pH-responsive antibacterial film based polyvinyl alcohol/poly (acrylic acid) incorporated with aminoethyl-phloretin and application to pork preservation.

This study demonstrates a pH-responsive antibacterial film based on polyvinyl alcohol/poly (acrylic acid) incorporated with aminoethyl-phloretin (PVA/PAA-AEP) for intelligent food packaging. The thermal, mechanical, barrier and light transmittance properties of PVA/PAA are enhanced by PAA presence of ≤6%. The interactions between PVA and PAA were hydrogen and ester bonds. The pH-responsive characteristic is dependent on the protonation/deprotonation tendency of the carboxylic groups on PAA in acidic/alkaline environment. The PVA/PAA3 is selected for the incorporation of AEP and its pH-responsive swelling follows Ritger-Peppas and Schott second-order models. The AEP is hydrogen bonded with the matrix of PVA/PAA3 and the release of AEP is pH-responsive and a rate-limiting step following the First-order model. With pH decrease, the predominant release control was gradually changing from polymer relaxation to Fick diffusion. The PVA/PAA3-AEP films demonstrate AEP content dependent antioxidant and antimicrobial activities. Furthermore, the antibacterial efficiency against Listeria monocytogenes and Staphylococcus aureus is significantly better than Escherichia coli. The target film PVA/PAA3-AEP3 can effectively prolong the shelf-life of pork (TVB-N < 25 mg/100 g) by 4 days at 25 °C, suggesting its great potential in intelligent food packaging.

Evaluation of Orchid-Like Aroma Between Different Grades of Taiping Houkui Tea by Solid-Phase Microextraction and Comprehensive Two-Dimensional Gas Chromatography Coupled with Time-of-Fight Mass Spectrometry.

BACKGROUND: Taiping houkui tea won the title of "the King of Green Tea" at the International Tea Exposition in 2004, which had an orchid fragrance but the material basis of the orchid fragrance had not been revealed yet. OBJECTIVE: To investigate the material basis of the orchid fragrance and identify the quality grade of Taiping houkui tea. METHODS: A method was developed for evaluating orchid-like aroma between different grades Taiping houkui tea by solid-phase micro extraction (SPME) and comprehensive two-dimensional gas chromatography coupled with time-of-fight mass spectrometry (GC×GC-TOFMS). The crushed tea sample in a 25 mL headspace bottle was incubated at 90°C for 10 min. Then the gaseous sample was extracted by SPME divinylbenzene/polydimethylsiloxane fiber for 50 min and thermally desorpted at 250°C for 2 min. RESULTS: In total, 735 volatile compounds were determined by SPME- GC×GC-TOFMS and 15 compounds were related to orchid-like aroma. CONCLUSIONS: The high-quality Taiping houkui tea has the orchid fragrance and the low-grade one does not; thus orchid-like aroma can be used as a reference of grades of Taiping houkui tea. HIGHLIGHTS: Thirty different grades of Taiping houkui tea samples were compared and analyzed experimentally, and the results showed that the special aroma substances, which played a key role in grade discrimination of Taiping houkui tea, were found by the statistical method.

Microwave-Assisted Extraction Combined with Ultra-High-Performance Liquid Chromatography and Quadrupole/Q-Exactive High-Resolution Mass Spectrometry for the Determination of Main Flavor Substances in Green Tea.

BACKGROUND: In order to make up for the efficacy of sensory assessment of green tea quality and taste, main flavor substances should be quantitatively characterized based on efficient extraction and frontier determination scheme. METHODS: A new, relatively simple sample preparation and detection workflow has been developed for the quantification and confirmation of flavor substances in green tea by microwave-assisted extraction combined with ultra-high-performance LC and quadrupole/Q-Exactive high-resolution MS system. The method identified main flavor substances (i.e., catechins, free amino acids, and caffeine) that were the main components in the formation of tea taste. After crushing pretreatment, tea was extracted and purified simultaneously in a microwave extraction jar by matrix-dispersed solid-phase extraction. The extracting solution was determined by LC coupled to high-resolution Orbitrap mass spectrometry. RESULTS: In this study, extraction solvent and purification materials were selected, extraction temperature and time were optimized, and the amount and proportion of solid-phase extraction packing were optimized. CONCLUSIONS: The method enabled detection of multiple catechins, amino acids, and caffeine in tea by LC coupled to high-resolution Orbitrap MS, and a database of tea flavor substances was established. HIGHLIGHTS: The method could be used for screening and confirmation of the main flavor substances in tea as well as assisting the sensory evaluation to grade the quality of tea.

Construction of an integration vector carrying hygromycin B resistance gene and its genetic transformation in Rhizopus oryzae / 生物工程学报

To construct a system of genetic transformation suitable for Rhizopus oryzae, we constructed a single-exchange vector pBS-hygro carrying hygromycin B resistance gene (hph) as its selective marker using gene splicing by overlap extension PCR (SOE PCR) technique. We introduced this recombinant vector into Rhizopus oryzae AS 3.819 by PEG/CaCl2-mediated transformation of protoplast, electroporation of protoplast and germinated spores; and we studied the effects of hydrolysis time, field strength and spore germination time on transformation frequency. We conducted quantitative real-time PCR (qPCR) assay to determine the gene copy number of ldhA integrated in the genome of R. oryzae transformants and its effect on the stability of transformants. We successfully achieved R. oryzae transformants integrated with pBS-hygro-ldhA vector. The optimal hydrolysis time for protoplast production was 140 min, and the optimal field strength of electroporation pulse for protoplast was 13 kV/cm. The optimal germination time of spores for electroporation was 2.5 h, and the optimal field strength of electroporation pulse was 14 kV/cm. The transformation frequency of method based on germinated spores was generally higher than the methods based on protoplast. The qPCR test results suggested that transformants with high copy number of integration in a certain range were relatively stable. Our results provided basis and support for metabolic regulation and genetic engineering breeding of R. oryzae.

Effects of germanium on cell growth, polysaccharide production and cellular redox status in suspension cultures of protocorm-like bodies of Dendrobium huoshanense / 生物工程学报

To solve the problem of low growth rate and metabolism level in suspension cultures of protocorm-like bodies (PLBs) of Dendrobium huoshanense. The effects of germanium on PLB proliferation and accumulation of polysaccharides together with nutrient utilization were investigated and the contents of reducing sugars, soluble proteins, the activities of antioxidant enzymes and redox status of the cells of PLB were analyzed. The results indicated that the optimum concentration of germanium dioxide (4.0 mg/L) significantly enhanced the cell growth and accumulation of polysaccharides, greatly improved contents of reducing sugars and soluble proteins, increased the activities of superoxide dismutase (SOD) and catalase (CAT) but decreased the activity of peroxidase(POD). The cell dry weight and production of polysaccharides were 32.6 g/L and 3.78 g/L, respectively. The analysis of cellular redox status showed that the ratio of reduced glutathione (GSH) to oxidized glutathione (GSSG) in cells and the activity of glutathione reductase were significantly increased by the addition of germanium dioxide. The suitable concentration of germanium dioxide was beneficial to the cell growth and the accumulation of polysaccharides.

Repeated intermittent L-lactic acid fermentation technology by self-immobilized Rhizopus oryzae / 生物工程学报

In order to carry out the process of the repeated intermittent L-lactic acid fermentation by self-immobilized Rhizopus oryzae, we investigated the effect of medium compositions on the morphology of Rhizopus oryzae. In submerged culture, fungi can be grown as broths of freely suspended mycelia and pellets or clumps, the pellets can be immobilized by themselves. The optimum medium composition for the first patch of fermentation was: 120 g/L glucose, 3 g/L NH4NO3, 0.14 g/L KH2PO4, 0.16 g/L NaH2PO4 (the concentration ratio of K+ and Na+ was 1:1). After 72 h fermentation, the physical form of Rhizopus oryzae was mostly uniform pellet with the diameter of 1.0-2.0 mm, the concentration of L-lactic acid was 100.8 g/L, and the conversion rate of glucose was 84%. During 16 batches of repeated fermentation, the L-lactic acid level was above 60.0 g/L and the conversion rate of glucose was 75%. The fermentation time of every batch was 24 h.

Breeding of Actinobacillus succiniogenes mutants with improved succinate production based on metabolic flux analysis / 生物工程学报

It is very important to obtain high yield mutant strains on the base of metabolic flux analysis of Actinobacillus succinogenes S.JST for the industrial bioconversion of succinic acid. The metabolic pathway was analized at first and the flux of the metabolic networks was calculated by matrix. In order to decrease acetic acid flux, the strains mutated by soft X-ray of synchronous radiation were screened on the plates with high concentration of fluoroacetic acid. For decreasing the metabolic flux of ethanol the site-directed mutagenesis was carried out for the reduction of alcohol dehydrogenase(Adh) specific activity. Then the enzyme activity determination and the gene sequence analysis of the mutant strain was compared with those of the parent strain. Metabolic flux analysis of the parent strain indicated that the flux of succinic acid was 1.78(mmol/g/h) and that the flux of acetic acid and ethanol were 0.60 (mmol/g/h) and 1.04( mmol/g/h), respectively. Meanwhile the metabolic pathway analysis showed that the ethanol metabolism enhanced the lacking of H electron donor during the synthesis of succinic acid and that the succinic acid flux was weakened by the metabolism of byproducts ethanol and acetic acid. Compared with the parent strain, the acetic acid flux of anti-fluoroacetic mutant strain S.JST1 was 0.024 (mmol/g/h), decreasing by 96%. Then the enzyme determination showed that the specific activity unit of phosphotransacetylase(Pta) decreased from 602 to 74 and a mutated site was founded in the pta gene of the mutant strain S.JST1. Compared with that of the parent strain S.JST1 the ethanol flux of adh-site-directed mutant strain S.JST2 was 0.020 (mmol/g/h), decreasing by 98%. Then the enzyme determination showed that the specific activity unit of Adh decreased from 585 to 62 and the yield of end product succinic acid was 65.7 (g/L). The interdiction of Adh and Pta decreased the metabolism of byproducts and the H electron donor was well balanced, thus the succinic acid flux was strengthened by the redundant carbon flux from these byproducts. The mutant strain S.JST2 obtained in this paper deserves being extended to application of industrial fermentation.