investigate the effects temperature (25.9 to 54.1 °C) and amount of enzyme (0.17 to 0.32% w/w) at 1, 2, and 4 h of reaction on the efficiency of lactose hydrolysis by Aspergillus oryzae β-galactosidase, preservation of major goat whey oligosaccharides, and on the de-novo formation of oligosaccharides. Lactose hydrolysis above 99% was achieved at 1, 2, and 4 h, not being significantly affected by temperature and amount of enzyme within the tested conditions. Formation of 4 Hexose (Hex) and 4 Hex 1 Hex and an increased de-novo formation of 2 Hex 1 N-Acetyl-Neuraminic Acid (NeuAc) and 2 Hex 1 N-Glycolylneuraminic acid (NeuGc) was observed in all treatments. Overall, processing conditions using temperatures ≤40 °C and enzyme concentration ≤0.25% resulted in higher preservation/formation of goat whey oligosaccharides.

The article describes the characteristics and classification of partial (hypolactasia) and complete (alactasia) lactose intolerance; the classification of dairy products for people with lactose intolerance is considered, the lactose content requirements for low-lactose and lactose-free products are indicated. The parameters of lactose hydrolysis in milk using the enzyme β-galactosidase HA-Lactase 2100 of the Danish company Christian Hansen are given.. Lactulose has been characterized as a prebiotic used in dairy product technologies to prevent dysbacteriosis. The mass fraction of lactulose in pasteurized low-lactose milk is substantiated. The expediency of production of pasteurized low-lactose milk with a lactulose content of 0.3% is shown, which will provide the human body with 30.0–50.0% lactulose when consuming the daily norm of the product - 500 cm3. The change of quality indicators in pasteurized low-lactose milk in hermetically sealed containers - plastic bottles with a capacity of 1000 cm3, produced under production conditions at Hormolzavod LLC (Odesa), during the storage process - was determined. It was noted that during 25 days of storage, the quality indicators of the target product meet the requirements of the current regulatory documents for drinking milk, in particular, according to the "titrated acidity" indicator, and during further storage, this indicator exceeds the normative value Recommendations are provided regarding the storage parameters of pasteurized low-lactose milk with lactulose in a hermetic container: taking into account the reserve ratio, which for drinking milk should be 1.3, the maximum storage period of pasteurized low-lactose milk with lactulose, produced at Hormolzavod LLC (Odesa), should not exceed 19.2 days at a temperature of 2–6 ºС. Taking into account the fact that the storage of the product at the enterprise during experimental research was carried out at a temperature of 2 ºС, it was decided to recommend establishing a maximum storage period of pasteurized low-lactose milk with lactulose at a temperature of 2–6 ºС for no more than 18 days to prevent spoilage of the product in trade networks. where its storage temperature can be 2–6 ºС. [ABSTRACT FROM AUTHOR]

ABSTRACT: β-Galactosidase (lacA) from Aspergillus oryzae is widely used in the dairy industry. Its acidic pH optimum and severe product inhibition limit its application for lactose hydrolysis in milk. In the present study, structure-based sequence alignment was conducted to determine the candidate mutations to shift the pH optimum of lacA to the neutral range. The Y138F and Y364F mutants shifted the pH optimum of lacA from 4.5 to 5.5 and 6.0, respectively. The acid dissociation constant (pKa) values of catalytic acid/base residues with upwards shift were consistent with the increased pH optimum. All variants in the present study also alleviated galactose inhibition to various extents. Molecular dynamics demonstrated that the less rigid tertiary structures and lower galactose-binding free energy of Y138F and Y364F might facilitate the release of the end product. Both Y138F and Y364F mutants exhibited better hydrolytic ability than lacA in milk lactose hydrolysis. The higher pH optimum and lower galactose inhibition of Y138F and Y364F may explain their superiority over lacA. The Y138F and Y364F mutants in the present study showed potential in producing low-lactose milk, and our studies provide a novel strategy for engineering the pH optimum of glycoside hydrolase.

Preparation of low lactose milk and milk products have attracted the attention of consumers due to increasing prevalence of lactose intolerance. However, the physico-chemical properties of product prepared from the single enzyme application (β-galactosidase) possess a significant concern of Maillard browning. While previous studies adjusted spray drying conditions to address Maillard browning in low lactose powder, our research introduces a multi-enzyme system for lactose hydrolysis in concentrated skim milk to produce a spray-dried powder with reduced browning. Glucose oxidase (GOX) converts glucose into gluconic acid that lowers down the concentration of Maillard reaction products (MRP's) formed during manufacture of lactose hydrolysed milk products. The activity of GOX was enhanced when used along with catalase (CAT). Use of GOX + CAT in buffer solution (containing 9 % glucose) exhibited 61.44 % glucose reduction after 22 h of reaction. GOX (70 U/ml) along with CAT (25 U/ml) when added to milk after 5 h of β-galactosidase (9.53 U/ml) action resulted in significantly higher reduction of glucose (53.95 %) after 20 h with final milk pH of 6.5. Spray dried powder prepared using optimised multi-enzymes (β-galactosidase and GOX + CAT) treated milk revealed reduced browning characteristics i.e., 12.85 browning index as compared to the powder prepared from milk treated with β-galactosidase alone (16.37). Industrial relevance: The use of GOX + CAT during lactose hydrolysis could serve as a potential approach for mitigating browning characteristics in low-lactose milk and milk products. The multi-enzymatic system used to produce low-lactose milk can be easily adapted by large-scale industries to produce a powder with physico-chemical properties similar to traditional skim milk powder. • Use of multi-enzyme system for preparation of lactose hydrolysed milk (LHM) products. • Multi-enzyme system includes β-galactosidase, glucose oxidase (GOX), and catalase (CAT). • Multi-enzyme approach reduced glucose content by converting glucose into gluconic acid. • Preparation of LHM powder with reduced Maillard browning characteristics. [ABSTRACT FROM AUTHOR]

The current research demonstrates the synthesis of zinc oxide nanoparticles (ZnO-NPs) via green nanotechnology approach (Azatirachta indica leaves). The size of the synthesized ZnO-NPs was confirmed as 27 nm by TEM. Glutaraldehyde was used to modify the surface of the developed ZnO-NPs in order to promote covalent binding of Aspergillus oryzae β-galactosidase. Enzyme activity was achieved as 93% on glutaraldehyde modified ZnO-NPs. The immobilized enzyme exhibited significant enhancement in activity under extreme temperature and pH variations, as compared to the soluble β-galactosidase (SβG). It was further observed that the immobilized enzyme retained 58% activity at 5% galactose concentration. However, under similar experimental conditions, SβG showed 27% activity. Reusability of immobilized enzyme revealed that it retained 89% activity even after fifth repeated use, and hence could be recovered easily by centrifugation for repeated use in biotechnological applications. Batch reactor experiment indicates that the immobilized enzyme displayed 81% and 70% lactose hydrolysis at 50 °C and 60 °C, respectively as compared to 70% and 58% lactose hydrolysis by soluble enzyme under identical conditions after 9 h.

BACKGROUND: The enzyme β‐galactosidase (EC 3.2.1.2.3) obtained from yeast is produced intracellularly. Cell extraction consists of the treatment of a cell suspension with chemical agents, thus obtaining the enzyme with biological activity. After obtaining the extracted enzyme, it is concentrated by ultrafiltration and dried. Lyophilization can be used in this step. The objective of this work was to study the production and extraction of β‐galactosidase from Saccharomyces fragilis IZ 275 using lactose from cheese whey. RESULTS: Under optimum extraction conditions, 2% (v/v) chloroform, 37 °C for 6 h of extraction, 35% of lactose hydrolysis was achieved using 1% (v/v) β‐galactosidase during 10 h of hydrolysis. Extracted enzyme activity resulted in 81% lactose hydrolysis using 12% (v/v) enzyme at pH 6.0 and 37 °C. Enzymatic kinetics were evaluated using 12% of extracted enzyme in 5 h of hydrolysis reaction, and 100% lactose hydrolysis was obtained. The enzyme activity after extraction, concentration and lyophilization was 100%, 60% and 45.3% of lactose hydrolysis, respectively, after 5 h of hydrolysis reaction at 37 °C and pH 6.0. CONCLUSION: The yeast S. fragilis IZ 275 grown in cheese whey produces intracellular β‐galactosidase and its extraction is efficient with the use of 2% chloroform at 37 °C for 6 h, allowing its use for industrial and pharmaceutical purposes. The extracted enzyme must be used at 37 °C and pH 6.0 at a concentration of 12% (v/v) of enzyme to obtain the total hydrolysis of lactose in 5 h of reaction. © 2022 Society of Chemical Industry (SCI). [ABSTRACT FROM AUTHOR]

A major challenge in isolating oligosaccharides from dairy streams is to enrich oligosaccharides while simultaneously reducing the content of simple sugars (mono- and disaccharides) that do not possess the desired prebiotic functions. An integrated approach based on optimized conditions that favor maximum lactose hydrolysis, monosaccharide fermentation and oligosaccharides recovery by nanofiltration was developed. Upon complete lactose hydrolysis and fermentation of the monosaccharides by yeast, nanofiltration of fermented whey permeate from colostrum enabled the recovery of 95% of the oligosaccharides at high purity. While the number of commercially available standards has limited the quantification of only a few sialylated oligosaccharides, the application of both high performance anion-exchange chromatography with pulsed amperometric detection and mass spectrometry provided a complete profile of the final product. Approximately 85% of the oligosaccharides in the final concentrate were sialylated, with the remainder being neutral.

The purification of caprine milk oligosaccharides (COS) by membrane filtration has been hampered by the low concentration of target COS and high concentration of lactose. In addition, their molecular weight proximity hinders the recovery of a COS fraction with high degree of purity and recovery yield. In this work, the recovery of a high purity COS concentrate was obtained by the optimization of an integrated approach including complete lactose hydrolysis, fermentation of the resulting monosaccharides and nanofiltration. All carbohydrates were quantified using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC PAD). Defatted goat whey was ultrafiltered with discontinuous diafiltrations to increase the recovery of COS in the whey permeate which was then subsequently concentrated by nanofiltration. COS recovery yields of 75% with negligible amounts of monosaccharides (0.3% of the initial amount of lactose in the whey permeate) were achieved. A final retentate containing 67.6 and 34.4% of acidic and neutral oligosaccharides respectively was obtained from caprine milk.

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 K m and V max 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. [Display omitted] [ABSTRACT FROM AUTHOR]

[Display omitted] • Cheese whey permeate (CWP) and crude glycerol (CG) are agro-food derived biomasses. • M-βGal is a β-galactosidase capable of hydrolyzing lactose in complex matrices. • CWP and CG are combined in a E. coli fed-batch culture to produce recombinant M-βGal. • Galactose, a by-product of the culture, can be extracted from the culture supernatant. • Recombinant enzymes and galactose can be efficiently produced from agro-food wastes. Responsible use of natural resources and waste reduction are key concepts in bioeconomy. This study demonstrates that agro-food derived-biomasses from the Italian food industry, such as crude glycerol and cheese whey permeate (CWP), can be combined in a high-density fed-batch culture to produce a recombinant β-galactosidase from Marinomonas sp. ef1 (M-βGal). In a small-scale process (1.5 L) using 250 mL of crude glycerol and 300 mL of lactose-rich CWP, approximately 2000 kU of recombinant M-βGal were successfully produced along with 30 g of galactose accumulated in the culture medium. The purified M-βGal exhibited high hydrolysis efficiency in lactose-rich matrices, with hydrolysis yields of 82 % in skimmed milk at 4 °C and 94 % in CWP at 50 °C, highlighting its biotechnological potential. This approach demonstrates the effective use of crude glycerol and CWP in sustainable and cost-effective high-density Escherichia coli cultures, potentially applicable to recombinant production of various proteins. [ABSTRACT FROM AUTHOR]

β-Galactosidase (lacA) from Aspergillus oryzae is widely used in the dairy industry. Its acidic pH optimum and severe product inhibition limit its application for lactose hydrolysis in milk. In the present study, structure-based sequence alignment was conducted to determine the candidate mutations to shift the pH optimum of lacA to the neutral range. The Y138F and Y364F mutants shifted the pH optimum of lacA from 4.5 to 5.5 and 6.0, respectively. The acid dissociation constant (p K a) values of catalytic acid/base residues with upwards shift were consistent with the increased pH optimum. All variants in the present study also alleviated galactose inhibition to various extents. Molecular dynamics demonstrated that the less rigid tertiary structures and lower galactose-binding free energy of Y138F and Y364F might facilitate the release of the end product. Both Y138F and Y364F mutants exhibited better hydrolytic ability than lacA in milk lactose hydrolysis. The higher pH optimum and lower galactose inhibition of Y138F and Y364F may explain their superiority over lacA. The Y138F and Y364F mutants in the present study showed potential in producing low-lactose milk, and our studies provide a novel strategy for engineering the pH optimum of glycoside hydrolase. [ABSTRACT FROM AUTHOR]

Protein entrapment has multiple applications in enzymatic hydrolysis, drug delivery, etc. Here, we report the studies that successfully utilized the Box–Behnken design to model and optimize the parameters of β-galactosidase entrapment in sol–gel-derived silica composites. We have also demonstrated the influence of polymer–polydimethylsiloxane as a composite modifying agent on the activity of entrapped enzymes. We have determined how different sol-gel process parameters influence the activity of entrapped enzymes. The highest impact on β-galactosidase activity was exerted by the water:tetramethoxysilane ratio, followed by polydimethylsiloxane content. Optimized synthesis parameters have been utilized to obtain a composite with maximum β-galactosidase activity. Performed porosity studies have shown that the addition of polydimethylsiloxane increased the pore diameter. Microscopy studies demonstrated that polydimethylsiloxane-modified composites are softer and less rough. Studies of β-galactosidase activity using the o-NPG test showed statistically significant shifts in the enzyme temperature and pH profiles compared to the soluble form. An improvement in the reusability of the enzyme and a significant increase in the thermal stability was also observed. When lactose was used, a strong correlation was observed between the substrate concentration and the type of the catalyzed reaction. Moreover, we have demonstrated that the yields and rates of both lactose hydrolysis and galactooligosaccharides formation were correlated with reaction temperature and with the presence of polydimethylsiloxane. All these findings provide the opportunity for industrial use of optimized PDMS-modified silica composites in lactose elimination from dairy products, e.g., milk or whey. [ABSTRACT FROM AUTHOR]

This study aimed to develop single-step purification and immobilization processes on cellulosic supports of β-galactosidase from Kluyveromyces sp. combined with the Cellulose-Binding Domain (CBD) tag. After 15 min of immobilization, with an enzymatic load of 150 U/g support , expressed activity values reached 106.88 (microcrystalline cellulose), 115.03 (alkaline nanocellulose), and 108.47 IU/g (acid nanocellulose). The derivatives produced were less sensitive to the presence of galactose in comparison with the soluble purified enzyme. Among the cations assessed (Na+, K+, Mg2+, and Ca2+), magnesium provided the highest increase in the enzymatic activity of β-galactosidases immobilized on cellulosic supports. Supports and derivatives showed no cytotoxic effect on the investigated cell cultures (HepG2 and Vero). Derivatives showed high operational stability in the hydrolysis of milk lactose and retained from 53 to 64% of their hydrolysis capacity after 40 reuse cycles. This study obtained biocatalyzers with promising characteristics for application in the food industry. Biocatalyzers were obtained through a low-cost one-step sustainable bioprocess of purification and immobilization of a β-galactosidase on cellulose via CBD. [ABSTRACT FROM AUTHOR]

The kinetic model of the hydrolysis of lactose by β-galactosidase from Aspergillus niger immobilized on a commercial ceramic monoliths was estimated in the attendance of lactose and its hydrolysis reaction products galactose and glucose. The aim of this work was to developing kinetic model of lactase hydrolysis by Aspergillus niger. The variables in this study are temperature, pH, enzyme concentration, substrate concentration and final product. The optimum temperature used to achieve the best hydrolysis performance in the kinetic model selection was 55 and 60 °C. The optimum pH used for enzyme activity was about 3.5 to 4. Five kinetic models were proposed to confirm experimental data the enzymatic reaction of the lactose hydrolysis by the β-galactosidase. The kinetics of lactose hydrolysis by both Immobilized and soluble lactases were scrutinized in a batch reactor system in the lack of any mass conduction restriction. In both instance the galactose inhibition kinetic models predicted the experimental data. The model is capable to fit the experimental data correctly in the extensive experimental span studied.

β-Galactosidase plays an important role in medicine and dairy industry. In this study, a new glycoside hydrolase family 42 (GH42) β-galactosidase-encoding gene, gal42, was cloned from a newly isolated marine bacterium Bacillus sp. BY02 and expressed in Escherichia coli. Structural characterization indicated that the encoding β-galactosidase, Gal42, is a homotrimer in solution, and homology modeling indicated that it retains the zinc binding sites of the Cys cluster. The reaction activity of Gal42 was significantly increased by Zn2+ (229.6%) and other divalent metal ions (Mn2+, Mg2+, and Co2+), while its activity was inhibited by EDTA (53.9%). Meanwhile, the thermo-stability of the Gal42 was also significantly enhanced by 5 and 10 mM of zinc ion supplement, which suggested that the “Cys-Zn” motif played important roles in both structural stability and catalytic function. Furthermore, Gal42 showed effective lactose hydrolysis activity, which makes the enzyme hydrolyze the lactose in milk effectively. These properties make Gal42 a potential candidate in food technology.

β-Galactosidase plays an important role in medicine and dairy industry. In this study, a new glycoside hydrolase family 42 (GH42) β-galactosidase-encoding gene, gal42 , was cloned from a newly isolated marine bacterium Bacillus sp. BY02 and expressed in Escherichia coli. Structural characterization indicated that the encoding β-galactosidase, Gal42, is a homotrimer in solution, and homology modeling indicated that it retains the zinc binding sites of the Cys cluster. The reaction activity of Gal42 was significantly increased by Zn2+ (229.6%) and other divalent metal ions (Mn2+, Mg2+, and Co2+), while its activity was inhibited by EDTA (53.9%). Meanwhile, the thermo-stability of the Gal42 was also significantly enhanced by 5 and 10 mM of zinc ion supplement, which suggested that the "Cys-Zn" motif played important roles in both structural stability and catalytic function. Furthermore, Gal42 showed effective lactose hydrolysis activity, which makes the enzyme hydrolyze the lactose in milk effectively. These properties make Gal42 a potential candidate in food technology. [ABSTRACT FROM AUTHOR]

Most β-galactosidases reported are sensitive to the end product (galactose), making it the rate-limiting component for the efficient degradation of lactose through the enzymatic route. Therefore, there is ongoing interest in searching for galactose-tolerant β-galactosidases. In the present study, the predicted galactose-binding residues of β-galactosidase from Bacillus coagulans , which were determined by molecular docking, were selected for alanine substitution. The asparagine residue at position 148 (N148) is correlated with the reduction of galactose inhibition. Saturation mutations revealed that the N148C, N148D, N148S, and N148G mutants exhibited weaker galactose inhibition effects. The N148D mutant was used for lactose hydrolysis and exhibited a higher hydrolytic rate. Molecular dynamics revealed that the root mean square deviation and gyration radius of the N148D-galactose complex were higher than those of wild-type enzyme-galactose complex. In addition, the N148D mutant had a higher absolute binding free-energy value. All these factors may lead to a lower affinity between galactose and the mutant enzyme. The use of mutant enzyme may have potential value in lactose hydrolysis. [Display omitted] [ABSTRACT FROM AUTHOR]

Multitudinous people suffer from lactose intolerance which is bound up with lactose malabsorption. Enzymatic hydrolysis is the most common approach for the preparation of lactose-free products. The recycling of hydrolytic enzymes is an effective way to produce low-cost lactose-free products. Herein, a facile strategy for the preparation of the novel Fe3O4 magnetic nanoparticles-β-galactosidase-CaHPO4 hybrid nanocomplexes (β-gal MNCs) was established. β-gal MNCs were composed of amino-modified Fe3O4 magnetic nanoparticles, β-galactosidase and crystalline calcium hydrogen phosphates. Meanwhile, under the external magnetic field, the magnetic β-gal MNCs can be easily and rapidly separated from the reaction medium. In addition, the β-galactosidase maintained most of its initial activity after immobilization into the β-gal MNCs. In a continuous hydrolysis reaction, β-gal MNCs exhibited excellent reusability and good stability. Therefore, β-gal MNCs hold promising prospects in the fast and low-cost preparation of lactose-free milk. [ABSTRACT FROM AUTHOR]

Context: The Novelty of the research is to formulate the probiotic product with lowcost submerged fermentation for the mitigation of lactose intolerance. The utilization of substrate enhances the production of ß-galactosidase from the potent strain Lactobacillus plantarum by optimizing the media using the response surface methodology tool. This research is expected to result in innovative scientific outcomes leading to the development of lactose-free products cheaply. Aim: The current research is focused on the optimization of cultural conditions using a D-optimal experimental design to enhance the yield of ß-galactosidase. Materials and Methods: Interaction of two physical and chemical factors such as lactose 0.5-2%, beef extract 0.5-2%, pH 6-8, and temperature 30-40°C, were studied in 3D interaction. ß-galactosidase production was estimated using the hydrolysis of o-Nitrophenyl ß-D-galactopyranoside as the substrate. Further, the enzyme was purified by 70% precipitation, fractionated with 50-kDa ultra-filtration, and separated in gel permeation chromatography with the matrix Sephadex-G100. The extracted ß-galactosidase from Lactobacillus plantarum has been immobilized in alginate and hardened with calcium chloride for lactose hydrolysis using Glucose oxidase-Peroxidase assay. Results: The molecular sequencing of VITDM15 was confirmed as Lactobacillus plantarum in 16srRNA sequencing. Using the optimized conditions there was a six-fold increase in the yield of ß-galactosidase. The purified ß-galactosidase from Lactobacillus plantarum KX838907 yielded 31.75% with 67.73 fold and specific activity of 1705IU/mg. The molecular weight of the enzyme was found to be 112kDa at denaturing conditions in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In GOD-POD assay the glucose dosage increased up to a maximum level of 4.891mg/l in the 4th cycle. Conclusion: Based on the results obtained we claim that this might be the first report on the enhanced yield of ß-galactosidase and lactose hydrolysis. Thus novel strain Lactobacillus plantarum KX838907 can be used in the commercial production of ß-galactosidase as it is considered safe to use as a probiotic microorganism. [ABSTRACT FROM AUTHOR]

A novel galactosidase gene (gal3149) was identified from Bacillus velezensis SW5 and heterologously expressed in Escherichia coli BL21 (DE3). The novel galactosidase, Gal3149, encoded by gal3149 in an open reading frame of 1,299 bp, was 433 amino acids in length. Protein sequence analysis showed that Gal3149 belonged to family 4 of glycoside hydrolases (GH4). Gal3149 displayed higher enzyme activity for the substrate 2-nitrophenyl-β- d -galactopyranoside (oNPG) than for 4-nitrophenyl-α- d -galactopyranoside (pNPαG). This is the first time that an enzyme belonging to GH4 has been shown to exhibit β-galactosidase activity. Gal3149 showed optimal activity at pH 8.0 and 50°C, and exhibited excellent thermal stability, with retention of 50% relative activity after incubation at a temperature range of 0 to 50°C for 48 h. Gal3149 activity was significantly improved by K+ and Na+, and was strongly or completely inhibited by Ag+, Zn2+, Tween-80, Cu2+, carboxymethyl cellulose, and oleic acid. The rate of hydrolyzed lactose in 1 mL of milk by 1 U of Gal3149 reached about 50% after incubation for 4 h. These properties lay a solid foundation for Gal3149 in application of the lactose-reduced dairy industry. [ABSTRACT FROM AUTHOR]

Cheese whey (CW) constitutes a dairy industry by-product, with considerable polluting impact, related mostly with lactose. Numerous bioprocessing approaches have been suggested for lactose utilization, however, full exploitation is hindered by strain specificity for lactose consumption, entailing a confined range of end-products. Thus, we developed a CW valorization process generating high added-value products (crude enzymes, nutrient supplements, biopolymers). First, the ability of Aspergillus awamori to secrete β-galactosidase was studied under several conditions during solid-state fermentation (SSF). Maximum enzyme activity (148 U/g) was obtained at 70% initial moisture content after three days. Crude enzymatic extracts were further implemented to hydrolyze CW lactose, assessing the effect of hydrolysis time, temperature and initial enzymatic activity. Complete lactose hydrolysis was obtained after 36 h, using 15 U/mL initial enzymatic activity. Subsequently, submerged fermentations were performed with the produced hydrolysates as onset feedstocks to produce bacterial cellulose (5.6–7 g/L). Our findings indicate a novel approach to valorize CW via the production of crude enzymes and lactose hydrolysis, aiming to unfold the output potential of intermediate product formation and end-product applications. Likewise, this study generated a bio-based material to be further introduced in novel food formulations, elaborating and conforming with the basic pillars of circular economy. [ABSTRACT FROM AUTHOR]

β-Galactosidase was isolated from Ranunculus arvensis seeds using DEAE-cellulose, Sephadex G-100, and Con A sepharose-4B chromatography. The enzyme was purified to electrophoretic homogeneity with a specific activity of 50 U/mg of protein and a yield of 7.1%. The molecular mass of the isolated β-galactosidase, as estimated by SDS-PAGE, was 18 kDa, indicating that it was a monomeric form The purified β-galactosidase has a glycoproteinic nature when applied to Con-A-Sepharose-4B chromatography. An activation energy (Ea) of 11 kcal/mol of lactose, pH 5.0, and 50 °C were found to be the optimum parameters to purify β-galactosidase from R. arvensis seeds. The residual activity test was carried at 55–75 °C, allowing calculating the half-lives of 533–48 min, enthalpy (ΔH° = 110.38–110.21 kJ/mol), free energy (ΔG° = 109.88–109.77 kJ/mol), and entropy (ΔS° = 1.52–1.26 J/mol·K). The β-galactosidase produced from this species is better than the previously described enzyme due to its kinetic and thermodynamic properties, and it could be used in various industrial applications. Purified β-galactosidase, when incubated with high lactose concentration, showed transgalactosylation activity, leading to trisaccharides as a major product of total galactooligosaccharide (GOS). Therefore, the purified β-galactosidase could be used as a potential alternative in the food industry and would be further explained for trisaccharide synthesis. Ranunculus arvensis β-galactosidase: insights into its purification, kinetics, thermodynamic characterization, and trisaccharide synthesis. [Display omitted] • β-Galactosidase from Ranunculus arvensis was purified 185 fold with 7.1% yield. • The enzyme was found thermostable upto 50 °C. • The maximum turnover V max for ortho -NPG is 96 μmol/min/mg of protein and a K m of 0.4 mM. • β-Galactosidase is used for the synthesis of galacto-oligosaccharides. • It is a highly thermophilic enzyme that could be suitable for biotechnological applications. [ABSTRACT FROM AUTHOR]

The present study aimed to evaluate the lactose hydrolysis conditions from "coalho" cheese whey using β-galactosidase (β-gal) produced by Kluyveromyces lactis immobilized with sodium alginate. Three sodium alginate-based immobilization systems were evaluated (0.5, 0.7, and 1% w/v) for maximizing the immobilization yield (Y), efficiency (EM), and recovered activity (ar). The lactose hydrolysis capacity of the immobilized form of β-gal was determined, and simulated environments were used to assess the preservation of the immobilized enzyme in the gastrointestinal tract. The results showed that β-gal immobilization with 1% (w/v) sodium alginate presented the best results (EM of 66%, Y of 41%, and ar of 65%). The immobilization system maintained the highest pH stability in the range between 5.0 and 7.0, with the highest relative activity obtained under pH 5 conditions. The temperature stability was also favored by immobilization at 50 °C for 30 min was obtained a relative activity of 180.0 ± 1.37%. In 6 h, the immobilized β-gal was able to hydrolyze 46% of the initial lactose content. For the gastrointestinal simulations, around 40% of the activity was preserved after 2 h. Overall, the results described here are promising for the industrial applications of β-galactosidase from K. lactis. [ABSTRACT FROM AUTHOR]

The present study demonstrates the synthesis of lactose-free dairy items by Kluyveromyces lactis β-galactosidase bound to polyvinyl alcohol (PVA)-modified gold nanoparticles (AuNPs). The size of AuNPs was analyzed by dynamic light scattering experiment. The developed AuNPs served as a stable matrix for enzyme immobilization which was observed by obtaining 88% immobilization yield. Km and Vmax were determined for soluble and immobilized enzyme by incubating them with varying concentrations of substrate. Our findings indicate that immobilization leads to an increase of Km and a decline in Vmax values for the enzyme attached to PVA-functionalized AuNPs. Moreover, the enzyme conjugated to surface functionalized AuNPs displayed exceptional conversion of lactose hydrolysis in batch reactors at 40°C in contrast to its hydrolysis at 50o C. Hence, the developed nanosystem [β-galactosidase-(PVA-modified AuNPs)] serves as an excellent model for suggesting its application in other biomedical applications, particularly for constructing lactose based biosensors. [ABSTRACT FROM AUTHOR]

Yogurt is a well-known nutritious and probiotic food and is traditionally fermented from milk using the symbiotic starter culture of Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus. However, yogurt consumption may cause health problems in lactose-intolerant individuals, and the demand for lactose-free yogurt has been increasing. The standard method to prepare lactose-free yogurt is to hydrolyze milk by lactase; however, this process has been reported to influence the fermentation properties of starter strains. This study aimed to investigate the fermentation properties of an industrial starter culture of L. bulgaricus 2038 and S. thermophilus 1131 in lactose-hydrolyzed milk and to examine the metabolic changes induced by glucose utilization. We found that the cell number of L. bulgaricus 2038, exopolysaccharide concentration, and viscosity in the coculture of L. bulgaricus 2038 and S. thermophilus 1131 was significantly increased in lactose-hydrolyzed milk compared with that in unhydrolyzed milk. Although the cell number of S. thermophilus 1131 showed no difference, production of formic acid and reduction of dissolved oxygen were enhanced in lactose-hydrolyzed milk. Further, in lactose-hydrolyzed milk, S. thermophilus 1131 was found to have increased the expression of NADH oxidase, which is responsible for oxygen reduction. These results indicated that glucose utilization promoted S. thermophilus 1131 to rapidly reduce the dissolved oxygen amount and produce a high concentration of formic acid, presumably resulting in the increased cell number of L. bulgaricus 2038 in the coculture. Our study provides basic information on the metabolic changes in starter strains in lactose-hydrolyzed milk, and demonstrates that lactose-free yogurt with increased cell number of L. bulgaricus can be prepared without delay in fermentation and decrease in the cell number of S. thermophilus. [ABSTRACT FROM AUTHOR]

A phospho-β-galactosidase gene (BsGal1332) was cloned from Bacillus velezensis and successfully expressed in Escherichia coli BL21(DE3). The active BsGal1332 was identified to be a homodimer with a combined molecular mass of approximately 113 kDa, and it belonged to the glycoside hydrolase family 1. The BsGal1332 displayed relative strict substrate specificity for galactosyl compounds compared with the other phospho-β-galactosidases. The purified BsGal1332 showed the maximum activity at pH 8.0 and 50 °C for 2-nitrophenyl-β- d -galactopyranoside (o NPGal) and at 40 °C for lactose. BsGal1332 was slightly activated by K+ and Na+, but not strongly affected by Ca2+, and was stable at pH 6.0–7.0 and 40 °C or below it. The activity of BsGal1332 decreased quickly after incubation at 50 °C or higher temperature, suggesting it was a cold-adapted enzyme. Moreover, BsGal1332 could hydrolyze lactose and o NPGal with K m values of 23.68 and 2.36 mM and k cat values of 117.55 and 155.61 s−1 at 4 °C, respectively. Additionally, 1 U of the BsGal1332 could thus be capable of hydrolyzing about 38% of the lactose in 1 mL of milk after incubating at 4 °C for 4 h. Taken together, these properties of BsGal1332 made it a new promising industrial biocatalyst for efficient lactose hydrolysis in milk. [ABSTRACT FROM AUTHOR]

A psychrophilic strain Cryobacterium sp. LW097 was isolated from the subglacial sediments and discovered to show considerable β -galactosidases activity at low temperatures. To provide access to genes predicted to encode cold-adapted glycoside hydrolases with biotechnological relevance, we have sequenced the genome of Cryobacterium sp. LW097. Annotation with CAZy database revealed four β -galactosidase genes, bgal322 , bgal435 , bgal436, and bgal2567 belonging to the GH-42 family and GH-35 family. All the four β -galactosidases recombinantly expressed retained a high level of relative activity at 5 °C and showed different optimum temperatures ranging from 25 °C to 40 °C. The enzyme kinetics proved that Bgal322, Bgal436, and Bgal2567 had lower K m to both o NPG and lactose at 5 °C, further proving their adaption to low temperature. Substrate specificity analysis showed that these four β -galactosidases owned different preferences. The novel GH-42 β -galactosidases Bgal435 showed β -D-glucosidase activity (33.67 ± 0.28%) in addition to β -D-galactosidase activity. Bgal322 preferred β -D-(1,4)-galactobiose, whereas the other three preferred lactulose. Bgal435 showed the highest k cat value of 68.2 ± 1.7 s −1 at 5 °C toward lactose among these four enzymes. The exquisite substrate specificity of Bgal436 in milk made it a potential candidate for applications in milk lactulose quantification. • Cryobacterium sp. LW097 produced four cold-adapted β -galactosidase with different optimum temperatures. • The substrate specificity of the four novel cold-adapted β -galactosidases is variable. • Bgal435 showed the highest k cat at 5°C toward lactose among these four enzymes and a minor β -D-glucosidase activity. • Bgal436 displayed hydrolytic activity toward lactulose but no detectable activity toward lactose in milk. [ABSTRACT FROM AUTHOR]

Lactose‐free dairy products undergo several chemical modifications during shelf life because of the reactivity of glucose and galactose produced by the lactose enzymatic hydrolysis. In this study, proton transfer reaction‐mass spectrometry (PTR‐MS), coupled with a time‐of‐flight (TOF) mass analyzer, was applied to get an insight on the phenomena occurring during the shelf life of ultrahigh‐temperature (UHT) lactose‐free milk (LFM). UHT LFMs produced by three different commercial lactase preparations were evaluated during storage at 20°C over a 150 days period, sampling the milk every 30 days. Production was repeated three times, on three consecutive weeks, in order to take milk variability into consideration. Principal component analysis applied to the whole “volatilome” data demonstrated the capability of PTR‐TOF‐MS in detecting the milk batch‐to‐batch variability: Freshly produced milk samples were distinguished based on the week of production at the beginning of shelf life. Additionally, a clear evolution of the volatiles organic compounds (VOCs) profiling during storage was highlighted. Further statistical analysis confirmed VOCs temporal evolution, mostly because of changes in methyl ketones concentration. Differences caused by the commercial lactases did not emerged, except for benzaldehyde. Altogether, data demonstrated PTR‐TOF‐MS analysis as a valuable and rapid method for the detection of changes in the VOCs profiling of UHT LFM. [ABSTRACT FROM AUTHOR]

Permeate is the by‐product of the process of ultrafiltration in a kind of cheese making process in which a semipermeable membrane filters the liquid. It mainly contains 4.5%–4.8% lactose and 0.44%–0.47% mineral salts which make it a safe disposal issue. This study was conducted to use permeate and its lactose as an alternative to sugar, and to use these useful permeate compounds in an optimized orange juice formulation. Milk permeate, as a waste disposal of dairy companies, was applied in lactose hydrolyzed form as the cost effective sugar and water substitution in production of orange juice. The RSM optimization method was applied for formulating beverage mixture. The heated and nonheated permeate samples were incubated with β‐glycosidase enzyme in three thermal ranges (35, 40, and 45°C), 3 time intervals (60,150, and 240 min), and 3 enzyme levels (0%, 0.1%, and 0.2%). The degree of hydrolysis was determined by MilkoScan analyzer. In the next step, optimization of orange juice was accomplished with a mixture of sugar (10%–40%) and hydrolyzed permeate (10%–40%) with specific Brix through RSM statistical design. The physicochemical properties and sensory evaluation were measured during 8 weeks of storage. At the first stage of the study, the heated sample with 0.1% enzyme density, which was incubated for 150 min at 40°C, was yielded the best result. At the second stage, which was the juice production and evaluation, the statistical analysis showed increasing trend of pH and sugar content, but density and vitamin C showed a decreasing trend during storage time (p <.05). The optimal condition was obtained in taking 35% permeate and 41 days of storage in which the values of formalin, vitamin C, and sensory tests were in the highest levels. [ABSTRACT FROM AUTHOR]