Potential of engineered nanostructured biopolymer based coatings for perishable fruits with Coronavirus safety perspectives.

Fresh fruits are prioritized needs in order to fulfill the required health benefits for human beings. However, some essential fruits are highly perishable with very short shelf-life during storage because of microbial growth and infections. Thus improvement of fruits shelf-life is a serious concern for their proper utlization without generation of huge amount of fruit-waste. Among various methods employed in extension of fruits shelf-life, design and fabrication of edible nanocoatings with antimicrobial activities have attracted considerable interest because of their enormous potential, novel functions, eco-friendly nature and good durability. In recent years, scientific communities have payed increased attention in the development of advanced antimicrobial edible coatings to prolong the postharvest shelf-life of fruits using hydrocolloids. In this review, we attempted to highlight the technical breakthrough and recent advancements in development of edible fruit coating by the application of various types of agro-industrial residues and different active nanomaterials incorporated into the coatings and their effects on shelf-life of perishable fruits. Improvements in highly desired functions such as antioxidant/antimicrobial activities and mechanical properties of edible coating to significantly control the gases (O2/CO2) permeation by the incorporation of nanoscale natural materials as well as metal nanoparticles are reviewed and discussed. In addition, by compiling recent knowledge, advantages of coatings on fruits for nutritional security during COVID-19 pandemic are also summarized along with the scientific challenges and insights for future developments in fabrication of engineered nanocoatings.

Characterization and toxicity of a phosphate-binding exobiopolymer produced by Acinetobacter haemolyticus MG606.

A novel, phosphate-binding exobiopolymer (EBP) produced by Acinetobacter haemolyticus MG606 was characterized and its biocompatibility evaluated in RAW 264.7 cells and in mice. EBP was identified as a 50 kDa heteropolysaccharide composed of pentose and hexose sugars. EBP exhibited cytotoxicity, stimulation of free radical production and loss of mitochondrial and lysosomal integrity in RAW 264.7 cells at 500 µg/mL concentration while lower concentrations exhibited no significant (p > 0.05) effect on these parameters. EBP exhibited dose-dependent mortality, body weight reduction, hypothermia and clinical signs of toxicity in mice following intraperitoneal administration. The LD50 of EBP was determined to be 92.31 mg/kg. Overall, the results of our study suggest that composition of EBP produced by A. haemolyticus MG606 is distinct from EBP produced by other Acinetobacter spp. The high biocompatibility supports application of EBP as a safe biosorbent for phosphate remediation.

Biofilm forming ability of Sphingomonas paucimobilis isolated from community drinking water systems on plumbing materials used in water distribution.

Sphingomonas paucimobilis, an oligotroph, is well recognized for its potential for biofilm formation. The present study explored the biofilm forming ability of a strain isolated from municipal drinking water on plumbing materials. The intensity of biofilm formation of this strain on different plumbing materials was examined by using 1 × 1 cm2 pieces of six different pipe materials, i.e. polyvinyl chloride (PVC), polypropylene (PP), polyethylene (PE), aluminium (Al), copper (Cu) and rubber (R) and observing by staining with the chemical chromophore, Calcofluor. To understand whether biofilm formation occurs under flow through conditions, a laboratory-scale simulated distribution system, comprised of the above materials was fabricated. Biofilm samples were collected from the designed system at different biofilm ages (10, 40 and 90 hours old) and enumerated. The results indicated that the biofilm formation occurred on all plumbing materials with Cu and R as exceptions. The intensity of biofilm formation was found to be maximum on PVC followed by PP and PE. We also demonstrated the chemical chromophore (Calcofluor) successfully for rapid and easy visual detection of biofilms, validated by scanning electron microscope (SEM) analysis of the plumbing materials. Chlorination has little effect in preventing biofilm development.

Antimicrobial efficacy and in vivo toxicity studies of a quaternized biopolymeric flocculant.

This study evaluated the antibacterial spectrum and safety of a chemically modified biopolymeric flocculant (TMB) against waterborne pathogens. The biopolymer previously characterized as polysaccharide with flocculating activity is produced extracellularly by the bacterium Klebsiella terrigena. The amino sugars on the polymer were chemically modified by quaternization, which resulted in N,N,N trimethyl biopolymer (TMB). Quaternization was effective in imparting biocidal activity to TMB against five selected waterborne pathogens, namely, Aeromonas hydrophila, Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes and Escherichia coli O157:H7. 99.999% inactivation was achieved with S. typhimurium at a dose of 60 µg ml(-1) of TMB within 60 min at the ambient temperature, followed by other pathogens. Haemotological, histopathological and general examinations indicated no adverse effects in Swiss albino mice fed with the quaternized biopolymer (120 mg kg(-1) body weight(-1) day(-1)) over a period of 30 days. These results suggested that TMB was tolerated well without any signs of toxicity and may have potential application as a safe, antimicrobial bioflocculant for both removing and inactivating waterborne pathogens.

Modeling in vitro cholesterol reduction in relation to growth of probiotic Lactobacillus casei.

Lactobacillus casei LA-1 isolated from a nondairy fermented source was evaluated for its in vitro ability to reduce cholesterol. The bacterium tested positive for bile salt deconjugation in relation to cholesterol removal. Tested growth-associated physiological variables such as pH, temperature and inoculum size were all found to have significant effects on in vitro cholesterol reduction and biomass production (both P < 0.005). Furthermore, a central composite design was used to evaluate the effects of significant variables and their interactions. A linear regression model was developed for in vitro cholesterol reduction as a function of growth-associated variables. Maximum cholesterol reduction achieved was 45% whereas maximum biomass yield of 2.34 optical density was observed at the central point. Our study possibly indicates that the growth of L. casei LA-1 depends on its cholesterol removing ability.

Development of a quaternized chitosan with enhanced antibacterial efficacy.

The antibacterial activity of a water-soluble chitosan derivative prepared by chemical modification to quaternary ammonium compound N,N,N-trimethylchitosan (TC) was investigated against four selected waterborne pathogens: Aeromonas hydrophila ATCC 35654, Yersinia enterocolitica ATCC 9610, Listeria monocytogenes ATCC 19111 and Escherichia coli O157:H7 ATCC 32150. An inactivation of 4 log CFU/ml of all waterborne pathogens was noted for the quaternized chitosan as compared with chitosan over a short contact time (30 min) and low dosage (4.5 ppm) at ambient temperature. A marked increase in glucose level, protein content and lactate dehydrogenase (LDH) activity was observed concurrently in the cell supernatant to be a major bactericidal mechanism. The results suggest that the TC derivative may be a promising commercial substitute for acid-soluble chitosan for rapid and effective disinfection of water.

Use of Lactococcus lactis to enrich sourdough bread with γ-aminobutyric acid.

Fried sourdough bread (bhatura) with an elevated amount of γ-aminobutyric acid (GABA) was produced using lactic acid bacteria (LAB). The LAB starter was screened and isolated from pickled yam showing highest GABA content and was identified as Lactococcus lactis subsp. lactis. The maximum GABA production in de Man Rogosa Sharpe (MRS) media supplemented with monosodium glutamate (MSG) was 110 mg/100 ml at pH 5, and 1-3% NaCl did not change the production of GABA significantly (p>0.05). When MSG was replaced with Vigna mungo in sourdough, the amount of GABA for bhatura was 226.22 mg/100 g representing about 10-fold increase. A sensory evaluation resulted as the overall general acceptability of bhatura to be 4.91 ± 0.03 on a five-point hedonic scale. Thus, the results indicated the potential of L. lactis as a LAB starter for the production of GABA-enriched bhatura. Although other physiological effects can be expected in the product, animal and clinical studies are mandatory prior to application of this food.

Development of exobiopolymer-based biosensor for detection of phosphate in water.

The present study was conducted to develop a biosensor by exploiting phosphate-binding capacity of exobiopolymer (EBP) produced by Acinetobacter sp. An environmental isolate of EBP-producing Acinetobacter sp. was subjected to transposon (Tn5) mutagenesis to overproduce EBP and afford improved phosphate selectivity. A mutant producing the highest amount of EBP with high phosphate-binding capacity was selected for biosensor probe fabrication. Phosphate samples were filtered through EBP-coated membranes and phosphate retained on membranes was determined by molybdenum blue method. The color produced was read using a LED 690 nm/photodiode detection system linked to an amplifier and signals were converted to appropriate phosphate concentrations. The biosensor had a limit of detection of 0.5 mg/L and a limit of quantification 1 mg/L. The biosensor as well as the probe were found to be stable for at least 28 days. In conclusion, we believe that the biosensor may have applications in monitoring of wastewater and environmental samples. Further, the enrichment of phosphate levels by EBP can help in analysis of very low phosphate concentrations.

Modeling of permeabilization process in Pseudomonas putida G7 for enhanced limonin bioconversion.

A facile process of enhanced whole cell biotransformation to debitter the triterpenoid limonin in citrus juices was optimized in this work. To maximize bioconversion, permeabilization conditions were modeled using response surface methodology. A central composite rotatable design with four significant variables (concentration, temperature, pH, and treatment time) was employed. The second order polynomial equations with R² values above 0.9 showed good correspondence between experimental and predicted values. The concentration, temperature, pH, and treatment time as well as their interactions had significant effects (p < 0.001) on limonin bioconversion. The optimum operating conditions for permeabilization were observed at a Na2EDTA concentration of 1.5 µM, treatment time of 15 min, temperature of 28 °C, and pH 8. A maximum reduction of 76.71% in the limonin content was achieved within 150 min under selected conditions. The results are promising for refining permeabilization technique for whole cell biocatalysts thereby improving the debittering of citrus juices significantly.

Enhancement of bioconversion efficiency of limonin by Pseudmonas putida G7.

The biocatalytic activity of periplasmic dehydrogenase of Pseudomonas putida G7 strain to catalyse limonin was enhanced when whole cells were permeabilized with EDTA (1 µM) lysozyme (100 µg/ml). The treated cells were entrapped in dialysis membranes to increase the stability. Permeabilized cells (1 g dry weight) entrapped in dialysis membrane could biotransform 73.67% of limonin in unpasteurized mandarin juices in a single-batch cycle of 3 h. Furthermore, permeabilized cells stored for 45 days in phosphate buffered saline (at 4 or 30°C) retained enzyme activity and were reusable for up to eight batch cycles of limonin reduction. The results of this study suggest a potential application of permeabilized P. putida G7 cells for reducing limonin levels in mandarin juices.

Mitogenic response and probiotic characteristics of lactic acid bacteria isolated from indigenously pickled vegetables and fermented beverages.

Lactic acid bacteria from indigenous pickled vegetables and fermented beverages (fermented rice and Madhuca longifolia flowers) were isolated and investigated for their functional characteristics in vitro as potential new probiotic strains. Four isolates (all Lactobacillus spp.) selected on the basis of high tolerance to bile (0.2%) were identified by standard and molecular methods (16S rDNA) as L. helveticus, L. casei, L. delbrueckii and L. bulgaricus from pickled vegetables and fermented beverages respectively. These selected strains had antibiotic resistance, tolerance to artificial gastric juice and phenol (0.4%), enzymatic profile, and antagonistic activity against enteric pathogens (Enterobacter sakazakii, Salmonella typhimurium, Shigella flexneri 2a, Listeria monocytogenes, Yersinia enterocolitica and Aeromonas hydrophila). All strains survived well in artificial gastric juice at low pH (3.0) values for 4 h, possessed bile salt hydrolase activity and were susceptible to most antibiotics including vancomycin. Additionally, the isolates exhibited high tolerance to phenol, high cell surface hydrophobicity (>60%) and induced proliferation of murine splenocytes. All the four strains of present study suppressed the Con A-stimulated proliferation of the mouse spleen cells, although L. casei had the strongest suppressive effect. The results of this study suggest a potential application of the strains (following human clinical trials), for developing probiotic foods.

Microbial Polymers as Sustainable Agents for Mitigating Health Risks of Plant-Based Endocrine Disruptors in Surface Water.

This study investigated the binding abilities of extracellular polymers produced by an environmentally isolated strain of Enterococcus hirae towards phytoestrogen endocrine disruptors-biochanin A, formonetin, genistein and daidzein. The extracellular biopolymer exhibited notable binding and removal for all four phytoestrogens, with a maximum removal of daidzein (87%) followed by genistein (72%) at a 1-1.5 mg/mL concentration. Adsorption proceeded rapidly at ambient temperature. The adsorption data fitted well with the Langmuir isotherm. Based on the adsorption energy, the biopolymer binding of phytoestrogens was inferred as daidzein > genistein > biochanin A > formononetin. Toxicity of the biopolymer (5-250 µg/mL) evaluated using RAW 264.7 cell lines indicated no significant (p < 0.05) changes in viability. In biopolymer-challenged Caenorhabditis elegans previously exposed to daidzein, complete protection to developmental toxicity, such as reduced egg-laying capacity, egg viability and progeny counts of the worm, was observed. The results of this study offer valuable insights into understanding the potential role of microbial extracellular biopolymers in binding and removal of phytoestrogens with sustainable technological implications in modulating the toxic effect of high levels of endocrine disruptors in the environment.

Evaluation of sanitizing efficacy of acetic acid on Piper betle leaves and its effect on antioxidant properties.

The sanitizing efficacy of acetic acid and its effect on health beneficial properties of Piper betle leaves were determined. Betel leaves artificially inoculated with Aeromonas, Salmonella and Yersinia were subjected to organic acid (citric acid, acetic acid and lactic acid) treatment. Pathogen populations reduced by 4 log upon individual inoculation and up to 2 log in a mixed cocktail following treatment with 2% acetic acid during storage up to 20 h at 28 degrees C, indicating a residual antimicrobial effect on pathogen during storage. Antioxidant potential ethanolic extracts of both raw and treated P. betle leaves were assayed for free radical scavenging activities against 2,2-diphenyl-1-picryhydrazyl. Polyphenols, flavonoids and the reducing power of treated and untreated P. betle were also compared. No significant (P>0.05) changes were observed in antioxidant status; flavonoids, polyphenols and reducing power of treated betel leaves. Results indicate the feasibility of a simple intervention strategy for inactivating pathogens in edible leaves of P. betle.

Effective removal of Cryptosporidium by a novel bioflocculant.

Extracellular biopolymer produced from Klebsiella terrigena was found to have excellent flocculating ability over a wide range of colloid particles (0.5 to 25micro). The biopolymer was thermostable, with an optimum temperature for flocculation of 30 degrees C. Analysis with Fourier transform infrared spectrophotometry (FT-IR) shows that the biopolymer mainly possesses hydroxyl, carboxyl, and methoxyl groups, with neutral sugar and uronic acid as its major and minor components, and the structure of a polysaccharide. The average molecular weight of the biopolymer was greater than 2 x 10(3) kilodalton (KDa), as determined by gel permeation chromatography. Scanning electron microscopy indicated a porous morphology of the biopolymer. At a dosage of 2 mg/L, the purified biopolymer could remove 62.3% of Cryptosporidium oocysts (1 x 10(6)) spiked in tap water samples. Calcium (5mM) was required for effective removal. The removal efficiency of Cryptosporidium oocysts by the biopolymer remained unaltered over a pH range of 6 to 8. The results of this study indicates a possible utility of the Klebsiella terrigena biopolymer as an alternative to typically used chemical flocculants for removal of Cryptosporidium oocysts from water.

Application of a novel biopolymer for removal of Salmonella from poultry wastewater.

This study evaluated the potential of an extracellular, novel biopolymeric flocculant produced by a strain of Klebsiella terrigena for removal of Salmonella, a potent pathogen prevalent in poultry wastewater. The purified biopolymer was applied to poultry wastewater containing 3 log CFU cells of Salmonella. An optimized dosage of 2 mg L(-1) of the purified bioflocculant was sufficient to remove 80.3% Salmonella spp. within 30 min, at ambient temperature. Also this bioflocculant showed high flocculating activity (90%) against kaolin particles and proved to be far more effective than the other synthetic flocculants used in this study. Fluorescent in situ hybridization (FISH) with the genus specific Sal3 probe hybridized with the Salmonella present in the agglomerated matrix of the bioflocculant. Confocal laser scanning micrographs (CLSM) allowed a clear visualization of the spatial distribution of the total flocculated bacterial population (with DAPI and Eub338 probe) as well as Salmonella (with the Sal3 probe), indicating that the removed Salmonella remained bound and embedded within the flocculant matrix. Scanning electron microscopic (SEM) analysis exhibited a porous surface morphology. The bioflocculant was characterized to be a polysaccharide by FTIR, HPLC, CHN and chemical analysis. A viable alternative treatment technology of poultry wastewater using this novel bioflocculant is suggested.

Inactivation of Escherichia coli O157:H7 by cinnamon extracts in carrot-kinnow mandarin blends.

We investigated the antimicrobial efficacy of cinnamon extracts in laboratory prepared Kinnow-mandarin carrot blends challenged with Escerichia coli O157:H7. Freshly squeezed carrot and kinnow-mandarin juices were mixed to obtain a typical blend, inoculated with E. coli O157:H7 cultures at 10(2) CFU/mL with and without cinnamon extracts (0.3%) and stored at 4, 8 and 28°C for up to 10 hours. Counts on tryptic soy agar (TSA) selective medium (Mac conkey sorbitol agar) and thin agar layer (TAL) were determined at every 2 hour. The TAL method was used for recovery of sublethally injured cells. Inactivation of E. coli O157:H7 in blends containing 0.3% cinnamon extracts were observed, with killing effects being more pronounced at 28°C, similar trends were evident with blends stored under refrigeration conditions (4 and 8°C).The decrease in counts were attributed to several factors namely, pH, storage temperature and addition of cinnamon. The results of our study indicate that cinnamon extracts could be used as an effective, natural antimicrobial for assuring consumer safety at the point of preparation of carrot-kinnow mandarin blends, which is a popular, nutritional beverage consumed in India.

Monitoring of biofilm aging in a Sphingomonas sp. strain from public drinking water sites through changes in capacitance.

This study reports the applicability of a capacitance-based technique for evaluating the biofilm progression of Sphingomonas sp. One hundred and forty isolates of Sphingomonas were screened from public drinking water sites, and one potential strain with biofilm-forming ability was used for the study. The biofilm production by this strain was established in microtiter plates and aluminum coupons. The standard biofilm-forming strain Sphingomonas terrae MTCC 7766 was used for comparison. Changes in biofilm were analyzed by energy-dispersive X-ray spectroscopy (EDX) and scanning electron microscope (SEM). Capacitance values were measured at 1, 100 and 200 kHz frequency; however, 1 kHz was selected since resulted in reproducible values, which could be correlated to biofilm age measured as dry weight over a time of 96 h (4 days) depicting the biofilm growth/progression over time. The EDX, SEM and capacitance values obtained in parallel indicated the related physiological profile usually displayed by biofilms upon growth, suggesting authenticity to the observed capacitance profile. The results of this study demonstrated the feasibility of a capacitance-based method for analyzing biofilm development/progression by Sphingomonas sp. and suggested a simple approach for developing an online system to detect biofilms by this opportunistic pathogen of concern in drinking water.

Antimicrobial efficacy and safety of mucoadhesive exopolymer produced by Acinetobacter haemolyticus.

This study evaluated five extracellular polymers of bacterial origin possessing mucoadhesive properties for their antimicrobial properties and toxicological characteristics. Of the five tested mucoadhesive biopolymers, the extracellular polymer produced by a strain of Acinetobacter haemolyticus exhibited broad antimicrobial efficacy towards Yersinia enterocolitica, Salmonella typhimurium, Listeria monocytogenes, Escherichia coli O157:H7 and Bacillus subtilis. Significant (p<0.05) inhibition of gram negative bacterial pathogens followed by gram positives were observed with the biopolymer at a dose of 40-60µg ml-1 at ambient temperature. The cytotoxicity under in vitro conditions and oral toxicity in murine models was also evaluated. The biopolymer did not elicit either haemolytic activity or toxicity in RAW 264.7 cell lines. Haemotological, histopathological and general examinations indicated no adverse effects in Swiss albino mice fed with the biopolymer (120mg kg-1 body weight-1 day1) over a period of 30 days. These results suggested that the biopolymer was well tolerated without any signs of toxicity and may have several potential biomedical applications where disinfection is desired.

Evidence of indigenous NAH plasmid of naphthalene degrading Pseudomonas putida PpG7 strain implicated in limonin degradation.

A well characterized naphthalene-degrading strain, Pseudomonas putida PpG7 was observed to utilize limonin, a highly-oxygenated triterpenoid compound as a sole source of carbon and energy. Limonin concentrations evidenced a 64% reduction over 48 h of growth in batch cultures. Attempts were made to acquire a plasmid-less derivative via various methods (viz. Ethidium Bromide, SDS, elevated temperature & mitomycin C), among which the method involving mitomycin C (20 ug/ml) proved successful. Concomitant with the loss of plasmid in P. putida PpG7 strain, the cured derivative was identified as a lim- phenotype. The lim+ phenotype could be conjugally transferred to the cured derivative. Based on the results of curing with mitomycin C, conjugation studies and presence of ndo gene encoding naphthalene 1,2 dioxygenase, it was demonstrated that genes for the limonin utilization were encoded on an 83 kb indigenous transmissible Inc. P9 NAH plasmid in Pseudomonas putida PpG7 strain.

Characterization and upregulation of bifunctional phosphoglucomutase/phosphomannomutase enzyme in an exobiopolymer overproducing strain of Acinetobacter haemolyticus.

Several members of the Acinetobacter spp. produce exobiopolymer (EBP) of considerable biotechnological interest. In a previous study, we reported phosphate removal capacity of EBP produced by Acinetobacter haemolyticus. Insertional mutagenesis was attempted to develop EBP-overproducing strains of A. haemolyticus and mutant MG606 was isolated. In order to understand the underlying mechanism of overproduction, the EBP overproducing mutant MG606 was analyzed and compared with the wild type counterpart for its key EBP synthetic enzymes. The EBP produced by MG606 mutant was 650 mg/L compared to 220 mg/L in its wild type counterpart. Significantly high (p<0.05) levels of phosphoglucomutase/phosphomannomutase (PGM/PMM) in MG606 mutant was noted, whereas activities of other enzymes responsible for EBP synthesis showed no significant change (p>0.05). The up-regulation of PGM/PMM expression in mutant was further confirmed by real time reverse transcriptase (RT)-PCR of PGM/PMM transcripts. The optimal conditions for PGM/PMM activity were found to be 35 °C and pH 7.5; PGM/PMM activity was inhibited by ions such as lithium, zinc, nickel. Further, incubation of cells with a PGM inhibitor (lithium) resulted in a concentration-dependent decrease in EBP production further confirming the role of PGM/PMM overexpression in enhanced EBP production by the mutant. Overall the results of our study indicate a key role of PGM/PMM in enhanced EBP production, as evident from enhanced enzyme activity, increased PGM/PMM transcripts and reduction in EBP synthesis by a PGM inhibitor. We envisage a potential exploitation of the insights so obtained to effectively engineer strains of Acinetobacter for overproducing phosphate binding EBP.