Efficient removal of pharmaceutical contaminants from water and wastewater using immobilized laccase on activated carbon derived from pomegranate peels.

In this study, pomegranate peels (PPs) as an abundant fruit processing waste was used to produce cost-effective, eco-friendly, and high-quality activated carbon. The produced carbon (fossil free activated carbon) was used for immobilizing laccase to remove a range of emerging pollutants namely diclofenac, amoxicillin, carbamazepine, and ciprofloxacin from water and wastewater. The loaded activated carbon by laccase (LMPPs) and the unloaded one (MPPs) were characterized using advanced surface chemistry analysis techniques. MPPs was found to have a porous structure with a large surface area and an abundance of acidic functional groups. Laccase immobilization reduced surface area but added active degradation sites. The optimal immobilization parameters were determined as pH 4, 35 °C, and a laccase concentration of 2.5 mg/mL resulting in a 69.8% immobilization yield. The adsorption of the emerging pollutant onto MPPs is best characterized as a spontaneous endothermic process that adheres to the Langmuir isotherm and first-order kinetics. Using synergistic adsorption and enzymatic degradation, the target pollutants (50 mg/L) were eliminated in 2 h. In both water types, LMPPs outperformed MPPs. This study shows that pomegranate peels can effectively be harnessed as an enzyme carrier and adsorbent for the removal of emerging pollutants even from a complex sample matrix. The removal of contaminants from wastewater lasted five cycles, whereas it continued up to six cycles for water.

Development of a novel SNP assay to detect lactase persistence associated genetic variants.

BACKGROUND: In adulthood the activity of the lactase enzyme is inherited as autosomal dominant form associated to Single nucleotide polymorphisms (SNPs). The present research was aimed to develop a novel genetic method to test lactase non persistence more powerfully. METHODS AND RESULTS: In our study, we selected eight different SNPs that are associated with lactase persistence from Caucasian, Arabian Bedouins, sub-Saharian Africans and Asian populations to set up an approach to detect all the eight different SNPs at the same time in the same sample. This technique is centred on the identification of SNPs with a single nucleotide primer extension method using Sanger sequencing and capillary electrophoresis. CONCLUSIONS: Our method allowed us to check the genotype asset of eight SNPs related to lactase persistence simultaneously and in a very efficient manner. It could be applied to a higher number of SNPs in a single reaction.

Detection of ricin activity and structure by using novel galactose-terminated magnetic bead extraction coupled with mass spectrometric detection.

Ricin is a toxic protein derived from the castor bean plant (Ricinus communis) and has potential for bioterrorism or criminal use. Therefore, sensitive and rapid analytical methods are needed for its confirmatory detection in environmental samples. Our laboratory previously reported on the development of a confirmatory method to detect ricin involving antibody capture of ricin followed by mass spectrometric detection of ricin's enzymatic activity and of tryptic fragments unique to ricin. Here, we describe a novel ricin capture method of magnetic beads coated with 4-aminophenyl-1-thiol-ß-galactopyranoside, using ricin's lectin characteristics. The assay has been adapted for use on a simple, benchtop MALDI-TOF MS mass spectrometer common in clinical microbiology laboratories. Validation of the novel assay includes establishment of a limit of detection, and an examination of assay selectivity. The limit of detection of the enzymatic activity method is 8 ng/mL and 500 ng/mL for the confirmatory tryptic fragment assay. The assay is highly selective with no cross-reactivity from near neighbors and highly specific with a panel of 19 cultivars all testing positive. Additionally, there were no interferences found during testing of a panel of white powders. This allows for a confirmatory detection method for ricin in laboratories lacking expensive, sophisticated mass spectrometers.

Molecular and isotopic evidence for milk, meat, and plants in prehistoric eastern African herder food systems.

The development of pastoralism transformed human diets and societies in grasslands worldwide. The long-term success of cattle herding in Africa has been sustained by dynamic food systems, consumption of a broad range of primary and secondary livestock products, and the evolution of lactase persistence (LP), which allows digestion of lactose into adulthood and enables the milk-based, high-protein, low-calorie diets characteristic of contemporary pastoralists. Despite the presence of multiple alleles associated with LP in ancient and present-day eastern African populations, the contexts for selection for LP and the long-term development of pastoralist foodways in this region remain unclear. Pastoral Neolithic (c 5000 to 1200 BP) faunas indicate that herders relied on cattle, sheep, and goats and some hunting, but direct information on milk consumption, plant use, and broader culinary patterns is rare. Combined chemical and isotopic analysis of ceramic sherds (n = 125) from Pastoral Neolithic archaeological contexts in Kenya and Tanzania, using compound-specific δ13C and Δ13C values of the major fatty acids, provides chemical evidence for milk, meat, and plant processing by ancient herding societies in eastern Africa. These data provide the earliest direct evidence for milk product consumption and reveal a history of reliance on animal products and other nutrients, likely extracted through soups or stews, and plant foods. They document a 5,000-y temporal framework for eastern Africa pastoralist cuisines and cultural contexts for selection for alleles distinctive of LP in eastern Africa.

Application of PTR-TOF-MS for the quality assessment of lactose-free milk: Effect of storage time and employment of different lactase preparations.

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.

Human Health Effects of Lactose Consumption as a Food and Drug Ingredient.

Lactose is a reducing sugar consisting of galactose and glucose, linked by a ß (1→4) glycosidic bond, considered as an antioxidant due to its α-hydroxycarbonyl group. Lactose is widely ingested through the milk and other unfermented dairy products and is considered to be one of the primary foods. On the other hand, lactose is also considered as one of the most widely used excipients for the development of pharmaceutical formulations. In this sense, lactose has been related to numerous drug-excipient or drug-food pharmacokinetic interactions. Intolerance, maldigestion and malabsorption of carbohydrates are common disorders in clinical practice, with lactose-intolerance being the most frequently diagnosed, afflicting 10% of the world's population. Four clinical subtypes of lactose intolerance may be distinguished, namely lactase deficiency in premature infants, congenital lactase deficiency, adult-type hypolactasia and secondary lactase intolerance. An overview of the main uses of lactose in human nutrition and in the pharmaceutical industry and the problems derived from this circumstance are described in this review.

Interplay between Residual Protease Activity in Commercial Lactases and the Subsequent Digestibility of ß-Casein in a Model System.

One of the conventional ways to produce lactose-hydrolyzed (LH) milk is via the addition of commercial lactases into heat-treated milk in which lactose is hydrolyzed throughout storage. This post-hydrolysis method can induce proteolysis in milk proteins due to protease impurities remaining in commercial lactase preparations. In this work, the interplay between lactose hydrolysis, proteolysis, and glycation was studied in a model system of purified ß-casein (ß-CN), lactose, and lactases using peptidomic methods. With a lactase presence, the proteolysis of ß-CN was found to be increased during storage. The protease side-activities mainly acted on the hydrophobic C-terminus of ß-CN at Ala, Pro, Ile, Phe, Leu, Lys, Gln, and Tyr positions, resulting in the formation of peptides, some of which were N-terminal glycated or potentially bitter. The proteolysis in ß-CN incubated with a lactase was shown to act as a kind of "pre-digestion", thus increasing the subsequent in vitro digestibility of ß-CN and drastically changing the peptide profiles of the in vitro digests. This model study provides a better understanding of how the residual proteases in commercial lactase preparations affect the quality and nutritional aspects of ß-CN itself and could be related to its behavior in LH milk.

Full-factorial central composite rotational design for the immobilization of lactase in natural polysaccharide-based hydrogels and hydrolysis of lactose.

A full-factorial central composite rotational design (FFCCRD) was applied for studying the immobilization of lactase in Arabic gum-based and chitosan-based hydrogels, and hydrolysis of lactose. The optimal immobilization capacities of both hydrogels aiming to obtain high immobilized enzyme activity and low released fraction were determined at 25.0 °C, 39.88 mg mL-1 initial enzyme concentration and pH 6.5. The immobilized enzyme activity and released fraction from the Arabic gum-based hydrogel were 0.322 U mg-1 and 0.193, respectively, during the hydrolysis of lactose contained in UHT milk. These values were 0.289 U mg-1 and 0.136, respectively, using a chitosan-based hydrogel. The immobilized enzyme activity and released fraction from these hydrogels during the hydrolysis of standard lactose were 0.246 U mg-1 and 0.407, and 0.211 U mg-1 and 0.245, respectively. The best conditions for the immobilization of lactase and hydrolysis of lactose were achieved by applying FFCCRD, which were compared with experimental results.

Dispersible Biopolymer Particles Loaded with Lactase as a Potential Delivery System To Control Lactose Hydrolysis in Milk.

To deliver lactase in milk, dispersible capsules were fabricated by anti-solvent precipitation of zein to form a zein-lactase core and a shell of low-methoxyl sugar beet pectin cross-linked by Ca2+, involving electrostatic, hydrophobic, hydrogen-bonding, and Ca2+-bridging forces. At optimal conditions, an encapsulation efficiency of 93.0% and Z-average diameter of 652.7 nm were observed, and spherical particles smaller than 200 nm were observed in scanning electron microscopy. Contrasting with complete hydrolysis within 1 week by unencapsulated lactase, 50 units/mL encapsulated lactase resulted in 33.1 and 40.0% lactose hydrolysis in whole and skim milk, respectively, after 3 weeks of storage at 4 °C. In separate in vitro digestion assays, 50 units/mL encapsulated lactase resulted in 100% lactose hydrolysis in milk, contrasting with negligible activity by free lactase. These findings suggest the potential of the studied biopolymer particles to incorporate lactase in milk, prevent lactose hydrolysis during storage, and hydrolyze lactose in milk after ingestion.

Measuring Enzymatic Stability by Isothermal Titration Calorimetry.

This work demonstrates a new method for measuring the stability of enzyme activity by isothermal titration calorimetry (ITC). The peak heat rate observed after a single injection of the substrate solution into an enzyme solution is correlated with enzyme activity. Multiple injections of the substrate into the same enzyme solution over time show the loss of enzyme activity. The assay is autonomous, requiring very little personnel time, and is applicable to most media and enzymes.

Measuring Lactase Enzymatic Activity in the Teaching Lab.

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and biomedical sciences. This easy to follow protocol was developed for first year undergraduate pharmacy students and provides an entry-level introduction to enzyme reactions and analytical procedures for enzyme analysis. The enzyme of choice is lactase, as this represents an example of a commercially available enzyme relevant to human disease/pharmaceutical practice. Lactase is extracted from dietary supplement tablets, and assessed using a colorimetric assay based upon hydrolysis of an artificial substrate for lactase (ortho-nitrophenol-beta-D-galactopyranoside, ONPG). Release of ortho-nitrophenol following the hydrolytic cleavage of ONPG by lactase is measured by a change in absorbance at 420 nm, and the effect of the temperature on the enzymatic reaction is evaluated by carrying out the reaction on ice, at room temperature and at 37 °C. More advanced analysis can be implemented using this protocol by assessing the enzyme activity under different conditions and using different reagents.

Application of a commercial digestive supplement formulated with enzymes and probiotics in lactase non-persistence management.

Strategies to avoid lactose malabsorption, which affects 70% of the world's population, are focused on the restriction of milk and dairy products or the use of non-human ß-galactosidases or probiotics endowed with ß-galactosidase activity added at mealtime. Our evaluation of a commercial blend of probiotics and enzymes (protease, lactase, lipase and amylase) and its potential application in lactase non-persistence management is described in this work. Recommended amounts (460-1000 mg) of the commercial probiotics-enzyme blend were shown to be adequate for performing in vitro lactose hydrolysis in standard solutions (0.25-5%) and commercial dairy products, namely milks (5% lactose) and yogurts (3% lactose), reaching hydrolysis values between 44 and 96%. According to these percentages, the use of the enzymatic preparation would guarantee the intake of less than 12 g, the recommendation of the EFSA for lactose intolerance. Furthermore, formation of prebiotic galactooligosaccharides was also detected, increasing the potential benefits of the enzymatic preparation in the gastrointestinal system.

Simple Strategy to Protect Lactase Activity in Solid Formulation.

BACKGROUND: Lactose intolerance is characterized by the absence of the enzyme lactase (beta-galactosidase) and affects two thirds of the world adult population. Our aim was to evaluate a lactase gastro-resistant formulation to ensure increased activity in the action site of the enzyme (lumen of the small intestine). Simultaneously, we also evaluated the commercial product stability and enzyme activity, because the product containing beta-galactosidase is classified as food supplement according to the Food and Drug Administration (FDA), so it is free to pass quality testing, efficacy and stability. So, it is possible that contain some irregularities as to the content and enzymatic activity. METHODS: The dissolution assay was performed using a dissolution test system and commercial product and the gastro-resistant formulation were evaluated according to a method adapted to the conditions recommended by United States Pharmacopeia (US Pharmacopeia) for gastro-resistant formulations. For the assessment of enzymatic activity throughout the dissolution test was employed the official method of lactase assay described in US Pharmacopoeia. This method is based on a colorimetric reaction which the substrate reacts with the enzyme generate a colored product further analyzed by UVVisible spectrophotometry. RESULTS: When carrying out dissolution test in commercial product it is noted that the existing formulation is not able to protect the enzyme from degrading action of gastric environment (a loss of 86.0 ± 0.8% of lactase activity was observed). Our proposed gastro-resistant pharmaceutical form there was no loss of activity during the acid step and the end of the dissolution test the found activity was 95 ± 1.3%. CONCLUSION: The formulations proposed in this work using hypromellose capsules ensure the enzymatic activity of lactase, preventing its contact with the acid medium. For the other side, the results of commercial tablets for lactase release indicate a significant loss of enzyme activity due to the immediate release of the enzyme in the simulated gastric fluids.

Gums induced microstructure stability in Ca(II)-alginate beads containing lactase analyzed by SAXS.

Previous works show that the addition of trehalose and gums in ß-galactosidase (lactase) Ca(II)-alginate encapsulation systems improved its intrinsic stability against freezing and dehydration processes in the pristine state. However, there is no available information on the evolution in microstructure due to the constraints imposed by the operational conditions. The aim of this research is to study the time course of microstructural changes of Ca(II)-alginate matrices driven by the presence of trehalose, arabic and guar gums as excipients and to discuss how these changes influence the diffusional transport (assessed by LF-NMR) and the enzymatic activity of the encapsulated lactase. The structural modifications at different scales were assessed by SAXS. The incorporation of gums as second excipients induces a significant stabilization in the microstructure not only at the rod scale, but also in the characteristic size and density of alginate dimers (basic units of construction of rods) and the degree of interconnection of rods at a larger scale, improving the performance in terms of lactase activity.

Freeze-dried capsules prepared from emulsions with encapsulated lactase as a potential delivery system to control lactose hydrolysis in milk.

The objective of this work was to study solid/oil/water (S/O/W) emulsions as delivery systems with retained lactase in milk and controlled release during in vitro digestion. Spray-dried lactase powder was suspended in anhydrous milk fat/Span® 80 emulsified by sodium caseinate and lecithin (5:1). The S/O/W emulsion had an encapsulation efficiency of 75%, a hydrodynamic diameter of 292nm, and a zeta potential of -17.37mV. Cross-linking the dialyzed emulsion with transglutaminase eliminated the detection of free lactase after freeze-drying emulsions and the addition of sodium caseinate further preserved lactase activity. The hydrolysis of lactose in full-fat or skim milk after 3-week storage reduced from>75% for free lactase to<15% for encapsulated lactase. The encapsulated lactase was released gradually during the simulated digestions to hydrolyze lactose in milk more efficiently than free lactase. The present findings suggest S/O/W emulsions are potential delivery systems to incorporate lactase in milk products.

Solid-in-Oil-in-Water Emulsions for Delivery of Lactase To Control in Vitro Hydrolysis of Lactose in Milk.

There is an established need to deliver lactase in milk to retain activity during storage and hydrolyze lactose after ingestion. In this work, spray-dried lactase powder was encapsulated in solid-in-oil-in-water (S/O/W) emulsions to fabricate delivery systems. The adoption of Span 80 in milk fat and lecithin in protein solution enabled the encapsulation of ∼76% lactase and <400 nm droplets. Additional cross-linking of proteins on droplets by transglutaminase and addition of sodium caseinate effectively reduced the amount of free lactase after spray drying emulsions. Compared to the data for free lactase, encapsulation significantly improved the thermal stability of lactase, reduced the level of lactose hydrolysis during a 14 day refrigeration (from ∼70 to <20%), enabled the gradual release of lactose during the simulated gastric and intestinal digestions, and resulted in the hydrolysis of most lactose during the simulated digestions. Therefore, the studied S/O/W emulsions have the potential to deliver lactase in milk for lactose-intolerant consumers.

Encapsulation of lactase in Ca(II)-alginate beads: Effect of stabilizers and drying methods.

The purpose of the present work was to analyze the effect of trehalose, arabic and guar gums on the preservation of ß-galactosidase activity in freeze-dried and vacuum dried Ca(II)-alginate beads. Freezing process was also studied as a first step of freeze-drying. Trehalose was critical for ß-galactosidase conservation, and guar gum as a second excipient showed the highest conservation effect (close to 95%). Systems with Tg values ~40°C which were stables at ambient temperature were obtained, being trehalose the main responsible of the formation of an amorphous matrix. Vacuum dried beads showed smaller size (with Feret's diameter below 1.08±0.09mm), higher circularity (reaching 0.78±0.06) and large cracks in their surface than freeze-dried beads, which were more spongy and voluminous. Ice crystallization of the beads revealed that the crystallization of Ca(II)-alginate system follows the Avrami kinetics of nucleation and growth. Particularly, Ca(II)-alginate showed an Avrami index of 2.03±0.07, which means that crystal growing is bidimensional. Neither the addition of trehalose nor gums affected the dimension of the ice growing or its rate. These results open an opportunity in the development of new lactic products able to be consumed by lactose intolerance people.

Effect of Storage on Lactase-Treated ß-Casein and ß-Lactoglobulin with Respect to Bitter Peptide Formation and Subsequent in Vitro Digestibility.

Using active lactose to hydrolyze lactose during storage is a common process to produce lactose-hydrolyzed (LH) milk. Proteolysis induced by residual proteases in commercial lactase was studied in a system using purified ß-casein or ß-lactoglobulin during a 60-day storage period at 22 or 38 °C. The proteolysis of ß-casein by residual proteases occurred more extensively than that of ß-lactoglobulin. Peptidomic analysis by LC-ESI-MS/MS revealed that Ile, Leu, Tyr, and Phe residues near the C-terminus of ß-casein were the main sites of cleavage by the residual proteases, generating assumed bitter peptides. In the subsequent in vitro digestion study, proteolysis during storage was shown to greatly affect the subsequent digestibility of ß-casein, leading to an elevated degree of hydrolysis and the formation of new digested peptides. This study highlights the potential influence of residual proteases in commercial lactase on the storage quality and digestibility of LH milk containing active lactase.

Alginate Beads Containing Lactase: Stability and Microstructure.

ß-Galactosidase (lactase) is a widely used enzyme in the food industry; however, it has low stability against thermal and mechanical treatments. Due to this, the purpose of the present research was to analyze the encapsulation of lactase in alginate-Ca(II) beads in order to maintain its enzymatic activity toward freezing, freezing/thawing, and storage. Also, the effect of the addition of trehalose, and arabic and guar gums and their influence on the microstructure as well as on thermal properties and molecular mobility were studied. Lactase was successfully encapsulated in alginate-Ca(II) beads, and the inclusion of trehalose was critical for activity preservation toward treatments, being improved in guar gum-containing systems. The gums increased the Tm' values, which represents a valuable technological improvement. Finally, the presence of secondary excipients affected the microstructure, showing rods with smaller outer diameter and with lower compactness than alginate-Ca(II) beads. Also, bead composition greatly affects the size, shape, and relaxation times.

Controlling the molecular architecture of lactase immobilized in Langmuir-Blodgett films of phospholipids to modulate the enzyme activity.

In this present work, the adsorption of the enzyme lactase onto Langmuir monolayers of the phospholipid dimyristoylphosphatidic acid (DMPA) was investigated and characterized with surface pressure-area isotherms, surface potential-area isotherms and polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS). The adsorption of the enzyme at the air-water interface expanded the lipid monolayer and increased the film compressibility at high surface pressures. Amide bands in the PM-IRRAS spectra were identified, with the CN and CO dipole moments lying parallel to the monolayer plane, revealing that the structuring of the enzyme into ß-sheets was kept in the mixed monolayer. The enzyme-lipid films were transferred from the floating monolayer to solid supports as Langmuir-Blodgett (LB) films and characterized with fluorescence spectroscopy and atomic force microscopy. The catalytic activity of the films was measured and compared to the homogenous medium. The enzyme accommodated in the LB films preserved more than 80% of the enzyme activity after 20days, in contrast for the homogeneous medium, which preserved less than 60% of the enzyme activity. The method presented in this present work not only allows for an enhanced catalytic activity toward lactose, but also can help explain why certain film architectures exhibit better performance.