AFM imaging of extracellular ice nucleators.

Ice nucleators are substances that can initiate nucleation of pure water at and above -10 °C. Some plant pathogens possess a gene which encodes for a protein that acts as an ice nucleator, activity of which is enhanced when it is combined with the sugar and lipid components from the cell membrane. This matter retains its ice nucleation activity even after it is detached from the cell wall, and is termed extracellular ice nucleator (ECIN). In this paper, surface morphology of ECINs was investigated using atomic force microscopy (AFM) in tapping mode. ECINs were immobilized onto polyelectrolyte multilayers using the layer by layer deposition method. Effect of layer build-up, method of ECIN production, and polyelectrolytes used for multilayer fabrication were investigated. Globular and rod-like structures were observed on the AFM images of the nano-thin ECIN layers. Activity of ECINs, tested in food solutions in earlier studies, was retained when applied as a nano-thin layer onto a silicon wafer surface. Protein aggregate sizes decreased when higher centrifugation speeds were applied, and ice nucleation activity also decreased. Nucleation occurred faster and at higher temperatures when substrates were immersed in solutions of higher ECIN concentration, whereas number of bilayers formed did not have a significant effect. Higher concentration ECIN dipping solutions also led to the formation of thicker and denser ECIN layers as observed via AFM imaging. PRACTICAL APPLICATION: This study demonstrates the properties of nano-thin ECIN layers, which can crystallize pure water into ice at higher temperatures and in shorter time. Accelerating ice nucleation can potentially be utilized to freeze liquids in shorter time hence using less energy, or improve frozen foods' stability against the risk of cold chain breakage.

Improved freeze drying efficiency by ice nucleation proteins with ice morphology modification.

This study aims to use ice nucleation proteins (INPs) as a novel approach to improve the efficiency of freeze drying process and investigate the related mechanism of ice morphology. Our results show that INPs can significantly improve freeze drying efficiency with increased primary drying rate under the increase of INP concentration from 0 to 10-2mg/mL. Moreover, such improvement was more significant at higher subzero freezing temperatures with the addition of INPs, when the control samples were unable to freeze. Those improvements further lead to reduced total drying time, which suggests an estimated total energy saving of 28.5% by INPs. Our ice morphology results indicate the ability of INPs to alter ice morphology with lamellar ice structure and larger crystal size, which both show linear relationships with primary drying rate. The results further suggest that these ice morphology characteristics induced by INPs are very likely to facilitate the water vapor flow and improve the sublimation rate. Additionally, the increase of freeze drying efficiency can also be achieved by INPs in other food systems like coffee and milk with elevated primary drying rate. The results of this study suggest great potential of using INPs to improve the efficiency of freeze drying process for a wide range of food products and other related applications. This study also provides new insights into the relationship between process efficiency and ice morphology.

A Novel Approach To Improve the Efficiency of Block Freeze Concentration Using Ice Nucleation Proteins with Altered Ice Morphology.

Freeze concentration is a separation process with high success in product quality. The remaining challenge is to achieve high efficiency with low cost. This study aims to evaluate the potential of using ice nucleation proteins (INPs) as an effective method to improve the efficiency of block freeze concentration while also exploring the related mechanism of ice morphology. Our results show that INPs are able to significantly improve the efficiency of block freeze concentration in a desalination model. Using this experimental system, we estimate that approximately 50% of the energy cost can be saved by the inclusion of INPs in desalination cycles while still meeting the EPA standard of drinking water (<500 ppm). Our investigative tools for ice morphology include optical microscopy and X-ray computed tomography imaging analysis. Their use indicates that INPs promote the development of a lamellar structured ice matrix with larger hydraulic diameters, which facilitates brine drainage and contains less brine entrapment as compared to control samples. These results suggest great potential for applying INPs to develop an energy-saving freeze concentration method via the alteration of ice morphology.

Extruded rice fortified with micronized ground ferric pyrophosphate reduces iron deficiency in Indian schoolchildren: a double-blind randomized controlled trial.

BACKGROUND: Iron fortification of rice could be an effective strategy for reducing iron deficiency anemia in South Asia. OBJECTIVE: We aimed to determine whether extruded rice grains fortified with micronized ground ferric pyrophosphate (MGFP) would increase body iron stores in children. DESIGN: In a double-blind, 7-mo, school-based feeding trial in Bangalore, India, iron-depleted, 6-13-y-old children (n = 184) were randomly assigned to receive either a rice-based lunch meal fortified with 20 mg Fe as MGFP or an identical but unfortified control meal. The meals were consumed under direct supervision, and daily leftovers were weighed. All children were dewormed at baseline and at 3.5 mo. Iron status and hemoglobin were measured at baseline, 3.5 mo, and 7 mo. RESULTS: At baseline, the prevalences of iron deficiency and iron deficiency anemia in the total sample were 78% and 29%, respectively. After 7 mo of feeding, there was a significant increase in body iron stores in both study groups (P < 0.001), with a greater increase in the iron group than in the control group (P < 0.05). There was a significant time x treatment interaction for iron deficiency, which fell from 78% to 25% in the dewormed iron group and from 79% to 49% in the dewormed control group. Iron deficiency anemia decreased from 30% to 15% (NS) in the iron group but remained virtually unchanged in the control group (28% and 27%). In sensory tests, the MGFP-fortified rice (fortified at 3 and 5 mg Fe/100 g) was indistinguishable from natural rice, in both cooked and uncooked form. CONCLUSIONS: Extruded rice fortified with MGFP has excellent sensory characteristics. Fed in a school lunch meal, it increases iron stores and reduces the prevalence of iron deficiency in Indian children.

The contribution of glutenin macropolymer depolymerization to the deterioration of frozen steamed bread dough quality.

Depolymerization of glutenin macropolymers (GMP) widely exists in the frozen dough with little effort in elucidating its effects on steamed bread quality. To clarify this, GMP was fractionated from wheat flour and reconstituted to yeast and chemical leavened dough (YLD/CLD). Results showed that with supplementary GMP fraction, depolymerization degree was alleviated in frozen dough. The bread quality loss from freezing was partially counteracted along with better preserved GMP content. Both of dough elasticity and gas retention capability were enhanced in GMP-enriched frozen dough. Gassing power in frozen YLD decreased while remained constant in CLD. Addition of GMP did not affect the gassing power, which allowed interpreting the improved bread qualities from the enhanced dough elasticity and gas retention capability. Based on the improved facts of frozen steamed bread dough quality with additional GMP fractions, this study revealed the pivotal role of GMP depolymerization on the frozen steamed bread dough quality.

Kinetic studies of lycopene isomerization in a tributyrin model system at gastric pH.

A semi-preparative HPLC method was developed in order to isolate and purify the 13-cis-lycopene isomer in tomato-based materials. The result was compared with the naturally predominant all-trans-lycopene isomer, in terms of stability to gastric pH at physiological temperature in a tributyrin model system. Kinetic experiments confirmed that lycopene isomerization is a reversible reaction, and under these conditions the all-trans isomer is more stable than the 13-cis isomer. In addition, it was found that at gastric pH 13-cis-lycopene would predominantly isomerize to the all-trans form rather than undergo oxidation/breakdown. A simulation based on the rate constants calculated in the kinetic study indicated that at gastric pH the lycopene isomeric distribution aimed toward an equilibrium characterized by approx 16% 13-cis-, 16% 9-cis-, and 68% all-trans-lycopene. This study suggests that pH-driven isomerization in the stomach is at least partially responsible for the relatively high cis-lycopene proportion found in vivo.

Engineering functional nanothin multilayers on food packaging: ice-nucleating polyethylene films.

Polyethylene is the most prevalent plastic and is commonly used as a packaging material. Despite its common use, there are not many studies on imparting functionalities to those films which can make them more desirable for frozen food packaging. Here, commercial low-density polyethylene (LDPE) films were oxidized by UV-ozone (UVO) treatment to obtain a negatively charged hydrophilic surface to allow fabrication of functional multilayers. An increase in hydrophilicity was observed when films were exposed to UVO for 4 min and longer. Thin multilayers were formed by dipping the UVO-treated films into biopolymer solutions, and extracellular ice nucleators (ECINs) were immobilized onto the film surface to form a functional top layer. Polyelectrolyte adsorption was studied and confirmed on silicon wafers by measuring the water contact angles of the layers and investigating the surface morphology via atomic force microscopy. An up to 4-5 °C increase in ice nucleation temperatures and an up to 10 min decrease in freezing times were observed with high-purity deionized water samples frozen in ECIN-coated LDPE films. Films retained their ice nucleation activity up to 50 freeze-thaw cycles. Our results demonstrate the potential of using ECIN-coated polymer films for frozen food application.

Structure and physical properties of zein/pluronic f127 composite films.

Triblock copolymer Pluronic F127 has been used to form composites with zein, a corn protein and coproduct of the bioethanol industry, to alleviate its natural brittleness. At low F127 loadings (0-35%), the plasticizing effect was dominant, and the elongation at break of zein composite film containing 35% F127 was about 8-fold higher than that of the zein film with 10% F127. At high F127 loadings (50-100%), a large number of lamellae crystals were formed in the film matrix as verified by a combination of differential scanning calorimetry (DSC), atomic force microscopy (AFM), and small-angle X-ray scattering (SAXS). The F127 crystallization surpassed its plasticizing effect, resulting in an increased brittleness of zein film with the further increase of F127 loading. Compared with the flat-on lamellae of pure F127, F127 chains folded into branch-like lamellar structures in the zein composite film containing 50% F127 due to the confinement of amorphous zein. Besides, the crystals in zein films were composed of extended chain integral folding (IF = 0) and once-folded chain (IF = 1) polyethylene oxide (PEO) crystals, and the portion of once-folded chain (IF = 1) PEO crystals increased with F127 loading. Thus, through investigation of the competition of plasticizing effect and crystal formation under different F127 loadings, the optimized F127 loading in zein/F127 composite film with a good overall performance was determined to be at around 35%.

Improving ice nucleation activity of zein film through layer-by-layer deposition of extracellular ice nucleators.

Zein protein has been of scientific interest in the development of biodegradable functional food packaging. This study aimed at developing a novel zein-based biopolymer film with ice nucleation activity through layer-by-layer deposition of biogenic ice nucleators, that is, extracellular ice nucleators (ECINs) isolated from Erwinia herbicola , onto zein film surface. The adsorption behaviors and mechanisms were investigated using quartz crystal microbalance with dissipation monitoring (QCM-D). On unmodified zein surface, the highest ECINs adsorption occurred at pH 5.0; on UV/ozone treated zein surface followed by deposition of poly(diallyldimethylammonium chloride) (PDADMAC) layer, the optimum condition for ECINs adsorption occurred at pH 7.0 and I 0.05 M, where the amount of ECINs adsorbed was also higher than that on unmodified zein surface. QCM-D analyses further revealed a two-step adsorption process on unmodified zein surfaces, compared to a one-step adsorption process on PDADMAC-modified zein surface. Also, significantly, in order to quantify the ice nucleation activity of ECINs-coated zein films, an empirical method was developed to correlate the number of ice nucleators with the ice nucleation temperature measured by differential scanning calorimetry. Calculated using this empirical method, the highest ice nucleation activity of ECINs on ECINs-modified zein film reached 64.1 units/mm(2), which was able to elevate the ice nucleation temperature of distilled water from -15.5 °C to -7.3 °C.

Improved mechanical property and water resistance of zein films by plasticization with tributyl citrate.

Pure zein film is intrinsically rigid and brittle and lacks necessary mechanical properties for industrial processing. In addition, pure zein film is sensitive to high relative humidity, which limits its application in food packaging. To improve these properties, tributyl citrate (TBC) was incorporated into zein film to the mass ratios from 10% to 50%. A significant decrease in Young's modulus was observed, from 409.86 MPa in pure zein films to 136.29 MPa in zein films with 50% TBC. Among all films, those containing 10% TBC are most flexible and toughest. Both DSC and microscopy methods suggested that the TBC may be loaded up to 20% to avoid microsized phase separation. Through modeling with experimental data, incorporating 50% TBC reduced the water absorption capacity to 12.94% compared to 31.78% by pure zein film. More importantly, the integrity of zein/TBC film was maintained at high relative humidity and even after immersion in water. However, more than 20% TBC in zein films led to microsized phase separation, which was harmful to mechanical properties.

Reducing the brittleness of zein films through chemical modification.

Zein protein is a major coproduct of biofuel from corn. To reduce the brittleness of zein films, a new type of zein-based biomaterial, was synthesized by chemical modification of zein with lauryl chloride through an acylation reaction. The final products were confirmed by (1)H NMR, FT-IR analysis, and SDS-PAGE. Thermal analysis detected no microphase separation in the synthesized polymer matrix. As the content of lauryl moiety increased, the glass transition temperatures of modified zein decreased by as large as 25.8 °C due to the plasticization effect of the lauryl moiety. In addition, mechanical and surface properties of cast films from acylated zein were also investigated. The elongation at break of modified zein sheet was increased by about 7-fold at the high modification level with some loss of mechanical strength. The surfaces of modified zein films were as uniform as unmodified zein film but more hydrophobic, further suggesting that no microphase separation happened during the film formation process. This work indicated the potential of these new biomaterials in the development of biodegradable food packaging materials and delivery systems.

Recombinant expression of pleurocidin cDNA using the Pichia pastoris expression system.

This research utilized the Pichia pastoris expression system for recombinant expression of cDNA of pleurocidin, a small (2.7 kd) antimicrobial peptide isolated from winter flounder (Pleuronectes americanus). The Pichia vector contains the alcohol oxidase gene promoter (AOX 1), which under the induction of methanol allows for expression of heterologous protein gene inserted downstream in the vector. Two strains of P pastoris were used as host cells, the wild type (P pastoris X-33((mut(+)))) and the mutant (P pasatoris KM71H((mut(s))) ). Polymerase chain reaction (PCR) and DNA sequencing showed that pleurocidin cDNA was successfully integrated into the P pastoris genome. Reverse transcription (RT)-PCR showed that pleurocidin was transcribed by both Pichia host strains. Affinity chromatography, SDS-PAGE, and immunological techniques were used for purification and detection of recombinant peptide. Although there was strong evidence of transcription of pleurocidin cDNA, the Pichia system requires further optimization to obtain detectable levels of this small peptide.

Enhanced production of extracellular ice nucleators from Erwinia herbicola.

The effects of growth conditions and chemical or physical treatments on the production of extracellular ice nucleators (ECINs) by Erwinia herbicola cells were investigated. The spontaneous release of ECINs, active at temperatures higher than -4 degrees C, into the environment depended on culture conditions, with optimal production when cells were grown in yeast extract to an early stationary phase at temperatures below 22 degrees C. ECINs were vesicular, released from cell surfaces with sizes ranging from 0.1 to 0.3 &mgr;m as determined by ultrafiltration and transmission electron microscopy. Protein profiles of ECIN fractions during bacterial growth were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Ina proteins were detected by Western blotting. ECIN production was enhanced 5-fold when cells were treated with EDTA and 20- to 30-fold when subjected to sonication. These conditions provide a means for large-scale preparationage> ECINs by E. herbicola.