Ultra-small magnetic Candida antarctica lipase B nanoreactors for enzyme synthesis of bixin-maltitol ester.

Bixin has desirable bioactivities but poor water solubility, which limits its practical applications. Enzymatic transesterification of methyl to alditol groups in bixin by Candida antarctica lipase B (CALB) improves bixin water solubility. Herein, magnetic CALB nanoreactors with diameter of 11.7 nm and CALB layer thickness of 3.5 nm were developed by covalently linking CALB onto silicon covered Fe3O4 nanoparticles. The CALB loading capacity in nanoreactors achieved 30%. The Michaelis constant (Km) and maximum reaction rate of magnetic CALB nanoreactors were 56.1 mmol/L and 0.2 mmol/(L·min). Magnetic CALB nanoreactors could circularly catalyze bixin-maltitol ester synthesis and keep catalytic efficiency of 62.6% after eight repetitive enzymatic reactions. Additionally, the optimal bixin-maltitol ester synthesis procedure was heating bixin-maltitol mixture at molar ratio of 1:7 in anhydrous 2-methyl-2-butanol-dimethylsulfoxide (8:2, v/v) at 50 °C for 24 h. Bixin-maltitol ester showed improved water solubility at pH 5.5 and 7.0.

Development of nonionic nanoemulsions to improve hydrophobic 9'-cis-bixin stability in acidic aqueous medium with in vitro cytosis and nanosafety evaluation.

Nonionic oil-in-water (O/W) nanoemulsion provides potential to stabilize hydrophobic bio-functional components in aqueous medium. Understanding safety of nanoemulsion droplets via investigating in vitro cell uptake and cellular substructural changes is important to achieve their practical applications. Herein, we developed a nonionic O/W nanoemulsion to stabilize representative bio-functional hydrophobic component of 9'-cis-bixin at pH 3-7 and ultraviolet (UV)-induced degradation at 365, 302, and 254 nm. In vitro cell uptake demonstrated that Caco-2 cells adequately enriched 9'-cis-bixin through fast uptake of nanoemulsion droplets within 15 min. However, excessive nanoemulsion droplets greatly decreased cell survival rate, which was due to the potential destruction of cellular substructures of mitochondria, nuclear membrane, and cell membrane. Lower nanoemulsion concentration provided no significant effects on Caco-2 cell survival. This work provided objective understanding on bio-functional component stability by nanoemulsion with in vitro safety evaluation.

Inactivation of Escherichia coli K12 on raw almonds using supercritical carbon dioxide and thyme oil.

Raw almonds could be contaminated by pathogens, but the current pasteurization practice using propylene oxide in the U.S. has flammability and carcinogenicity concerns. Supercritical carbon dioxide (scCO2) is a water-free technology and is a solvent of essential oils that are effective antimicrobial preservatives. The objective of this study was to investigate the possibility of combining scCO2 and thyme oil (TO) to reduce Escherichia coli K12 inoculated on raw almonds. Raw almonds inoculated with ∼6 log CFU/g E. coli K12 were batch-treated with scCO2 alone or the combination of presoaking in pure TO followed by scCO2 treatments at different combinations of temperature, pressure, and duration. Compared to scCO2 alone treatments, the combination of TO and scCO2 treatments significantly improved the disinfection effectiveness. Temperature had the most significant effect on the log reduction. At 70 °C, the log reduction by the combination treatment was over 4-log CFU/g and the maximum reduction was 5.16 log CFU/g. The findings suggest that the combination of TO and scCO2 may be a potential water-free technology to meet the requirement of over 4-log reduction of target microorganism for almond and other tree nut products.

Nanoemulsified Carvacrol as a Novel Washing Treatment Reduces Escherichia coli O157:H7 on Spinach and Lettuce.

ABSTRACT: Fresh produce continues to be the main source of foodborne illness outbreaks in the United States, implicating bacterial pathogens such as Escherichia coli O157:H7 (EHEC). The efficacy of nanoemulsified carvacrol (NCR) as a washing treatment in reducing EHEC on fresh produce was investigated. Fresh baby spinach, romaine lettuce, and iceberg lettuce leaves (2.5-cm-diameter cores) were spot inoculated with a five-strain cocktail of nalidixic acid-resistant EHEC at ∼6 log CFU/cm2. After air drying for 1 h, 20 pieces of each inoculated produce leaf were immersed in water-based treatment solutions (200 mL per group), including water alone, 25 or 50 ppm of free chlorine, and 0.25 or 0.75% NCR for 2 min. Inoculated produce leaves without any treatment served as baseline. Produce leaves were stored at 10°C, and surviving EHEC populations were enumerated on days 0, 2, 7, and 14. The viability of EHEC following NCR treatments on the fresh produce was visualized under a fluorescence microscope. NCR treatment at 0.75% immediately reduced EHEC populations on iceberg lettuce by 1.3 log CFU/cm2 as compared with the produce treated with water alone (P < 0.05). Antimicrobial activity of NCR against EHEC was comparable to chlorine treatments on day 0 for all produce (P > 0.05). After 14 days of storage at 10°C, populations of EHEC on 0.75% NCR-treated romaine lettuce were reduced by 2.3 log CFU/cm2 compared with the recovery from 50 ppm of chlorine-treated samples (P < 0.05). Microscopic images revealed that EHEC cells were observed to be clustered on the baseline samples, indicating the development of cell aggregation, compared with the scattered cells seen on NCR-treated leaf surfaces. Treatments with NCR did not significantly affect the color of the fresh produce leaves during 14 days of storage at 10°C. Results of this study support the potential use of NCR as a water-soluble natural antimicrobial wash treatment for controlling EHEC on fresh produce.

An entrapped metal-organic framework system for controlled release of ethylene.

A novel gas storage and release system was developed for ethylene, an exogenous plant hormone that regulates fruit ripening and senescence. This system consists of a metal organic framework (MOF) core and an alginate-based shell. The MOF comprises a coordination complex of Al and [btc]3- ligands, which formed hexagonal structure (P63/mmc) with unit cell of 14.28 × 14.28 × 31.32 Å3, as revealed by the X-ray diffraction analysis. Ethylene absorption isotherm exhibited an absorption capacity of 41.0 cm3/g MOF at 25 °C and 101.3 kPa. After charging with ethylene, the MOFs are further entrapped in a close-knit bead formed with alginate-Fe(III) matrix, observed under a scanning electron microscopy coupled with energy dispersive spectroscopy (SEM-EDS). The alginate shell is degraded by exposing to 200 mM sodium citrate aqueous solution, triggering a continuous release of ethylene. With 20 mg of MOF, ethylene concentration reached 0.41-0.46 mg/L per mg MOF after 2.5 h. This is the first report regarding a controlled release of ethylene through degrading alginate-Fe(III) matrix rather than by changing the interfacial pore size of MOF under extreme conditions. This technology can enable precisely controlled and targeted applications of ethylene for food processing and agricultural applications.

An immune magnetic nano-assembly for specifically amplifying intercellular quorum sensing signals.

Quorum sensing (QS) enables intercellular communication after bacterial cells sense the autoinducers have reached or exceeded a critical concentration. Selectively amplifying specific bacterial "quorum" activity at a lower cell density is still a challenge. Here, we propose a novel platform of immune magnetic nano-assembly to amplify specific bacterial QS signaling via improving the bioavailability of autoinducers-2 (AI-2, furanosyl borate) from sender (wide-type, WT cells) to receiver (reporter cells). Antibody coated magnetic nanoparticle (MNPAB) was fabricated with an average diameter of 12 nm and a specific surface area of 96.5 m2/g. The distribution efficiency of the antibody on the surface was 25.8 µg/m2 of magnetic nanoparticles. It was found that more than 3 × 108 of K12 serotype Escherichia coli (E. coli) reporter or WT cells were collected using 1 mg fabricated MNPAB at a saturated condition. The MNPAB not only captured E. coli WT cells but also brought them into proximity of E. coli (CT104, pCT6+pET-DsRed) reporter cells via magnetic attraction. The amplified QS signaling of the reporter cells by this immune magnetic nano-assembly was approximately 3 times higher than the nature QS signaling in cell suspension at optical density (OD) 0.08. This study foresees potential applications in amplifying specific biological QS signals based on a preprogrammed design.

Catechol-chitosan redox capacitor for added amplification in electrochemical immunoanalysis.

Antibodies are common recognition elements for molecular detection but often the signals generated by their stoichiometric binding must be amplified to enhance sensitivity. Here, we report that an electrode coated with a catechol-chitosan redox capacitor can amplify the electrochemical signal generated from an alkaline phosphatase (AP) linked immunoassay. Specifically, the AP product p-aminophenol (PAP) undergoes redox-cycling in the redox capacitor to generate amplified oxidation currents. We estimate an 8-fold amplification associated with this redox-cycling in the capacitor (compared to detection by a bare electrode). Importantly, this capacitor-based amplification is generic and can be coupled to existing amplification approaches based on enzyme-linked catalysis or magnetic nanoparticle-based collection/concentration. Thus, the capacitor should enhance sensitivities in conventional immunoassays and also provide chemical to electrical signal transduction for emerging applications in molecular communication.

Focusing quorum sensing signalling by nano-magnetic assembly.

Quorum sensing (QS) exists widely among bacteria, enabling a transition to multicellular behaviour after bacterial populations reach a particular density. The coordination of multicellularity enables biotechnological application, dissolution of biofilms, coordination of virulence, and so forth. Here, a method to elicit and subsequently disperse multicellular behaviour among QS-negative cells is developed using magnetic nanoparticle assembly. We fabricated magnetic nanoparticles (MNPs, ∼5 nm) that electrostatically collect wild-type (WT) Escherichia coli BL21 cells and brings them into proximity of bioengineered E. coli [CT104 (W3110 lsrFG- luxS- pCT6 + pET-DsRed)] reporter cells that exhibit a QS response after receiving autoinducer-2 (AI-2). By shortening the distance between WT and reporter cells (e.g., increasing local available AI-2 concentrations), the QS response signalling was amplified four-fold compared to that in native conditions without assembly. This study suggests potential applications in facilitating intercellular communication and modulating multicellular behaviours based on user-specified designs.

Silver Nanocluster-Embedded Zein Films as Antimicrobial Coating Materials for Food Packaging.

Highly efficient antimicrobial agents with low toxicity and resistance have been enthusiastically pursued to address public concerns on microbial contamination in food. Silver nanoclusters (AgNCs) are known for their ultrasmall sizes and unique optical and chemical properties. Despite extensive studies of AgNCs for biomedical applications, previous research on their application as antimicrobials for food applications is very limited. Here, for the first time, by incorporating AgNCs (∼2 nm in diameter) into zein films that are widely used as food packaging materials, we developed a novel coating material with potent antimicrobial activity, low toxicity to human cells, and low potential to harm the environment. In addition, we systematically evaluated the antimicrobial activities and cytotoxicity of AgNCs-embedded zein films and compared them to zein films embedded with AgNO3 or Ag nanoparticles with diameters of 10 and 60 nm (AgNP10 and AgNP60, respectively). At equivalent silver concentrations, AgNCs and AgNO3 solutions exhibited considerably higher antimicrobial activities than those of AgNP10 and AgNP60 solutions. Moreover, AgNCs exhibited less cytotoxicity to human cells than AgNO3, with a half maximal inhibitory concentration (IC50) of 34.68 µg/mL for AgNCs, compared to 9.14 µg/mL for AgNO3. Overall, the novel AgNCs coating developed in this research has great potential for antimicrobial applications in food packaging materials due to its high antimicrobial efficacy, ultrasmall size, and low cytotoxicity.

Encapsulation of ferulic acid ethyl ester in caseinate to suppress off-flavor formation in UHT milk.

Phenolic compounds can principally suppress the off-flavor development in ultrahigh temperature (UHT) treated milk, but little has been studied for lipophilic phenolic compounds that are to be encapsulated for even distribution in milk. The objective of this work was to study physicochemical properties of ferulic acid ethyl ester (FAEE) encapsulated in sodium caseinate and the inhibition of volatile formation after UHT processing. The capsules had an average hydrodynamic diameter of 246.2±10.9nm, a polydispersity index of 0.26±0.01, and a zeta-potential of -31.72±0.74mV. The capsules and the encapsulated FAEE were stable after heating at 138°C for 16min and UV radiation at 365nm for 32h. The encapsulated FAEE at a level of 0.18-1.42mg/mL suppressed the formation of 2-acetyl-2-thiazoline in model UHT milk by 32.8-63.2% after 30-day storage at 30°C. Therefore, FAEE encapsulated in caseinate can be potentially used to improve the quality of UHT milk.

Eugenol improves physical and chemical stabilities of nanoemulsions loaded with ß-carotene.

Food-grade nanoemulsions are potential vehicles of labile lipophilic compounds such as ß-carotene, but much work is needed to improve physical and chemical stabilities. The objective of this work was to study impacts of eugenol on physical and chemical stabilities of ß-carotene-loaded nanoemulsions prepared with whey protein and lecithin. The combination of whey protein and lecithin resulted in stable nanoemulsions with eugenol added at 10% mass of soybean oil. Nanoemulsions, especially with eugenol, drastically reduced the degradation of ß-carotene during ambient storage, heating at 60 and 80°C, and UV radiation at 254, 302, and 365nm. The droplet diameter of the nanoemulsion without eugenol increased from 153.6 to 227.3nm after 30-day ambient storage, contrasting with no significant changes of nanoemulsions with eugenol. Heating or UV radiation up to 8h did not significantly change the droplet diameter. Therefore, eugenol can be used to improve the stability of nanoemulsion delivery systems.

Microemulsions based on a sunflower lecithin-Tween 20 blend have high capacity for dissolving peppermint oil and stabilizing coenzyme Q10.

The objectives of the present study were to improve the capability of microemulsions to dissolve peppermint oil by blending sunflower lecithin with Tween 20 and to study the possibility of codelivering lipophilic bioactive compounds. The oil loading in microemulsions with 20% (w/w) Tween 20 increased from 3% (w/w) to 20% (w/w) upon gradual supplementation of 6% (w/w) lecithin. All microemulsions had particles of <12 nm that did not change over 70 d of storage at 21 °C. They had relatively low Newtonian viscosities and were physically and chemically stable after 50-200-fold dilution in water, resulting from similar hydrophile-lipophile-balance values of the surfactant mixture and peppermint oil. Furthermore, the microemulsions were capable of dissolving coenzyme Q10 and preventing its degradation at UV 302 nm, more significant for the microemulsion with lecithin. Therefore, natural surfactant lecithin can reduce the use of synthetic Tween 20 to dissolve peppermint oil and protect the degradation of dissolved lipophilic bioactive components in transparent products.

Yeast mannoproteins improve thermal stability of anthocyanins at pH 7.0.

Anthocyanins are food colourants with strong antioxidant activities, but poor thermal stability limits their application in neutral foods. In the present study, impacts of yeast mannoproteins on the thermal stability of anthocyanins were studied at pH 7.0. The degradation of anthocyanins at 80 and 126 °C followed first order kinetics, and the addition of mannoproteins reduced the degradation rate constant and increased the half-life by 4 to 5-fold. After heating at 80 and 126 °C for 30 min, mannoproteins improved the colour stability of anthocyanins by 4 to 5-fold and maintained the antioxidant capacity of anthocyanins. Visible light absorption, fluorescence spectroscopy, and zeta-potential results suggest that anthocyanins bound with the protein moiety of mannoproteins by hydrophobic interactions, and that the inclusion of anthocyanins in complexes effectively reduced the thermal degradation at pH 7.0. Therefore, mannoproteins may expand the application of anthocyanins as natural colours or functional ingredients.

Gum arabic and Fe²âº synergistically improve the heat and acid stability of norbixin at pH 3.0-5.0.

Thermal and acid stabilities of norbixin are challenges for its application as a food colorant. In this work, gum arabic and Fe(2+) were studied for the possibility to improve the thermal and acid stabilities of norbixin. Norbixin was dissolved at 0.004% w/v in deionized water with and without 0.2% w/v gum arabic and/or 0.15 mM ferrous chloride, adjusted to pH 3.0-5.0, and heated at 90 or 126 °C for 30 min. Before heating, norbixin precipitated at pH 3.0-4.0, which was prevented by gum arabic. The thermal stability of norbixin was improved by the combination of gum arabic and Fe(2+). Fluorescence analyses indicated the complex formation between norbixin and gum arabic with and without Fe(2+). Particle size and atomic force microscopy results suggested Fe(2+) and gum arabic synergistically prevented the aggregation of norbixin at acidic pH and during heating. It was hypothesized that the core of gum arabic-norbixin complexes was strengthened by Fe(2+) to enable the synergy.

Preparation of antioxidants from sugarcane molasses.

Supercritical carbon dioxide fluid extraction with piecewise distillation separation was used to obtain antioxidants from sugarcane molasses. Extraction pressure, time, temperature, flow rate of CO2 and ethanol content were selected as the independent variables. Oxygen radical absorbance capacity was used to evaluate the antioxidant activity of the extract. Results showed that conditions to obtain the highest total oxygen radical absorbance capacity value of sugarcane molasses extract were determined to be an extraction pressure of 33.3 MPa, temperature of 43.3 °C, time of 86.7 min, 90% ethanol content of sugarcane molasses and flow rate of CO2 of 20 L/h. Under the conditions stated above, the experimental value was 2584.9. This study indicated that supercritical carbon dioxide fluid extraction with piecewise distillation separation can effectively extract antioxidants from sugarcane molasses.

Developing an effective means to reduce 5-hydroxymethyl-2-furfural from caramel colour.

Supercritical carbon dioxide fluid extraction was used to extract 5-hydroxymethyl-2-furfural from caramel colour (solid content was about 75%). The procedure was carried out by response surface methodology using a quadratic polynomial model. Extraction pressure, time, temperature and ethanol content were selected as the independent variables. Conditions to obtain the highest extraction ratio of 5-hydroxymethyl-2-furfural were determined to be an extraction pressure of 21.65MPa, time of 46.7min, temperature of 35°C and 70% ethanol content of caramel colour. The predicted 5-hydroxymethyl-2-furfural extraction ratio was 87.42%. Under the conditions stated above, the experimental value of 5-hydroxymethyl-2-furfural extraction ratio was 86.98%, which was similar to the predicted value by the model. This study indicated that supercritical carbon dioxide fluid extraction can effectively reduce 5-hydroxymethyl-2-furfural from caramel colour, which can help food industry to improve the safety of the food material, as well as provide more healthy caramel colour for human beings.

Simultaneous analysis of Nε-(carboxymethyl)lysine, reducing sugars, and lysine during the dairy thermal process.

A new analytical method allowing the simultaneous quantification of Nε-(carboxymethyl)lysine (CML), lysine, and reducing sugars (glucose, lactose, and galactose) is described. It is based on high performance anion-exchange chromatography with pulsed amperometric electrochemical detection. This method demonstrated a low limit of quantification (0.385 to 0.866 mg/L), excellent linear correlation (R(2)>0.997), and desired calibration range (3.125 to 25 mg/L). In addition, lactose-lysine solutions containing sulfite (4 to 400 mmol/L) were heated at 110°C for 2h. The results showed that sulfite inhibited the formation of CML and promoted the consumption of reducing sugars and lysine in the Maillard reaction model. The method proved to be useful for simultaneous analysis of CML, lysine, and reducing sugars (glucose, galactose, and lactose) in the Maillard reaction system. Moreover, sulfite was an effective inhibitor of CML formation.

Indirect competitive ELISA based on monoclonal antibody for the detection of 5-hydroxymethyl-2-furfural in milk, compared with HPLC.

In this study, a method for rapid detection of 5-hydroxymethyl-2-furfural (HMF) was investigated. Monoclonal antibody (anti-HMF) was prepared and evaluated by an indirect competitive ELISA (ic-ELISA) format. The optimized standard curve was y=-0.2097x+1.0432 [where x is the logarithm (base 10) of the values of the HMF concentration and y is the absorbance of ic-ELISA results tested at 490 nm] and the linear detection range was 0.008 to 32.768 mg/L. The percentage of cross-reactivity of HMF with 5 major furfural derivatives was less than 2.92%. Finally, the established ic-ELISA format was used to test HMF in milk, and compared with the result obtained by HPLC, which produced an error of about 0.3%. Based on the data in this experiment, we concluded that the established ic-ELISA format was reliable with a high specificity.

Short communication: possible mechanism for inhibiting the formation of polymers originated from 5-hydroxymethyl-2-furaldehyde by sulfite groups in the dairy thermal process.

5-Hydroxymethyl-2-furaldehyde can undergo polymerization to form high-molecular weight molecules via the Maillard reaction during dairy thermal treatment. In this study, the effect of sulfite group on polymer formation, especially in inhibiting the formation of high-molecular weight polymers has been described. Results showed that the sulfite group significantly inhibited the increase of polymer molecular weight via prevention of the polymerization of 5-hydroxymethyl-2-furaldehyde. The formation of an intermolecular dimer based on the glucose molecule through Schiff base cyclization can lead to a competitive reaction with 1,2-enolization to reduce 5-hydroxymethyl-2-furaldehyde formation, which might be another factor in reducing the formation of high-molecular weight polymers.

Effects of pressure on the glucose-ammonium sulphite caramel solutions.

Effects of pressure on glucose-ammonium sulphite solutions were investigated. The reactant (i.e. glucose), intermediate products content, and browning intensity of advanced stages were tested using an UV-Vis spectrophotometer and a high performance liquid chromatograph to gain a better understanding of the influence of pressure on the glucose-ammonium model Maillard reaction system. This study indicates that pressure could promote the first step of the reaction, i.e. condensation reaction between SO(3)(2-)/ammonium and glucose, but inhibit the increase of A(294), A(420) and 5-hydroxymethyl-2-furaldehyde content. The mechanism of inhibiting the glucose-ammonium model Maillard reaction might be that pressure increases dissociative SO(3)(2-) content in solutions and inhibits the degradation of the Amadori rearrangement product.