Enzymatic Hydrolysis Research Articles

Maximizing microbial activity and synergistic interaction to boost biofuel production from lignocellulosic biomass.

Addressing global environmental challenges and meeting the escalating energy demands stand as two pivotal issues in the current landscape. Lignocellulosic biomass emerges as a promising renewable bio-energy source capable of fulfilling the world's energy requirements on a large scale. One of the most important steps in lowering reliance on fossil fuel and lessening environmental effect is turning lignocellulosic biomass into biofuel. As carbon-neutral substitutes for traditional fuel, biofuel offer a solution to environmental concerns compared to conventional fuel. Effective utilization of lignocellulosic biomass is imperative for sustainable development. Ongoing research focuses on exploring the potential of various microorganisms and their co-interactions to synthesize diverse biofuels from different starting materials, including lignocellulosic biomass. Co-culture techniques demonstrate resilience to nutrient scarcity and environmental fluctuations. By utilising a variety of carbon sources, microbes can enhance their adaptability to environmental stressors and potentially increase productivity through their symbiotic interactions. Furthermore, compared to single organism involvement, co-interactions allow faster execution of multistep processes. Lignocellulosic biomass serves as a primary substrate for pre-treatment, fermentation, and enzymatic hydrolysis processes. This review primarily delves into the pretreatment, enzymatic hydrolysis process and the biochemical pathways involved in converting lignocellulosic biomass into bioenergy.

Exploring the competitive inhibition of α-glucosidase by citrus pectin enzymatic hydrolysate and its mechanism: An integrated experimental and simulation approach

The endo-polygalacturonase D (PgaD) from Aspergillus niger JL15 was recombinantly expressed in Escherichia coli BL21, exhibiting an optimal activity at 55 °C and pH 4.0. Hydrolysis products of citrus pectin by recombinant PgaD included galacturonic acid (GalA), digalacturonic acid (GalA2), trigalacturonic acid (GalA3), and tetragalacturonic acid (GalA4). The hydrolysates exhibited significant antioxidant capacity and dose-dependent competitive inhibition of α-glucosidase. GalA2 and GalA3 acted as competitive inhibitors of α-glucosidase, with inhibition constant of 0.0589 mmol.L−1 and 0.6732 mmol.L−1, respectively. Molecular dynamics (MD) simulations revealed that both GalA2 and GalA3 penetrated the catalytic pocket of α-glucosidase and formed stable hydrogen bonds with key catalytic residues D352 and D215. The binding free energies of GalA2-α-glucosidase and GalA3-α-glucosidase complexes were − 10.3 ± 0.6 kcal·mol−1 and -10.8 ± 0.7 kcal·mol−1, respectively. These findings might offer new ideas for the development of α-glucosidase inhibitors sourced from citrus pectin, as well as enhance utilization of the renewable plant polysaccharide resources.

Comparative study of physicochemical and structural properties of plant proteins hydrolyzed by different

This study used trypsin to hydrolyze soybean protein isolate (SPI), Cyperus esculentus protein (CEP), Wheat gluten protein (WGP), and flaxseed protein (FP) to varying extents, comparing the structure and physicochemical properties of four plant proteins as well as their enzymatic hydrolysates. Results indicate SPI's surface hydrophobicity rises with hydrolysis, whereas CEP's decreases. Additionally, all four plant proteins exhibit improved essential amino acid content and oil-holding capacity as hydrolysis progresses. The CEP with a 20% degree of hydrolysis achieved the optimal oil holding capacity, reaching 10.2 g/g. SPI demonstrated superior foam capacity. As the degree of hydrolysis increased, foam stability varied among the four proteins. The CEP hydrolyzed to 10% exhibited the highest emulsifying activity index, achiving a value of 60 m2/g. Additionally, the emulsifying stability index of CEP hydrolysates significantly improved as the degree of hydrolysis increased. According to the DPPH• assay, both CEP and their enzymatic hydrolysate had the highest free radical scavenging rate. These findings provided a theoretical basis and empirical evidence for the use and adaptation of plant protein hydrolysates as a promising and environmentally sustainable source of plant-derived peptides in food processing reaprocessing.

Enhancing the Nutritional and Bioactive Properties of Bee Pollen: A Comprehensive Review of Processing Techniques.

Bee pollen is recognized as a superfood due to its high content of nutrients and bioactive compounds. However, its bioavailability is restricted by a degradation-resistant outer layer known as exine. Physical and biotechnological techniques have recently been developed to degrade this layer and improve pollen's nutritional and functional profile. This review examines how processing methods such as fermentation, enzymatic hydrolysis, ultrasound, and drying affect pollen's chemical profile, nutrient content, and bioactive compounds. The review also considers changes in exine structure and possible synergistic effects between these methods. In addition, the challenges associated with the commercialization of processed bee pollen are examined, including issues such as product standardization, stability during storage, and market acceptance. The objective was to provide an understanding of the efficacy of these techniques, their physicochemical conditions, and their effect on the nutritional value of the pollen. The work also analyzes whether pollen transformation is necessary to maximize its benefits and offers conclusions based on the analysis of available methods, helping to determine whether pollen transformation is a valid strategy for inclusion in functional foods and its impact on consumer health. Although the literature reports that pollen transformation influences its final quality, further studies are needed to demonstrate the need for pollen exine modification, which could lead to greater market availability of pollen-based products with functional properties.

Preparation and tissue structure analysis of horse bone collagen peptide

Horse bone is rich in collagen, with a composition similar to that of human collagen. Collagen peptides supply nutrients needed for human growth that act as antioxidants, lower blood pressure. This study explored the extraction of collagen and the preparation of collagen short peptides from Mongolian horse bones. Bones were collected from horses of varying ages, and the collagen content along with calcium salt distribution were observed through staining and imaging analyses. Next, the bones were processed into a powder and then subjected to ultra-high-pressure processing for degreasing. The degreasing conditions were optimised by single-factor and orthogonal tests. Following this, collagen was extracted using an acid-enzymatic method, and its structural characteristics and thermal stability were assessed. The collagen short peptides were extracted from the collagen samples, and the effects of the enzymatic hydrolysis time, temperature, pH, and enzyme amount on the extraction rate were evaluated. Finally, the resulting collagen peptides were analysed for antioxidant activity. In summary, this experiment optimised the extraction conditions for horse bone collagen, demonstrating that the ultra-high-pressure method minimally affects collagen structure, and the extraction rate was high. Hence our method has significant development potential.

Biomass Demineralization and Pretreatment Strategies to Reduce Inhibitor Concentrations in Itaconic Acid Fermentation by Aspergillus terreus

Itaconic acid (IA) is a platform chemical, derived from non-petroleum sources, produced through the fermentation of glucose by Aspergillus terreus. However, producing IA from alternative sugar sources (e.g., lignocellulose) has been shown to be problematic, requiring post-hydrolysis mitigation to allow growth and IA production by the fungus. It is well known that the side products of lignocellulosic biomass conversion to sugars act as microbial growth inhibitors. An uncommon feature of fungal organic acid fermentations is production inhibition caused by mineral ions in biomass hydrolysate after pretreatment and enzymatic hydrolysis. To minimize mineral introduction during pretreatment and hydrolysis, we determined the sources of growth and production inhibitors at each of these steps. Biomass demineralization and four pretreatment strategies were evaluated for inhibitor introduction. Dilution assays determined the approximate degree of inhibition for each hydrolysate. An ammonium hydroxide pretreatment of demineralized wheat straw presented the lowest concentration of inhibitors and concomitant lowest inhibition: subsequent fermentations produced 35 g L−1 IA from wheat straw hydrolysate (91 g L−1 sugar) without post-hydrolysis mitigation.

Effects of Dietary Inclusion of Enzymatically Hydrolyzed Compound Soy Protein on the Growth Performance and Intestinal Health of Juvenile American Eels (Anguilla rostrata).

The enzymatic hydrolysis of soybeans could enhance their application as an ingredient and alternative to fishmeal in aquafeeds. Here, a 10-week feeding trial was conducted to evaluate the impacts of different dietary inclusion levels of enzymatically hydrolyzed compound soy protein (EHCS) on the growth performance and intestinal health of juvenile American eels (Anguilla rostrata). Five experimental diets were formulated with graded EHCS inclusion levels at 0% (EHCS0), 8% (EHCS8), 16% (EHCS16), 24% (EHCS24), and 32% (EHCS32). Each diet was randomly assigned to four replicate tanks. The results showed that eels fed the EHCS8 diet exhibited superior growth performance, decreased serum lipid content, and increased immunity compared to those fed the EHCS0 diet. Eels fed the EHCS8 diet also displayed improved intestinal histology, enhanced antioxidant capacity and balance of intestinal microbiota as well as an enhanced proliferation of probiotics compared to those receiving the EHCS0 diet. Compared with eels fed the EHCS0 diet, those fed the EHCS16 diet exhibited comparable growth performance and values for the aforementioned markers. The quadratic regression analysis of weight gain rate and feed efficiency against the dietary EHCS inclusion levels determined the maximum levels of dietary EHCS inclusion for American eels range from 17.59% to 17.77%.

Distinct effects of dilute acid prehydrolysate inhibitors on enzymatic hydrolysis and yeast fermentation.

The effects of dilute acid prehydrolysate from poplar were investigated and compared in the enzymatic hydrolysis, fermentation, and simultaneous saccharification fermentation (SSF) in this study. The improvement of enzymatic hydrolysis and fermentation with resin adsorption and surfactant addition has also been represented. A total of 16 phenolic alcohols, aldehydes, acids and 3 furan derivatives in the prehydrolysates were identified and quantified by gas chromatography/mass spectrometry (GC/MS). The degree of inhibition from the phenolic compounds (26.55%) in prehydrolysate on the enzymatic hydrolysis was much higher than carbohydrates-derived inhibitors (0.52-4.64%). Around 40% degree of inhibition was eliminated in Avicel enzymatic hydrolysis when 75% of prehydrolysates phenolic compounds were removed by resin adsorption. This showed distinguishing inhibition degrees of various prehydrolysate phenolic compounds. Inhibition of prehydrolysate on enzymatic hydrolysis was more dosage-dependent, while their suppression on the fermentation showed a more complicated mode: fermentation could be terminated by the untreated prehydrolysate, while a small number of prehydrolysate inhibitors even improved the glucose consumption and ethanol production in the fermentation. Correlated with this distinct inhibition modes of prehydrolysate, the improvement of Tween 80 addition in SSF was around 7.10% for the final ethanol yield when the glucose accumulation was promoted by 76.6%.

Corn biomass as a feedstock for biorefinery: a bibliometric analysis of research on bioenergy, biofuel and value‐added products

AbstractLimited research has been dedicated to exploring the potential reuse and creation of value‐added products from corn stover. Existing gaps in current research underscore the need for waste biorefineries that adhere to the principles of a circular bioeconomy to maximize the value of corn stover. This review uses bibliometric analysis from the past decade to examine the potential and pathways for converting corn stover into energy and value‐added products within the biorefinery framework. The data were processed using the Web of Science® database and analyzed with the VosViewer® bibliometric software's, Bibliometrix package in R and Gephi, generating keyword‐based maps. A total of 2557 experimental articles and 30 reviews on corn stover research were analyzed. The bibliometric study revealed that the primary research focuses on pretreatment technologies for converting corn stover into value‐added products, bioenergy and biofuels. The main technologies employed include acid, alkaline and enzymatic hydrolysis, as well as torrefaction anaerobic digestion and steam explosion. The pretreatment processes yield sugars, xylooligosaccharides and organic acids. Additionally, the studies explored the use of corn stover biochar for soil remediation and adsorption processes. This review aims to enhance the understanding of technological pathways explored in previous research, contributing to the evaluation of sustainable processes for utilizing corn stover byproducts. Ultimately, it promotes environmentally conscious agroindustry practices that align with circular economy principles and sustainable development.

Nucleotide-mediated modulation of chemoselective protein functionalization in a liquid-like condensed phase

Liquid-like protein condensates are ubiquitous in cellular system and are increasingly recognized for their roles in physiological processes. Condensed phase harbors distinctive chemical microenvironment, markedly different than dilute aqueous phase. Herein, we demonstrate chemoselective modification pattern of nucleophilic canonical amino acid sidechains (namely – cysteine, tyrosine and lysine) of the protein towards 4-chloro-7-nitrobenzofurazan in the dilute and condensed phase. We also delineate how the effect of nucleotides and their in situ enzymatic dissociation temporally modulate the protein condensate’s pH and the protein’s corresponding chemoselective modification. We have shown that the pH of the condensate decreases in the presence of nucleoside triphosphate, whereas it increases in the presence of nucleoside monophosphates or phosphate ion. For instance, we find lysine-specific modification gets inhibited in the presence of adenosine triphosphate (ATP), but significantly enhanced in the presence of monophosphates. This feature enables us to gain temporal control over dynamic change in protein functionalization via enzymatic ATP hydrolysis. Overall, this work substantiates the alteration in pH-responsiveness of Brønsted basicity of a protein’s ε-amine in the condensed phase. Furthermore, this environment sensitivity in chemoselective protein functionalization in condensed phase will be important in adaptable protein engineering to the chemical biology of protein phase separation.

Olive pomace upcycling: Eco-friendly production of cellulose nanofibers by enzymatic hydrolysis and application in starch films.

Olive pomace (OP) waste, produced in large quantities, contains significant amounts of cellulose and fibers, making it a valuable resource for developing reinforcing ingredients in biodegradable packaging materials. This study aimed to produce nanofibers from OP using enzymatic hydrolysis with hemicellulases and cellulases, and to incorporate these nanofibers into starch films as a reinforcing agent. Cellulose nanofibers (CNFs) were prepared by alkaline pretreatment followed by enzymatic hydrolysis (with hemicellulases and cellulases) from olive pomace and applied as reinforcement in starch films in concentrations of 0.5%-5% (w/v). The nanofibers were analyzed according to composition, structural, and thermal properties. The nanofibers' suspension presented a cloudy and white color in aqueous suspension, the X-ray diffraction (XRD) analysis showed the increase of crystallinity, and the fibers' range was no wider than 100nm (according to Scherer equation). The composition analysis showed the decrease of carbonyl groups of hemicellulose and lignin. The starch films presented a homogenous surface. The solubility from these biodegradable films significantly reduced after the incorporation of CNF, and the nanomaterial's presence improved the degradation temperature (from 310°C to 322°C) and the mechanical resistance because the tension of rupture increased from 3.79 to 6.21MPa. PRACTICAL APPLICATION: The utilization of waste from the olive pomace for cellulose nanofiber production holds promise, given the nanofibers' ability to readily integrate into various materials, including starches used in biodegradable film production. Within these matrices, nanofibers act as structure reinforcers and significantly reduce the solubility of films. Although biodegradable films ensure the shelf life, safety, and quality of food, their properties currently do not match those of traditional petroleum-based materials at an industrial scale, indicating a need for further enhancement.

Hydrolysates of mare's milk proteins. Immunochemical and physico-chemical characteristics

In recent years, the attention of specialists has been attracted by a significant increase in allergic diseases, especially among children. Enzymatic hydrolysis with the participation of peptidases is considered the most effective measure to reduce the allergenicity of milk proteins. The study of the features of protein proteolysis by various enzymes, aimed at establishing optimal process parameters, is the subject of research in the production of hydrolysates with specified properties: a certain peptide and amino acid profile, reduced antigenic properties. At the same time, for a correct preclinical assessment of the safety and potential effectiveness of specialized food products, it is not enough only to determine the molecular weight distribution of peptide fractions in the composition. A necessary additional and informative indicator of reducing the potential allergenicity of the hydrolysate is the degree of preservation of the antigenic properties of the initial protein (residual antigenicity), determined by immunochemical methods. The preparation and subsequent comprehensive assessment of enzymatic hydrolysates of mare's milk proteins is of considerable interest, since, according to the results of clinical studies, it was suggested that upon allergy to cow's milk proteins mare's milk can be considered hypoallergenic. The purpose of this work was to obtain enzymatic hydrolysates of mare's milk proteins using domestic enzymes and their further physico-chemical and immunochemical characteristics. The object of the study was mare's milk protein obtained from skimmed mare's milk powder as a protein substrate. Two samples of enzymes from various manufacturers of the Russian Federation were used in the work. The results of the conducted studies show that the peptide profiles of hydrolysates vary greatly depending on the enzyme used, however, the multiplicity of antigenicity reduction practically does not depend on the nature of the enzyme, and does not depend on an increase in the enzyme/substrate ratio. The peptide profile and the multiplicity of reducing the antigenicity of hydrolysates of mare's milk proteins are influenced by ultrafiltration treatment of the obtained hydrolysates, which allows reducing the content of antigenic structures in the final product by several orders of magnitude.

Valorization of Residual Fractions from Defatted Rice Bran Protein Extraction: A Carbohydrate-Rich Source for Bioprocess Applications

Defatted rice bran (DRB) is the by-product of rice bran oil extraction and presents approximately 66% carbohydrates and 15% proteins, a composition with the potential to integrate biorefinery systems. This study aimed to investigate the feasibility of residual fractions from ultrasound-assisted protein extraction from DRB as sources of carbohydrates in bioprocesses. First, DRB was exposed to protein extraction in an alkaline medium assisted by ultrasound. The residual fractions, including the precipitate from the extraction process (P1) and the supernatant from protein precipitation (S2), were combined and autoclaved to gelatinize the starch. Enzyme activity tests showed that a temperature of 70 °C was optimal for the simultaneous application of α-amylase and amyloglucosidase (AMG). To study enzymatic hydrolysis, a Full Factorial Design (FFD) 22 was employed, with α-amylase and AMG concentrations ranging from 0.12 to 0.18 mL∙L−1 and a substrate concentration (P1/S2 ratio) between 30 and 70 g∙L−1, resulting in a maximum of 18 g∙L−1 of reducing sugars (RS). Fermentation assays with Saccharomyces cerevisiae demonstrated that the hydrolysate of the residual fractions was effective for ethanol production (8.84 g∙L−1 of ethanol; YP/S: 0.614 gethanol∙gRS−1; η: 120.24%), achieving results comparable to control media (with sucrose as the substrate), indicating its potential for application in bioprocesses. These outcomes highlight a promising technological approach for utilizing DRB in integrated biorefineries.

Influences of superfine-grinding and mix enzymolysis alone or combined with hydroxypropylation or acetylation on the hypolipidemic and hypoglycemic properties of coconut endosperm residue fiber.

Coconut endosperm residue is an abundant and low-cost resource of dietary fiber, but the low soluble fiber content limits its functional properties and applications in the food industry. To improve the hypolipidemic and hypoglycemic properties, coconut endosperm residue fiber (CERF) was modified by superfine-grinding and mix enzymatic hydrolysis alone, or combined with acetylation or hydroxypropylation. The effects of these modifications on the structure and functional properties were studied using scanning electron microscopy, Fourier-transformed infrared spectroscopy, and in vitro tests. After these modifications, the microstructure of CERF became more porous, and its soluble fiber content, surface area, water adsorption, and expansion capacities were all improved (p < 0.05). Moreover, superfine-grinding and mix enzymolysis combined with acetylation treated CERF showed the highest surface hydrophobicity (48.96) and cholesterol and cholate adsorption abilities (33.72 and 42.04mg∙g‒1). Superfine-grinding-, mix enzymolysis-, and hydroxypropylation-treated CERF exhibited the highest viscosity (17.84 cP), glucose adsorption capacity (29.61µmol∙g‒1), and glucose diffusion inhibition activity (73.96%), and water-expansion ability (8.60mL∙g‒1). Additionally, superfine-grinding and mix enzymatic hydrolyzed CERF had the highest α-amylase inhibiting activity (42.76%). Therefore, superfine-grinding and mix enzymolysis alone or combined with hydroxypropylation were better choices to improve hypoglycemic properties of CERF; meanwhile, superfine-grinding and mix enzymolysis combined with acetylation can effectively improve its hypolipidemic properties. PRACTICAL APPLICATION: This study offered three composite modification methods to improve the soluble fiber content and in vitro hypolipidemic and hypoglycemic properties of coconut endosperm residue fiber. These modification methods were practicable and low-cost. Moreover, it provides good choices to improve the functional properties and applications of other dietary fibers in the food industry.

The Molecular Bases of Anti-Oxidative and Anti-Inflammatory Properties of Paraoxonase 1

The anti-oxidative and anti-inflammatory properties of high-density lipoprotein (HDL) are thought to be mediated by paraoxonase 1 (PON1), a calcium-dependent hydrolytic enzyme carried on a subfraction of HDL that also carries other anti-oxidative and anti-inflammatory proteins. In humans and mice, low PON1 activity is associated with elevated oxidized lipids and homocysteine (Hcy)-thiolactone, as well as proteins that are modified by these metabolites, which can cause oxidative stress and inflammation. PON1-dependent metabolic changes can lead to atherothrombotic cardiovascular disease, Alzheimer’s disease, and cancer. The molecular bases underlying these associations are not fully understood. Biochemical, proteomic, and metabolic studies have significantly expanded our understanding of the mechanisms by which low PON1 leads to disease and high PON1 is protective. The studies discussed in this review highlight the changes in gene expression affecting proteostasis as a cause of the pro-oxidative and pro-inflammatory phenotypes associated with attenuated PON1 activity. Accumulating evidence supports the conclusion that PON1 regulates the expression of anti-oxidative and anti-inflammatory proteins, and that the disruption of these processes leads to disease.

Enhancing PET degradation efficiency through fusing carbohydrate-binding modules in LCC-ICCG

Polyethylene terephthalate (PET) is a largely-produced polymer worldwide. However, its extensive waste generation and resistance to degradation pose significant environmental concerns. Consequently, there is considerable interest in researching enzymatic degradation of PET. Relevant studies have shown that the addition of a carbohydrate binding module (CBM) can increase the affinity between the enzyme and the substrate, enhancing the enzyme's degradation ability. In order to develop more efficient PET hydrolytic enzymes, this study introduced carbohydrate binding domains (CBMs) from different families with different substrate affinities into the PET-degrading enzyme LCC-ICCG. High crystallinity PET powder and amorphous PET film were used as substrates to characterize the degradation efficiency of the modified enzymes, aiming to explore the enzyme with the optimal degradation ability. The results showed that the fusion of type B CBM reduced the degradation rate and Tm value of the enzyme towards PET, while the introduction of type A and type C CBMs significantly improved the degradation rate of the enzyme towards the film-like substrate. The degradation rate and Tm value of PET were also enhanced, especially with the fusion enzyme LCC-ICCG-CBM9-2, which showed an over 10-fold increase in the degradation rate compared to the original enzyme LCC-ICCG. Therefore, this study demonstrates that by introducing type A and type C CBMs, the degradation rate and thermal stability of LCC-ICCG towards film-like PET can be improved, addressing the issue of its low activity and enabling more effective PET degradation. This research provides support for plastic degradation technology and contributes to environmental conservation efforts.

Identification of umami peptides and mechanism of the interaction with umami receptors T1R1/T1R3 in pigeon meat.

Pigeon meat offer an ideal source for extracting fresh flavor peptides. These peptides not only enhance the taste of food but also have potential health benefits, including providing low-sugar, low-sodium, and low-calorie options for individuals with conditions like diabetes, hypertension, and obesity. Therefore, further research into the pigeon industry holds promise for addressing both economic and nutritional needs. To explore umami peptides and their molecular binding mechanisms with umami receptor type 1 member 1 in pigeon meat, an enzymatic hydrolysate product is isolated, analyzed, and subjected to sensory evaluation. Fifteen peptides with high freshness characteristics are separated and identified by ultrafiltration, gel separation, reverse performance liquid chromatography, and nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). The molecular docking results show that the amino acid residue Glu128 is a common ligand binding site for all of the fresh-flavored peptides to taste T1R1/T1R3 receptors and it exerts freshness-presenting effects with 15 fresh-flavored peptides through hydrogen bonding, electrostatic interactions, salt bridges, and hydrophobic interactions. This study provides a theoretical basis and technical support for the subsequent development of flavor peptide products in pigeon meat.

Nanostructure Formation in Glycerolipid Films during Enzymatic Hydrolysis: A GISAXS Study.

Responsive nanostructured films from food-grade lipids can be valuable for food, pharmaceutical, and biotechnological science. Lyotropic liquid crystalline structures that respond to enzymes in their environment can, for instance, be innovated as drug delivery platforms or biosensors. However, the structural changes that such films undergo during enzymatic reactions with lipase are not yet understood. This work demonstrates the preparation of mesostructured lipid films from the food-grade lipids glycerol monooleate (GMO) and triolein on silicon wafers and their digestion with pancreatic lipase using time-resolved synchrotron grazing incidence small-angle X-ray scattering (GISAXS). The film structure is compared with the corresponding GMO/triolein bulk phases in excess water. Increasing the GMO/triolein ratio in the film makes it possible to modulate the structure of the films from oil coatings to inverse hexagonal and inverse bicontinuous cubic films. Pancreatic lipase triggered swelling of the internal film nanostructure and eventually structural transformation inside the film. Orientation and reorientation of the internal film structure relative to the silicon wafer surface were observed during the preparation of the films and their digestion. The findings contribute to the understanding of self-assembly in thin films and guide the development of enzyme-responsive coatings for the functional modification of various substrates.

Measurement Uncertainty and Validation for Quantification of Marijuana Metabolite: (−)-11-nor-9-Carboxy-Δ9-THC in Human Urine by GC-MS/MS

Background: Since the International Olympic Committee (IOC) was established, sports doping control analyses have revealed a high rate of positive cases for cannabinoids. Cannabinoids were banned in all sports where they were used in competition as per the Prohibited List published annually. Further, it was also included in the threshold drug category. Consequently, developing a reliable method for urine Cannabinoids metabolite quantification plays a pivotal role in sports dope testing. Objective: This work aimed to develop and validate a reliable, cost-effective, robust gas chromatography-tandem mass spectrometry method for detecting (−)-11-nor-9-Carboxy-Δ9- THC component in human urine samples, in compliance with ICH and WADA guidelines. Method: The sample preparation was done by enzymatic hydrolysis for deconjugation, further proceeded with solid phase extraction (SPE), liquid-liquid extraction (LLE), and using an XAD2 column, and N-methyl trimethylsilyl trifluoroacetamide (MSTFA) for derivatization. Results: The linearity was obtained in a range of 50–300 ng/mL, and the correlation coefficient was found to be higher than 0.99. Throughout the entire validation study, the difference in Retention Time (RT) for the analyte, including the Internal Standard (IS), was shown to be less than 1.0%. The quantification limit (LOQ) was calculated at a level of 50 ng/mL in human urine samples for the 3 most abundant ion transitions. The detection limit (LOD) was established at 4 ng/mL. Conclusion: The accuracy, precision, linearity, recovery, quantification limit, and selectivity by GC-MS/MS technique were found acceptable and well satisfactory while following the ICH guidelines. The developed method has been proven to be fit for purpose in accordance with the enforced Guidelines of WADA.

Cell Disruption and Hydrolysis of Microchloropsis salina Biomass as a Feedstock for Fermentation

Microalgae are a promising biomass source because of their capability to fixate CO2 very efficiently. In this study, the potential of Microchloropsis salina biomass as a feedstock for fermentation was explored, focusing on biomass hydrolysis by employing various mechanical and chemical cell disruption strategies in combination with enzymatic hydrolysis. Among the mechanical cell disruption methods investigated on a lab scale, namely ultrasonication, bead milling, and high-pressure homogenization, the most effective was bead milling using stainless-steel beads with a diameter of 2 mm. In this way, 87–97% of the cells were disrupted in 40 min using a mixer mill. High-pressure homogenization was also effective, achieving 86% disruption efficiency after four passes on a 30–200 L scale using biomass with 15% (w/w) solids content. Enzymatic hydrolysis of the disrupted cells using a mixture of cellulases and mannanases yielded up to 25% saccharification efficiency after 72 h. Acidic hydrolysis of undisrupted cells followed by enzymatic treatment yielded around 30% saccharification efficiency but was coupled with significant dilution of the resulting hydrolysate. Microalgal biomass hydrolysate produced was determined to have ~8.1 g L−1 sugars and 2.5% (w/w) total carbon, as well as sufficient nitrogen and phosphorus content as a fermentation medium.