Biological control of postharvest pear diseases using a bacterium, Pantoea agglomerans CPA-2.

Epiphytic microorganisms isolated from the fruits and leaf surfaces of apples and pears were screened for antagonistic activity against Penicillium expansum on pears. From 247 microorganisms tested for antagonistic properties against P. expansum, a bacterium strain identified as Pantoea agglomerans (CPA-2) was selected. This bacterium was very effective against Botrytis cinerea, P. expansum and Rhizopus stolonifer. Complete control at the three tested concentrations (2 x 10(7), 8 x 10(7) and 1 x 10(8) CFU ml(-1)) was obtained on wounded pears inoculated with 10(3), 10(4) and 10(5) conidia ml(-1) of P. expansum and R. stolonifer. At 8 x 10(7) CFU ml(-1), Pan. agglomerans reduced B. cinerea decay by more than 80% at the three concentrations of the pathogen. In over 3 years of experiments in semicommercial trials, Pan. agglomerans provided excellent control against B. cinerea and P. expansum under cold storage, either in air or in low oxygen atmospheres. Equal control was obtained with Pan. agglomerans at 8 x 10(7) CFU ml(-1), as with the fungicide imazalil at commercial doses, against both pathogens. Pan. agglomerans grew well inside wounds on pears at both room and cold temperatures and under modified atmospheres. In contrast, it grew poorly on the surface of intact fruit.     

Combining Pantoea agglomerans (CPA-2) and curing treatments to control established infections of Penicillium digitatum on lemons

The effectiveness of the strain CPA-2 of Pantoea agglomerans alone or in combination with a curing treatment at 33 degrees C for 65 h to control green mold was evaluated on lemons stored at ambient temperature and in cold storage. An application of P. agglomerans at 2 x 10(8) CFU/ml effectively reduced green mold incidence on recently inoculated lemons stored at temperatures from 5 to 25 degrees C. Moreover, a 30-s immersion of lemons in a P. agglomerans suspension at 2 x 10(8) CFU/ml significantly reduced green mold incidence, even when delayed up to 15 h after inoculation with Penicillium digitatum at either 20 degrees C or while in cold storage. However, it failed to control established infections of P. digitatum of more than 24 h. Curing P. agglomerans-treated lemons at 33 degrees C for 65 h completely controlled 24-h-old infections on artificially inoculated lemons stored at 20 degrees C for 14 days and on naturally infected lemons stored at 10 degrees C for 3 weeks plus 7 additional days at 20 degrees C. When applied before curing, population growth of P. agglomerans in wounds was similar to that within wounds of control fruits at 20 degrees C. In contrast, when it was applied immediately after curing treatment, P. agglomerans populations within wounds did not increase.     

Influence of diluent and sample processing methods on the recovery of the biocontrol agent Pantoea agglomerans CPA-2 from different fruit surfaces

Determining the populations of biocontrol agents applied as a postharvest treatment on fruit surfaces is fundamental to the assessment of the microorganisms' ability to colonise and persist on fruit. To obtain maximum recovery, we must develop a methodology that involves both diluent and processing methods and that does not affect the viability of the microorganisms. The effect of diluent composition was evaluated using three diluents: phosphate buffer, peptone saline and buffered peptone saline. An additional study was performed to compare three processing methods (shaking plus sonication, stomaching and shaking plus centrifugation) on the recovery efficiency of Pantoea agglomerans strain CPA-2 from apples, oranges, nectarines and peaches treated with this biocontrol agent. Overall, slight differences occurred among diluents, although the phosphate buffer maintained the most ideal pH for CPA-2 growth (between 5.2 and 6.2). Stomaching, using the phosphate buffer as diluent, was the best procedure for recovering and enumerating the biocontrol agent; this fact suggested that no lethal effects from naturally occurring antimicrobial compounds present on the fruit skins and/or produced when the tissues were disrupted affected the recovery of the CPA-2 cells, regardless of fruit type. The growth pattern of CPA-2 on fruits maintained at 20°C and under cold conditions was similar to that obtained in previous studies, which confirms the excellent adaptation of this strain to conditions commonly used for fruit storage.     

成团泛菌苯丙氨酸氨基变位酶的热稳定性改造

来源于原核生物成团泛菌(Pantoea agglomerans)的苯丙氨酸氨基变位酶(Pa PAM)可催化α-苯丙氨酸转变为药用价值更高的(S)-β-苯丙氨酸,然而其酶活较低,热稳定性较差,限制其工业应用。本研究突变苯丙氨酸氨基变位酶酶活中心上方loop环上的氨基酸及与其相互作用位点的氨基酸,降低loop环的柔性,以期稳定酶活中心的结构,提高酶的热稳定性。筛选得到热稳定性显著提高的突变体I91M,50℃保温1 h后,剩余酶活达到83%(野生型酶酶活仅剩余30%左右)。该结果有助于苯丙氨酸氨基变位酶的理论研究和工业应用。     

Survival of bifidobacteria after spray-drying

To investigate the survival of bifidobacteria after spray-drying, Bifidobacterium infantis CCRC 14633, B. infantis CCRC 14661, B. longum ATCC 15708, B. longum CCRC 14634 and B. longum B6 were first spray-dried with different carrier media including 10% (w/w) gelatin, gum arabic and soluble starch. B. infantis CCRC 14633 and B. longum were also determined in skim milk. It was found that survival of bifidobacteria after spray-drying varied with strains and is highly dependent on the carriers used. Among the test organisms, B. longum B6 exhibited the least sensitivity to spray-drying and showed the highest survival of ca. 82.6% after drying with skim milk. Comparisons of the effect of carrier concentrations revealed that spray-drying at 10% (w/w) gelatin, gum arabic or soluble starch resulted in the highest survival of bifidobacteria. In addition, among the various outlet-air temperatures tested, bifidobacteria showed the highest survival after drying at 50 degrees C. Elevation of outlet-air temperature caused increased inactivation of bifidobacteria. However, the inactivation caused by increased outlet-air temperature varied with the carrier used, with the greatest reduction observed using soluble starch and the least with skim milk.     

喷雾干燥对双歧杆菌存活影响的研究

为了研究喷雾干燥后双歧杆菌的存活情况，选择了三种B．longtma和两种B．infantis菌与分别含有明胶、树胶和可溶性淀粉的载体一起喷雾干燥。结果发现，喷雾干燥后双歧杆菌的存活因其种类不同而不同，同时很大程度上也取决于所用载体。比较不同的载体浓度对存活的影响，发现双歧杆菌在与10％的明胶、树胶或可溶性淀粉喷雾干燥后其存活率最高。经50℃干燥后双歧杆菌表现最大存活，温度升高则失活升高。然而温度升高引起的失活程度因所用载体不同而不同，采用可溶性淀粉失活程度最大，采用脱脂乳则最小。     

肉桂精油对成团泛菌和腐生葡萄球菌的抑菌活性及其机理

本文利用滤纸片法和二倍稀释法分别测定了肉桂精油对成团泛菌及腐生葡萄球菌的抑菌圈直径、最小抑菌浓度(MIC)以及最小杀菌浓度(MBC)来评估肉桂精油对其的抑菌活性,通过扫描电镜、透射电镜探究肉桂精油对成团泛菌和腐生葡萄球菌的形态影响,以及通过测定细胞膜的通透性、细胞膜的完整性和膜电位来共同阐释肉桂精油对两种菌活性抑制的机理。结果表明肉桂精油对成团泛菌和腐生葡萄球菌的生长均有较强的抑制作用,且对腐生葡萄球菌的抑菌作用明显强于成团泛菌的抑制作用;肉桂精油改变细胞的形态、增加膜的通透性、破坏了膜的完整性、并造成了细胞内容物的泄露、膜电位的降低,从而导致细菌的死亡。      

喷雾干燥对发酵乳杆菌KLDS1.0709存活影响的研究

研究喷雾干燥对高抗氧化活性发酵乳杆菌KLDS1.0709存活的影响。以脱脂乳为菌体悬浮基质,研究不同出风温度对该菌喷雾干燥后菌体存活率的影响,然后在脱脂乳中添加不同比例的海藻糖和蔗糖,比较不同基质对菌体存活率的影响。结果表明,随出风温度的升高,菌体存活率和水分含量均降低;当出风温度为75℃时,菌体存活率为62.98%,残留水分含量为5.80%。以10%脱脂乳+5%海藻糖+5%蔗糖为基质进行喷雾干燥,菌体存活率最高达73.90%,表明海藻糖和蔗糖与脱脂乳混合作为基质时可提高该菌喷雾干燥后的存活率。     

Microencapsulation of β-galactosidase with different biopolymers by a spray-drying process

The aim of this work was to investigate the possibility of producing microparticles containing β-galactosidase, using different biopolymers (arabic gum, chitosan, modified chitosan, calcium alginate and sodium alginate) as encapsulating agents by a spray-drying process. This study focused on the enzyme β-galactosidase, due to its importance in health and in food processing. Encapsulation of β-galactosidase can increase the applicability of this enzyme in different processes and applications. A series of β-galactosidase microparticles were prepared, and their physicochemical structures were analyzed by laser granulometry analysis, zeta potential analysis, and by scanning electron microscopy (SEM). Microparticles with a mean diameter around 3 μm have been observed, for all the biopolymers tested. The microparticles formed with chitosan or arabic gum presented a very rough surface; on the other hand, the particles formed with calcium or sodium alginate or modified chitosan presented a very smooth surface. The activity of the enzyme was studied by spectrophotometric methods using the substrate ONPG (O-nitrophenyl-β,d-galactopyranoside). The microencapsulated β-galactosidase activity decreases with all the biopolymers. The relative enzyme activity is 37, 20, 20 and 13%, for arabic gum, modified chitosan, calcium alginate and sodium alginate, respectively, when compared with the free enzyme activity. The enzyme microparticles formed with arabic gum shows the smallest decrease of Vmax, followed by the calcium alginate, sodium alginate, and modified chitosan.     

Effect of biocontrol agents Candida sake and Pantoea agglomerans on Penicillium expansum growth and patulin accumulation in apples

Penicillium expansum is the major responsible of fruit pome decaying in cold storage. Apples spoiled by P. expansum are expected to contain patulin, a mycotoxin which is proven to affect human health. The use of chemicals is the most common procedure to prevent rots in postharvest but legislation is becoming more and more restrictive. The use of biocontrol agents (BCA) as an alternative tool is currently being proposed. The aim of this study was to evaluate the effect of two BCA (Candida sake CPA-2 and Pantoea agglomerans CPA-1) on P. expansum growth and patulin accumulation in cold storage and further deck (ambient) storage. Wounded apples were inoculated with a cell suspension of either C. sake or P. agglomerans and with a P. expansum conidial suspension. Apples were cold stored at 1 degrees C until lesion diameter reached 2 or 4 cm. Half the apples of each treatment were further stored at 20 degrees C for three days before patulin analyses. Both BCA tested controlled blue rot and patulin accumulation during cold storage. The control of P. expansum growth was enhanced in C. sake treated apples. On the other side, control of patulin accumulation in P. agglomerans treated apples seemed to be more efficient. BCA treatment could not control blue rot and patulin accumulation during further storage at room temperature and in some cases, an increase in P. expansum aggressiveness was observed.     

Control of Penicillium expansum and Botrytis cinerea on apples and pears with the combination of Candida sake and Pantoea agglomerans.

The effectiveness of Candida sake (CPA-1) in combination with Pantoea agglomerans (CPA-2) for controlling Penicillium expansum and Botrytis cinerea on pears and apples was determined. The concentrations tested were 2 x 10(6) and 2 x 10(7) CFU/ml for C. sake and 2 x 10(7) and 8 x 10(7) CFU/ml for P. agglomerans. At room temperature, the two antagonists were combined in proportions of 0 to 100% in 25% increments. At the proportion of 50:50, no rot development was observed in pears, and the greatest control of blue mold in apples was observed at this proportion for all the tested concentrations. Under cold temperature on pears, the highest effectiveness of the mixture was observed when C. sake at 2 x 10(7) CFU/ml was combined with P. agglomerans at 2 x 10(7) or at 8 x 10(7) CFU/ml at the proportion 50:50. Under these conditions, no rot development of blue mold was reported, and gray mold lesion size was reduced by more than 95%. On apples, the mixture of C. sake at 2 x 10(7) CFU/ml and P. agglomerans at 8 x 10(7) CFU/ml at the proportion 50:50 reduced blue and gray mold incidence by 90%. Populations of the two antagonists had the same growth pattern at 20 degrees C when they were applied individually or in combination, but the population level was always higher when they grew alone. In contrast, at 1 degrees C, the population of both antagonists in combination formed a stable community with the same levels as individual application during the first 30 days; after that, C. sake dominated, and P. agglomerans decreased on apples and pears. At both temperatures, the maximum population level of C. sake was observed in apples, and themaximum population level of P. agglomerans was observed in pears.     

Application of flow cytometry for the assessment of preservation and recovery efficiency of bioaerosol samplers spiked with Pantoea agglomerans

Exposure assessment of biological aerosols requires trade-offs between efficient sampling of airborne microorganisms as either particles or viable units. The main objective of this work was to characterize aspects of bioaerosol measurement efficiency. A known concentration of the vegetative bacteria Pantoea agglomerans was spiked onto different samplers (AGI-30, BioSampler, and membrane filters) and then run for increasing time periods using HEPA filtered air. Measurement efficiency was evaluated based on total, viable, and culturable counts. Total and viable counts were determined by flow-cytometry (FCM); culturable counts were evaluated by standard plating. FCM as a method for assaying viability showed excellent agreement with known proportions of live/dead organisms (slope = 0.82, R(2) = 0.99). P. agglomerans recoveries (total, viable, and culturable) in order of best sampler performance included the BioSampler (75%, 52%, and 50%), filtration (50%, 13%, and 2%), and the AGI-30 (<30%, 15%, and 5%). The difference between viability and culturability provided an indication of viable but nonculturable (VBNC) cells. VBNC efficiency for sampling by filter, AGI-30, and BioSampler was 80%, 50%, and 100%, respectively. This research helps characterize recovery, survival, and culturability efficiencies while sampling environmentally sensitive airborne bacteria for purposes of exposure assessment, epidemiologic studies, and homeland security.     

Efficient synthesis of hydroxystyrenes via biocatalytic decarboxylation/deacetylation of substituted cinnamic acids by newly isolated Pantoea agglomerans strains

BACKGROUND: Decarboxylation of substituted cinnamic acids is a predominantly followed pathway for obtaining hydroxystyrenes—one of the most extensively explored bioactive compounds in the food and flavor industry (e.g. FEMA GRAS approved 4‐vinylguaiacol). For this, mild and green strategies providing good yields with high product selectivity are needed. RESULTS: Two newly isolated bacterial strains, i.e. Pantoea agglomerans KJLPB4 and P. agglomerans KJPB2, are reported for mild and effective decarboxylation of substituted cinnamic acids into corresponding hydroxystyrenes. Key operational parameters for the process, such as incubation temperature, incubation time, substrate concentration and effect of co‐solvent, were optimized using ferulic acid as a model substrate. With strain KJLPB4, 1.51 g L^?1 4‐vinyl guaiacol (98% yield) was selectively obtained from 2 g L^?1 ferulic acid at 28 °C after 48 h incubation. However, KJPB2 provided vanillic acid in 85% yield after 72 h following the oxidative decarboxylation pathway. In addition, KJLPB4 was effectively exploited for the deacetylation of acetylated α‐phenylcinnamic acids, providing corresponding compounds in 65–95% yields. CONCLUSION: Two newly isolated microbial strains are reported for the mild and selective decarboxylation of substituted cinnamic acids into hydroxystyrenes. Preparative‐scale synthesis of vinyl guaiacol and utilization of renewable feedstock (ferulic acid extracted from maize bran) have been demonstrated to enhance the practical utility of the process. Copyright © 2011 Society of Chemical Industry     

Changes of volatiles' attribute in durian pulp during freeze- and spray-drying process

Changes of volatile profile from durian (Durio zibethinus) pulp during freeze-drying and spray-drying process were studied using headspace SPME coupled to fast GC-TOFMS. Results showed that 4 esters diminished in the volatiles' composition of freeze-dried pulp and spray drying caused losses of 14 volatile constituents from the profile. Formation of new volatiles was induced in spray-dried durian powder, comprising 5 aldehydes, 1 ketone, 1 furan and 1 pyrrole compounds. Furthermore, dramatic decline was observed during freeze drying for the amount of major durian aroma included propanethiol, ethyl propanoate, propyl propanoate, ethyl 2-methylbutanoate and diethyl disulfide, ranging from 71% to 97% while 98% to 99% amount of such volatiles were vanished during spray-drying process.     

成团泛菌胞外多糖吸湿保湿和抗辐射活性

成团泛菌KFS发酵产胞外多糖(EPS)是由葡萄糖和甘露糖组成的均一性杂多糖,重均分子量约为756kDa。以透明质酸和甘油为对照,分别在2种相对湿度环境下考察了EPS的吸湿性和保湿性。基于该EPS较强的抗氧化活性,进一步考察了其对不同菌体的抗辐射保护作用。结果表明:在相对湿度43%和81%的环境中,EPS均显示出较好的保湿活性,吸湿性与透明质酸相似,略低于甘油;EPS对菌体的辐射保护作用与糖溶液浓度和紫外辐照时间呈相关性,当糖溶液浓度为4 mg/mL、紫外辐照5 min时,大肠杆菌、白色念球菌以及金黄色葡萄球菌的存活率分别是48.49%、36.73%、47.5%,明显高于空白对照组的13.68%、13.47%和12.25%。所以,EPS作为天然的保湿剂和抗辐射剂有应用前景。     

Biological control of Monilinia laxa and Rhizopus stolonifer in postharvest of stone fruit by Pantoea agglomerans EPS125 and putative mechanisms of antagonism

Treatment of stone fruits (apricot, peach and nectarine) with Pantoea agglomerans strain EPS125 decreased the incidence and diameter of lesions of brown rot caused by Monilinia laxa and soft rot caused by Rhizopus stolonifer. Root control was achieved on fruits either wounded and subsequently inoculated with the pathogens or non-wounded and naturally infected from orchards. The efficacy of biocontrol was dependent on the concentration of the biocontrol agent and pathogen. At medium to low pathogen dose, optimal EPS125 concentrations were above 10(7) CFU ml(-1). The median effective dose (ED(50)) of EPS125 was 4.5x10(4) in M. laxa and 2.2x10(5) CFU ml(-1) in R. stolonifer. However, EPS125 was more effective in M. laxa than in R. stolonifer as indicated by the ratio between ED(50) of the biocontrol agent and pathogen (K(z)/K(x)) which was 166 and 1263, respectively. Interactions between the strain EPS125 and the fruit surface, and M. laxa and R. stolonifer, were studied to determine the mechanisms of protection from postharvest rots. The strain EPS125 colonizes, grows and survives on stone fruit wounds. Significant inhibition of conidial germination and hyphal growth of R. stolonifer and M. laxa was achieved when the fungal and EPS125 cells were cocultivated on peel leachate or nectarine juice. However, no effect was observed when the antagonist and the pathogen cells were physically separated by a membrane filter which permits nutrient and metabolite interchange. Therefore, a direct interaction between the strain and the pathogen cells is necessary for antagonism, without a significant contribution of the production of antibiotic substances or nutrient competition. Preemptive exclusion by wound colonization and direct interaction with the pathogen is proposed as the mechanism of biocontrol.     

Spontaneous ignition of milk powders in a spray-drying plant

A company operating a spray-drying plant for skimmed milk and fat-filled powders asked the Fire Research Station for help in determining the cause of a series of fires. The subsequent investigation showed that the powders could undergo spontaneous ignition in certain circumstances. The thicknesses of powder deposit on the spray-drier walls necessary for there to be a risk of self-ignition were calculated. These were sufficiently close to those encountered in practice to suggest that self-ignition represents a real problem. Mechanisms for the occurrence of fires in the fluid-bed instantizers are also put forward. Preventive measures are suggested.     

Biochemical characterization of l-arabitol 2-dehydrogenase from Pantoea ananatis

Two oxidoreductases, XDH and LAD, were found in the same operon that was involved in sugar metabolism in Pantoea ananatis. LAD, whose endogenous substrate was unknown, was recombinantly prepared and biochemically analyzed. Consequently, LAD was identified as l-arabitol 2-dehydrogenase and its substrate specificity was complementary to that of XDH.