Highly efficient production and simultaneous purification of d-tagatose through one-pot extraction-assisted isomerization of d-galactose.

A one-pot extraction-assisted d-galactose-to-d-tagatose isomerization strategy was proposed based on the selective extraction of d-tagatose by phenylborate anions. 4-Vinylphenylboronic acid was selected with high extraction efficiency and selectivity towards d-tagatose. The extracted sugars could be desorbed through a two-staged stripping process with the purity of d-tagatose significantly increased. In-situ extraction-assisted d-galactose-to-d-tagatose isomerization was implemented for the first time ever reported, and the effect of boron-to-sugar ratio (boron: sugar) was investigated. The conversion yield of d-tagatose at 60 °C increased from âˆ¼ 39 % (boron: sugar = 0.5) to âˆ¼ 56 % (boron: sugar = 1) but then decreased to âˆ¼ 44 % (boron: sugar = 1.5). With temperature increased to 70 °C, the conversion yield of d-tagatose was further improved to âˆ¼ 61 % (boron: sugar = 1.5), with the minimized formation of byproducts. Moreover, high purity (∼83 %) and concentrated d-tagatose solution (∼40 g/L) was obtained after sequential desorption. The proposed extraction-assisted isomerization strategy achieved improving the yield and purity of d-tagatose, proving its feasibility in industrial applications.

A precise swaying map for how promiscuous cellobiose-2-epimerase operate bi-reaction.

Promiscuous enzymes play a crucial role in organism survival and new reaction mining. However, comprehensive mapping of the catalytic and regulatory mechanisms hasn't been well studied due to the characteristic complexity. The cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) with complex epimerization and isomerization was chosen to comprehensively investigate the promiscuous mechanisms. Here, the catalytic frame of ring-opening, cis-enediol mediated catalysis and ring-closing was firstly determined. To map the full view of promiscuous CE, the structure of CsCE complex with the isomerized product glucopyranosyl-ß1,4-fructose was determined. Combined with computational calculation, the promiscuity was proved a precise cooperation of the double subsites, loop rearrangement, and intermediate swaying. The flexible loop was like a gear, whose structural reshaping regulates the sway of the intermediates between the two subsites of H377-H188 and H377-H247, and thus regulates the catalytic directions. The different protonated states of cis-enediol intermediate catalyzed by H188 were the key point for the catalysis. The promiscuous enzyme tends to utilize all elements at hand to carry out the promiscuous functions.

Efficient Secretory Production of δ-Tocotrienol by Combining Pathway Modularization and Transportation Engineering.

The vitamin E component δ-tocotrienol has shown impressive activities in radioprotection, neuroprotection, and cholesterol reduction. Its production is limited by the low content in plants and difficulty in separation from other tocotrienols. Fermentative production using a microbial cell factory that exclusively produces and secretes δ-tocotrienol is a promising alternative approach. Assembly of the δ-tocotrienol synthetic pathway in Saccharomyces cerevisiae followed by comprehensive pathway engineering led to the production of 73.45 mg/L δ-tocotrienol. Subsequent addition of 2-hydroxypropyl-ß-cyclodextrin (CD) and overexpression of the transcription factor PDR1 significantly elevated δ-tocotrienol titer to 241.7 mg/L (63.65 mg/g dry cell weight) in shake flasks, with 30.4% secreted. By properly adding CD and the in situ extractant olive oil, 181.12 mg/L of δ-tocotrienol was collected as an extracellular product, accounting for 85.6% of the total δ-tocotrienol production. This process provides not only a promising δ-tocotrienol cell factory but also insights into yeast engineering toward secretory production of other terpenoids.

Precise Photothermal Treatment of Methicillin-Resistant S. aureus Infection via Phage Lysin-Cell Binding Domain-Modified Gold Nanosheets.

The increasing spread of antibiotic resistance in bacterial pathogens poses a huge threat to global human health. Precise targeting of bacterial pathogens while avoiding collateral damage to healthy tissues has become the overriding goal for bacterial infection treatment. Inspired by the host specificity of bacteriophages, a biomimetic intelligent platform was designed for highly precise photothermal treatment herein. As proof-of-concept, the lysin cell-binding domain (CBD) from a newly discovered virulent methicillin-resistant S. aureus (MRSA) phage Z was applied to the functionalization of gold nanosheets. Our results demonstrated that the bionanocomposite gold particles (Au@PEG-CBDz) could be effectively delivered directly to MRSA and kill them effectively under near infrared irradiation in vitro, while displaying good in vivo biocompatibility. This work is the first to report the combination of phage lysin navigatory function with photothermal effect-induced bactericidal activity from Au nanosheets, providing a novel therapeutic mode for the precision treatment of antibiotic-resistant bacterial infections.

Pulsed electric field as a promising technology for solid foods processing: A review.

A pulsed electric field (PEF) induces cell electroporation for solid foods, thereby allowing PEF to be used as a pretreatment method for extraction, drying, peeling, freeze-thawing, and cooking by increasing mass transfer. Currently, popular research mainly focuses on the process and results of the application of PEF to solid food processing. Therefore, this review summarizes the impact of PEF on the quality of solid food, the evaluation techniques of PEF-treated tissues and the characteristics of PEF treatment chambers. Furthermore, other pretreatments, including freezing and peeling, typically used in the meat and vegetable sectors, are also discussed alongside PEF to evaluate the impact on its effects. Finally, this article examined the main obstacles and prospects of PEF in solid food processing. This evaluation is anticipated to expand future PEF research paths in the solid food industry.

Reshaping the Binding Pocket of Cellobiose 2-Epimerase for Improved Substrate Affinity and Isomerization Activity for Enabling Green Synthesis of Lactulose.

Enzymatic isomerization of lactose into lactulose via cellobiose 2-epimerase (CE) could provide an eco-friendly route for the industrial production of lactulose, a valuable food prebiotic. However, poor substrate affinity for lactose and preference for epimerization over isomerization hinder this application. Previous studies on CE improvement have focused on random mutagenesis or active site rational design; little is known about the relationship between substrate binding and enzyme efficacy, which was hence the subject of this study. First, residues 372W and 308W were identified as key for disaccharide recognition in CEs based on crystal structure alignment of the N-acetyl-glucosamine 2-epimerase superfamily and site-directed mutation. This binding domain was then reshaped through site saturation mutagenesis, resulting in seven mutants with enhanced isomerization activity. The optimal mutant CsCE/Q371E had significantly enhanced substrate affinity (Km, 269.65 mM vs Km, 417.5 mM), reduced epimerization activity, and 3.3-fold increased isomerization activity over the original CsCE. Molecular dynamics simulation further revealed that substituting Gln-371 with Glu strengthened the hydrogen-bonding network and altered the active site-substrate interactions, increasing the substrate stability and shifting the catalytic direction. This study uncovered new information about the substrate binding region and its mechanisms and impact on CE catalytic performance, paving the way for potential commercial applications.

Reduction of intestinal fat digestion and absorption by ß-glucan secreted by Rhizobium pusense via interference in triglyceride hydrolysis.

In this study, the ability of ß-glucan extracted from Rhizobium pusense to reduce digestion and absorption of ingested fat was systematically investigated via in vitro and in vivo experiments. Specifically, in in vitro gastrointestinal simulations, when ß-glucan was added to a high-fat food model, the concentration of free fatty acids (FFAs) were remarkably decreased. An in vitro intestinal epithelial cell model demonstrated that addition of ß-glucan can significantly reduce the translocation of triglycerides (TGs) and FFAs. In in vivo experiments, a high-fat food model with the addition of ß-glucan showed a significant reduction in postprandial serum TG elevation. Confocal laser scanning microscopy analysis showed that when ß-glucan was added to a cream sample, both coalescence and disappearance of lipid droplets were reduced, and the distribution of oil droplets was very consistent with the distribution of ß-glucan positions. This suggests the main mechanism for this effect: the coagulation of lipid droplets by ß-glucan leads to a reduction in the available surface area for lipase binding. All these findings suggest that ß-glucan could potentially be used as a food additive or supplement to reduce the absorption of ingested fat and thus aid in weight loss and the treatment of diseases caused by TGs.

De Novo Production of Hydroxytyrosol by Saccharomyces cerevisiae-Escherichia coli Coculture Engineering.

Hydroxytyrosol is a valuable plant-derived phenolic compound with excellent pharmacological activities for application in the food and health care industries. Microbial biosynthesis provides a promising approach for sustainable production of hydroxytyrosol via metabolic engineering. However, its efficient production is limited by the machinery and resources available in the commonly used individual microbial platform, for example, Escherichia coli, Saccharomyces cerevisiae. In this study, a S. cerevisiae-E. coli coculture system was designed for de novo biosynthesis of hydroxytyrosol by taking advantage of their inherent metabolic properties, whereby S. cerevisiae was engineered for de novo production of tyrosol based on an endogenous Ehrlich pathway, and E. coli was dedicated to converting tyrosol to hydroxytyrosol by use of native hydroxyphenylacetate 3-monooxygenase (EcHpaBC). To enhance hydroxytyrosol production, intra- and intermodule engineering was employed in this microbial consortium: (I) in the upstream S. cerevisiae strain, multipath regulations combining with a glucose-sensitive GAL regulation system were engineered to enhance the precursor supply, resulting in significant increase of tyrosol production (from 17.60 mg/L to 461.07 mg/L); (II) Echpabc was overexpressed in the downstream E. coli strain, improving the conversion rate of tyrosol to hydroxytyrosol from 0.03% to 86.02%; (III) and last, intermodule engineering with this coculture system was performed by optimization of the initial inoculation ratio of each population and fermentation conditions, achieving 435.32 mg/L of hydroxytyrosol. This S. cerevisiae-E. coli coculture strategy provides a new opportunity for de novo production of hydroxytyrosol from inexpensive feedstock.

Structural characterization of a water-soluble and antimicrobial ß-glucan secreted by Rhizobium pusense.

In this study, a bacterium with the ability to extracellularly produce a water-soluble polysaccharide (with high molecular mass of 743 kDa) was obtained from saline soils. This strain named as ZB01 was identified as Rhizobium punsense by 16S rRNA sequence analysis. The monomer composition and structure of extracellular polysaccharides were investigated by high-performance anion-exchange chromatography-pulsed amperometric detector, Fourier-transform infrared, methylation and nuclear magnetic resonance spectroscopy analysis. In addition, the morphological characterization of extracellular polysaccharides was performed by scanning electron microscopy analysis. As a result, the polysaccharide is found to be a novel water-soluble type of glucan most likely consisting of repeating two ß-d-Glcp-(1 â†’ 3) and one α-d-Glcp-(1 â†’ 3). Furthermore, it showed pronounced antimicrobial effects against Escherichia coli and Bacillus subtilis, suggesting it has the potential to be used as a natural antibacterial agent in the future.

Lactulose production from lactose isomerization by chemo-catalysts and enzymes: Current status and future perspectives.

Lactulose, a semisynthetic nondigestive disaccharide with versatile applications in the food and pharmaceutical industries, has received increasing interest due to its significant health-promoting effects. Currently, industrial lactulose production is exclusively carried out by chemical isomerization of lactose via the Lobry de Bruyn-Alberda van Ekenstein (LA) rearrangement, and much work has been directed toward improving the conversion efficiency in terms of lactulose yield and purity by using new chemo-catalysts and integrated catalytic-purification systems. Lactulose can also be produced by an enzymatic route offering a potentially greener alternative to chemo-catalysis with fewer side products. Compared to the controlled trans-galactosylation by ß-galactosidase, directed isomerization of lactose with high isomerization efficiency catalyzed by the most efficient lactulose-producing enzyme, cellobiose 2-epimerase (CE), has gained much attention in recent decades. To further facilitate the industrial translation of CE-based lactulose biotransformation, numerous studies have been reported on improving biocatalytic performance through enzyme mediated molecular modification. This review summarizes recent developments in the chemical and enzymatic production of lactulose. Related catalytic mechanisms are also highlighted and described in detail. Emerging techniques that aimed at advancing lactulose production, such as the boronate affinity-based technique and molecular biological techniques, are reviewed. Finally, perspectives on challenges and opportunities in lactulose production and purification are also discussed.

De Novo Production of Hydroxytyrosol by Metabolic Engineering of Saccharomyces cerevisiae.

Hydroxytyrosol is an olive-derived phenolic compound of increasing commercial interest due to its health-promoting properties. In this study, a high-yield hydroxytyrosol-producing Saccharomyces cerevisiae cell factory was established via a comprehensive metabolic engineering scheme. First, de novo biosynthetic pathway of hydroxytyrosol was constructed in yeast by gene screening and overexpression of different phenol hydroxylases, among which paHD (from Pseudomonas aeruginosa) displayed the best catalytic performance. Next, hydroxytyrosol precursor supply was enhanced via a multimodular engineering approach: elimination of tyrosine feedback inhibition through genomic integration of aro4K229L and aro7G141S, construction of an aromatic aldehyde synthase (AAS)-based tyrosine metabolic pathway, and redistribution of metabolic flux between glycolytic pathway and pentose phosphate pathway (PPP) by introducing the exogenous gene Bbxfpkopt. As a result, the titer of hydroxytyrosol was improved by 6.88-fold. Finally, a glucose-responsive dynamic regulation system based on GAL80 deletion was implemented, resulting in the final hydroxytyrosol yields of 308.65 mg/L and 167.98 mg/g cell mass, the highest known from de novo production in S. cerevisiae to date.

Radio frequency as an innovative method to produce low-fat French fries.

BACKGROUND: A large amount of evidence shows that excessive fat intake can increase the risk of obesity, type 2 diabetes, non-alcoholic fatty liver disease, and cardiovascular disease. The main purpose of this study was to use radio frequency (RF) technology to prepare low-fat French fries. RESULTS: RF treatment for 10 min significantly decreased the force required to cut potatoes and inhibited the enzymatic browning of fresh-cut potatoes. Moreover, RF treatment increased the hardness, gumminess, and chewiness of French fries from 388.55 g, 85.67, and 33.27 to 776.93 g, 159.36, and 70.11, respectively. Furthermore, RF treatment for 10 min reduced the oil content of French fries by 28.0% compared to that of the control group. This result was related to the pre-gelatinized potato starch content after RF treatment. Pre-gelatinized starch forms a 'protective film', that prevents oil from entering the fries during frying. CONCLUSION: Moderate RF treatment (10 min) reduced the oil content of French fries without making their texture significantly different from that of commercial French fries. These findings may provide a new perspective for the application of RF technology in the development of low-fat fried foods. © 2022 Society of Chemical Industry.

Effects of pulse electric field pretreatment on the frying quality and pore characteristics of potato chips.

The main purpose of this work was to investigate the effect of pulsed electric field (PEF) treatment on the oil absorption capacity of potato chips, evaluated via changes to microstructure and pore characteristics. Our results showed that as electric field strength increased from 0 kV/cm (no pretreatment) to 5 kV/cm, the oil content of potato chips decreased by up to 20.6%. Furthermore, at higher the electric field strengths (5 ~ 20 kV/cm), most of the potato cell walls collapsed, and dense pores could be observed in the horizontal profile of the chips. Moreover, some smaller pores (10-50 nm) in the potato chips were disrupted and merged into larger pores (50-100 nm), thus increasing the total volume and average diameter of the pores, accelerating moisture evaporation and reducing oil absorption during frying. Our findings provide a novel perspective on the application of PEF towards the development of lower-fat and healthier fried foods.

Biotechnological advances for improving natural pigment production: a state-of-the-art review.

In current years, natural pigments are facing a fast-growing global market due to the increase of people's awareness of health and the discovery of novel pharmacological effects of various natural pigments, e.g., carotenoids, flavonoids, and curcuminoids. However, the traditional production approaches are source-dependent and generally subject to the low contents of target pigment compounds. In order to scale-up industrial production, many efforts have been devoted to increasing pigment production from natural producers, via development of both in vitro plant cell/tissue culture systems, as well as optimization of microbial cultivation approaches. Moreover, synthetic biology has opened the door for heterologous biosynthesis of pigments via design and re-construction of novel biological modules as well as biological systems in bio-platforms. In this review, the innovative methods and strategies for optimization and engineering of both native and heterologous producers of natural pigments are comprehensively summarized. Current progress in the production of several representative high-value natural pigments is also presented; and the remaining challenges and future perspectives are discussed.

Key determinants of misdiagnosis of tracheobronchial tuberculosis among senile patients in contemporary clinical practice: A retrospective analysis.

BACKGROUND: Tracheobronchial tuberculosis (TBTB) is a common subtype of pulmonary tuberculosis. Concomitant diseases often obscure the diagnosis of senile TBTB. AIM: To characterize senile patients with TBTB and to identify the potential causes of misdiagnosis. METHODS: One hundred twenty patients with senile TBTB who were admitted to the Anhui Chest hospital between May 2017 and May 2019 were retrospectively analyzed. Patients were classified as diagnosed group (n = 58) and misdiagnosed group (n = 62). Clinical manifestations, laboratory results, radiographic data, and endoscopic findings were compared between the two groups. RESULTS: Patients in the misdiagnosed group were most commonly diagnosed as pulmonary tuberculosis (non-TBTB, 29/62, 46.8%), general pneumonia (9/62, 14.5%), chronic obstructive pulmonary disease (8/62, 12.9%), and tracheobronchial carcinoma (7/62, 11.3%). The time elapsed between disease onset and confirmation of diagnosis was significantly longer in the misdiagnosed group [median (first quartile, third quartile): 6.32 (4.94, 16.02) mo vs 3.73 (2.37, 8.52) mo]. The misdiagnosed group had lower proportion of patients who underwent bronchoscopy [33.87% (21/62) vs 87.93% (51/58)], chest computed tomography (CT) scan [69.35% (43/62) vs 98.28% (57/58)], and those who showed CT signs of tuberculosis [27.91% (12/62) vs 50% (29/58)] as compared to that in the diagnosed group (P < 0.05). There were no significant between-group differences with respect to age, gender, occupation, clinical manifestations, or prevalence of comorbid chronic diseases (P > 0.05). CONCLUSION: Insufficient or inaccurate radiographic or bronchoscopic assessment was the predominant cause of delayed diagnosis of TBTB. Increased implementation and better interpretation of CT scan and early implementation of bronchoscopy can help reduce misdiagnosis of senile TBTB.

Screening of a Bacillus subtilis strain producing both nattokinase and milk-clotting enzyme and its application in fermented milk with thrombolytic activity.

Bacillus subtilis is a generally recognized as safe probiotic, which is used as a starter for natto fermentation. Natto is a functional food with antithrombus function due to nattokinase. Compared with natto, fermented milk is a more popular fermented food, which is commonly fermented by Lactobacillus bulgaricus and Streptococcus. However, there is no report on B. subtilis-fermented milk. In this study, to produce a functional fermented milk with antithrombus function, a B. subtilis strain (B. subtilis JNFE0126) that produced both nattokinase and milk-clotting enzyme was isolated from traditionally fermented natto and used as the starter for the functional fermented milk. In liquid fermentation culture, the peak values of thrombolytic activity and milk-clotting activity were 3,511 U/mL at 96 h and 874.5 Soxhlet unit/mL at 60 h, respectively. The optimal pH and temperature were pH 7.0 at 40°C for nattokinase and pH 6.5 and 55°C for milk-clotting enzyme, respectively. The thrombolytic activity in the fermented milk reached 215.1 U/mL after 8 h of fermentation. Sensory evaluation showed that the acceptance of the milk fermented by B. subtilis JNFE0126 was similar to the traditional milk fermented by L. bulgaricus and S. thermophilus. More importantly, oral intake of the fermented milk by the thrombosis-model mice prevented the development of thrombosis. Our results suggest that B. subtilis JNFE0126-fermented milk has potential as a novel, functional food in the prevention of thrombosis-related cardiovascular diseases.

Active Delivery of CRISPR System Using Targetable or Controllable Nanocarriers.

Among programmable nuclease-based genome editing tools, the clustered regularly interspaced short palindromic repeats (CRISPR) system with accuracy and the convenient operation is most promising to be applied in gene therapy. The development of effective delivery carriers for the CRISPR system is the major premise to achieve practical applications. Although many nanocarrier-mediated deliveries have been reported to be safer and cheaper over the physical and viral delivery, the accumulation at disease sites or controllability with the spatial or temporal resolution are still desired on nanocarriers to reduce side effects and off-target from the CRISPR system. Therefore, the targetable and controllable nanocarriers to actively deliver the CRISPR system are summarized. The cell or even organ selective nanocarriers are introduced first, followed by the discussion of nanocarriers controlled by biochemical or physical signals. At last, the potential challenges faced by existing nanocarriers are discussed.

Enhancement of the Isomerization Activity and Thermostability of Cellobiose 2-Epimerase from Caldicellulosiruptor saccharolyticus by Exchange of a Flexible Loop.

Cellobiose 2-epimerase (CE) offers a promising enzymatic approach to produce lactulose. However, its application is limited by the unsatisfactory isomerization activity and thermostability. Our study attempted to optimize the catalytic performances of CEs by flexible loop exchange, for which four mutants were constructed using CsCE (CE from Caldicellulosiruptor saccharolyticus) as a template. As a result, all mutants maintained the same catalytic directions as the templates. Mutant RmC displayed a 2.2- and 1.34-fold increase in the isomerization activity and catalytic efficiency, respectively. According to the results of molecular dynamics (MD) simulations, it was revealed that the loop exchange in RmC enlarged the entrance of the active site for substrate binding and benefited proton transfer involved in the isomerization process. Besides, the t1/2 of mutant StC at 70 °C was increased from 29.07 to 38.29 h, owing to the abundance of rigid residues (proline) within the flexible loop of StC. Our work demonstrated that the isomerization activity and thermostability of CEs were closely related to the flexible loop surrounding the active site, which provides a new perspective to engineer CEs for higher lactulose production.

Structure and chain conformation characterization of arabinoglucan from by-product of peanut oil processing.

A neutral polysaccharide (NPP) from peanut sediment of aqueous extraction process was purified via anion-exchange and gel-filtration chromatography. The weight-average molecular weight and polydispersity index were 3.36 × 104 Da and 1.06. Composition of glucose (82.66 %, molar percentage) and arabinose (17.34 %) suggested an arabinoglucan structure. Multiple medium-length chains consisting of many 1,4-linked α-Glcp and a few 1,5-linked α-Araf maintained the main chain structure. The backbone was substituted at O-6 and O-3 positions, attached by side chains consisting of two to six α-Glcp and terminated with Araf and Glcp. Degree of branching was 42.50 %. Aggregates formed in NPP aqueous solution. They were eliminated by DMSO combining with sonication. Consequently, the average radius of gyration (Rg), hydrodynamic radius (Rh), and Rg/Rh ratio were 17.0 nm, 5.8 nm and 2.93, respectively, indicating extended rigid chain conformation. The backbone substituted at O-3 and short branching chains probably together induced this conformation.

Insight into the potential factors influencing the catalytic direction in cellobiose 2-epimerase by crystallization and mutagenesis.

Cellobiose 2-epimerase (CE) is commonly recognized as an epimerase as most CEs mainly exhibit an epimerization activity towards disaccharides. In recent years, several CEs have been found to possess bifunctional epimerization and isomerization activities. They can convert lactose into lactulose, a high-value disaccharide that is widely used in the food and pharmaceutical industries. However, the factors that determine the catalytic direction in CEs are still not clear. In this study, the crystal structures of three newly discovered CEs, CsCE (a bifunctional CE from Caldicellulosiruptor saccharolyticus), StCE (a bifunctional CE from Spirochaeta thermophila DSM 6578) and BtCE (a monofunctional CE from Bacillus thermoamylovorans B4166), were determined at 1.54, 2.05 and 1.80 Šresolution, respectively, in order to search for structural clues to their monofunctional/bifunctional properties. A comparative analysis of the hydrogen-bond networks in the active pockets of diverse CEs, YihS and mannose isomerase suggested that the histidine corresponding to His188 in CsCE is uniquely required to catalyse isomerization. By alignment of the apo and ligand-bound structures of diverse CEs, it was found that bifunctional CEs tend to have more flexible loops and a larger entrance around the active site, and that the flexible loop 148-181 in CsCE displays obvious conformational changes during ligand binding. It was speculated that the reconstructed molecular interactions of the flexible loop during ligand binding helped to motivate the ligands to stretch in a manner beneficial for isomerization. Further site-directed mutagenesis analysis of the flexible loop in CsCE indicated that the residue composition of the flexible loop did not greatly impact epimerization but affects isomerization. In particular, V177D and I178D mutants showed a 50% and 80% increase in isomerization activity over the wild type. This study provides new information about the structural characteristics involved in the catalytic properties of CEs, which can be used to guide future molecular modifications.