Search Results (62)

Pulmonary nocardiosis (PN) is a rare, opportunistic, and potentially life-threatening infection, especially in disseminated cases. This retrospective study aimed to characterize the clinical features of PN and assess the diagnostic utility of metagenomic next-generation sequencing (mNGS). We reviewed data from 19 patients diagnosed with PN between September 2019 and August 2022, including 3 with disseminated disease. Common symptoms included fever, cough, and sputum production, while chest imaging frequently revealed nodules, consolidations, exudates, cavities, and pleural effusions. The sensitivity of mNGS for detecting Nocardia was significantly higher than that of culture (100% vs. 36.84%, p < 0.001). mNGS successfully identified Nocardia species and co-infected pathogens. The most common species was Nocardia farcinica. Four PN cases were co-infected with Rhizomucor pusillus, Cryptococcus neoformans, Lichtheimia ramosa, and Aspergillus spp. Eighteen patients (94.7%) received trimethoprim-sulfamethoxazole (TMP-SMZ). Sixteen cases (84.2%) were improved or cured. Misdiagnosis is common due to the nonspecificity of clinical and imaging presentations of pulmonary nocardiosis. The timely combination of mNGS represents a promising approach to enhance the diagnosis of pulmonary nocardiosis and inform targeted antimicrobial therapy. TMP-SMZ is the first line of treatment. Full article

The sustainable production of healthy structured lipids (SLs) using oils extracted from agro-industry by-products or non-conventional lipid sources is of utmost importance in the framework of a circular bioeconomy, toward a zero-waste goal. In this study, low-calorie triacylglycerols (TAGs) containing a long-chain (L) fatty acid (FA) at position sn-2 and medium-chain (M) FAs at positions sn-1,3 (MLM type SL) were obtained from virgin cold-pressed milk thistle (51.55% linoleic acid; C18:2), grapeseed (66.62% C18:2), and apricot kernel (68.61% oleic acid; C18:1) oils. Lipase-catalyzed acidolysis with capric acid (C10:0) or interesterification with ethyl caprate (C10 Ethyl) in solvent-free media were performed. In batch reactions, immobilized Rhizomucor miehei lipase (Lipozyme RM) was used as a biocatalyst. For all tested oils, new TAG (SL) yields, varying from 61 to 63%, were obtained after 6 h of interesterification. Maximum new TAG yields were reached after 6, 24, and 30 h of acidolysis with grapeseed (64.7%), milk thistle (56.1%), or apricot kernel (69.7%) oils, respectively. Continuous acidolysis and interesterification of grapeseed oil were implemented in a packed-bed bioreactor, catalyzed by immobilized Thermomyces lanuginosus lipase (Lipozyme TL IM). Throughout 150 h of continuous operation, no lipase deactivation was observed, with average SL yields of 79.2% ± 4.1 by interesterification and 61.5% ± 5.91 by acidolysis. Full article

The concept of combi-lipases is herein explored in the full hydrolysis of babassu oil. The commercially immobilized lipases from Candida antarctica (form B) (Novozym^® 435), Rhizomucor miehei (Lipozyme^® RM-IM), and Thermomyces lanuginosus (Lipozyme^® TL-IM) were evaluated as single and combined biocatalysts by a mixture design with triangular surface. As a result, after evaluating the response desirability profiling for all biocatalysts, the best biocatalyst in the reaction was the combi-lipases composed of 75% of Lipozyme^® RM-IM, 17% of Novozym^® 435, and 8% of Lipozyme^® TL-IM, reaching full hydrolysis (>99%) after 4 h of reaction. Subsequently, such combi-lipases were employed as biocatalysts in the optimization of the reaction in a shorter reaction time (3 h). After optimization by the Taguchi method, full hydrolysis (>99%) was reached under optimized reaction conditions (9 wt.% of biocatalyst content, 1:2 (oil/water), 40 °C, and 180 rpm). Under such conditions, the combi-lipases maintained 70% of their initial activity after 10 reaction cycles. The antimicrobial activity against some of the most common environmental bacteria of the obtained free fatty acids (FFAs) was also evaluated. The FFAs inhibited more than 90% of the growth of S. aureus, E. coli, and P. aeruginosus when using 10 mg FFAs/mL. Full article

Plant-derived rennet sources have been recognized as effective alternatives to traditional animal-derived rennet in cheese processing, producing various cheeses for vegetarians and Halal consumers. The aim of the present study was to assess the quality and identify potential chemical and sensory markers of Canestrato cheese made from pasteurized mixed cow/sheep milk (50:50 ratio) using a kiwifruit extract (prepared in the laboratory) as a rennet source of vegetable origin. The milk was curdled with an aqueous kiwi extract (cK group), with commercial calf rennet (cT group), or with commercial microbial rennet obtained from the controlled fermentation of Rhizomucor miehei (cH group). For each cheesemaking process, 30 L of mixed milk was used, resulting in 10 forms, each weighing approximately 1 kg. Cheese produced using kiwifruit rennet exhibited improved nutritional characteristics, such as lower total lipid, cholesterol, and sodium content and greater levels of polyphenols, potassium, and phytosterols compared with the cheese of the cT group. The cheese of the cK group had a slightly more pronounced bitterness compared to cheeses made with conventional rennets (cH and cT). Overall, the use of kiwifruit extract as a milk coagulant has proved to be a promising alternative, improving both the nutritional profile and functional properties of the resulting cheese. Full article

Lipase, a green biocatalyst, finds extensive application in the food sector. Enhancing the thermal stability of lipase presents both challenges and opportunities within the food industry. This research employed multiple rounds of cross-screening using tools like FoldX and I-Mutant 3.0 to strategically design mutations for Rhizomucor miehei lipase (RML), resulting in eight unique single-point mutation designs. E230I, N120M, and N264M have been confirmed experimentally to be potential combination mutation candidates. The resulting triple mutant N120M/E230I/N264M showed a higher thermal stability, with an optimum temperature of 55 °C, 10 °C higher than that of the wild-type RML. The half-life was extended from 46 to 462 min at 50 °C. Furthermore, the catalytic activity of N120M/E230I/N264M on camphor tree seed oil increased by 140% to 600 U/mg, which aids in the production of novel structured lipids. Using molecular docking and molecular dynamics simulations, we analyzed the molecular mechanism of enhanced thermal stability. This study validated the efficacy and dependability of computer-aided design to generate heat-resistant RML mutants and indicated that RML N120M/E230I/N264M lipase can be used as an effective biocatalyst for fat processing in the food industry. Full article

Triacylglycerols containing medium-chain fatty acids at the sn-1,3 positions and a long-chain fatty acid at the sn-2 position (MLM-TAG) are of nutritional interest. However, they are scarce in common food sources and are usually synthesized by chemical or enzymatic methods. In this work, the enzymatic synthesis of MLM-TAG was attempted using sn-2 monoacylglycerols (sn-2 MAG) from the ethanolysis of an arachidonic acid-rich fraction from ARASCO and fatty acid ethyl esters from the ethanolysis of coconut oil as substrates. The highest yield of sn-2 MAG (23.3 mol%) was obtained after 1 h of ethanolysis with Novozym 435 lipase at 25 °C, and the best profile of the ethanolysis products of coconut oil was obtained after 24 h of reaction catalyzed by the lipase from Thermomyces lanuginosus. Regarding the enzymatic synthesis of structured TAG, the lipase from Rhizopus oryzae gave better results than those from Thermomyces lanuginosus and Rhizomucor miehei, with the sn-2 position mainly esterified with arachidonic acid (34.8%) and the sn-1,3 positions mainly esterified with capric and lauric acids (35.1%). This work focuses on a simple process for the enzymatic production of structured TAG without prior purification of the sn-2 MAG. Full article

In the modern world, the principles of the bioeconomy are becoming increasingly important. Recycling and reusability play a crucial role in sustainable development. Green chemistry is based on enzymes, but immobilized biocatalysts are still often designed with synthetic polymers. Insoluble carriers for immobilized biocatalysts, particularly those derived from agro-industrial waste such as mesoporous lignocellulosic materials, offer a promising alternative. By using waste materials as support for enzymes, we can reduce the environmental impact of waste disposal and contribute to the development of efficient bioprocessing technologies. The current study aimed to assess the possibility of using apple and chokeberry pomace as carriers for the immobilization of Palatase 20000L (lipase from Rhizomucor miehei). The analysis of lignocellulosic materials revealed that chokeberry pomace has a higher neutral detergent fiber (NDF) and lignin contents than apple pomace. Moreover, Scanning Electron Microscopy (SEM) observations indicated similar compact structures in both pomaces. The lipase activity assays demonstrated that immobilization of lipase from R. miehei onto apple and chokeberry pomace improves their properties, especially the synthetic activity. The findings highlight the potential of utilizing fruit pomaces not only as a source of bioactive compounds but also in enhancing enzyme stability for industrial applications. Full article

The enzyme-assisted approaches for plant phenolics extraction are more eco-friendly methods compared to acid or alkaline hydrolysis. Carbohydrase enzymes can release free phenolics from plant materials by cleaving the glycosidic bonds between phenolic compounds and cell wall polymers. In this study, the efficiency of carbohydrase-assisted treatment approaches was evaluated to extract bioactive phenolics from hawthorn (Crataegus orientalis) fruit residues. Enzymatic treatment of the fruits was operated by using a crude cellulolytic enzyme cocktail from Rhizomucor miehei NRRL 5282 and a pectinase preparate from Aspergillus niger. Both cellulase and combined cellulase–pectinase treatments improved the total phenolic content (TPC) and antioxidant activity of extracts. The TPC increased to 1899 ± 27 mg gallic acid equivalents/100 g dry matter during the combined enzyme treatment, showing a strong correlation with the average antioxidant capacity determined by ferric-reducing antioxidant power (1.7-fold increment) and 2,2-diphenyl-1-picrylhydrazyl (1.15-fold increment) reagents. The major phenolics in enzyme-treated extracts were vanillic and ferulic acids, the concentrations of which increased 115.6-fold and 93.9-fold, respectively, during carbohydrase treatment. The planktonic growth of Escherichia coli, Bacillus subtilis, Pseudomonas aeruginosa, and Chromobacterium violaceum was slightly inhibited by the extracts with minimum inhibitory concentration values between 15.0 and 77.9 mg/mL, while the yeasts tested were quite resistant to the samples. B. subtilis and yeast biofilms were sensitive to the enzyme-treated extracts, which also showed quorum-sensing inhibitory effects against C. violaceum. The obtained bioactive hawthorn extracts hold potential as a natural source of antioxidants and antimicrobials. Full article

Strong-flavor Daqu, as a fermentation agent, plays a significant role in shaping the quality of strong-flavor baijius, and fungal species in Daqu are important factors affecting the quality of Daqu. Therefore, we selected strong-flavor Daqu from seven different origins to study the fungal composition and the effects of the fungal composition on the physicochemical properties and volatile organic compounds (VOCs). It was found that the fungal composition influences the physicochemical properties of Daqu. Specifically, there was a positive link between Rhizomucor, Rhizopus, Thermomyces, and liquefying activity and a positive correlation between Aspergillus and fermenting activity. Furthermore, the relationships between esterifying activity and Thermomyces, Rhizomucor, Aspergillus, Pichia, and Saccharomycopsis were found to be positive. The VOCs in Daqu were affected by Aspergillus, Issatchenkia, Pichia, and Thermoascus. Issatchenkia was significantly positively correlated with benzeneethanol as well as Aspergillus and pentadecanoic acid ethyl ester, ethyl myristate. Pichia and Thermoascus were significantly negatively correlated with benzaldehyde and 2-furaldehyde. This study deepens our understanding of the relationship between VOCs, the physicochemical properties with microbial communities, and reference significance for the production of better-quality strong-flavor Daqu. Full article

Low levels of docosahexaenoic acid (DHA) in the brain have been related to neurological disorders, like Alzheimer’s disease (AD). After ingestion, dietary DHA must cross the blood–brain barrier, where it is absorbed as lysophosphatidylcholine (LPC), due to its role as a preferential DHA carrier in the brain. This work aimed at the production of LPC-DHA extracts to be used in supplementation/food fortification intended neural enrichment in DHA. As it is rich in DHA, especially its phospholipids (PL), Atlantic mackerel (Scomber scombrus, caught in Spring/2022) was used as a raw material. The polar lipids fraction was separated and hydrolysed with Rhizomucor miehei lipase, to enzymatically convert phosphatidylcholine (PC) into LPC. The fish (muscle and by-products) lipids fraction was used for total lipids (TL) content, lipid classes (LC) and fatty acid (FA) profile evaluation, whilst polar lipids extracts were studied for LC production and FA analysis. Muscle TL ranged between 1.45 and 4.64 g/100 g (WW), while by-products accounted for 7.56-8.96 g/100 g, with the highest contents being found in March. However, PL were more abundant in muscle (22.46–32.20% of TL). For polar lipids extracts, PL represented 50.79% of TL, among which PC corresponded to 57.76% and phosphatidylethanolamine to 42.24%. After hydrolysis, nearly half of this PC was converted into LPC. When compared to the initial PC, DHA relative content (33.6% of total FA) was significantly higher after hydrolysis: 55.6% in PC and 73.6% in LPC. Such extract, obtained from this undervalued species, may represent a promising strategy to increase DHA uptake into brain cells while allowing this species to upgrade. Full article

Biomimetic ligands are synthetic compounds that mimic the structure and binding properties of natural biological ligands. The first uses of textile dyes as pseudo-affinity ligands paved the way for the rational design and de novo synthesis of low-cost, non-toxic and highly stable triazine-scaffolded affinity ligands. A novel method to assess and enhance protein stability, employing triazine-based biomimetic ligands and using cutinase from Fusarium solani pisi as a protein model, has been previously reported. This innovative approach combined the concepts of molecular modeling and solid-phase combinatorial chemistry to design, synthesize and screen biomimetic compounds able to bind cutinase through complementary affinity-like interactions while maintaining its biological functionality. The screening of a 36-member biased combinatorial library enabled the identification of promising lead ligands. The immobilization/adsorption of cutinase onto a particular lead (ligand 3′/11) led to a noteworthy enhancement in thermal stability within the temperature range of 60–80 °C. In the present study, similar triazine-based compounds, sourced from the same combinatorial library and mimicking dipeptides of diverse amino acids, were selected and studied to determine their effectiveness in binding and/or improving the thermal stability of several lipases, enzymes which are closely related in function to cutinases. Three ligands with different compositions were screened for their potential thermostabilizing effect on different lipolytic enzymes at 60 °C. An entirely distinct enzyme, invertase from Saccharomyces cerevisiae, was also assessed for binding to the same ligands and functioned as a ‘control’ for the experiments with lipases. The high binding yield of ligand 3′/11 [4-({4-chloro-6-[(2-methylbutyl)amino]-1,3,5-triazin-2-yl}amino)benzoic acid] to cutinase was confirmed, and the same ligand was tested for its ability to bind lipases from Aspergillus oryzae (AOL), Candida rugosa (CRL), Chromobacterium viscosum (CVL), Rhizomucor miehei (RML) and Rhizopus niveus (RNL). The enzymes CRL, CVL, RNL and invertase showed significant adsorption yields to ligand 3′/11—32, 29, 36 and 94%, respectively, and the thermal stability at 60 °C of free and adsorbed enzymes was studied. CVL and RNL were also stabilized by adsorption to ligand 3′/11. In the case of CRL and invertase, which bound but were not stabilized by ligand (3′/11), other ligands from the original combinatorial library were tested. Between the two alternative ligands, one was effective at stabilizing C. rugosa lipase, while none stabilized invertase. Full article

Ionic additives affect the structure, activity and stability of lipases, which allow for solving common application challenges, such as preventing the formation of protein aggregates or strengthening enzyme–support binding, preventing their desorption in organic media. This work aimed to design a biocatalyst, based on lipase improved by the addition of ionic additives, applicable in the production of ethyl esters of fatty acids (EE). Industrial enzymes from Thermomyces lanuginosus (TLL), Rhizomucor miehei (RML), Candida antárctica B (CALB) and Lecitase^®, immobilized in commercial supports like Lewatit^®, Purolite^® and Q-Sepharose^®, were tested. The best combination was achieved by immobilizing lipase TLL onto Q-Sepharose^® as it surpassed, in terms of %EE (70.1%), the commercial biocatalyst Novozyme^® 435 (52.7%) and was similar to that of Lipozyme TL IM (71.3%). Hence, the impact of ionic additives like polymers and surfactants on both free and immobilized TLL on Q-Sepharose^® was assessed. It was observed that, when immobilized, in the presence of sodium dodecyl sulfate (SDS), the TLL derivative exhibited a significantly higher activity, with a 93-fold increase (1.02 IU), compared to the free enzyme under identical conditions (0.011 IU). In fatty acids ethyl esters synthesis, Q-SDS-TLL novel derivatives achieved results similar to commercial biocatalysts using up to ~82 times less enzyme (1 mg/g). This creates an opportunity to develop biocatalysts with reduced enzyme consumption, a factor often associated with higher production costs. Such advancements would ease their integration into the biodiesel industry, fostering a greener production approach compared to conventional methods. Full article

Shaoxing rice wine is a notable exemplar of Chinese rice wine. Its superior quality is strongly correlated with the indigenous natural environment. The results indicated that Firmicutes (75%), Actinobacteria (15%), Proteobacteria (5%), and Bacteroidetes (3%) comprised the prevailing bacterial groups. Among the main bacterial genera, Lactobacillus was the most abundant, accounting for 49.4%, followed by Lactococcus (11.9%), Saccharopolyspora (13.1%), Leuconostoc (4.1%), and Thermoactinomyces (1.1%). The dominant fungal phyla were Ascomycota and Zygomycota. Among the dominant genera, Saccharomyces (59.3%) prevailed as the most abundant, followed by Saccharomycopsis (10.7%), Aspergillus (7.1%), Thermomyces (6.2%), Rhizopus (4.9%), Rhizomucor (2.2%), and Mucor (1.3%). The findings demonstrate that the structure of the bacterial and fungal communities remains stable in the environment, with their diversity strongly influenced by climatic conditions. The continuous fluctuations in environmental factors, such as temperature, air pressure, humidity, rainfall, and light, significantly impact the composition and diversity of microbial populations, particularly the dominant bacterial community. Full article

Due to its effectiveness, ibuprofen is one of the most popular anti-inflammatory drugs worldwide. However, the poor water solubility of this active ingredient severely limits its spectrum of pharmaceutical formulations (and often results in severe adverse effects due to high administered doses). To overcome these limitations, in this work, we enzymatically synthesized more hydrophilic derivatives of ibuprofen through its covalent attachment to two biobased polyalcohols: erythritol and glycerol. Herein, we report the optimized reaction conditions to produce an IBU–erythritol ester (82% ± 4% of conversion) by using Candida antarctica lipase B (CalB). Furthermore, we also report the enantioselective solventless esterification of (S)-ibuprofen with glycerol (83% ± 5% of conversion), exploiting immobilized Rhizomucor miehei lipase as a biocatalyst. The full NMR characterizations of the prodrug esters were performed via ¹H, ¹³C-NMR, DEPT, COSY, HSQC, and HMBC-NMR. The approach reported in this work can be extended to a large variety of poorly water-soluble active pharmaceutical ingredients (APIs). Full article

Green leaf volatiles (GLVs), including short chain volatile aldehydes, are widely used in the flavor and food industries because of their fresh aroma. To meet the growing demand for natural GLVs with high added value, the use of biocatalytic processes appears as a relevant application. In such processes, vegetable oils are bioconverted into GLVs. First, the triacylglycerols of the oils are hydrolyzed by a lipase. Then, the free polyunsaturated fatty acids are converted by a lipoxygenase. Finally, volatile C6 or C9 aldehydes and 9- or 12-oxoacids are produced with a hydroperoxide lyase. Optimization of each biocatalytic step must be achieved to consider a scale-up. In this study, three oils (sunflower, hempseed, and linseed oils) and three lipases (Candida rugosa, Pseudomonas fluorescens, and Rhizomucor miehei lipases) have been tested to optimize the first step of the process. The experimental design and response surface methodology (RSM) were used to determine the optimal hydrolysis conditions for each oil. Five factors were considered, i.e., pH, temperature, reaction duration, enzyme load, and oil/aqueous ratio of the reaction mixture. Candida rugosa lipase was selected as the most efficient enzyme to achieve conversion of 96 ± 1.7%, 97.2 ± 3.8%, and 91.8 ± 3.2%, respectively, for sunflower, hempseed, and linseed oils under the defined optimized reaction conditions. Full article