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Foods (Basel, Switzerland) Dec 2023With the increasing number of people affected by gluten consumption-related diseases, adhering to a gluten-free (GF) diet is the most effective preventive measure....
With the increasing number of people affected by gluten consumption-related diseases, adhering to a gluten-free (GF) diet is the most effective preventive measure. Herein, we aimed to isolate and characterize the functional properties of autochthonous lactic acid bacteria (LAB) and yeast from various GF sourdoughs to determine their suitability in starter cultures for sourdough preparation. Three LAB, BAQ2, AQ2, YC2, and BW1, were identified. The isolated LAB exhibited greater TTA, faster acidification rates, and higher acid tolerance than commercial LAB. BAQ2 exhibited the highest EPS production, BAQ2 and AQ2 showed high maltose utilization, and BW1 exhibited the highest CO production rate. Accordingly, all four microbial strains were mixed for the starter culture. The sourdough prepared with starter cultures exhibited differences in gas production depending on fermentation time, which influenced the volume of GF bread dough. GF bread prepared with fermented sourdough exhibited a 16% higher specific volume and enhanced crumb firmness and elasticity than that prepared using non-fermented sourdough. Thus, autochthonous LAB strains isolated from various GF sourdoughs can be used together to improve the quality of sourdough bread, demonstrating their potential for use in starter cultures for GF sourdough production.
PubMed: 38231883
DOI: 10.3390/foods12234367 -
Water content, transition temperature and fragility influence protection and anhydrobiotic capacity.BBA Advances 2024Water is essential for metabolism and all life processes. Despite this, many organisms distributed across the kingdoms of life survive near-complete desiccation or...
UNLABELLED
Water is essential for metabolism and all life processes. Despite this, many organisms distributed across the kingdoms of life survive near-complete desiccation or anhydrobiosis. Increased intracellular viscosity, leading to the formation of a vitrified state is necessary, but not sufficient, for survival while dry. What properties of a vitrified system make it desiccation-tolerant or -sensitive are unknown. We have analyzed 18 different vitrified systems, composed of one of three protective disaccharides (trehalose, sucrose, or maltose) and glycerol, quantifying their enzyme-protective capacity and their material properties in a dry state. Protection conferred by mixtures containing maltose correlates strongly with increased water content, increased glass-transition temperature, and reduced glass former fragility, while the protection of glasses formed with sucrose correlates with increased glass transition temperature and the protection conferred by trehalose glasses correlates with reduced glass former fragility. Thus, different vitrified sugars confer protection through distinct material properties. Next, we examined the material properties of a dry desiccation tolerant and intolerant life stage from three different organisms. The dried desiccation tolerant life stage of all organisms had an increased glass transition temperature and reduced glass former fragility relative to its dried desiccation intolerant life stage. These results suggest in nature organismal desiccation tolerance relies on a combination of various material properties. This study advances our understanding of how protective and non-protective glasses differ in terms of material properties that promote anhydrobiosis. This knowledge presents avenues to develop novel stabilization technologies for pharmaceuticals that currently rely on the cold-chain.
STATEMENT OF SIGNIFICANCE
For the past three decades the anhydrobiosis field has lived with a paradox, while vitrification is necessary for survival in the dry state, it is not sufficient. Understanding what property(s) distinguishes a desiccation tolerant from an intolerant vitrified system and how anhydrobiotic organisms survive drying is one of the enduring mysteries of organismal physiology. Here we show the enzyme-protective capacity of different vitrifying sugars can be correlated with distinct material properties. However, diverse desiccation tolerant organisms appear to combine these material properties to promote their survival in a dry state.
PubMed: 38318251
DOI: 10.1016/j.bbadva.2024.100115 -
International Journal of Biochemistry... 2023The diverse nature of carbohydrate structures and linkages requires a variety of enzymes responsible for sugar degradation. The periplasmic protein encoded by the gene...
BACKGROUND
The diverse nature of carbohydrate structures and linkages requires a variety of enzymes responsible for sugar degradation. The periplasmic protein encoded by the gene has been assigned to glycoside hydrolase family 3 and is predicted to function as a β-glucosidase.
OBJECTIVES
We investigated the catalytic properties of the protein BglX and identified two functionally important amino acid residues.
METHODS
The gene was cloned into a pET20b(+) vector, and three mutants, D111N, D287G, and E293Q, were generated using site-directed mutagenesis. Kinetic studies were performed on the wild-type and mutant enzymes.
RESULTS
Substrate specificity tests indicated that the BglX enzyme hydrolyzes β-glycosidic bonds in nitrophenyl-β-glycosides and demonstrates greater activity towards galactose-containing substrates compared to glucose derivatives. Monomeric glucose and galactose inhibit enzyme activity to a different degree in a substrate-dependent manner. In addition, BglX can hydrolyze lactose but not cellobiose, maltose, or laminarin. Subsequently, cells overexpressing active BglX have a growth advantage on minimal media supplemented with lactose as a carbon source. Mutation of D287 or D111 residues negatively affected the activity of BglX indicating their involvement in catalysis. Overexpression of BglX by cells did not increase biofilm formation.
CONCLUSIONS
The low activity towards glucose-containing substrates and significantly elevated activity towards galactosides suggests that β-glucosidase activity may not be the primary function of the BglX enzyme.
PubMed: 37736388
DOI: No ID Found -
Food Science & Nutrition Mar 2024The kefir-based smoothies with kale and spinach were designed as a ready-to-drink and innovative functional snack. Microbiological, physicochemical, as well as pre- and...
The kefir-based smoothies with kale and spinach were designed as a ready-to-drink and innovative functional snack. Microbiological, physicochemical, as well as pre- and postgastrointestinal total antioxidant capacity (TAC; CUPRAC, DPPH, and FRAP) analyses were conducted. It was determined that the kefir-based smoothies with vegetables had higher ash, carbohydrate, and dietary fiber values. Fructose and glucose contents of smoothy with kale were high, while smoothy with spinach included high sucrose and maltose. The microbiology results revealed that kefir-based vegetable smoothies had minimum Lactobacillaceae viability (>log 7 cfu g) for the required functional effect after 14-day storage. Moreover, the addition of kale significantly increased ( < .01) the level of initial TAC (CUPRAC, DPPH, and FRAP) and total phenolic content (TPC) values. After in vitro gastric digestion analysis, smoothie with spinach demonstrated higher TAC and TPC values and the control sample had higher TAC and TPC values compared with a predigestion step. It was found that in vitro intestinal DPPH values were higher for the sample with spinach samples, while the sample with kale had the highest FRAP values. It was also found that the bioaccessibility indexes of plain kefir were determined to be the highest in both in vitro gastric and intestinal procedures. The present study provided novel insights into the in vitro digestion properties of kefir fortified with vegetables. Nevertheless, further studies are needed to identify the functional properties of the milk and plant matrices mixture using in vitro and in vivo trials.
PubMed: 38455206
DOI: 10.1002/fsn3.3917 -
AMB Express Sep 2023The biosynthetic process of selenium nanoparticles (SeNPs) by specific bacterial strain, whose growth directly affects the synthesis efficiency, has attracted great...
The biosynthetic process of selenium nanoparticles (SeNPs) by specific bacterial strain, whose growth directly affects the synthesis efficiency, has attracted great attentions. We previously reported that Bacillus paralicheniformis SR14, a SeNPs-producing bacteria, could improve intestinal antioxidative function in vitro. To further analyze the biological characteristics of SR14, whole genome sequencing was used to reveal the genetic characteristics in selenite reduction and sugar utilization. The results reviewed that the genome size of SR14 was 4,448,062 bp, with a GC content of 45.95%. A total of 4300 genes into 49 biological pathways was annotated to the KEGG database. EC: 1.1.1.49 (glucose-6-phosphate 1-dehydrogenase) and EC: 5.3.1.9 (glucose-6-phosphate isomerase), were found to play a potential role in glucose degradation and EC:2.7.1.4 (fructokinase) might be involved in the fructose metabolism. Growth profile and selenite-reducing ability of SR14 under different sugar supplements were determined and the results reviewed that glucose had a better promoting effect on the reduction of selenite and growth of bacteria than fructose, sucrose, and maltose. Moreover, RT-qPCR experiment proved that glucose supplement remarkably promoted the expressions of thioredoxin, fumarate reductase, and the glutathione peroxidase in SR14. Analysis of mRNA expression showed levels of glucose-6-phosphate dehydrogenase and fructokinase significantly upregulated under the supplement of glucose. Overall, our data demonstrated the genomic characteristics of SR14 and preliminarily determined that glucose supplement was most beneficial for strain growth and SeNPs synthesis.
PubMed: 37665384
DOI: 10.1186/s13568-023-01598-9 -
International Journal of Molecular... Apr 2024Intelectins belong to a family of lectins with specific and transitory carbohydrate interaction capabilities. These interactions are related to the activity of...
Intelectins belong to a family of lectins with specific and transitory carbohydrate interaction capabilities. These interactions are related to the activity of agglutinating pathogens, as intelectins play a significant role in immunity. Despite the prominent immune defense function of intelectins, limited information about its structural characteristics and carbohydrate interaction properties is available. This study investigated an intelectin transcript identified in RNA-seq data obtained from the South American lungfish (), namely LpITLN2-B. The structural analyses predicted LpITLN2-B to be a homo-trimeric globular protein with the fibrinogen-like functional domain (FReD), exhibiting a molecular mass of 57 kDa. The quaternary structure is subdivided into three monomers, A, B, and C, and each domain comprises 11 β-sheets: an anti-parallel β-sheet, a β-hairpin, and a disordered β-sheet structure. Molecular docking demonstrates a significant interaction with disaccharides rather than monosaccharides. The preferential interaction with disaccharides highlights the potential interaction with pathogen molecules, such as LPS and Poly(I:C). The hemagglutination assay inhibited lectins activity, especially maltose and sucrose, highlighting lectin activity in samples. Overall, our results show the potential relevance of LpITLN2-B in immune defense against pathogens.
Topics: Animals; Lectins; Immunity, Innate; Fishes; Fish Proteins; Molecular Docking Simulation; Amino Acid Sequence; GPI-Linked Proteins
PubMed: 38732017
DOI: 10.3390/ijms25094798 -
The Protein Journal Oct 2023The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical...
The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas.
Topics: Hydrolysis; Thermotoga maritima; Glycogen Debranching Enzyme System; alpha-Amylases; Substrate Specificity
PubMed: 37464145
DOI: 10.1007/s10930-023-10136-2 -
Journal of Dairy Science Mar 2024This study aimed to identify coagulase-positive staphylococci (CPS) species from 21 samples of clandestine Minas Frescal cheese, investigate the potential for...
This study aimed to identify coagulase-positive staphylococci (CPS) species from 21 samples of clandestine Minas Frescal cheese, investigate the potential for deterioration in psychrotrophic and mesophilic conditions, verify the toxigenic potential of Staphylococcus aureus, and determine the antimicrobial susceptibility profile of toxigenic S. aureus. Species determination was performed based on the detection of β-hemolysis in 5% ovine blood agar; fermentation of mannitol, maltose, and trehalose sugars; and production of acetoin. After species determination, DNA extraction and analysis was performed for S. aureus colonies for genes encoding staphylococcal toxins (eta, etb, tst, sea, seb, sec, sed, and see) using 2 multiplex PCR assays. Isolates identified as toxigenic S. aureus were tested for antimicrobial susceptibility to tetracycline, erythromycin, clindamycin, gentamicin, ciprofloxacin, sulfazotrim, trimethoprim, streptomycin, cefoxitin, vancomycin and enrofloxacin. Elevated CPS counts were observed with an average of >6 log cfu/g. Of the 355 isolates, 177 (49.86%) were identified as S. aureus. Staphylococcus hyicus, Staphylococcus intermedius, Staphylococcus delphini, and Staphylococcus coagulans were identified in 3 (0.84%), 2 (0.56%), 2 (0.56%), and 1 (0.28%) isolates, respectively. Of the total number of S. aureus, 25 (52.08%) were positive for the gene that encodes for toxic shock toxin (TSST-1). Another 16 (33.33%) were positive for the sea gene, and 4 isolates (8.33%) were positive for see and one isolate each was positive for seb (2.08%), sec (2.08%), and etb (2.08%) genes. All isolates demonstrated lipolytic activity under mesophilic and psychrotrophic conditions. S. intermedius and S. hyicus had the most prominent proteolytic potential. Multidrug resistance was observed in most of the potentially toxigenic isolates, with clindamycin having the lowest efficiency (40%), whereas the aminoglycosides (gentamicin and streptomycin) had the highest effectiveness demonstrating inhibition in all evaluated isolates. Methicillin-resistant S. aureus (MRSA) was detected. Minas Frescal cheeses, marketed in the north of Tocantins in the Brazilian Amazon region, do not comply with legal quality standards and pose a public health risk due to the enterotoxigenic potential of multiresistant isolates, in addition to low shelf life of the samples given the high spoilage potential of this microbiota.
Topics: Animals; Sheep; Staphylococcus aureus; Coagulase; Methicillin-Resistant Staphylococcus aureus; Clindamycin; Cheese; Staphylococcus; Anti-Bacterial Agents; Streptomycin; Gentamicins
PubMed: 37944805
DOI: 10.3168/jds.2023-23747 -
Microorganisms Nov 2023A novel cellulose microfibril swelling (Cms) gene of sp. AY8 was successfully cloned and sequenced using a set of primers designed based on the conserved region of the...
A novel cellulose microfibril swelling (Cms) gene of sp. AY8 was successfully cloned and sequenced using a set of primers designed based on the conserved region of the gene from the genomic database. The molecular cloning of the Cms gene revealed that the gene consisted of 679 bp sequences encoding 225 amino acids. Further in silico analysis unveiled that the Cms gene contained the NlpC/P60 conserved region that exhibited a homology of 98% with the NlpC/P60 family proteins found in both the strains, sp. and . The recombinant Cms enzyme had a significant impact on the reduction of crystallinity indices (CrI) of various substrates including a 3%, a 3.97%, a 4.66%, and a substantial 14.07% for filter paper, defatted cotton fiber, avicel, and alpha cellulose, respectively. Additionally, notable changes in the spectral features were observed among the substrates treated with recombinant Cms enzymes compared to the untreated control. Specifically, there was a decrease in band intensities within the spectral regions of 3000-3450 cm, 2900 cm, 1429 cm, and 1371 cm for the treated filter paper, cotton fiber, avicel, and alpha cellulose, respectively. Furthermore, the recombinant Cms enzyme exhibited a maximum cellulose swelling activity at a pH of 7.0 along with a temperature of 40 °C. The molecular docking data revealed that ligand molecules, such as cellobiose, dextrin, maltose 1-phosphate, and feruloyated xyloglucan, effectively bonded to the active site of the Cms enzyme. The molecular dynamics simulations of the Cms enzyme displayed stable interactions with cellobiose and dextrin molecules up to 100 ns. It is noteworthy to mention that the conserved region of the Cms enzyme did not match with those of the bioadditives like expansins and swollenin proteins. This study is the initial report of a bacterial cellulose microfibril swellase enzyme, which could potentially serve as an additive to enhance biofuel production by releasing fermentable sugars from cellulose.
PubMed: 38138001
DOI: 10.3390/microorganisms11122857 -
BioRxiv : the Preprint Server For... Jan 2024Proteins are the workhorses of biology, orchestrating a myriad of cellular functions through intricate conformational changes. Protein allostery, the phenomenon where...
Proteins are the workhorses of biology, orchestrating a myriad of cellular functions through intricate conformational changes. Protein allostery, the phenomenon where binding of ligands or environmental changes induce conformational rearrangements in the protein, is fundamental to these processes. We have previously shown that transition metal Förster resonance energy transfer (tmFRET) can be used to interrogate the conformational rearrangements associated with protein allostery and have recently introduced novel FRET acceptors utilizing metal-bipyridyl derivatives to measure long (>20 Å) intramolecular distances in proteins. Here, we combine our tmFRET system with fluorescence lifetime measurements to measure the distances, conformational heterogeneity, and energetics of maltose binding protein (MBP), a model allosteric protein. Time-resolved tmFRET captures near-instantaneous snapshots of distance distributions, offering insights into protein dynamics. We show that time-resolved tmFRET can accurately determine distance distributions and conformational heterogeneity of proteins. Our results demonstrate the sensitivity of time-resolved tmFRET in detecting subtle conformational or energetic changes in protein conformations, which are crucial for understanding allostery. In addition, we extend the use of metal-bipyridyl compounds, showing Cu(phen) can serve as a spin label for pulse dipolar electron paramagnetic resonance (EPR) spectroscopy, a method which also reveals distance distributions and conformational heterogeneity. The EPR studies both establish Cu(phen) as a useful spin label for pulse dipolar EPR and validate our time-resolved tmFRET measurements. Our approach offers a versatile tool for deciphering conformational landscapes and understanding the regulatory mechanisms governing biological processes.
PubMed: 37873384
DOI: 10.1101/2023.10.09.561594