Determination of spore inactivation during thermal and pressure-assisted thermal processing using FT-IR spectroscopy

The efficacy of microbial inactivation techniques is currently tested using time-consuming and labor-intensive plate count methods, which are the principal rate-limiting steps in developing inactivation kinetic parameters for alternative food processing technologies. Fourier transform infrared (FT-IR) spectroscopy combined with multivariate analysis was used to quantify viable spores and identify some biochemical changes in samples treated by autoclaving, pressure-assisted thermal processing (PATP), and thermal processing (TP). Spore suspensions ( approximately 109 CFU/mL) of Bacillus amyloliquefaciens TMW 2.479 Fad 82, B. amyloliquefaciens TMW 2.482 Fad 11/2, B. sphaericus NZ 14, B. amyloliquefaciens ATCC 49764, and Clostridium tyrobutyricum ATCC 25755 were treated by PATP (121 degrees C and 700 MPa) for 0, 10, 20, and 30 s and by TP (121 degrees C) for 0, 10, 20, and 30 s. The concentrations of spores in treated samples were determined by plating (reference method). Models developed using partial least-squares regression (PLSR) for predicting spore levels in treated samples had correlation coefficients (r) of >0.99 and standard errors of cross-validation ranging between 100.2 and 100.5 CFU/mL. Changes in dipicolinic acid (DPA) and secondary structure of proteins were found to occur during inactivation of spores by PATP and TP. FT-IR spectroscopy could rapidly estimate viable bacterial spore levels in PATP- and TP-treated spore suspensions, providing an accurate analytical tool for monitoring the efficacy of sterilization techniques in inactivating spore-forming microorganisms.     

Combined pressure-temperature effects on carotenoid retention and bioaccessibility in tomato juice

This study highlights the changes in lycopene and β-carotene retention in tomato juice subjected to combined pressure-temperature (P-T) treatments ((high-pressure processing (HPP; 500-700 MPa, 30 °C), pressure-assisted thermal processing (PATP; 500-700 MPa, 100 °C), and thermal processing (TP; 0.1 MPa, 100 °C)) for up to 10 min. Processing treatments utilized raw (untreated) and hot break (～93 °C, 60 s) tomato juice as controls. Changes in bioaccessibility of these carotenoids as a result of processing were also studied. Microscopy was applied to better understand processing-induced microscopic changes. TP did not alter the lycopene content of the tomato juice. HPP and PATP treatments resulted in up to 12% increases in lycopene extractability. all-trans-β-Carotene showed significant degradation (p < 0.05) as a function of pressure, temperature, and time. Its retention in processed samples varied between 60 and 95% of levels originally present in the control. Regardless of the processing conditions used, <0.5% lycopene appeared in the form of micelles (<0.5% bioaccessibility). Electron microscopy images showed more prominent lycopene crystals in HPP and PATP processed juice than in thermally processed juice. However, lycopene crystals did appear to be enveloped regardless of the processing conditions used. The processed juice (HPP, PATP, TP) showed significantly higher (p < 0.05) all-trans-β-carotene micellarization as compared to the raw unprocessed juice (control). Interestingly, hot break juice subjected to combined P-T treatments showed 15-30% more all-trans-β-carotene micellarization than the raw juice subjected to combined P-T treatments. This study demonstrates that combined pressure-heat treatments increase lycopene extractability. However, the in vitro bioaccessibility of carotenoids was not significantly different among the treatments (TP, PATP, HPP) investigated.     

Bacteria associated with Glomus clarum spores influence mycorrhizal activity

The effects of bacteria associated with arbuscular mycorrhizal fungal (AMF) spores on spore germination, growth in vitro and on the pea-AMF symbiosis were evaluated. Bacterial colonies were recovered from untreated Glomus clarum NT4 spores and NT4 spores decontaminated with 5% chloramine-T for 30, 45 and 60 min on five different media. Both G+ and G− bacteria were recovered from untreated NT4 spores, whereas only G+ bacteria were isolated from decontaminated spores. An in vitro assessment of the effect of spore-associated bacteria on clean, decontaminated NT4 spores revealed that (i) most of the bacteria isolated from untreated spores generally did not significantly alter spore function, (ii) some bacteria isolated from clean, decontaminated spores inhibited or stimulated NT4 spore germination, (iii) stimulation of spore germination occurred only when bacteria were in contact with spores, and (iv) inhibition of spore germination was the result of volatile bacterial metabolites. A stimulatory bacterial isolate, Bacillus pabuli LA3, significantly (P<0.05) enhanced the shoot growth, AMF-colonization, shoot N content and P use efficiency of NT4-inoculated 6 week-old pea plants over that of plants co-inoculated with an inhibitory bacterial isolate, Bacillus chitinosporus LA6a and NT4.     

Rapid detection and identification of bacterial strains by Fourier transform near-infrared spectroscopy

The use of Fourier transform near-infrared (FT-NIR) spectroscopy and multivariate pattern recognition techniques for the rapid detection and identification of bacterial contamination in liquids was evaluated. The complex biochemical composition of bacteria yields FT-NIR vibrational transitions (overtone and combination bands) that can be used for classification and identification. Bacterial suspensions (Escherichia coli HB101, E. coli ATCC 43888, E. coli 1224, Bacillus amyloliquifaciens, Pseudomonas aeruginosa, Bacillus cereus, and Listeria innocua) were filtered to harvest the cells and eliminate the matrix, which has a strong NIR signal. FT-NIR measurements were done using a diffuse reflection-integrating sphere. Principal component analysis showed tight clustering of the bacterial strains at the information-rich spectral region of 6000-4000 cm(-1). The method reproducibly distinguished between different E. coli isolates and conclusively identified the relationship between a new isolate and one of the test species. This methodology may allow for the rapid assessment of potential bacterial contamination in liquids with minimal sample preparation.     

Effect of spore inoculum and agricultural practices on the vertical distribution of the biocontrol plant-growth-promoting bacterium <Emphasis Type="Italic">Pasteuria penetrans</Emphasis> and growth of <Emphasis Type="Italic">Meloidogyne incognita</Emphasis>-infected tomato

Three concentrations of Pasteuria spores applied to soil and some agricultural practices were evaluated for their effects on spore attachment to nematodes and biocontrol of Meloidogyne incognita on tomato in a microplot experiment. Applications of Pasteuria at concentrations of 5᎒¹⁰ spores/m² increased tomato fruit yield per plant by 46% compared to non-Pasteuria treatments but also increased nematode densities in soil at harvest time. M. incognita juveniles recovered from plots where Pasteuria was applied at 5᎒¹⁰ spores/m² showed greater spore attachment than those with application rates of 2.5᎒⁹ spores/m² or 5᎒⁹ spores/m². Pasteuria spores penetrated to 30-40 cm soil depth in a volcanic ash sandy soil after application of spore suspensions to the soil surface. Densities of over 2.5᎒⁴ spores/g of soil were reached at 0-30 cm soil depth only when the application rate was 5᎒¹⁰ spores/m², but at harvest and after fallow densities of about 2.5᎒⁴ spores/g of soil were also reached in the top 10 cm of soil at 2.5᎒⁹ and 5᎒⁹ spores/m² application rates. Spore densities in soil decreased after 6 months of fallow when densities at harvest time were higher than 10⁵ spores/g of soil. Tillage and additional watering 2 days after spore application increased spore densities in soil at harvest throughout the soil depth (0-40 cm).     

Heat-induced changes in myofibrillar protein structures and myowater of two pork qualities. A combined FT-IR spectroscopy and low-field NMR relaxometry study

Low-field NMR T(2) and Fourier transform infrared (FT-IR) measurements were performed on meat samples of two qualities (normal and high ultimate pH) during cooking from 28 degrees C to 81 degrees C. Pronounced changes in both T(2) relaxation data and FT-IR spectroscopic data were observed during cooking, revealing severe changes in the water properties and structural organization of proteins. The FT-IR data revealed major changes in bands in the amide I region (1700-1600 cm(-)(1)), and a tentative assignment of these is discussed. Distributed NMR T(2) relaxation data and FT-IR spectra were compared by partial least-squares regression. This revealed a correlation between the FT-IR peaks reflecting beta-sheet and alpha-helix structures and the NMR relaxation populations reflecting hydration water (T(2B) approximately 0-10 ms), myofibrillar water (T(21) approximately 35-50 ms), and also expelled "bulk" water (T(2) relaxation times >1000 ms). Accordingly, the present study demonstrates that definite structural changes in proteins during cooking of meat are associated with simultaneous alterations in the chemical-physical properties of the water within the meat.     

Determination of secondary structural changes in gluten proteins during mixing using Fourier transform horizontal attenuated total reflectance spectroscopy

Fourier transform horizontal attenuated total reflectance (FT-HATR) was used to examine changes in the secondary structure of gluten proteins in a flour-water dough system during mixing. Midinfrared spectra of mixed dough revealed changes in four bands in the amide III region associated with secondary structure in proteins: 1317 (alpha-helix), 1285 (beta-turn), 1265 (random coil), and 1242 cm (-1) (beta-sheet). The largest band, which also showed the greatest change in second derivative band area (SDBA) during mixing, was located at 1242 cm (-1). The bands at 1317 and 1285 cm (-1) also showed an increase in SDBA over time. Conversely, the band at 1265 cm (-1) showed a corresponding decrease over time as the doughs were mixed. All bands reached an optimum corresponding to the minimum mobility of the dough as determined by the mixograph. Increases in alpha-helix, beta-turn, and beta-sheet secondary structures during mixing suggest that the dough proteins assume a more ordered conformation. These results demonstrate that it is possible, using infrared spectroscopic techniques, to relate the rheological behavior of developing dough in a mixograph directly to changes in the structure of the gluten protein system.     

Discrimination of intact and injured Listeria monocytogenes by Fourier transform infrared spectroscopy and principal component analysis

Fourier transform infrared spectroscopy (FT-IR, 4000-600 cm(-)(1)) was used to discriminate between intact and sonication-injured Listeria monocytogenes ATCC 19114 and to distinguish this strain from other selected Listeria strains (L. innocua ATCC 51742, L. innocua ATCC 33090, and L. monocytogenes ATCC 7644). FT-IR vibrational overtone and combination bands from mid-IR active components of intact and injured bacterial cells produced distinctive "fingerprints" at wavenumbers between 1500 and 800 cm(-)(1). Spectral data were analyzed by principal component analysis. Clear segregations of different intact and injured strains of Listeria were observed, suggesting that FT-IR can detect biochemical differences between intact and injured bacterial cells. This technique may provide a tool for the rapid assessment of cell viability and thereby the control of foodborne pathogens.     

Factors that Influence the Transport of Bacillus cereus Spores through Sand

The goal of this study is to clarify the surface-chemical and microphysical variables that influence bacterial spore transport through soil, thereby defining the factors that may affect spore transport velocity. Bacillus cereus spores were continuously monitored in a soil column under saturated conditions with experimental variations in soil grain size (0.359 and 0.718 mm), pH (7.2 and 8.5), and water flow rate (1.3 and 3.0 mL/min). Increasing soil grain size, flow rate, and pH resulted in enhanced spore movement. Spore transport increased 82% when soil grain size was doubled. An increase in effluent flow rate from 1.3 to 3.0 mL/min increased spore movement by 71%. An increase in pH increased spore transport by 53%. The increase in hydrodynamic forces resulting from the larger grain size soil and higher flow rate functioned to overcome the hydrophobic nature of the spore’s coat, and the interparticle bonding forces between the spore and soil particles.     

用高压脉冲电场杀细菌孢子

在高压脉冲电场中，大部分细菌孢子能被杀死。其效率与脉冲电场处理参数有关，处理时间长，杀死的孢子数多；脉冲频率与脉冲宽度均对处理结果有影响     

Using Fourier transform infrared (FT-IR) absorbance spectroscopy and multivariate analysis to study the effect of chlorine-induced bacterial injury in water

The effect of chlorine-induced bacterial injury on spectral features using Fourier transform infrared (FT-IR) absorbance spectroscopy was studied using a mixed bacterial culture of (1:1) ca. 500 CFU/mL each Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 15442 in 0.9% saline. Bacterial cells were treated with 0, 0.3, or 1.0 ppm of initial free chlorine (21 degrees C, 1 h of contact time). Chlorine-injured and dead bacterial cells retained the ATR spectral properties of uninjured or live cells in the region of C-O-C stretching vibrations of polysaccharides, indicative of the cell wall peptidoglycan layer and lipopolysaccharide outer leaflet. This confirms the observations of others that extensive bacterial membrane damage is not a key factor in the inactivation of bacteria by chlorine. The bactericidal effect of chlorine caused changes in the spectral features of bacterial ester functional groups of lipids, structural proteins, and nucleic acids, with apparent denaturation reflected between 1800 and 1300 cm (-1) for injured bacterial cells. Three-dimensional principal component analysis (PCA) showed distinct segregation and clustering of chlorine-treated and untreated cells. Cells exposed to chlorine at 0.3 or 1.0 ppm could be distinguished from the untreated control 73 and 80% of the time, respectively, using soft independent modeling of class analogy (SIMCA) analysis. This study suggests that FT-IR spectroscopy may be applicable for detecting the presence of injured and viable but not culturable (VBNC) waterborne pathogens that are underestimated or not discernible using conventional microbial techniques.     

FT-IR investigation of cell wall polysaccharides from cereal grains. Arabinoxylan infrared assignment

The FT-IR fingerprint of wheat endosperm arabinoxylan (AX) was investigated using a set of polysaccharides exhibiting variation of their degree of substitution and xylo-oligosaccharides comprising xylose units mono- or disubstituted by arabinose residues. Substitution of the xylose backbone by arabinose side units was more particularly studied in the 1000-800 cm(-1) spectral region, by taking advantage of second-derivative enhancement. The 920-1020 cm(-1) spectral region revealed two absorption bands at 984 and 958 cm(-1), the intensities of which varied according to the degree of substitution. Whereas the intensity of the band at 958 cm(-1) increased with the degree of substitution, that at 984 cm(-1) decreased. The second-derivative spectral data of xylo-oligosaccharides indicated that these changes could be attributed to substitution of the xylan backbone by arabinose residues, and the band at 958 cm(-1) was ascribed to the presence of disubstituted xylose residues. Principal component analysis of FT-IR spectra of model mixtures of AX, beta-glucans, and arabinogalactans suggested that it is possible to evaluate the relative proportions of the polymers and degree of substitution of AX in complex mixtures such as the cell wall of cereal grains.     

Study of oat globulin conformation by Fourier transform infrared spectroscopy.

The conformation of oat globulin dispersions (10% in D2O) under the influence of pH, chaotropic salts, protein structure perturbants, and heating conditions was studied by Fourier transform infrared (FTIR) spectroscopy. The FTIR spectrum of oat globulin showed major bands from 1670 to 1634 cm(-1), corresponding to the four major types of secondary structures, that is, beta-turns, beta-sheets, alpha-helices, and random coils. At extreme acidic and alkaline pH conditions, there were changes in intensity in the bands attributed to beta-sheet structures (1626, 1634, and 1682 cm(-1)), and shifts of the bands to higher or lower wavenumbers, indicating changes in conformation. In the presence of some chaotropic salts, the 1626 and 1634 cm(-1) bands were shifted upward, with a marked decrease in the intensity of the 1634 cm(-1) peak. The addition of several protein structure perturbants led to a slight shift in the alpha-helix/random coil bands and a marked reduction in the beta-sheet peaks, suggesting protein unfolding. Heating under aggregating conditions led to slight shifts in all of the major bands and progressive changes in the intensity of the alpha-helix, beta-sheet, and beta-turn peaks, suggesting protein denaturation. This was accompanied by marked increases in intensity of the two intermolecular beta-sheet bands (1682 and 1624-1626 cm(-1)) associated with the formation of aggregated strands. The IR spectra of soluble and insoluble aggregates showed a redistribution of native and extensively denatured proteins in the two fractions.     

Optimizing conditions for thermal processes of soy milk

Mathematical and kinetic models were set up for heat-induced quality changes in soy milk, including inactivation of trypsin inhibitor activity (TIA) and degradation of thiamin, riboflavin, color, and flavor over a wide range of time-temperature combinations with particular interest in the ultrahigh-temperature (UHT) range. On the basis of these models, multiresponse optimization of the thermal processes for soy milk was carried out to obtain the following effects simultaneously: (1) maximum destruction of bacterial spores, (2) maximum inactivation of TIA, and (3) minimum degradation of sensory and nutritional qualities. By a suitable selection of high temperatures and extended heating times, for example, 143 degrees C/60 s, it is possible to use a single-step UHT process to produce a commercially sterile soy milk with satisfactory TIA inactivation, highly acceptable color and flavor, and thiamin retention between 90 and 93%.     

基于图像处理的小麦条锈病菌夏孢子模拟捕捉的自动计数

利用孢子捕捉器进行气传植物病原真菌孢子捕捉,实现田间病原真菌孢子数量的监测,对于气传植物真菌病害的预测预报和防治决策具有重要意义。目前对捕捉到的孢子多采用传统显微镜孢子计数方法,由于孢子个体小、数量大,利用这种计数方法费时费力,易造成较大计数误差。为了获得一种孢子捕捉器捕捉孢子的自动计数方法,提高计数的准确性和工作效率,本研究利用透明胶带、凡士林玻片和Eppendorf离心管3种方法模拟捕捉小麦条锈病菌夏孢子,利用显微镜照相技术获得孢子图像,在MATLAB软件环境下,对图像进行基于最近邻插值法的缩放处理、基于K-means聚类算法的分割处理、形态学操作修饰和分水岭分割等一系列的处理,实现夏孢子的自动计数和标记。结果表明,3种模拟方法获得的孢子图像经过处理后,均可获得较好的孢子计数结果。透明胶带、凡士林玻片、Eppendorf离心管模拟捕捉条锈病菌夏孢子的平均计数准确率最低分别为98.5%、98.7%、99.9%,Eppendorf离心管模拟捕捉条锈病菌夏孢子和小麦白粉病菌分生孢子的平均计数准确率为99.8%。本研究为实现田间利用孢子捕捉器捕捉孢子的自动计数提供了一种简便、快捷、准确、高效的方法。     

Biopriming in bean seeds

The incorporation of biological control organisms in seeds may help control pathogens and improve seedling performance. The aim of this study was to determine the effects of biopriming bean seeds with Trichoderma spp. and Bacillus subtilis using physiological conditioning, suspensions of biological structures and film coating techniques. Biopriming treatments with suspensions of biological structures were performed using the commercial products Agrotrich plus^® and Rhizoliptus^®. Water restriction was achieved using a PDA?+?Manitol (?0.7?MPa) medium for both Trichoderma spp. and Bacillus subtilis. Fifty disinfected bean seeds were placed in each petri dish. When the first radicle protrusion appeared in a seed, the other seeds were removed and dried in the laboratory environment for 48?h. Film coating was performed with the addition of the Color Seed^® (150?mL kg^?1) polymer to a treatment solution containing either the Trichoderma spp. or the Rhizoliptus product. Seeds were dried for 48?h in the laboratory. Seeds were covered and treated with one or both organisms. Biopriming with spore or bacterial cell suspensions promoted bean seedling growth, and the other techniques were not required to potentiate these benefits. Covering the primed bean seeds reduced seed quality. Both Trichoderma spp. and Bacillus subtilis promote bean seedling growth, benefitting the seedling even after it becomes independent of the seed reserves.     

利用付里叶转换──红外光谱（FT－IR）鉴别大豆根瘤菌初探

利用付里叶转换──红外分光光度计制作了标准大豆根瘤菌菌株２２－１０，２７－５０，ＵＳ－ＤＡ１１０及１５００６的ＦＴ－ＩＲ图谱。结果表明，所得图谱分辨率高、重复性好，并且不同菌株的图谱在指纹区（波数８００～１０００）有明显差别，这说明各菌株的ＦＴ－ＩＲ图谱是高度特异的，这种特异性可用于菌株鉴别．     

Depth transfer of fecal indicator organisms in an arable clayey soil

In clayey soils, shrinking cracks formed during dry periods in the summer are the most important form of macropores. They may contribute under disadvantageous conditions to the movement of fecal microorganisms and therefore to the contamination of groundwater. For this reason, a field experiment was carried out on a highly clayey arable soil to investigate the effect of the factors slurry addition, sampling date, depth, and season. Under worst case conditions, i.e. a soil at field capacity, irrigated directly after application of fresh cattle slurry with high rates of water, the numbers of spores, total coliforms, fecal coliforms and fecal streptococci were not significantly increased at depths of 15 cm, 45 cm and 75 cm in spring (with less shrinking cracks) or in autumn (with more shrinking cracks). The close correlations between the numbers of spores, total coliforms, aerobic bacteria and microbial biomass C indicate that spore forming organisms and most total coliforms belong to the autochthonous soil microflora and cannot indicate fecal pollution.     

利用PCR-DGGE技术研究长期自然恢复、种植作物和裸地条件下黑土土壤细菌群落结构变化

Soil microbial biomass and community structures are commonly used as indicators for soil quality and fertility. A investigation was performed to study the effects of long-term natural restoration, cropping, and bare fallow managements on the soil microbial biomass and bacterial community structures in depths of 0--10, 20--30, and 40--50 cm in a black soil (Mollisol). Microbial biomass was estimated from chloroform fumigation-extraction, and bacterial community structures were determined by analysis of 16S rDNA using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Experimental results showed that microbial biomass significantly declined with soil depth in the managements of restoration and cropping, but not in the bare fallow. DGGE profiles indicated that the band number in top 0--10 cm soils was less than that in depth of 20--30 or 40--50 cm. These suggested that the microbial population was high but the bacterial community structure was simple in the topsoil. Cluster and principle component analysis based on DGGE banding patterns showed that the bacterial community structure was affected by soil depth more primarily than by managements, and the succession of bacterial community as increase of soil depth has a similar tendency in the three managements. Fourteen predominating DGGE bands were excised and sequenced, in which 6 bands were identified as the taxa of Verrucomicrobia, 2 bands as Actinobacteria, 2 bands as α-Proteobacteria, and the other 4 bands as δ-Proteobacteria, Acidobacteria, Nitrospira, and unclassified bacteria. In addition, the sequences of 11 DGGE bands were closely related to uncultured bacteria. Thus, the bacterial community structure in black soil was stable, and the predominating bacterial groups were uncultured.     

内生环状芽孢杆菌Jcxy8对番茄灰霉病的防病机制研究

为明确从番茄植株体内筛选出的内生环状芽孢杆菌(Bacillus circulan)Jcxy8对番茄灰霉病菌的抑菌作用及防病的生理生化机制,采用平板打孔法测定了菌株Jcxy8对灰霉病菌(Botrytiscinerea)的拮抗力。结果表明:菌株Jcxy8对灰霉病菌的抑菌圈直径为35.6mm,抑菌圈边缘的产孢抑制率达到66.9%。当菌株培养滤液浓度为40%时,病菌孢子萌发完全被抑制。镜检发现抑菌圈周围的菌丝(或芽管)细胞消融,生长扭曲,中间或顶端膨大成泡囊状。Jcxy8菌株与灰霉病菌同时处理的番茄果体内可溶性蛋白含量比清水对照处理高12.7%,比单独接种灰霉病菌处理高39.1%;SOD、POD、CAT活性均较只经病菌处理低;O2-产生速率比清水对照和病菌处理低,而比菌株Jcxy8处理高。说明菌株Jcxy8对番茄果实有明显的诱导抗病作用。