Immobilization of subtilisin on polycaprolactam for antimicrobial food packaging applications

Subtilisin was immobilized on polycaprolactam and used for food packaging applications to reduce the transference of microorganisms from the packaging material to the packaged food material. The optimized conditions for subtilisin immobilization was as follows: pH, 8; temperature, 4 °C; glutaraldehyde, 0.5%; incubation time, 25 h; and subtilisin concentration, 600 μL. The formation of -CH═N- at 1576 cm(-1) in the Fourier transform infrared (FTIR) spectrum confirmed the immobilization. Subtilisin-immobilized polycaprolactam (SIP) exhibited the highest residual activity of 106.67 ± 4.41% and 104.67 ± 0.88% at 40 °C and pH 8 and retained residual activity of 94% at the end of 56 days when compared to 21.33 ± 4.10% in the case of free subtilisin. SIP significantly (p < 0.05) lowered the colony forming units (CFU), dry weight, and protein and carbohydrate contents in bacterial and fungal biofilm. Practical application of the SIP on ham steaks at 4 and 20 °C showed a 2-3 times reduction of Staphylococcus aureus as well as Escherichia coli cells in the range of p < 0.05.     

Enzyme immunoassay for hen egg white lysozyme used as a food additive

An indirect competitive enzyme immunoassay for hen egg white lysozyme (HEL) used as a food additive was investigated. Anti-HEL antibodies were obtained from B10A mouse ascites immunized by intraperitoneal injection of HEL. HEL samples to be assayed were extracted from foods with 1% gelatin in borate buffer. Goat anti-mouse IgG (H+L)-peroxidase complex was used as a second antibody, and 3,3',5,5'-tetramethylbenzidine was used as a substrate for the peroxidase. The working range for quantitative analysis was 1-50 ng/mL, because in this range the binding inhibition curve of anti-HEL antibodies to HEL-coated plates by HEL was linear. Even after losing the lysozyme activity by heat treatment, HEL could be detected by indirect competitive enzyme immunoassay. Recoveries of HEL by this assay were greater than 85% for Japanese noodles and Japanese traditional-style confectioneries, 53-95% for Miso and cooked beans, and 30-85% for fried fish pastes. HEL contents of 55 commercial foods were determined; HEL was detected in 19 samples in the range 25-20,000 ng/g. HEL as a food additive was detected more frequently in plant-derived foods than in foods of animal origin.     

Human lysozyme possesses novel antimicrobial peptides within its N-terminal domain that target bacterial respiration

Human milk lysozyme is thought to be a key defense factor in protecting the gastrointestinal tract of newborns against bacterial infection. Recently, evidence was found that pepsin, under conditions relevant to the newborn stomach, cleaves chicken lysozyme (cLZ) at specific loops to generate five antimicrobial peptide motifs. This study explores the antimicrobial role of the corresponding peptides of human lysozyme (hLZ), the actual protein in breast milk. Five peptide motifs of hLZ, one helix-loop-helix (HLH), its two helices (H1 and H2), and two helix-sheet motifs, H2-β-strands 1-2 (H2-S12) or H2-β-strands 1-3 (H2-S13), were synthesized and examined for antimicrobial action. The five peptides of hLZ exhibit microbicidal activity to various degrees against several bacterial strains. The HLH peptide and its N-terminal helix (H1) were significantly the most potent bactericidal to Gram-positive and Gram-negative bacteria and the fungus Candida albicans . Outer and inner membrane permeabilization studies, as well as measurements of transmembrane electrochemical potentials, provided evidence that HLH peptide and its N-terminal helix (H1) kill bacteria by crossing the outer membrane of Gram-negative bacteria via self-promoted uptake and are able to dissipate the membrane potential-dependent respiration of Gram-positive bacteria. This finding is the first to describe that hLZ possesses multiple antimicrobial peptide motifs within its N-terminal domain, providing insight into new classes of antibiotic peptides with potential use in the treatment of infectious diseases.     

Preparation and characterization of chitosan-based nanocomposite films with antimicrobial activity

Four different types of chitosan-based nanocomposite films were prepared using a solvent-casting method by incorporation with four types of nanoparticles, that is, an unmodified montmorillonite (Na-MMT), an organically modified montmorillonite (Cloisite 30B), a Nano-silver, and a Ag-zeolite (Ag-Ion). X-ray diffraction patterns of the nanocomposite films indicated that a certain degree of intercalation was formed in the nanocomposite films, with the highest intercalation in the Na-MMT-incorporated films followed by films with Cloisite 30B and Ag-Ion. Scanning electron micrographs showed that in all of the nanocomposite films, except the Nano-silver-incorporated one, nanoparticles were dispersed homogeneously throughout the chitosan polymer matrix. Consequently, mechanical and barrier properties of chitosan films were affected through intercalation of nanoparticles, that is, tensile strength increased by 7-16%, whereas water vapor permeability decreased by 25-30% depending on the nanoparticle material tested. In addition, chitosan-based nanocomposite films, especially silver-containing ones, showed a promising range of antimicrobial activity.     

Some characteristics of sugar ester nonionic microemulsions in view of possible food applications

This study explores some characteristics of microemulsions composed of sucrose monostearate (SMS), medium-chain triglycerides (MCT), or R-(+)-limonene, alcohols, and water. The systems are homogeneous, soft, and waxy solids at room temperature but liquefy and structure into homogeneous microemulsions when heated to >40 degrees C. The amount of solubilized water is enhanced as a function of the alcohol/oil ratio and is inversely proportional to the alcohol chain length. Over 60 wt % water can be solubilized in systems consisting of propanol/MCT/SMS at a weight ratio of 3:1:4 (initial weight ratio). These microemulsions are unique and differ from nonionic ethoxylated-based microemulsions in that their viscosity is very low and is reduced with increasing amounts of solubilized water. The electrical conductivity increases only slightly as a function of the water content and does not show typical bicontinuous or percolated behavior. The water in the core of the microemulsion strongly binds to the headgroups of the surfactant. Only at >15 wt % solubilization of water was free or bulk water detected in the core of the microemulsions. Such unique behavior of the core water might have a possible application in systems requiring monitoring of enzymatic (lipase) reactions carried out in the microemulsions as microreactors.     

Self-assembled pullulan-silica oxygen barrier hybrid coatings for food packaging applications

The scope of this study encompassed the evaluation of pullulan as a suitable biopolymer for the development of oxygen barrier coatings to be applied on poly(ethylene terephthalate) (PET), especially for food packaging applications. To enhance the oxygen barrier properties of the organic phase (pullulan) even at high relative humidity values, an inorganic phase (silica), obtained through in situ polymerization, was also utilized to obtain hybrid coatings via the sol-gel technique. Transmission electron microscopy (TEM) images and Fourier transform infrared (FT-IR) spectra showed that mixing the two phases yielded a three-dimensional hybrid network formed by self-assembly and mediated by the occurrence of new hydrogen-bond interactions at the intermolecular level, although the formation of new covalent bonds could not be excluded. The deposition of the hybrid coatings decreased the oxygen transmission rate (OTR) of the plastic substrate by up to 2 orders of magnitude under dry conditions. The best performance throughout the scanned humidity range (0%-80% relative humidity) was obtained for the formulation with the lowest amount of silica (that is, an organic/inorganic ratio equal to 3).     

Solubilized wheat protein isolate: functional properties and potential food applications

Solubility, foaming capacity/stability, water holding and fat absorption capacities, and emulsifying capacity/stability of a solubilized wheat protein isolate (SWPI) were compared with those of commercial protein, that is, sodium caseinate (NaCAS), dried egg white (DEW), nonfat dry milk (NFDM), and soy protein isolate (SPI). SWPI was highly soluble at pH 6.5-8.5. Foaming capacity of SWPI was superior to those of SPI, NFDM, and DEW, and its foaming stability was similar to those of the commercial proteins. Foaming properties of SWPI were greatly improved in the presence of 0.5% (w/v) CaCl(2). Water holding capacity of SWPI was greater than that of NaCAS, NFDM, and DEW, whereas its fat absorption capacity was comparable to that of SPI, NaCAS, and DEW. SWPI exhibited emulsifying properties similar to those of SPI. SWPI was incorporated at 5, 10, 15, or 20% into ice cream, chocolate chip cookies, banana nut muffins, and hamburger patties. Products containing <5% SWPI were acceptable to consumers.     

国外农产品及食品介电特性测量技术及应用

了解农产品和食品的介电特性对于微波设备和基于介电特性的物质组成成份检测仪的研发具有重要的意义。为了给国内农产品和食品介电特性的研究提供借鉴,该文介绍了目前国外常用的介电特性测量技术——平行极板技术、同轴探头技术、传输线技术、自由空间法和谐振腔技术;说明了各测量技术的特点、测试系统的组成和适用范围;综述了各技术在农产品和食品介电特性研究中的应用情况;并指出中国目前在该领域研究中存在的问题和今后的研究重点。     

核桃壳木醋液的制取、成分分析及抑菌试验（简报）

为了充分利用核桃壳资源,以核桃壳为原料,采用干馏的方法,分90～150℃、150～310℃、310～550℃ 3个温度段收集馏出液,经静置、木炭粉吸附焦油处理等过程,制得核桃壳木醋液。然后对所得木醋液的基本性质和抑菌活性进行了研究,并对抑菌活性最强的木醋液的化学成分进行了分析。结果表明：核桃壳木醋液产生的温度范围为90～550℃;各温度段所得木醋液精制后的得率随干馏温度的增加而降低,密度随温度增加而增加,pH值随温度升高呈增加趋势,有机酸含量则呈降低趋势;各温度段核桃壳木醋液对测试菌均有抑制作用,尤其是较高温度段（150～310℃、310～550℃）的抑菌效果较好。气相色谱-质谱（GC-MS）分析表明,温度段为150～310℃的木醋液中含有约43种化合物,其中主要成分为酸类、酚类、酮类、醛类和杂环化合物等。其中含量最多的是酚类化合物和有机酸,其含量占总检出成分的80.70％。初步分析其抑菌的有效成分为酚类物质。     

串联磁致伸缩生物传感器的制备和食品中大肠杆菌的检测

磁致伸缩生物传感器的制造成本、检测灵敏度、检测效率是影响传感器广泛应用的重要因素。为研究串联磁致伸缩生物传感器对食品中大肠杆菌的检测性能,该研究研制了一种新型串联磁致伸缩生物传感器,该传感器的换能器采用铁基非晶软磁合金,功能涂层采用环保亲水材料聚乙烯醇。将不同尺寸的单一传感器作为对照组,利用阻抗分析仪检测传感器的谐振参数。选取尺寸为4.8 mm×0.8 mm与7.2 mm×1.2 mm的传感器进行串联,将无抗体加载的传感器作为参考传感器,检测浓度为10~10⁸ CFU/mL大肠杆菌悬浮液。利用扫描电子显微镜观察传感器表面吸附致病菌情况。探索串联磁致伸缩传感器检测大肠杆菌食品溶液时谐振频率漂移量与浸泡时间的关系。研究发现：该串联磁致伸缩生物传感器相较于单一传感器具有更好的响应强度。通过对不同浓度大肠杆菌悬浮液的检测,得到该串联抗体传感器与大肠杆菌发生特异性结合,检测限为10² CFU/mL。利用扫描电子显微镜观察传感器表面,证实谐振频率偏移量均由吸附大肠杆菌引起。利用阻抗分析仪检测串联传感器在大肠杆菌食品溶液中浸泡不同时间后的谐振频率漂移量,得到传感器实现稳定检测时间为20 min。表明此新型串联磁致伸缩生物传感器具有较高的灵敏度、稳定性、检测效率且制造简单,成本较低,使用方便。该研究可为生物传感器在食品安全检测方面的广泛应用提供有力参考。     

Development and characterization of silver-based antimicrobial ethylene-vinyl alcohol copolymer (EVOH) films for food-packaging applications

The use of silver as an antimicrobial in the food area has raised wide interest in recent years. In the present work, 0.001-10 wt % silver ions was satisfactorily incorporated into an ethylene-vinyl alcohol (EVOH) copolymer matrix by a solvent casting technique. The antibacterial efficacy of the composite was evaluated under laboratory conditions and in contact with some foods. The ionic compound did not affect the crystallinity or the water-induced plasticization of the materials and was homogeneously distributed across the surface and thickness of the films. When immersed in water, sorption-induced release of 50-100% of the silver ions took place in <30 min. In the bacterial minimal growth medium M9, the minimal inhibitory concentration (MIC) of the film was in the range of 0.01-0.1 ppm. High protein content food samples displayed low susceptibility to the films (<1 log reduction in any case), whereas low protein content food samples exhibited no detectable bacterial counts for films with 1 and 10 wt % silver and about 2 log reduction for films with 0.1 wt % silver. These results represent a step forward in the understanding of silver antimicrobial efficacy and its possible application in the food-packaging industry, most likely as food coatings.     

Resistance screening essay of wine lactic acid bacteria on lysozyme: efficacy of lysozyme in unclarified grape musts

In wine making, the bacteriolytic activity of lysozyme has primarily been used to control the malolactic fermentation in wines. The use of lysozyme in musts before settling and the beginning of the alcoholic fermentation to inhibit the growth of lactic acid bacteria could be very beneficial. In a resistance test carried out in MT/b broth, lysozyme had greater antimicrobial activity toward Oenococcus oeni than Lactobacillus species. Several strains of wine bacteria belonging to Oenococcus proved sensitive to the bacteriolytic activity of lysozyme at low concentrations in both synthetic medium (MT/b) (50 mg/L), white must, or red must made with or without the skins (100 mg/L). Lactobacillus and Pediococcus strains survived at lysozyme concentrations of 200-500 and 500 mg/L, respectively, in MT/b and musts. Suspended solids in unclarified musts may strongly bind to lysozyme thereby causing its removal by filtration or centrifugation. One hour after lysozyme was added to musts, it was quantified by HPLC and found after centrifugation to be 40-50% and only 10% in musts made with or without the skins, respectively. Although appreciable amounts of lysozyme were bound to wine components, this did not appear to be a serious hindrance to lysozyme activity.     

减磷施肥配施生物炭对南方酸性稻作土壤磷素形态的影响

针对南方酸性稻作土壤专性吸附和固定磷素能力强的问题,采用微区试验,以正常施磷(75 kg/hm²)不施生物炭为对照,探讨不同磷肥水平(P2O5 75、60、45、30 kg/hm²)和生物炭配施(40、60、80 t/hm²)对土壤不同形态磷素含量的影响。结果表明：(1)减磷施肥配施生物炭后全磷、有效磷、树脂磷、碳酸氢钠无机磷及稀盐酸磷的含量显著增加,残留态磷的含量显著降低。(2)磷肥水平≥45 kg/hm²时,配施生物炭处理的全磷和有效磷含量较对照分别显著提高17.1%～46.7%和32.5%～76.3%。(3)磷肥水平≥45 kg/hm²时,配施生物炭处理的树脂磷和碳酸氢钠无机磷的含量较对照分别显著增加50.8%～160.3%和36.1%～118.3%。(4)减磷施肥导致氢氧化钠有机磷含量较对照下降38.4%～39.8%;处理间氢氧化钠无机磷的含量无显著差异。(5)配施生物炭后稀盐酸磷含量较对照显著提高57.9%～352.1%;减磷施肥处理的浓盐酸有机磷含量显著低于正常施磷...     

Detection and quantification of lysozyme in champagne wines

We describe here techniques to detect and quantify lysozyme in Pinot noir and Chardonnay Champagne wines. Using a dot-blot technique, lysozyme antibodies were able to recognize their antigens even when the concentration of lysozyme in wine was 75 mg/L. SDS-PAGE was the second technique used. After Coomassie Brilliant Blue (CBB) staining or antibody immunostaining was performed, the wine originating from the lysozyme-treated must gave only one band corresponding to the lysozyme. It is then possible to precisely determine the concentration of lysozyme in a must or a wine by densitometric measurement of this band. The control wine gave no band with the CBB staining, such as with the immunostaining. The quantification of lysozyme with HPLC is another useable technique because the lysozyme elution time is largely superior to that of all of the wine compounds. In wines, losses of lysozyme were higher when the enzyme was added at one time to the must (-34% for the Pinot noir and -37% for the Chardonnay) than when lysozyme is added in 2-fold both in the must and in the wine (around -26% for the two wines). The lowest diminution is observed when lysozyme was added to the wine only (-18%) in comparison to the addition to the must at 300 mg/L (-43%).     

Development of sensitive crop-specific polymerase chain reaction assays using 5S DNA: applications in food traceability

The 5S intergenic spacers were amplified using a common pair of primers and sequenced from four species (Brassica napus, Zea mays, Helianthus annuus, and Glycine max). Crop-specific assays were developed from primers designed from the spacers and tested to amplify corresponding DNAs in both conventional end-point and real-time polymerase chain reactions (PCRs). The high copy numbers of the 5S DNA in plants make it possible to detect very small amounts of DNA using this marker. This sensitivity made it possible to compare different DNA extraction methods for highly processed food products using 5S spacers, even allowing dilution of templates to overcome PCR inhibition.     

Using ATR-FTIR spectroscopy to design active antimicrobial food packaging structures based on high molecular weight chitosan polysaccharide

ATR-FTIR spectroscopy has been used in this study to characterize the molecular mechanisms and kinetic processes that take place when a chitosonium acetate thin coating is put in contact with water solutions, Staphylococcus aureus solutions, microbial nutrient solutions, and with a high water activity TSA hydrogel medium to simulate the effect of direct contact with high moisture foods such as fresh meats, fish, and seafood products or beverages. The results of this work suggest that the biocide carboxylate groups that form when chitosan is cast from acetic acid solutions are being continuously evaporated from the formed film in the form of acetic acid (mechanism I) in the presence of environmental humidity, rendering weak biocide film systems. On the other hand, upon direct contact of the cast chitosonium acetate film with liquid water, water solutions, or the high moisture TSA hydrogel, a positive rapid migration, with a diffusion coefficient faster than 3.7 x 10(-12) m2/s, of protonated glucosamine water soluble molecular fractions (mechanism II) takes place from the film into the liquid phase, yielding strong antimicrobial performance and leaving in the remaining cast film only the non-water soluble chitosan fractions. Finally, this study describes a refined spectroscopic methodology to predict the antimicrobial properties of chitosan and gives insight into the capacity of chitosan as a natural biocide agent.     

Sweetness and enzymatic activity of lysozyme

Hen egg lysozyme elicits a sweet taste sensation for human beings. Effects of reduction of disulfide bonds, heat treatment, and chemical modification of hen egg lysozyme on both sweetness and hydrolytic activity were investigated. Both the sweetness and enzymatic activities were lost when the intradisulfide linkage in a lysozyme molecule was reduced and S-3-(trimethylated amino) propylated. The sweetness and enzymatic activity of lysozyme were lost on heating at 95 degrees C for 18 h. These facts suggest that tertiary structures of lysozyme are indispensable for eliciting a sweet taste as well as enzymatic activity. Although the modification of carboxyl residues in a lysozyme by glycine methylester or aminomethansulfonic acid resulted in the loss of enzymatic activity by blocking the catalytic residues, the sweetness was fully retained. These results indicate that the sweetness of lysozyme was independent of its enzymatic activity. The lysozyme purified from goose egg white similarly elicited a sweet taste, although goose (g-type) lysozyme is quite different from hen egg lysozyme (c-type) on the basis of structural, immunological, and enzymatic properties. These findings indicate that a specific protein property of lysozyme is required for sweetness elicitation and that the enzymatic activity and carbohydrates produced by enzymatic reaction are not related to the sweet taste.     

Maize meal fortification is associated with improved vitamin A and iron status in adolescents and reduced childhood anaemia in a food aid-dependent refugee population

OBJECTIVE: To assess changes in the Fe and vitamin A status of the population of Nangweshi refugee camp associated with the introduction of maize meal fortification. DESIGN: Pre- and post-intervention study using a longitudinal cohort. SETTING: Nangweshi refugee camp, Zambia. SUBJECTS: Two hundred and twelve adolescents (10-19 years), 157 children (6-59 months) and 118 women (20-49 years) were selected at random by household survey in July 2003 and followed up after 12 months. RESULTS: Maize grain was milled and fortified in two custom-designed mills installed at a central location in the camp and a daily ration of 400 g per person was distributed twice monthly to households as part of the routine food aid ration. During the intervention period mean Hb increased in children (0.87 g/dl; P < 0.001) and adolescents (0.24 g/dl; P = 0.043) but did not increase in women. Anaemia decreased in children by 23.4% (P < 0.001) but there was no significant change in adolescents or women. Serum transferrin receptor (log10-transformed) decreased by -0.082 microg/ml (P = 0.036) indicating an improvement in the Fe status of adolescents but there was no significant decrease in the prevalence of deficiency (-8.5%; P = 0.079). In adolescents, serum retinol increased by 0.16 micromol/l (P < 0.001) and vitamin A deficiency decreased by 26.1% (P < 0.001). CONCLUSIONS: The introduction of fortified maize meal led to a decrease in anaemia in children and a decrease in vitamin A deficiency in adolescents. Centralised, camp-level milling and fortification of maize meal is a feasible and pertinent intervention in food aid operations.     

Development of an ELISA for quantifying lysozyme in hen egg white

An indirect enzyme-linked immunosorbent assay (ELISA) by inhibition was developed for quantifying lysozyme in hen egg white (HEW), a protein of value in not only the food and pharmaceutical industries but also for poultry research. Various experimental conditions (coating, antibodies dilutions, samples dilutions, preparations, blocking agents, and incubation times) were assayed to optimize this assay to the quantification of HEW in egg white samples. HEW samples were diluted 1:3000 to avoid matrix effects, possibly resulting from lysozyme interaction with other egg white proteins. Assay linearity for lysozyme ranged from 0.38 to 4.8 mug/mL, with intra- and interassay variations of 6.8% and 7.6%, respectively, and the lower detection limit was 0.264 mug/mL. We found that lysozyme concentrations in albumen from eggs laid by a hen cohort ranged from 2.2 to 4.5 mg/mL, thus underlining interhen variability. Overall, these data present an ELISA assay that is simple, quick, sensitive, accurate, and has been specifically designed to determine lysozyme concentrations in egg white samples.