Microscopic and Spectroscopic Evaluation of Inactivation of <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> by Pulsed UV Light and Infrared Heating

Pulsed UV light and infrared heat-treated Staphylococcus aureus cells were analyzed using transmission electron microscopy to identify the cell damage due to the treatment process. A 5-s treatment with pulsed UV light resulted in complete inactivation of S. aureus even after enrichment. The temperature increase during the pulsed UV light treatment was insignificant, which suggested a nonthermal treatment. S. aureus was also infrared heat treated using an infrared heating system with six infrared lamps. Five milliliters of S. aureus cells in phosphate buffer was treated at 700°C lamp temperature for 20 min. The microscopic observation clearly indicated that there was cell wall damage, cytoplasmic membrane shrinkage, cellular content leakage, and mesosome disintegration after both pulsed UV light and infrared treatments. Fourier transform infrared microspectrometry was successfully used to classify the pulsed UV light and infrared heat-treated S. aureus by discriminant analysis.     

Inactivation of Staphylococcus aureus by pulsed UV-light sterilization

Pulsed UV light is a novel technology to inactivate pathogenic and spoilage microorganisms in a short time. The efficacy of pulsed UV light (5.6 J/cm2 per pulse) for the inactivation of Staphylococcus aureus as suspended or agar seeded cells was investigated. A 12-, 24-, or 48-ml cell suspension in buffer was treated under pulsed UV light for up to 30 s, and 0.1 ml of sample was surface plated on Baird-Parker agar and incubated at 37 degrees C for 24 h to determine log reductions. Also, 0.1 ml of cell suspension in peptone water was surface plated on Baird-Parker agar plates, and the plates were treated under pulsed UV light for up to 30 s. The treated and untreated plates were incubated in the conditions described above. A 7- to 8-log CFU/ml reduction was observed for suspended and agar-seeded cells treated for 5 s or longer. In the case of suspended cells, the sample depth, time, treatment, and interaction were significant (P < 0.05). In the case of agar-seeded cells, the treatment time was significant (P < 0.05). Our results clearly indicate that pulsed UV technology has potential for the inactivation of pathogenic microorganisms.     

UV disinfection of Giardia lamblia cysts in water

The human and animal pathogen Giardia lamblia is a waterborne risk to public health because the cysts are ubiquitous and persistent in water and wastewater, not completely removed by physical-chemical treatment processes, and relatively resistant to chemical disinfection. Given the recently recognized efficacy of UV irradiation against Cryptosporidium parvum oocysts, the inactivation of G. lamblia cysts in buffered saline water at pH 7.3 and room temperature by near monochromatic (254 nm) UV irradiation from low-pressure mercury vapor lamps was determined using a "collimated beam" exposure system. Reduction of G. lamblia infectivity for gerbils was very rapid and extensive, reaching a detection limit of >4 log within a dose of 10 JM-2. The ability of UV-irradiated G. lamblia cysts to repair UV-induced damage following typical drinking water and wastewater doses of 160 and 400 JM(-2) was also investigated using experimental protocols typical for bacterial and eucaryotic DNA repair under both light and dark conditions. The infectivity reduction of G. lamblia cysts at these UV doses remained unchanged after exposure to repair conditions. Therefore, no phenotypic evidence of either light or dark repair of DNA damage caused by LP UV irradiation of cysts was observed at the UV doses tested. We conclude that UV disinfection at practical doses achieves appreciable (much greater than 4 log) inactivation of G. lamblia cysts in water with no evidence of DNA repair leading to infectivity reactivation.     

Impact of dried,creamed and cake supply formats on the genetic variation and ethanol tolerance of three Saccharomyces cerevisiae distilling strains

Scotch whisky fermentations typically employ high‐gravity fermentation practices to maximize product formation and to minimize both energy and water inputs. This approach increases ethanol concentrations at the end of fermentation, creating stressful conditions for the yeast. In this work we examined the relative tolerance of four Saccharomyces cerevisiae distilling yeast strains, supplied in dried, creamed, cake or slurry format, to ethanol under CO₂‐induced anaerobic conditions. The cells were assessed for their capacity to recover and grow on inhibition spot plates and to maintain cell viability in ethanol‐dosed suspensions. Variations in ethanol tolerance were observed between strains and between the same strain supplied in different formats. The creamed yeast format typically exhibited a higher tolerance to ethanol. One possible explanation for this observation is that cells surviving the dehydration and rehydration process might incur sub‐lethal genome damage. Thus the genetic integrity of the most ethanol‐tolerant strain was assessed as a function of supply format (two dried and one creamed). The mitochondrial DNA was examined using mitochondrial restriction fragment length polymorphism and the chromosomal DNA using pulsed field gel electrophoresis and polymerase chain reaction with both ITS and delta‐specific primers. In one dried yeast sample, genetic integrity was compromised, highlighting the requirement for yeast intake quality assurance programmes. Copyright © 2012 The Institute of Brewing & Distilling     

Characterization of UV-peroxide killing of bacterial spores

Advantage is taken in many sterilization processes, especially for food packaging materials, of the synergy between H2O2 and UV irradiation for spore killing. The nature of the synergy is currently not well defined in terms of targets and mechanisms. We found that under some experimental conditions, the synergistic killing of spores of Bacillus megaterium ATCC 19213 appeared to be mainly UV-enhanced peroxide killing, while under other conditions, it appeared to be mainly peroxide-enhanced UV killing. Lethal combinations of H2O2 and UV irradiation for spores resulted in only modest increases in auxotrophic mutations among survivors, indicative of little DNA damage, in contrast to higher mutation levels after dry-heat damage at 115 degrees C. However, the combination of UV light and peroxide did lead to major inactivation of glucose 6-phosphate dehydrogenase, an enzyme that was used to monitor the damage to bacterial protein. Synergistic UV-H2O2 killing was reduced by agents such as pyruvate, thiosulfate, and iron or copper cations, which appeared to act at least in part by reacting chemically with H2O2, and was only slightly affected by the use of UV light at a wavelength of 222 nn rather than 254 nm. Hydrogen peroxide treatment can precede UV irradiation for synergistic killing by some hours with an interim of drying for spores of Bacillus subtilis A, a spore type used commonly for the validation of aseptic processes. Synergistic killing of dried spores or those in suspensions was accelerated at higher temperatures (50 degrees C) rather than at lower temperatures (25 degrees C).     

Adaptive antioxidant response and photoaging

As the skin is always in contact with oxygen and is increasingly exposed to environmental and artificial ultraviolet (UV) irradiation the risk of photooxidative damage induced by reactive oxygen species – finally leading to phototoxicity, photoaging and skin cancer – has increased substantially. The term reactive oxygen species (ROS) includes oxygen centered radicals like the superoxide anion radical and the hydroxyl radical, but also non‐radical species such as hydrogen peroxide and singlet oxygen – all being produced in skin upon UV irradiation. In response to the attack of reactive oxygen species the skin has developed a complex antioxidant defense system including enzymatic and non‐enzymatic antioxidants. As a first line of the enzymatic antioxidative defense, superoxide dismutases reduce superoxide anion radicals to hydrogen peroxide which subsequently is detoxified to water by catalase and glutathione peroxidases. We were interested whether the antioxidant enzymes manganese superoxide dismutase (SOD2) and glutathion peroxidase (GPx1) are inducible upon UV irradiation and whether repetitive UV exposure, as practiced for the light‐hardening during phototherapy of photodermatoses, can even enhance the adaptive antioxidant response. To address this question skin fibroblasts and keratinocytes were exposed in vitro to single and repetitive UV low dose irradiation in different time intervals and afterwords challenged by high dose irradiation. The antioxidant response was measured in terms of steady state mRNA levels and activity changes of SOD2 or GPx1 as well as of the viability after challenge with high dose UV‐irradiation. Interstingly, only UVA but not UVB irradiation was able to induce the mRNA steady state levels and the activity of SOD2 in fibroblasts. However, fibroblasts incubated with the supernatants from UVB‐irradiated epidermal cells responded with an increase in SOD2. This increase on mRNA and activity levels was mediated by paracrine acting secreted factors produced by the keratinocytes. If fibroblasts were exposed repetitively to sublethal UVA doses the further up‐regulation of SOD2 correlated with the protection against high UV doses. Importantly, SOD2 basal levels of protein content and activity substantially differed within cultivated cells and skin biopsies from different individuals. These results provide evidence for an adaptive antioxidative UV response of the skin. Interindividual differences might account for differences in the susceptibility to develop photodermatologic disorders related to photosensitivity, photoaging, and skin cancer.     

Oral administration of bovine lactoferrin attenuates ultraviolet B-induced skin photodamage in hairless mice

Lactoferrin (LF) is recognized as a host defensive glycoprotein, especially for newborn infants. The aim of this study was to investigate whether orally administered LF had protective activity against UV-induced skin damage in hairless mice. Transepidermal water loss and skin hydration were evaluated in nonirradiated mice, UVB-irradiated mice, and UVB-irradiated and LF-administered mice. Supplementation with LF (1,600 mg/kg per day) effectively suppressed the increase in transepidermal water loss, reduction in skin hydration, aberrant epidermal hyperplasia, and cell apoptosis induced by UV irradiation. Although no significant changes in superoxide dismutase-like activity or malondialdehyde levels were observed in the skin with both UV irradiation and LF administration, UV-stimulated IL-1β levels in the skin were significantly suppressed by the administration of LF. Oral supplementation with LF has the potential to reduce IL-1β levels and prevent UV-induced skin damage. Further studies are needed to elucidate the relationships between the antiinflammatory effects and skin protective function of LF.     

Daily Ingestion of Aloe Vera Gel Powder Containing Aloe Sterols Prevents Skin Photoaging in OVX Hairless Mice

Estrogen deficiencies associated with menopause accelerate spontaneous skin aging and stimulate the ultraviolet (UV) irradiation‐induced photoaging of skin. However, food compositions with the potential to ameliorate the UV irradiation‐induced acceleration of skin aging with menopause have not yet been investigated in detail. In the present study, we examined the ability of plant sterols derived from Aloe vera gel to prevent the UV irradiation‐induced acceleration of skin aging in ovariectomized mice. Skin transepidermal water loss (TEWL) was significantly higher in the ovariectomy group than in the sham operation group following UVB irradiation, whereas skin elasticity was significantly lower. Ultraviolet B (UVB) irradiation induced greater reductions in skin hyaluronic acid levels and more severe collagen fiber damage in the derims in the ovariectomy group than in the sham group. The intake of AVGP significantly ameliorated this acceleration in skin aging by reducing the expression of matrix metalloproteinases (MMPs) and increasing that of epidermal growth factor (EGF) and hyaluronan synthase (HAS) in the skin. These results indicate that AVGP supplementation prevents skin damage induced by UVB irradiation and ovariectomy in part by inhibiting damage to the extracellular matrix.     

A bioactive complex to protect proteins from UV‐induced oxidation in human epidermis1

UV light induces multiple damages including protein oxidation on skin. Oxidized proteins if not degraded by the proteasome would eventually accumulate causing metabolic damage, elastosis and pigment formation such as lipofuscin. During ageing, the activity of the proteasome decreases dramatically together with enzymes that protect from oxidation and as a result oxidized proteins accumulate. We have investigated a combination of Panthenyl triacetate and Ethyl linoleate (bioactive complex) to fight against protein oxidation. This complex when tested at 3% on human skin biopsies showed statistically significant protection from UV (UVA + UVB)‐induced protein oxidation both in a 24‐h pre‐treatment before UV irradiation (72% protection, P < 0.05) and immediately after irradiation (78% protection, P < 0.05). UV light also induced a significant decrease of mRNA for protein repairing enzymes, such as Methionine Sulfoxide Reductase (MSR). The complex, given both pre‐ and post‐irradiation, stimulated the repairing enzyme expression. We can suggest utilization of this new complex to prevent accumulation of oxidized protein as a result of skin photo‐ageing and to prevent stratum corneum dehydration, skin elastosis and pigmentation formation (age spots).     

Adaptive response of the skin to UVB damage: role of the p53 protein

Different adaptation mechanisms like heat shock response, cell cycle arrest and DNA repair, melanin pigmentation and thickening of the epidermis are present in the human skin to protect against the adverse effects of solar UV irradiation. When DNA damage is beyond repair, cells undergo apoptosis to prevent their replication. We discuss the current knowledge on these different adaptation mechanisms to UVB damage, the most energetic fraction of solar UV that reaches the skin. As p53 protein, the guardian of the genome, plays a key role in protective response to genotoxic damage, its role in this adaptive response of the skin to UV will be further discussed.     

Improving skin function with CM-glucan, a biological response modifier from yeast

Preparations from yeast have been used for a long time for cosmetic and pharmaceutical purposes. Studies have identified glucan from the cell wall of baker's yeast as an immunologically active agent. Glucan is a poly beta-( 1-3)-linked glucopyranose of high molecular weight and belongs to the class of compounds known as biological response modifiers. Glucan preparations are involved in the activation of the body's natural defence mechanisms and in the acceleration of the skin's wound healing processes. In the skin, Langerhans' cells and keratinocytes are the immunologically competent cells. Recent studies indicate that UV irradiation can deplete the number and viability of these cells (immunosuppression). The use of non-specific immune-stimulators, such as glucan, is a new approach for improving the function of stressed skin. We have developed a process to modify pure glucan from baker's yeast to carboxymethyl glucan (CM-glucan), a water soluble product suitable for topical formulations. The functional properties of this new compound have been investigated in vitro and in vivo. Cell culture experiments showed that CM-glucan protects skin cells against the depletion of antioxidant molecules upon UV-A irradiation and promotes the growth of keratinocytes. In placebo controlled studies with healthy volunteers, the pretreatment of skin with CM-glucan offered substantial protection against skin damage caused by a detergent challenge or UV-A irradiation. In addition, CM-glucan enhanced the renewal rate of the stratum corneum.     

耐盐酵母尿嘧啶营养缺陷型菌株的构建

在酱油高盐稀态发酵过程中通常添加假丝酵母(T酵母)和鲁氏酵母(S酵母)来增强酱油的风味品质。但是目前对T酵母和S酵母进行遗传操作没有相应的营养缺陷型菌株。因此,对T酵母和S酵母分别进行甲基磺酸乙酯(ethylmethane sulfonate,EMS)诱变和紫外诱变,以酵母的致死率为诱变标准做单因素试验,得到最佳的EMS质量分数、EMS作用时间,紫外照射距离、紫外照射时长,酵母菌浓度。通过筛选、测序获得尿嘧啶营养缺陷型菌株,为后续耐盐酵母的分子生物学研究提供理论依据。     

A comparative study on the structure of Saccharomyces cerevisiae under nonthermal technologies: high hydrostatic pressure, pulsed electric fields and thermo-sonication

Nonthermal technologies are becoming more popular in food processing; however, little detailed research has been conducted on the study of the lethal effect of these technologies on certain microorganisms. Saccharomyces cerevisiae is a yeast related to spoilage of fruit products such as juices; novel technologies have been explored to inactivate this yeast. Three nonthermal technologies, high hydrostatic pressure (HHP), pulsed electric fields (PEF) and thermo-sonication (TS), were used to evaluate and to compare the structural damage of yeast cells after processing. Processing conditions were chosen based on previous experiments to ensure the death of cells; HHP was conducted at 600 MPa for 7 min (room temperature, 21 °C); for PEF, 30.76 kV/cm at 40 °C and 21 pulses (2 μs each), and finally for TS the conditions were 120 μm, 60 °C and 30 min in continuous and pulsed modes; all treatments were applied in apple juice. Cells were prepared for electron microscopy using an innovative and short microwave assisted dehydration technique. Scanning electron microscopy showed the degree of damage to the cells after processing and illustrated the important and particular characteristics of each technology. Cells treated with high hydrostatic pressure showed a total disruption of the cell membrane, perforation, and release of the cell wall; scars were also observed on the surface of the pressurized cells. PEF treated cells showed less superficial damage, with the main changes being the deformation of the cells, apparent fusion of cells, the formation of pores, and the breakdown of the cell wall in some cells. Finally, the thermo-sonicated cells showed a similar degree of cellular damage to their structure regardless of whether the TS was applied continuously or pulsed. The main characteristics of cellular death for this technology were the erosion and disruption of the cellular membrane, formation of orifices on the surface, lysis of cells causing the release of intracellular contents, roughness of the cell membrane, and displacement of cell debris to the surface of other cells. This study confirms some theories about cell inactivation and presents new and detailed results about nonthermal technologies, but also shows that after using the above mentioned conditions, recovery of cells, specifically those that are pressurized and thermo-sonicated, it is not possible to do it following the high extent of damage observed in the entire population. Furthermore, a faster methodology that was used in sample preparation for electron microscopy provided high quality resolution images, allowing closer study of the detail of structural lethal effects on treated cells.     

响应面法优化脉冲强光杀灭干豆腐细菌条件的研究

采用响应面法对影响脉冲强光杀灭干豆腐细菌效果的因素进行研究,得出影响灭菌率的大小顺序为:照射距离脉冲强光能量照射时间;最佳灭菌条件为:脉冲强光能量400 J,照射距离10 cm,照射时间15 s,此条件下的灭菌率可达98.83%。经过脉冲强光照射的干豆腐其感官品质与未处理样品相差甚微。     

Investigation on bonding quality of beech wood (Fagus orientalis L.) veneer during high temperature drying and aging

In the present study, the effects of high drying temperature and UV light induced aging on bonding quality of plywood manufactured from untreated and treated veneer layers were investigated. Rotary cut veneers with dimensions of 500 mm×500 mm×2 mm produced from beech (Fagus orientalis Lipsky) log were selected for topochemical, chemical and mechanical analyses. The veneer sheets were oven-dried at 100°C and 180°C after the peeling process. Afterwards, the surfaces were exposed to artificial UV irradiation in an UV chamber for 24 h, 48 h and 72 h representing natural sun irradiation of 2, 4 and 6 months, respectively. Topochemical distribution of lignin and phenolic extractives of the treated and untreated veneers was investigated on a cellular level using UV microspectrophotometry (UMSP). For the chemical characterization of accessory compounds high performance liquid chromatography (HPLC) was used. Furthermore, the shear and bending strengths of plywood manufactured from the treated samples are determined in order to study the bonding quality. The UV microscopic detection shows that after high drying temperature and aging treatment, lignin condensation occurs. With increasing drying temperature and aging duration, more phenolic extractives are situated in parenchyma cells and vessel lumens which can be proved by increased absorbance at 278 nm. The HPLC analysis of the treated tissue showed distinct signals of polymerized compounds such as catechin and 2,6-dimethoxybenzoquinone which are chromophoric compounds in discolored beech wood. The mechanical properties of plywood showed that with increasing drying temperature up to 180°C does not negatively affect shear and bending strengths of samples. After exposure of the veneers to UV irradiation (especially 6 months), decreasing shear and bending strengths of plywood samples can be observed.     

Inactivation of Staphylococcus aureus in milk using flow-through pulsed UV-light treatment system

ABSTRACT:  This study investigated the efficacy of pulsed UV-light for continuous-flow milk treatment for the inactivation of Staphylococcus aureus , a pathogenic microorganism frequently associated with milk safety concerns. Pulsed UV light is an emerging technology, which can be used for the inactivation of this pathogen in milk in a relatively short time. Pulsed UV light damages the DNA of the bacteria by forming thymine dimers that lead to bacterial death. The effect of sample distance from the quartz window of the UV-light source, number of passes, and flow rate was investigated. A response surface methodology was used for the design and analysis of experiments. Milk was treated at 5-, 8-, or 11-cm distance from a UV-light strobe at 20, 30, or 40 mL/min flow rate and treated up to 3 times by recirculation of milk to assess the effect of the number of passes on inactivation efficiency. Log₁₀ reductions varied from 0.55- to 7.26-log₁₀ CFU/mL. Complete inactivation was obtained in 2 cases and no growth was observed following an enrichment protocol. Predicted results were in agreement with the experimental data. Overall, this work demonstrates that pulsed UV-light has a potential for inactivation of milk pathogens.     

脉冲强光对枯草芽孢杆菌NG-2灭活机理

采用脉冲强光对枯草芽孢杆菌NG-2及芽孢进行灭活并研究其机理。在脉冲强光辐照枯草芽孢杆菌NG-2菌体及其芽孢后,利用扫描电子显微镜和透射电子显微镜观察形态特征变化,利用紫外分光光度法测定细胞内物质含量,利用荧光光谱法检测细胞内Ca~(2+)浓度和非特异性酯酶活力。结果表明,辐照距离为5 cm,闪照频率为5次/s,辐照15 s时,脉冲强光对NG-2菌体及其芽孢灭活率超过50%;辐照40 s时,脉冲强光对NG-2菌体及其芽孢灭活率超过99.98%。辐照枯草芽孢杆菌及芽孢后菌体结构损伤,芽孢壁破裂,细胞内核酸、蛋白质、Ca~(2+)和2,6-吡啶二羧酸含量显著减少,非特异性酯酶活力降低,研究结果为脉冲强光对枯草芽孢杆菌及芽孢灭活机理提供理论依据。     

脉冲强光对黑曲霉细胞膜损伤及胞内酶活性的影响

通过脉冲强光处理黑曲霉菌悬液,研究菌体细胞膜通透性的变化以及胞内酶活性的变化。结果表明：经脉冲强光处理后菌悬液的蛋白质溢出量、丙二醛的漏出量与对照相比均有显著增加,透射电子显微镜观察到细胞膜和细胞壁表面凹凸不平,出现褶皱。此外,脱氢酶活力与另外胞内3种主要抗氧化酶(超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物酶)活力也都有不同程度的下降。因此,霉菌屏障结构破坏所造成的细胞膜渗透压和通透性的变化可使霉菌胞内物质发生改变,甚至导致菌体死亡。     

DESIGN, CONSTRUCTION AND VALIDATION OF A SANITARY GLOVE BOX PACKAGING SYSTEM FOR PRODUCT SHELF-LIFE STUDIES

A glove box has been constructed as pan of an integrated pilot plant scale pulsed electric field processing and packaging system to facilitate studies of product shelf-life with selected packaging materials. The glove box was sanitized using combination of hydrogen peroxide and germicidal UV light. A HEPA air filter provided positive pressure of bacteria-free air. Nonselective nutrient broth was sterilized and filled into presanitized bottles inside the glove box. Negative and positive controls were included in the experiment. All bottles were incubated at 22C and 37C for two weeks and checked for rnicrobial growth by measuring optical density at 600 nm using a spectrophotometer and by plating on plate count agar and potato dextrose agar for total aerobic and, yeast and mold counts, respectively. No turbidity or microbial growth was observed in the media filled in the sanitized bottles using the sanitized glove box at 22 and 37C. PEF processed orange juice using this system had a shelf-life of more than 16 weeks at 4C.     

Genome‐wide map of Apn1 binding sites under oxidative stress in Saccharomyces cerevisiae

The DNA is cells is continuously exposed to reactive oxygen species resulting in toxic and mutagenic DNA damage. Although the repair of oxidative DNA damage occurs primarily through the base excision repair (BER) pathway, the nucleotide excision repair (NER) pathway processes some of the same lesions. In addition, damage tolerance mechanisms, such as recombination and translesion synthesis, enable cells to tolerate oxidative DNA damage, especially when BER and NER capacities are exceeded. Thus, disruption of BER alone or disruption of BER and NER in Saccharomyces cerevisiae leads to increased mutations as well as large‐scale genomic rearrangements. Previous studies demonstrated that a particular region of chromosome II is susceptible to chronic oxidative stress‐induced chromosomal rearrangements, suggesting the existence of DNA damage and/or DNA repair hotspots. Here we investigated the relationship between oxidative damage and genomic instability utilizing chromatin immunoprecipitation combined with DNA microarray technology to profile DNA repair sites along yeast chromosomes under different oxidative stress conditions. We targeted the major yeast AP endonuclease Apn1 as a representative BER protein. Our results indicate that Apn1 target sequences are enriched for cytosine and guanine nucleotides. We predict that BER protects these sites in the genome because guanines and cytosines are thought to be especially susceptible to oxidative attack, thereby preventing large‐scale genome destabilization from chronic accumulation of DNA damage. Information from our studies should provide insight into how regional deployment of oxidative DNA damage management systems along chromosomes protects against large‐scale rearrangements. Copyright © 2017 John Wiley & Sons, Ltd.