Expanding the Applicability of an Innovative Laccase TTI in Intelligent Packaging by Adding an Enzyme Inhibitor to Change Its Coloration Kinetics.

Enzymatic time-temperature indicators (TTIs) usually suffer from instability and inefficiency in practical use as food quality indicator during storage. The aim of this study was to address the aforementioned problem by immobilizing laccase on electrospun chitosan fibers to increase the stability and minimize the usage of laccase. The addition of NaN3, as and enzyme inhibitor, was intended to extend this laccase TTI coloration rate and activation energy (Ea) range, so as to expand the application range of TTIs for evaluating changes in the quality of foods during storage. A two-component time-temperature indicator was prepared by immobilizing laccase on electrospun chitosan fibers as a TTI film, and by using guaiacol solution as a coloration substrate. The color difference of the innovative laccase TTI was discovered to be <3, and visually indistinguishable when OD500 reached 3.2; the response reaction time was regarded as the TTI's coloration endpoint. Enzyme immobilization and the addition of NaN3 increased coloration Km and reduced coloration Vmax. The coloration Vmax decreased to 64% when 0.1 mM NaN3 was added to the TTI, which exhibited noncompetitive inhibition and a slower coloration rate. Coloration hysteresis appeared in the TTI with NaN3, particularly at low temperatures. For TTI coloration, the Ea increased to 29.92-66.39 kJ/mol when 15-25 µg/cm2 of laccase was immobilized, and the endpoint increased to 11.0-199.5 h when 0-0.10 mM NaN3 was added. These modifications expanded the applicability of laccase TTIs in intelligent food packaging.

Enzymatic Time-Temperature Indicator Prototype Developed by Immobilizing Laccase on Electrospun Fibers to Predict Lactic Acid Bacterial Growth in Milk during Storage.

Laccase was immobilized on a chitosan/polyvinyl alcohol/tetraethylorthosilicate electrospun film (ceCPTL) and colored with guaiacol to obtain a laccase time-temperature indicator (TTI) prototype. The activation energy (Ea) of coloration of the prototype was 50.89-33.62 kJ/mol when 8-25 µg/cm2 laccase was immobilized on ceCPTL, and that of lactic acid bacteria (LAB) growth in milk was 73.32 kJ/mol. The Ea of coloration of the TTI prototype onto which 8-10 µg/cm2 laccase was immobilized was in the required range for predicting LAB growth in milk. The coloration endpoint of the TTI prototype onto which 10 µg/cm2 (0.01 U) laccase was immobilized could respond to the LAB count reaching 106 colony-forming units (CFU)/mL in milk during a static temperature response test, and the prediction error was discovered to be low. In dynamic temperature response experiments with intermittent temperature changes between 4 and 25 °C, the coloration rate of the laccase TTI prototype was consistent with LAB growth. The results of this study indicate that the laccase TTI prototype can be applied as a visual monitoring indicator to assist in evaluating milk quality in cold chains.

Silver nanoparticle biosynthesis by using phenolic acids in rice husk extract as reducing agents and dispersants.

Rice husk extract, obtained using acid and alkali pretreatment extraction (AAPE), contains bioactive compounds and exhibits reducing abilities. Phenolic composition in rice husk extract was analyzed and the mechanism of silver nanoparticle (AgNP) biosynthesis by using AAPE rice husk extract was investigated in this study. Stable and spherically shaped AgNPs with a size of <15 nm were prepared under the following conditions: 0.001 M AgNO3, AAPE rice husk extract diluted 10 times, pH 10, and reacted at 25 °C for 60 min. Synergistic effects among phenolic acids contributed to the formation of AgNPs, with the acids acting as excellent reducing agents (owing to their abundant hydroxyl groups) and excellent dispersants (owing to their derived CO groups), which enhanced the NPs' stability. Caffeic acid (CA) was demonstrated to synthesize AgNPs independently and is suggested to be the most crucial compound for reducing Ag+ during the biosynthesis with rice husk extract. A possible mechanism and reaction process for the formation of AgNPs synthesized using CA in rice husk extracts is proposed.

Hurdle Effect of Antimicrobial Activity Achieved by Time Differential Releasing of Nisin and Chitosan Hydrolysates from Bacterial Cellulose.

We investigated the combined antimicrobial effect of nisin and chitosan hydrolysates (CHs) by regulating the antimicrobial reaction order of substances due to differential releasing rate from hydroxypropylmethylcellulose-modified bacterial cellulose (HBC). The minimum inhibitory concentration of nisin against Staphylococcus aureus and that of CHs against Escherichia coli were 6 IU and 200 µg/mL, respectively. Hurdle and additive effects in antimicrobial tests were observed when nisin was used 6 h before CH treatment against S. aureus; similar effects were observed when CH was used before nisin treatment against E. coli. Simultaneously combined treatment of nisin and CHs exhibited the low antimicrobial effect. HBC was then selected as the carrier for the controlled release of nisin and CHs. A 90% inhibition in the growth of S. aureus and E. coli was achieved when 30 IU-nisin-containing HBC and 62.5 µg/mL-CH-containing HBC were used simultaneously. The controlled release of nisin and CHs by using HBC minimized the interaction between nisin and CHs as well as increased the number of microbial targets.

The bioactive composite film prepared from bacterial cellulose and modified by hydrolyzed gelatin peptide.

The hydrolyzed gelatin peptides, obtained from the hydrolysis of Tilapia nilotica skin gelatin with alcalase and pronase E, were fractionated using an ultrafiltration system into hydrolyzed gelatin peptides-a (10 kDa membrane), hydrolyzed gelatin peptides-b1, and hydrolyzed gelatin peptides-b2 (5 kDa membrane) fractions. The highest oxygen radical absorbance capacity was observed in hydrolyzed gelatin peptides-b2, which contained more nonpolar amino acids than the other hydrolyzed gelatin peptides. Hydrolyzed gelatin peptides-b2 at a concentration of 12.5 mg/ml exhibited the highest proliferation ability and increased the expression of Type I procollagen mRNA, which indicated an enhanced collagen synthesis. Hydrolyzed gelatin peptides protected Detroit 551 cells from 2,2'-azobis(2-amidinopropane) dihydrochloride-induced oxidative damage and increased cell viability. Hydroxylpropylmethyl cellulose-modified bacterial cellulose and dried fabricated biofilm were less eligible for Detroit 551 cell proliferation than bacterial cellulose. The release of hydrolyzed gelatin peptides in bacterial cellulose film was slower than that in hydroxylpropylmethyl cellulose-modified bacterial cellulose and dried fabricated biofilm; thus, bacterial cellulose film and hydroxylpropylmethyl cellulose-modified bacterial cellulose and dried fabricated biofilm are suitable candidates for applications in delayed release type and rapid release type biofilms, respectively.

Preparation of thermally stable microcapsules with a chitosan-silica hybrid.

Addition of microcapsules with a high dielectric constant and low specific heat capacity to a battered layer was designed to create a higher temperature in the crust than in the prefried fish nuggets to prevent the water vapor in the fish nuggets from migrating to the crust during microwave heating. Therefore, chitosan-silica hybrids and soybean oil were utilized to prepare the shell and core of the thermally stable microcapsules (MC(CS)), respectively. The MC(CS) were prepared by sol-gel coacervation from an oil-in-water emulsion. The sodium silicate was hydrolyzed and coacervated through polymerization for 24 h at pH 5. The zeta potential analysis indicated that chitosan with a positive charge and silica with a negative charge interacted through electrostatic attraction to form a hybrid shell. The volume mean particle size and encapsulation efficiency of the MC(CS) were 9.6 ± 0.2 µm and 75.6% ± 1.3%, respectively, when oil/chitosan = 0.2 and chitosan/silica = 0.5 (w/w). In addition to H-bonding and electrostatic attraction, Si-O-N bonds were formed between chitosan and silica. Dehydration of the bound water in the MC(CS) was observed in the range of 25 to 250 °C in the differential scanning calorimetry thermal analysis, with the lack of apparent thermal peaks indicating its high thermal stability. The decrease of force to cut the crust observed by texture analysis as well as the increase of hedonic score by consumer acceptance test revealed the addition of 1% MC(CS) significantly improved the crispness of the crust in the microwave-reheated nuggets.

Molecular mechanism for telomere-binding proteins in regulating mammalian spermatogenesis / 上海交通大学学报（医学版）

Telomere attachment to the nuclear envelope and telomere-led chromosomal movement allow proper homologous pairing in mammalian meiotic prophase I. However, the molecular mechanisms for telomere meiosis-specific functions remain to be elucidated. The present review introduced the structure basis for telomere attachment to the nuclear envelope, and revealed a new mechanism by which telomere functions in mammalian spermatogenesis.

Molecular mechanism for telomere-binding proteins in regulating mammalian spermatogenesis / 上海交通大学学报（医学版）

Telomere attachment to the nuclear envelope and telomere-led chromosomal movement allow proper homologous pairing in mammalian meiotic prophase I. However, the molecular mechanisms for telomere meiosis-specific functions remain to be elucidated. The present review introduced the structure basis for telomere attachment to the nuclear envelope, and revealed a new mechanism by which telomere functions in mammalian spermatogenesis.

Influence of alanine uptake on Staphylococcus aureus surface charge and its susceptibility to two cationic antibacterial agents, nisin and low molecular weight chitosan.

Low molecular weight chitosan (LMWC) and nisin, recognized as cationic antibacterial agents (CAAs), inhibit bacterial growth by interacting with the anionically charged cell wall. In this study, alanine uptake significantly reduced the anionic cell surface charge, as determined by the zeta potential (ZP) measurements, of Staphylococcus aureus, resulting from the incorporation of d-alanine into the cell wall. Minimum inhibitory concentration (MIC) tests and growth inhibition curves revealed that LMWC and nisin possessed inverse antibacterial activity against three strains of S. aureus, depending on the strains' net charge. A twofold reduction in the MIC value of nisin was obtained against S. aureus, inoculated in a 1.0% d- or l-alanine-augmented trypticase soy broth medium. A flocculation test demonstrated that neutralizing the anionic surface charge using d-alanine reduced the adsorption of S. aureus onto LMWC. Furthermore, the reduced surface net charge could enhance the colonization capacity of S. aureus on glass.

In situ modification of bacterial cellulose network structure by adding interfering substances during fermentation.

In an attempt to obtain bacterial cellulose (BC) with improved rehydration ability, Tween 80, urea, fluorescent brightener, hydroxypropylmethyl cellulose (HPMC) and carboxymethyl cellulose (CMC) were introduced into BC fermentation medium. Measurements of the mechanical strength of the resulting BCs (TBC, UBC, FBC, HBC and CBC) showed a decline except for UBC. SEM images showed that, although the cellulose bundle widths of FBC, HBC and CBC increase, the cellulose network void in FBC grew, while those in HBC and CBC shrank. X-ray diffraction and FT-IR analysis demonstrated that the addition of HPMC and CMC reduced the degree of crystallinity in their corresponding MBCs from 70.54% to 52.23% and 45.38%, respectively. HBC and CBC also exhibited the highest rehydration ability among all MBCs as well as the lowest crystallinity. The in situ modification with HPMC and CMC during fermentation can effectively improve rehydration ability of BC by altering its network structure.