Lactobacillus and Bifidobacterium are the most known genera of the therapeutic niche of organisms that have developed various defense mechanisms through their antioxidative activity which help mitigate the abnormality caused by reactive oxygen species to cell components. Nevertheless, for these probiotic organisms to exert their beneficial effects, the need to enhance functionality and protect bacterial cells in the gastrointestinal tract requires an efficient method for a better selection of favorable fermentation conditions with a sufficient yield of desired property or product. Here we reported the potential and antioxidant activities of isolated probiotic strains, with a considerable viable cell count between 108 and 109 CFU/mL, as the pH of both fermented millet substrate decreased from 6.13 to 3.62 at 35 °C within 14 h using a statistical optimization approach. The identified strains Pediococcus pentosaceus RZ01 and Lacticaseibacillus paracasei RZ02 alongside the reference strain had an overall gastrointestinal tolerance of 60% under simulated conditions, with Pediococcus pentosaceus RZ01 showing the highest bile salt survival rate (123.07%). Lactobacillus paracasei RZ02 indicates a high hydrophobicity potential (86.48%), suggesting a better adhesion ability to the gastrointestinal tract. Concurrently, Pediococcus pentosaceus RZ01 and Lactobacillus paracasei RZ02 fermented substrate had 39.69 and 44.51% of 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity, with the release of bioactive compounds of health importance. These provide important references for formulating novel probiotic fermented cereal product, understanding of probiotic-prebiotic relationship, as well as pave way for biotechnological application of identified and related probiotics.
Quantitative real-time PCR is widely used to determine absolute abundance of microbes in food fermentation. However, it remains challenges in the application for quantification at the species level due to the difficulty in designing species-specific primer sets. This work, using Lactobacillaceae, a dominant family within the lactic acid bacteria that involved in diversity food fermentations, as a case, presents an extendable strategy to design species-specific primer sets for microbial quantitative analysis. 136,257 species-specific genes were obtained from all 307 species within Lactobacillaceae family through comparative genomics analysis. A total of 130,521 primer sets were designed using species-specific genes. Among them, 81,710 primer sets had 100% interspecific specificity and 100% intraspecific coverage, and were reserved to quantify all 307 individual Lactobacillaceae species. These primer sets had uniform melting temperature (57–63 ℃) and product size (100–300 bp), that allowed simultaneously quantify different Lactobacillaceae species with the same qPCR condition. We then established a Lactobacillaceae species quantitation primer database (LSQP-DB, http://lsqp-db.com) containing all 81,710 species-specific primer sets. The database would facilitate a fast and easy absolute quantitation analysis of all individual Lactobacillaceae species. This work represented the first ever large-scale integration of species-specific primer sets for microorganism, it can be extended to other bacterial and fungal genera to advance development of microbial absolute quantification.
Microbial fermentation could produce many active substances in green and pollution-free ways. In recent years, studies on the hypoglycemic ability of microorganisms had become a trending topic in the prevention and treatment of diabetes. Garlic is a food containing a variety of biologically active substances and physiological functions, but it is not accepted by the general public because of its strong pungent smell. In our study, the microorganisms with potential hypoglycemic effect were screened from strains resistant to garlic juice. As a result, 41 strains of Bacillus, 157 strains of lactic acid bacteria, and 23 strains of yeast with tolerance of garlic juice were screened from the selected 568 strains. After primary screening of α-amylase inhibitor, it was found that 15 Bacillus and 9 lactic acid bacteria had high α-amylase inhibitory activity. Besides, after α-glucosidase inhibitor re-screening, Pediococcus acidilactici Z1 with high α-amylase inhibitory activity and α-glucosidase inhibitory activity was screened out. The results of fermented garlic showed that the inhibition rate of α-amylase and α-glucosidase of garlic from different origins were significantly increased from 59.09 to 63.99% (p < 0.01) and from 52.71 to 71.93% (p < 0.01) after Z1 fermentation, respectively. The improvement rates of α-amylase and α-glucosidase were 107.69 to 287.32%, and 197.74 to 239.86%, respectively. In a word, this study provides a theoretical and experimental basis for the development of new functional garlic products.
Papaya is a rich source of nutrients, antioxidants, and vitamins; however, it is highly perishable and hence needs to be preserved using suitable unit operations. Drying is an economical method of papaya treatment, but results in the loss of essential nutrients and also degrades its pigments, color, and texture. Therefore, this work aims to utilize ultrasound and osmotic dehydration as pretreatment strategies to preserve the rich composition of papaya during conventional hot air drying. The dried papaya slices were evaluated for moisture content, water activity, total phenolic content, β-carotene, color, antioxidant activity, microstructure, and texture. Results showed that sample pretreatment with 33 kHz ultrasound frequency and dehydrated with 35°Brix osmotic solution resulted in lower moisture content (< 12% w.b.) and water activity (< 0.41), high phenolic content (88.5 mg GAE/mL), high β-carotene content (184.54 mg/g) and antioxidant capacity (48.3%), and preserved color after drying. The texture (hardness) of the pretreated samples was also more desirable from an industrial standpoint, which was further validated using SEM micrographs that showed a more porous structure. The study concludes that pretreatment of papaya slices with ultrasound and osmotic dehydration is recommended before drying.
Lactate dehydrogenase and malate dehydrogenase are the two main alpha-hydroxy acid dehydrogenases in the human body. We investigated the catalytic properties of human lactate dehydrogenase LDHC, LDHL6A and malate dehydrogenase MDH1 on aromatic α-keto acids phenylpyruvic acid, p-hydroxyphenylpyruvic acid and 3,4-dihydroxyphenylpyruvic acid. The optimum temperatures for LDHC, LDHL6A, and MDH1 are 37 °C, 35 °C, and 45 °C, respectively; and the optimum pH is 6.5, 6.5, and 5.5, respectively. The K m of LDHC for phenylpyruvic acid and 3,4-dihydroxyphenylpyruvic acid were 0.90 mM and 0.92 mM, respectively. LDHL6A has a high affinity for phenylpyruvic acid and 3,4-dihydroxyphenylpyruvic acid with K m of 0.77 mM and 0.80 mM, respectively; MDH1 has an extremely high affinity (K m = 0.46 mM) and catalytic efficiency (k cat/K m = 23.87 s−1·mM−1) for p-hydroxyphenylpyruvic acid. It also has a high affinity for 3,4-dihydroxyphenylpyruvic acid with a K m of 0.90 mM, but with a low affinity for phenylpyruvic acid (K m = 3.76 mM). The catalytic properties of human LDHC, LDHL6A, and MDH1 for the abovementioned aromatic α-keto acids may be one of the sources of L-phenyllactic acid, L-p-hydroxyphenyllactic acid, and L-3,4-dihydroxyphenyllactic acid in humans.
Ethyl (R)-4-chloro-3-hydroxybutyrate ((R)-CHBE), as a chiral intermediate, is widely used in the synthesis of various chiral drugs. In this study, we screened two aldo–keto reductases (LP-AKRs) from the probiotic Lactobacillus plantarum DSM20174, both with a molecular weight of approximately 31 kDa. Both enzymes could reduce 4-chloroacetoacetic acid ethyl ester (COBE) to produce (R)-CHBE with an enantioselectivity value of 99%. When determining the kinetic parameter, the K m, K cat, and V max of LP-AKR5 and LP-AKR9 were 9.5 mM, 7.6 U/mg, 3.96 s−1 and 8.7 mM, 8.59 U/mg, 4.47 s−1, respectively. Both LP-AKR5 and LP-AKR9 had an optimal reaction pH of 6 and could maintain a high level of stability at pH 6, allowing them to perform well in an acidic environment. LP-AKR5 and LP-AKR9 had optimal reaction temperatures of 30 °C and 40 °C, respectively. Metal ions had minimal influence on LP-AKR5 and LP-AKR9 enzyme activities. This series of enzymatic properties showed that LP-AKR5 and LP-AKR9 mined from Lactobacillus plantarum DSM20174 could asymmetrically catalyze the synthesis of (R)-CHBE under weakly acidic circumstances, which could maintain product stability and provide a good foundation for industrial production.
8-Prenylnaringenin (8-PN) is a valuable medical phytoestrogen, which is a precursor to many prenylated flavonoids. However, the availability of 8-PN is limited by inefficient prenyltransferases (PTs) and inadequate substrate precursor levels in microbial chassis. First, six PTs from different sources and their truncated cognates were expressed in a (2S)-naringenin producing strain. Only SfN8DT-1 derived from Sophora flavescens and its truncated cognate, tSfN8DT-1, could synthesize 8-PN. Second, tSfN8DT-1 was engineered by multiple sequence alignment and a mutant tSfN8DT-1$^{Q12E}$ with greater catalytic activity was obtained. Third, key genes, tHMGR and IDI1, of the mevalonate (MVA) pathway were overexpressed using a copy number combinatorial strategy, which greatly improved 8-PN titer by 368.75%. Fourth, a predicted structure of tSfN8DT-1$^{Q12E}$ was used for molecular docking and virtual saturation mutagenesis. Two key residues, P229 and N305, were identified and saturation mutagenesis on these two sites resulted in an improved mutant N305M. The best-performing mutant, tSfN8DT-1$^{Q12E N305M}$, produced 49.35 $\pm {}$ 0.05 mg/L (5.57 $\pm {}$ 0.01 mg/g DCW) 8-PN in a shaking flask. Finally, 101.40 $\pm {}$ 2.55 mg/L of 8-PN was obtained in a 5-L bioreactor, which is the greatest titer reported to date for 8-PN. This study combined metabolic engineering and protein engineering methods to enhance precursor supplements and improve the catalytic ability of SfN8DT-1. The production of 8-PN in Saccharomyces cerevisiae was greatly increased through these methods, which may provide a feasible strategy for the biosynthesis of prenylated flavonoids.
Chinese hamster ovary (CHO) cells are widely used in biopharmaceuticals because of their high-density suspension culture, high safety, and high similarity between expressed exogenous proteins and natural proteins. However, the level of exogenous protein expression decreases with increasing culture time; this phenomenon occurs due to the recombination of foreign genes into chromosomes through random integration. The present study integrated the foreign genes into a specific chromosomal site for stable expression based on CRISPR–Cas9 technology. The results showed that the exogenous proteins enhanced green fluorescent protein (EGFP) and human serum albumin (HSA) were successfully integrated into the vicinity of base 1969647 on chromosome NW_003613638.1 of CHO-K1 cells. The obtained positive monoclonal cell lines expressed all the corresponding exogenous proteins after 60 consecutive passages, and no significant differences in expression levels were observed. This study might provide a feasible method to construct a CHO cell line with long-term stable expression of exogenous proteins.
This study conducted experiments used for the development of both the regression model with uncertainty analysis and the adaptive neuro-fuzzy inference system (ANFIS) model for the prediction of the yield of biodiesel (YB) produced from castor oil in the presence of calcium oxide derived from the eggshell. Box Behnken design (BBD) was used to develop the experimental condition for five different variables while YB was the response. Uncertainty analysis was determined from Monte Carlo simulation (MCS). The model was optimized and validated before the generated data was applied in the three ANFIS modelling techniques. Root mean square error (RMSE), coefficient of correlation (R 2) and average percentage error (APE) were used to determine the accuracy of the models developed. The result of this modelling shows that the optimum YB (94.29%) was achieved at a methanol to oil ratio of 11.48, catalyst loading of 3.38 wt%, reaction time of 1.84 h, the temperature of 60.2 °C, and agitation of 343.5 rpm. The prediction from BBD, ANFIS and MCS agreed that the methanol to oil ratio was the most important parameter for investigation. The considered ANFIS model technique (subtractive clustering) for the modelling of YB outperformed BBD model. The novelty of this study are the determination of the optimum condition for the transesterification of castor oil in the presence of thermally treated anthill, the establishment of the use of ANFIS in modelling YB, the prediction of the influence of variables on YB using both statistical and, AI techniques and validation of the predictions from the two methods using MCS.
The depletion of fossil fuel reserves with increased fuel demand and global emissions has increased the search for eco-friendly renewable fuels with a low environmental impact. Biodiesel can be considered as mono-alkyl esters of long-chain fatty acids obtained from the transesterification of vegetable oils and animal fats. Economically low-cost biodiesel production has received considerable interest for blending with fossil-based diesel for a more sustainable future. Therefore, the current study focuses on synthesizing an efficient, low-cost heterogeneous CaO catalyst from waste egg and seashell using a solid-state method and applying it to the transesterification of Jatropha oil. The Ca2Fe2O5 solid catalyst was prepared by doping calcined CaO with iron in a 2:1 ratio using ferric oxide (Fe2O3). Furthermore, the catalyst was extruded and analytically characterized using XRD, FT IR, BET, and its basic strength was quantified by Hammett indicators. Later on, transesterification of Jatropha oil was optimized by varying reaction parameters, such as the molar ratio of methanol to Jatropha oil, reaction time, and catalyst loading. The maximum conversion yield was 96.3% at a 20:1 methanol-to-oil ratio and 80 bar N2 pressure using 5% (w/w) catalyst loading. Furthermore, the catalytic recycling study demonstrated that the Ca2Fe2O5 catalyst could retain > 70–80% of transesterification efficiency and stability up to 4 cycles under high acid value and moisture conditions.
Biodiesel production from microalgae depends on algal biomass production through autotrophic cultivation, and it has a lot of potential as a feasible alternative source of energy. The lethal and mutagenic effects of ethyl methanesulphonate (EMS) are investigated in the present study on the organism Chlorella pyrenoidosa for the generation of paler mutants. The C. pyrenoidosa wild-type strain was treated with 0.1, 0.2, and 0.5 M EMS doses and the colonies that appeared after 20 days of growth on a solid TAP medium were analyzed, and a total of two mutant colonies with less pigmentation were identified. The CPM1 (10.02 ± 0.03 and 3.12 ± 0.04 µg/ml) and CPM2 (9.55 ± 0.0 and 32.84 ± 0.03 µg/ml) mutants had lower chlorophyll a and b and carotenoids content than wild type. Mutants produced significant biomass (1.34- and 1.31-fold enhanced biomass in CPM1 and CPM2) and lipid content than the wild type. These findings suggest that genetic modification of C. pyrenoidosa has the potential to produce strains with increased biomass productivity and lipid content for biofuel production.
In this study, a total of 1125 actinobacteria were isolated from the selected mangrove species: Avicennia marina, Rhizopora mucronata and Ceriops tagal from three study stations viz., Minnie Bay, Carbyn’s Cove and Burmanallah. Among these three stations, the highest number of actinobacteria was recorded in Carbyn’s Cove (64.97%), followed by (25.51%) at Burmanallah and the minimum of (9.51%) was recorded in Minnie Bay. Maximum number of actinobacteria was recorded from Ceriops tagal (40.44%) than the other selected mangrove species Avicennia marina (34.13%) and Rhizopora mucronata (25.42%). Among the 1,125 mangrove-associated actinobacteria, 103 morphologically different isolates from the Minnie Bay station was selected for the further characterization studies. In antibacterial assay, 30.11% of the isolates revealed inhibitory activity against all tested clinical pathogens and 65% isolates displayed inhibitory activity against minimum of 04 tested clinical pathogens. Growth survival studies of the actinobacterial isolates also accomplished to withstand in varied NaCl and pH levels. Of 103 isolates, all were found to synthesize gelatinase enzyme, 73 isolates demonstrated amylolytic activity, 38 isolates exhibited proteolytic and 63 isolates displayed urease activity. Interestingly, 56 isolates exhibited excellent DNase activity and 71 isolates revealed positive for l-asparaginase production. To our recognition, 11 isolates exhibited constructive results in the production of 06 extracellular enzymes of industrial importance. Of 103 isolates, 48 isolates were confirmed by molecular level identification. Based on the phylogenetic analysis, the isolates were categorized under the genera: Streptomyces, Nocardiopsis, Salinispora and Actinomadura.
Degradation of pendimethalin by microorganisms is an approach seeking more attention nowadays. This study aims to isolate a new strain of Planococcus, which is capable of degrading pendimethalin from the soil in rice field. Identification of this coccus bacterium was done by phylogenetic analysis of 16S rRNA gene sequence. Strain PD6 was found to grow potentially on pendimethalin supplemented minimal salt medium and degraded 50 mg L−1 pendimethalin in monosubstrate system. Fourier transform infrared (FTIR) spectroscopy was performed to check degradation of pendimethalin by Planococcus, which was demonstrated by changing chemical bonding and stretching patterns. This study was further extended to predict possible first enzyme of pendimethalin-degradation pathway. Molecular docking was performed to check efficient binding of dehydrogenase with pendimethalin. This strain of Planococcus degraded pendimethalin with relatively high efficiency in minimal salt media and is presented as another possible bacterium other than species of Bacillus and Pseudomonas which are already demonstrated as potential in vitro pendimethalin-degrading bacteria. Moreover, docking reveals that dehydrogenases having more chance to prove as first regulatory enzyme of pendimethalin-degradation pathway.
D-xylose is an abundant sugar found in plant biomass and can be used as a renewable feedstock for the microbial production of diverse biofuels and bioproducts. However, D-xylose metabolism is slow in many industrial microorganisms, at least as compared to glucose metabolism. Not surprisingly, a number of approaches have been developed for improving D-xylose metabolism in diverse microorganisms. In this work, we applied a previously developed evolution strategy based on media-in-oil emulsions for improving the growth yield of Escherichia coli NCM3722 on D-xylose. After 30 rounds of evolutions, we isolated multiple mutants with increased growth yield on D-xylose. In addition, we also observed similar increases in the growth rate. Three mutants were selected for whole-genome sequencing. Two mutants had an amber stop mutation in adenylate cyclase, which truncates nearly 60% of the enzyme. However, the ability of this mutant to grow on xylose indicated that truncated enzyme, lacking the C-terminal regulatory domain, is still active. The other mutant had a point mutation in the cyclic AMP receptor protein (CRP), near the high affinity binding site for cyclic AMP. Both mutations, when introduced into wild type E. coli, were able to increase the growth yields at levels similar to the isolated mutants. In addition to D-xylose, these mutant strains and their genetic mimics also exhibited higher growth rates and yields on glucose, lactose, and L-arabinose. These results suggest that the improved growth rates and yields are due to changes in the production and sensing of intracellular cyclic AMP concentrations and also suggest native concentrations are suboptimal with respect to the growth rate and yield under the growth conditions tested. Collectively, these results may prove useful for engineering strains of E. coli for high-density fermentations or protein production.
As a bright red carotenoid pigment found in tomatoes and other red fruits, lycopene was efficiently produced by Escherichia coli transformed with the genes involving in lycopene biosynthesis. Our previous work showed that E. coli ΔwaaC and ΔwaaF with higher permeability of outer membrane were better chassis for lycopene biosynthesis. However, further work needed to improve lycopene synthesis in the aforementioned strains. In the current study, the exogenous crtEBI genes from C. glutamicum 14067 were first integrated into the chromosome of ΔwaaC and ΔwaaF. Compared to ΔwaaC/pWSK29-crtEBI and ΔwaaF/pWSK29-crtEBI (4.19 and 4.20 mg/g lycopene respectively), ΔwaaC lacZ::crtEBI (CWC01) and ΔwaaF lacZ::crtEBI (CWF01) produced higher lycopene levels at 16.14 mg/g and 15.81 mg/g, respectively. Later, the individual and combinational deletion of aceE and gdhA was conducted in CWC01 and CWF01, respectively. The double knock-out strains CWC04 and CWF04 showed the improved yield at 19.65 mg/g and 22.24 mg/g, respectively. Finally, the four genes involving in MEP pathway (dxs, dxr , ispA and idi) were further overexpressed. The highest lycopene yield was achieved at 25.82 mg/g by CWF04/pACYC184-dxs E289G -dxr K37N,K217N -ispA-idi. The current study showed that E. coli ΔwaaC and ΔwaaF were optimal chassis for ambitious metabolic modification to produce lycopene.
P-Coumaric acid was previously reported to contain antioxidant, antidiabetic, anti-inflammatory, antiplatelet, antiulcer and anticancer activities. Along with these, the present work has been conducted to study the antibacterial activity of p-coumaric acid. It could be used to control broad-spectrum microbiome-based inflammation or in cancer control. HPLC analysis of methanolic extract from the mushroom Termitomyces heimii has exhibited a rich fraction of p-coumaric acid (p-CA), which in the pure form showed significant bactericidal potentials. To predict the molecular interactions associated with the bactericidal mechanism of p-CA, the transmembrane protein sequences of Staphylococcus aureus and Escherichia coli were retrieved from the IMG–JGI database, screened and then aligned using Clustal X2 and PHYLIP 3.69 softwares. The common sequences were subjected to tertiary structure prediction using Phyre2 server, followed by quality assessment through the Ramachandran plot. Next, the 3D molecular structure of p-CA was downloaded from PubChem and docked with the selected tertiary structures using Patchdock and showed higher affinity towards 12 common transmembrane protein structures, amongst which CDP-diacylglycerol–glycerol-3-phosphate 3-phosphatidyl transferase (PgsA) exhibited best docking with p-CA on the basis of ACE value (– 249 kcal/mol). This fact revealed that p-CA can block the normal functioning of membrane-bound enzyme PgsA, consequently leading to the interruption in the synthesis and recycling of an essential membrane component phosphatidylglycerol (PG), resulting in membrane disruption followed by cell lysis. Here, for the first time we reported the molecular insights and fundamental biochemical events underlying the bactericidal action by p-CA, to explore new perspective to combat multidrug-resistant bacteria.
Succinic acid is one of the most useful intermediate chemicals that can be produced in a biorefinery approach. In this study, Actinobacillus succinogenes was immobilized to produce succinic acid using non-detoxified corn fiber hydrolysate (CFH) and a control mimicking the sugars in CFH. Tests were carried out in a hollow fiber membrane packed-bed biofilm reactor (HFM–PBR) operated in a continuous mode. Under steady-state conditions, the bioconversion process was characterized in terms of sugar consumption, succinic acid and other organic acid production. Steady states were obtained at dilution rates of 0.025, 0.05, 0.075, 0.1, 0.2, and 0.3 h−1. The optimal results were achieved at the dilution rate of 0.05 h−1 and recirculation rate of 50 ml/min with a maximum succinic acid concentration, yield and productivity of 31.1 g/L, 0.61 g/g and 1.56 g/L h, respectively, when control was used. Succinic acid concentration, yield and productivity of 23.4 g/L, 0.51 g/g and 1.17 g/L h, respectively, were obtained when CFH was used. Productivity in the HFM–PBR was between 1.3 and 1.9 times higher than productivities for succinic acid production from CFH stated in the literature. The results demonstrated that immobilized A. succinogenes has the potential for effective conversion of an inexpensive biomass feedstock to succinic acid. Furthermore, the process has the potential to serve as a means for value-added chemical biomanufacturing in an integrated corn biorefinery.