A new set of small,extrachromosomal expression vectors for Dictyostelium discoideum

A new set of extrachromosomal Dictyostelium expression vectors is presented that can be modified according to the experimental needs with minimal cloning efforts. To achieve this, the vector consists of four functional regions that are separated by unique restriction sites, (1) an Escherichia coli replication region, and regions for (2) replication, (3) selection and (4) protein expression in Dictyostelium. Each region was trimmed down to its smallest possible size. A basic expression vector can be constructed from these modules with a size of only 6.8 kb. By exchanging modules, a large number of vectors with different properties can be constructed. The resulting set of vectors allows most basic expression needs, such as immuno blotting, protein purification, visualization of protein localization and identification of protein–protein interactions. In addition, two genes can be simultaneously expressed on one vector, which yields far more synchronous levels of expression than when expressing two genes on separate plasmids.     

In vitro anti-rotavirus activity of polyphenol compounds isolated from the roots of Glycyrrhiza uralensis

We evaluated the ability of six polyphenols isolated from the roots of Glycyrrhiza uralensis to inactivate rotaviruses, specially G5P[7] and G8P[7]. Upon finding that all polyphenols possessed anti-rotavirus activity, we evaluated whether these properties were attributable to direct inhibition of the binding of rotavirus to cells and/or to inhibition of viral replication. Using the virucidal assay, we found that all six compounds directly inhibited rotavirus binding, with activity being dependent on the type of virus. The 50% effective inhibitory concentrations (EC₅₀) of the six compounds were 18.7–69.5 μM against G5P[7] and 14.7–88.1 μM against G8P[7], respectively. Five of the six compounds inhibited hemagglutination activity. Moreover, the CPE inhibition assay showed that five compounds inhibited viral replication with EC₅₀ values of 12.1–24.0 μM against G5P[7] and 12.0–42.0 μM against G8P[7], respectively. RT-PCR showed that the compounds suppressed viral RNA synthesis in TF-104 cells. Interestingly, the anti-rotavirus activities of four compounds were attributable to inhibition of both viral absorption and viral replication. These results suggest that compounds isolated from the roots of G. uralensis may be potent anti-rotavirus agents in vivo, acting by inhibiting both viral absorption and viral replication.     

Biocoagulation of dairy wastewater by Lactobacillus casei TISTR 1500 for protein recovery using micro-aerobic sequencing batch reactor (micro-aerobic SBR)

This study investigated biocoagulation of dairy process wastewater with a new system of the micro-aerobic sequencing batch reactor (micro-aerobic SBR) at a batch bench scale. Lactobacillus casei TISTR 1500 was inoculated to produce acid coagulants under non-sterile acid conditions. Colloidal proteins were removed by employing a solid–liquid separation step as a pre-treatment. The micro-aerobic SBR process had the efficiencies of organic reduction with 73.6 ± 5.9%, 90.1 ± 1.3%, and 85.7 ± 0.6% of chemical oxygen demand (COD), proteins, and sugars without adding external coagulant, and flocculant, respectively. Sustained acid fermentation was achieved for at least 150 cycles by applying an indigenous fill-react-settle-draw-idle sequence in the micro-aerobic SBR process and the use of different solid retention times at 3, 6, 9, 12 and 15 d, consecutively. The micro-aerobic SBR system was able to support lactic acid bacteria (LAB) growth with long SRT (12 and 15 d), due to at least 3 factors: the large inoculum size employed, relatively high concentration of lactic acid produced, and the change in pH during the restoring stage. Current process offered a possible alternative to the more costly chemical and other biological pre-treatments.     

Lead biosorption efficiency of Levilactobacillus brevis MZ384011 and Levilactobacillus brevis MW362779: A response surface based approach

Lead (Pb) is a substantial contaminant in the environment and a potent toxin for living organisms. Current study describes probiotic characteristics of Pb-biosorbing lactic acid bacteria (LAB), and response surface methodology (RSM) based optimization of physical conditions for maximum Pb biosorption. A total of 18 LAB, isolated from carnivore feces (n = 8) and human breast milk (n = 9), along with one reference strain Lactobacillus acidophilus ATCC4356 were included in the study. Pb biosorption was strain specific. Eight strains, demonstrating ≥ 70 % lead biosorption, were selected for further testing. The lactobacillus-Pb complex was found to be stable and strains had a negative surface charge. The strains displayed good probiotic properties with the survival rate of 71–90 % in simulated gastric environment, 36–69 % in intestinal condition (1.8 % bile salts) and 55–72 % hydrophobicity. On the basis of excellent probiotic ability, Levilactobacillus brevis MZ384011 and Levilactobacillus brevis MW362779 were selected for optimization of physical conditions of Pb biosorption through RSM. Maximum biosorption was observed at pH 6 in 60 min at a cell density of 1 g/L. L. brevis MZ384011 and L. brevis MW362779 are recommended for experimentation on Pb toxicity amelioration and safety evaluation in in-vivo setting.     

In vitro antiviral activity of phlorotannins isolated from Ecklonia cava against porcine epidemic diarrhea coronavirus infection and hemagglutination

Despite the prepdominat agent causing severe entero-pathogenic diarrhea in swine, there are no effective therapeutical treatment of porcine epidemic diarrhea virus (PEDV). In this study, we evaluated the antiviral activity of five phlorotannins isolated from Ecklonia cava (E. cava) against PEDV. In vitro antiviral activity was tested using two different assay strategies: (1) blockage of the binding of virus to cells (simultaneous-treatment assay) and (2) inhibition of viral replication (post-treatment assay). In simultaneous-treatment assay, compounds 2–5 except compound 1 exhibited antiviral activities of a 50% inhibitory concentration (IC₅₀) with the ranging from 10.8 ± 1.4 to 22.5 ± 2.2 μM against PEDV. Compounds 1–5 were completely blocked binding of viral spike protein to sialic acids at less than 36.6 μM concentrations by hemagglutination inhibition. Moreover, compounds 4 and 5 of five phlorotannins inhibited viral replication with IC₅₀ values of 12.2 ± 2.8 and 14.6 ± 1.3 μM in the post-treatment assay, respectively. During virus replication steps, compounds 4 and 5 exhibited stronger inhibition of viral RNA and viral protein synthesis in late stages (18 and 24 h) than in early stages (6 and 12 h). Interestingly, compounds 4 and 5 inhibited both viral entry by hemagglutination inhibition and viral replication by inhibition of viral RNA and viral protein synthesis, but not viral protease. These results suggest that compounds isolated from E. cava have strong antiviral activity against PEDV, inhibiting viral entry and/or viral replication, and may be developed into natural therapeutic drugs against coronavirus infection.     

The polymorphism and haplotype of TLR3 gene in grass carp (Ctenopharyngodon idella) and their associations with susceptibility/resistance to grass carp reovirus

Toll-like receptors (TLRs) have emerged as crucial sensors of invading microbes through recognition of pathogen-associated molecular patterns (PAMPs) in viruses, bacteria, fungi and protozoa. The polymorphisms in TLRs are closely associated with the resistance to pathogen infections. TLR3 involved in the recognition of double stranded RNA in humans, mice, pigs and fishes. In present study, the nucleotide sequence polymorphisms of TLR3 gene in grass carp (Ctenopharyngodon idella) (CiTLR3) were investigated to explore their association with susceptibility/resistance to grass carp reovirus (GCRV). Twelve single nucleotide polymorphisms (SNPs) and an ins/del mutation were detected in the complete sequence of CiTLR3. Ten of them were sited in the non-coding region. The two SNPs in exon were synonymous mutation. The ins/del mutation was coincidental at the start codon. To investigate the association between the polymorphism and the susceptibility/resistance to GCRV, we selected eight SNPs in the non-coding region and analyzed the genotype and allele distribution in susceptible and resistant groups with PCR-RFLP. The statistical results indicated that only ?764 G/T was significantly associated with the resistance of grass carp to GCRV both in genotype (P = 0.040) and allele (P = 0.025). Linkage disequilibrium analysis revealed ?543 A/G, ?488 G/T, 4116 G/T and 4731 C/T were linkage disequilibrium, and haplotype analysis revealed that haplotype GTTT frequency in susceptible group was significantly higher than that in the resistant group (OR = 2.01, 95% CI 0.996–4.043, P = 0.049). To further confirm the correlation, an additional infection experiment was carried out. The mortality in the ?764 GG genotype individuals was significantly lower than GT genotype (OR = 0.208, 95% CI 0.067–0.643, P = 0.011) and TT genotype (OR = 0.183, 95% CI 0.052–0.648, P = 0.015). All the results indicated that haplotype GTTT and genotype ?764 TT and ?764 GT individuals were susceptible to GCRV while ?764 GG was resistant, which could be the optional markers for selective breeding for the GCRV-resistant grass carp in future.     

Shigella flexneri type-specific antigen V: cloning,sequencing and characterization of the glucosyl transferase gene of temperate bacteriophage SfV

With lysogeny by bacteriophage SfV, Shigella flexneri serotype Y is converted to serotype 5a. The glucosyl transferase gene (gtr) from bacteriophage SfV of S. flexneri, involved in serotype-specific conversion, was cloned and characterized. The DNA sequence of a 3.7 kb EcoRI–BamHI fragment of bacteriophage SfV which includes the gtr gene was determined. This gene, encoding a polypeptide of 417 aa with 47.67 kDa molecular mass, caused partial serotype conversion of S. flexneri from serotype Y to type V antigen as demonstrated by Western blotting and the sensitivity of the hybrid strain to phage Sf6. The deduced protein of the partially sequenced open reading frame upstream of the gtr showed similarity to various glycosyl transferases of other bacteria. Orf3, separated from the gtr by a non-coding region and transcribed convergently, codes for a 167 aa (18.8 kDa) protein found to have homology with tail fibre genes of phage lambda and P2.     

Isolation and characterization of tetrahydrofuran-degrading Rhodococcus aetherivorans strain M8

We isolated tetrahydrofuran (THF)-degrading bacteria from waste sludge obtained from a chemical factory in Japan. The isolate designated as strain M8 was identified as Rhodococcus aetherivorans by sequence analysis of its 16S rRNA gene. It grew in a medium containing THF as the sole source of carbon and energy, and its optimal growth pH range and temperature were 6–9 and 37 °C, respectively. Strain M8 grew even in the presence of 35 mM THF. For its growth, this bacterium used 1,4-butanediol and γ-butyrolactone, which are supposed to be metabolites of THF. To elucidate the pathway involved in THF metabolism in strain M8, the resting cell reaction was performed, and the metabolites of THF were analyzed. In the resting cell reaction, 5 mM THF was completely degraded within 5 h. Cells were harvested at 2, 3, and 4 h after the initiation of the reaction; the intermediates accumulated in the cells were extracted using methanol and were derivatized using phenyl boronate. Gas chromatography–mass spectrometry (GC–MS) analysis of the derivatized products showed 4-hydroxybutyrate accumulating in the resting cells. This result suggests that R. aetherivorans strain M8 degrades THF via the oxidation pathway.     

Construction of small plasmid vectors for use in genetic improvement of the extremely acidophilic Acidithiobacillus caldus

The genetic improvement of biomining bacteria including Acidithiobacillus caldus could facilitate the bioleaching process of sulfur-containing minerals. However, the available vectors for use in A. caldus are very scanty and limited to relatively large broad-host-range IncQ plasmids. In this study, a set of small, mobilizable plasmid vectors (pBBR1MCS-6, pMSD1 and pMSD2) were constructed based on plasmid pBBR1MCS-2, which does not belong to the IncQ, IncW, or IncP groups. The function of the tac promoter on 5.8-kb pMSD2 was determined by inserting a kanamycin-resistant reporter gene. The resulting recombinant pMSD2-Km was successfully transferred by conjugation into A. caldus MTH-04 with transfer frequency of 1.38 ± 0.64 × 10^?5. The stability and plasmid copy number of pMSD2-Km in A. caldus MTH-04 were 75 ± 2.7% and 5–6 copies per cell, respectively. By inserting an arsABC operon into pMSD2, an arsenic-resistant recombinant pMSD2-As was constructed and transferred into A. caldus MTH-04 by conjugation. The arsenic tolerance of A. caldus MTH-04 containing pMSD2-As was obviously increased up to 45 mM of NaAsO₂. These vectors could be applied in genetic improvement of A. caldus as well as other bioleaching bacteria.     

Earthworm-mediated synthesis of silver nanoparticles: A potent tool against hepatocellular carcinoma,Plasmodium falciparum parasites and malaria mosquitoes

The development of parasites and pathogens resistant to synthetic drugs highlighted the needing of novel, eco-friendly and effective control approaches. Recently, metal nanoparticles have been proposed as highly effective tools towards cancer cells and Plasmodium parasites. In this study, we synthesized silver nanoparticles (EW–AgNP) using Eudrilus eugeniae earthworms as reducing and stabilizing agents. EW–AgNP showed plasmon resonance reduction in UV–vis spectrophotometry, the functional groups involved in the reduction were studied by FTIR spectroscopy, while particle size and shape was analyzed by FESEM. The effect of EW–AgNP on in vitro HepG2 cell proliferation was measured using MTT assays. Apoptosis assessed by flow cytometry showed diminished endurance of HepG2 cells and cytotoxicity in a dose-dependent manner. EW–AgNP were toxic to Anopheles stephensi larvae and pupae, LC₅₀ were 4.8 ppm (I), 5.8 ppm (II), 6.9 ppm (III), 8.5 ppm (IV), and 15.5 ppm (pupae). The antiplasmodial activity of EW–AgNP was evaluated against CQ-resistant (CQ-r) and CQ-sensitive (CQ-s) strains of Plasmodium falciparum. EW–AgNP IC₅₀ were 49.3 μg/ml (CQ-s) and 55.5 μg/ml (CQ-r), while chloroquine IC₅₀ were 81.5 μg/ml (CQ-s) and 86.5 μg/ml (CQ-r). EW–AgNP showed a valuable antibiotic potential against important pathogenic bacteria and fungi. Concerning non-target effects of EW–AgNP against mosquito natural enemies, the predation efficiency of the mosquitofish Gambusia affinis towards the II and II instar larvae of A. stephensi was 68.50% (II) and 47.00% (III), respectively. In EW–AgNP-contaminated environments, predation was boosted to 89.25% (II) and 70.75% (III), respectively. Overall, this research highlighted the EW–AgNP potential against hepatocellular carcinoma, Plasmodium parasites and mosquito vectors, with little detrimental effects on mosquito natural enemies.     

Warneckea parvifolia (Melastomataceae–Olisbeoideae), a new “sand-forest” endemic from northeastern KwaZulu-Natal (South Africa) and southernmost Mozambique,and a phylogenetic analysis of eastern and southern African representatives of W. section Warneckea

Warneckea populations from “sand-forest” or “sand-thicket” habitats in Tembe Elephant Park, South Africa, and Licuati Forest Reserve in adjacent southern Mozambique were previously thought to be a small-leaved form of W. sousae, which typically includes larger-leaved plants ranging from central Mozambique northward to Tanzania. We examine this hypothesis using molecular and morphological evidence. Maximum-likelihood phylogenetic analysis of combined nrDNA ETS and ITS sequence data failed to resolve W. sousae and the Maputaland populations as an exclusively monophyletic group. Instead, the Kenyan endemic W. mouririifolia was strongly supported as the sister species of W. sousae, and the Maputaland plants were resolved in a separate, strongly supported clade together with populations of an as-yet undetermined Warneckea species from northern Mozambique. A hypothesis of exclusive monophyly for the plants from Tembe and Licuati had moderate support in separate ETS and ITS1 analyses (bootstrap proportions of 88% and 81%, respectively). Statistically significant differences in leaf dimensions and internode length were found between the Maputaland plants and typical W. sousae. We conclude that the populations from Tembe and Licuati represent a distinct species, which we describe as W. parvifolia. The species differs from W. sousae in having shorter internodes (mostly 5–25 mm not 10–60 mm long), smaller leaves (mostly 14–32 × 8–19 mm not 40–76 × 22–52 mm), shorter petioles (mostly 1–1.5 mm not 1.5–6 mm long), smaller flowers (hypanthium 1 × 1.5–1.75 mm not 1.5–2 × 2 mm; calyx lobes 0.5 mm not 0.75 mm long; staminal filaments 3–4 mm not 5 mm long; style 4–5 mm not 9 mm long), and globose fruit (not obovoid). An IUCN conservation status of Endangered (EN) B1a, b(ii, iii) is indicated for W. parvifolia, due to its limited distribution and projected declines in its habitat quality and area of occupancy.     

Intestinal Lactobacillus sp. is associated with some cellular and metabolic characteristics of blood in elderly people

The higher counts or particular groups (Firmicutes/Bacteroidetes) of intestinal microbiota are related to host metabolic reactions, supporting a balance of human ecosystem. We further explored whether intestinal lactobacilli were associated with some principal cellular and metabolic markers of blood in 38 healthy >65-year-old persons. The questionnaire, routine clinical and laboratory data of blood indices as much as the oxidized low-density lipoprotein (ox-LDL) and baseline diene conjugates in low-density lipoprotein (BDC-LDL) of blood sera were explored. The PCR-based intestinal Lactobacillus sp. composition and counts of cultivable lactobacilli (LAB) were tested. The facultative heterofermentative lactobacilli (Lactobacillus casei and Lactobacillus paracasei) were the most frequent (89 and 97%, respectively) species found, while Lactobacillus acidophilus, Lactobacillus plantarum and Lactobacillus reuteri were present in almost half of the elderly persons. The number of species simultaneously colonizing the individuals ranged from 1 to 7 (median 4). In elderly consuming probiotics the LAB counts were significantly higher than in these not consuming (median 7.8, range 4.2–10.8 vs. median 6.3, range 3.3–9.7 log cfu/g; p = 0.005), adjusted (OR = 1.71, CI95 1.04–2.82; p = 0.035) for age and body mass index (BMI). The colonization by L. acidophilus was negatively related (r = ?0.367, p = 0.0275) to L. reuteri, staying significant after adjusting for age, sex and BMI (OR = 0.16, CI95 0.04–0.73; p = 0.018). However, the blood glucose concentration showed a tendency for a negative correlation for colonization with Lactobacillus fermentum (r = ?0.309, p = 0.062) adjusted for BMI (Adj. R² = 0.181; p = 0.013) but not for age and sex. The higher white blood cells (WBC) count was positively related (r = 0.434, p = 0.007) to presence of Lactobacillus reuteri adjusted for age, sex and BMI (Adj. R² = 0.193, p = 0.027). The lower values of ox-LDL were predicted by higher counts of cultivable lactobacilli adjusted by sex, age and BMI (r = ?0.389, p = 0.016; Adj. R² = 0.184 p = 0.029). In conclusion, the pilot study of elderly persons shows that the intestinal lactobacilli are tightly associated with WBC count, blood glucose and content of ox-LDL which all serve as risk markers in pathogenesis of inflammation, metabolic syndrome and cardiovascular disease (CVD).     

Pseudotyped recombinant adeno-associated viral vectors mediate efficient gene transfer into primary human CD34+ peripheral blood progenitor cells

Background and aimsBecause of their pluripotency, human CD34⁺ peripheral blood progenitor cells (PBPC) are targets of interest for the treatment of many acquired and inherited disorders using gene therapeutic approaches. Unfortunately, most current vector systems lack either sufficient transduction efficiency or an appropriate safety profile. Standard single-stranded recombinant adeno-associated virus 2 (AAV2)-based vectors offer an advantageous safety profile, yet lack the required efficiency in human PBPC.MethodsA panel of pseudotyped AAV vectors (designated AAV2/x, containing the vector genome of serotype 2 and capsid of serotype x, AAV2/1–AAV2/6) was screened on primary human granulocyte–colony-stimulating factor (G-CSF)-mobilized CD34⁺ PBPC to determine their gene transfer efficacy. Additionally, double-stranded self-complementary AAV (dsAAV) were used to determine possible second-strand synthesis limitations.ResultsAAV2/6 vectors proved to be the most efficient [12.8% (1.8–25.4%) transgene-expressing PBPC after a single transduction], being significantly more efficient (all P < 0.005) than the other vectors [AAV2/2, 2.0% (0.2–7.3%); AAV2/1, 1.3% (0.1–2.9%); others, <; 1% transgene-expressing PBPC]. In addition, the relevance of the single-to-double-strand conversion block in transduction of human PBPC could be shown using pseudotyped dsAAV vectors: for dsAAV2/2 [9.3% (8.3–20.3%); P < 0.001] and dsAAV2/6 [37.7% (23.6–61.0%); P < 0.001) significantly more PBPC expressed the transgene compared with their single-stranded counterparts; for dsAAV2/1, no significant increase could be observed.ConclusionsWe have shown that clinically relevant transduction efficiency levels using AAV-based vectors in human CD34⁺ PBPC are feasible, thereby offering an efficient alternative vector system for gene transfer into this important target cell population.     

Purification and characterization of recombinant Bacillus subtilis 168 catalase using a basic polypeptide from ribosomal protein L2

An efficient purification system for purifying recombinant Bacillus subtilis 168 catalase (KatA) expressed in Escherichia coli was developed. The basic region containing 252–273 amino acids derived from E. coli ribosomal protein L2 was used as an affinity tag while the small ubiquitin-like modifier (SUMO) was introduced as one specific protease cleavage site between the target protein and the purification tags. L2 (252–273)–SUMO fusion protein purification method can be effectively applied to purify the recombinant catalase using cation exchange resin. This purification procedure was used to purify the KatA and achieved a purification fold of 30.5, a specific activity of 48,227.2 U/mg and an activity recovery of 74.5%. The enzyme showed a Soret peak at 407 nm. The enzyme kept its activity between pH 5 and 10 and between 30 °C and 60 °C, with the highest activity at pH 8.0 and 37 °C. The enzyme displayed an apparent Km of 39.08 mM for hydrogen peroxide. These results agree well with the previous reports about B. subtilis catalase. L2 (252–273)–SUMO fusion protein purification technique provides a novel and effective fusion expression system for the production of recombinant proteins.     

Biosynthesis of tetraoxygenated phenylphenalenones in Wachendorfia thyrsiflora

The biosynthetic origin of 1,2,5,6-tetraoxygenated phenylphenalenones and the sequence according to which their oxygen functionalities are introduced during the biosynthesis in Wachendorfia thyrsiflora were studied using two approaches. (1) Oxygenated phenylpropanoids were probed as substrates of recombinant W. thyrsiflora polyketide synthase 1 (WtPKS1), which is involved in the diarylheptanoid and phenylphenalenone biosynthetic pathways, (2) Root cultures of W. thyrsiflora were incubated with ¹³C-labelled precursors in an ¹⁸O₂ atmosphere to observe incorporation of the two isotopes at defined biosynthetic steps. NMR- and HRESIMS-based analyses were used to unravel the isotopologue composition of the biosynthetic products, lachnanthoside aglycone and its allophanyl glucoside. Current results suggest that the oxygen atoms decorating the phenalenone tricycle are introduced at different biosynthetic stages in the sequence O-1 → O-2 → O-5. In addition, the incubation of W. thyrsiflora root cultures with ¹³C-labelled lachnanthocarpone established a direct biosynthetic precursor–product relationship with 1,2,5,6-tetraoxygenated phenylphenalenones.     

C-Terminal proline-rich sequence broadens the optimal temperature and pH ranges of recombinant xylanase from Geobacillus thermodenitrificans C5

Efficient utilization of hemicellulose entails high catalytic capacity containing xylanases. In this study, proline rich sequence was fused together with a C-terminal of xylanase gene from Geobacillus thermodenitrificans C5 and designated as GthC5ProXyl. Both GthC5Xyl and GthC5ProXyl were expressed in Escherichia coli BL21 host in order to determine effect of this modification. The C-terminal oligopeptide had noteworthy effects and instantaneously extended the optimal temperature and pH ranges and progressed the specific activity of GthC5Xyl. Compared with GthC5Xyl, GthC5ProXyl revealed improved specific activity, a higher temperature (70 °C versus 60 °C) and pH (8 versus 6) optimum, with broad ranges of temperature and pH (60–80 °C and 6.0–9.0 versus 40–60 °C and 5.0–8.0, respectively). The modified enzyme retained more than 80% activity after incubating in xylan for 3 h at 80 °C as compared to wild −type with only 45% residual activity. Our study demonstrated that proper introduction of proline residues on C-terminal surface of xylanase family might be very effective in improvement of enzyme thermostability. Moreover, this study reveals an engineering strategy to improve the catalytic performance of enzymes.     

Fermentation-based biotransformation of bioactive phenolics and volatile compounds from cashew apple juice by select lactic acid bacteria

Select lactic acid bacteria (LAB); Lactobacillus plantarum, L. casei and L. acidophilus were targeted for enhancing bioactives and flavor volatiles of cashew apple juice (CAJ) that is an underutilized byproduct from cashew nut processing in Tropical countries. Results indicated the vitamin C and phenolic metabolites such as condensed tannin can be increased at certain stages such as at 12 h over the 48 h fermentation period. Whereas antioxidant activity based on DPPH and ABTS radical scavenging activity generally decreased from initial unfermented stage range of (75%–95%) to consistently in the 50% range by 48 h of fermentation and this follows the decrease in viable counts. The fermentation process increased the condensed tannin contents in CAJ whereas hydrolysable tannins decreased. In this study the changes in flavor volatile types were also analyzed over the course of CAJ fermentation. The results indicated that LAB changed the flavor profiles of fermented CAJ and overall the fruity odor decreased, but the whiskey and acid odor increased. These results provide the foundation to further target the functional benefits of LAB-induced fermented CAJ for further human, animal, and plant health applications.     

High catalytic performances of Pseudomonas fluorescens lipase adsorbed on a new type of cyclodextrin-based nanosponges

Lipases are water-soluble enzymes that catalyze the hydrolysis of triacylglycerols (in aqueous media) or trans-esterification reactions (in microaqueous media) and are involved in a number of industrial applications. As a limit to lipase application is represented by the need for interfacial activation, the search for suitable solid supports able to fulfill this requirement is always ongoing. In the present work, we report the preliminary characterization of a system obtained by adsorbing Pseudomonas fluorescens lipase on a newly synthesized cyclodextrin-based carbonate nanosponge (CD–NS–1:4). The activity and structural stability of lipase adsorbed on this new support were evaluated by checking the effect of temperature, pH changes and organic solvents (methanol) on the enzyme structure and function, which were compared with those of the free enzyme in solution. Our data show that the non-covalent interaction of Ps. fluorescens lipase with CD–NS–1:4 results in enzyme structural and functional stabilization, as it was still active after 66 days of incubation at T ～ 18 °C. Stabilization with respect to T, pH and the presence of organic solvent was observed as well as, unlike the solubilized enzyme, the adsorbed lipase was active at T > 40 °C, at pH 5 and after 24-h incubation with 70% (v/v) methanol (13% residual activity).