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.     

Efficacy of sanitizing treatments against Penicillium expansum inoculated on six varieties of apples

The effectiveness of several wash treatments was evaluated against spores of Penicillium expansum inoculated on six varieties of apples (Red Delicious, Golden Supreme, Empire, Macintosh, Fuji, and Gala). The wash treatments were water, acidified water (pH 6.5), acidified sodium hypochlorite (pH 6.5), nonacidified sodium hypochlorite (pH 8.8, 9.3, and 9.7; 50, 100, and 200 ppm, respectively), and peracetic acid (50 and 80 ppm). Spores of P. expansum were dried on the surface of the apples for 2 h before exposure to the different sanitizer solutions. Each apple was submerged in 100 ml of each treatment solution for 30 s, and the number of spores remaining were recovered and enumerated. The efficacy of chlorine solutions was enhanced by decreasing the pH to 6.5 (up to 5-log reduction, depending on apple variety). Peracetic acid solutions (50 and 80 ppm) resulted in a reduction of less than 2 log spores per g and had the same efficacy (P < or = 0.05) as nonacidified chlorine solutions (50, 100, and 200 ppm). Control water solutions produced a reduction of 1.34 log spores per g. Chlorine solutions at pH 6.5 resulted in the largest reduction of P. expansum spores for all apple varieties tested.     

Effect of chitin on the antagonistic activity of Cryptococcus laurentii against Penicillium expansum in pear fruit

This study was designed to evaluate the impact of chitin on the antagonistic activity of Cryptococcus laurentii against the postharvest blue mold rot caused by Penicillium expansum in pear fruit. The results showed that the antagonistic activity of C. laurentii obtained from the culture media of nutrient yeast dextrose broth (NYDB) amended with chitin at 0.5-1.0% was improved greatly compared with the case that without chitin. The addition of chitin to NYDB did not influence the growth of C. laurentii, however, its population was found to increase rapidly thereafter in pear fruit wounds compared to that harvested from NYDB without chitin. Moreover, the cell-free filtrate of the chitin-supplement culture media in which the yeast was incubated for 24 h emerged a direct antifungal activity against P. expansum in pear fruit wounds, with the associated high level of chitinase activity. These results suggested that the use of chitin may be an effective method to induce the antagonistic activity of C. laurentii. To our knowledge, this is the first report regarding the chitin could enhance the efficacy of postharvest biocontrol yeasts.     

温度对苹果汁中扩展青霉生长和产毒的影响

在苹果汁培养基静置培养条件下,研究了培养温度为28、25、20、16、12、4℃时扩展青霉的生长和产毒情况。结果表明,在各贮藏温度下,扩展青霉均可在苹果汁中生长,并产生大量毒素。温度越高,生长速率和产毒速率越快,但生长高峰期和产毒高峰期发生的先后顺序差别很大。其中25℃是扩展青霉的最适生长和产毒温度,最大生物量和产毒量分别达到4.35 g/L和570.53 mg/L,在冷藏温度4℃时菌体仍能生长和产毒。因此在苹果汁的生产和贮藏过程中要充分考虑温度的影响,以减少果汁中展青霉素污染的风险。     

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.     

Integration of biocontrol agents and food-grade additives for enhancing protection of stored apples from Penicillium expansum

Forty-nine compounds currently used as additives in foods were tested in combination with three biocontrol agents, the yeasts Rhodotorula glutinis, Cryptococcus laurentii, and the yeastlike fungus Aureobasidium pullulans, to increase their antagonistic activity against Penicillium expansum, the causal agent of blue mold on apples. Twelve additives dramatically improved the antagonistic activity of one or more of the tested biocontrol agents. In a two-way factorial experiment with these selected additives the percentage of P. expansum rots on apples was significantly influenced by the antagonist and the additive as well as by their interaction. The combination of the biocontrol agents and some additives resulted in a significantly higher activity with respect to the single treatments applied separately, producing additive or synergistic effects. Some of the selected additives combined with a low yeast concentration (106 cells per ml) had comparable or higher efficacy than the biocontrol agents applied alone at a 100-fold higher concentration (10(8) cells per ml). Some organic and inorganic calcium salts, natural gums, and some antioxidants displayed the best results. In general, the effect of each additive was specific to the biocontrol isolate used in the experiments. Possible mechanisms involved in the activity of these beneficial additives and their potential application in effective formulations of postharvest biofungicides are discussed.     

Effects of the origins of Botrytis cinerea on earthy aromas from grape broth media further inoculated with Penicillium expansum

Earthy “off” aromas from wine and grape juice are highly detrimental to the production of quality grape products. These volatile compounds are produced on grapes by Botrytis cinerea, Penicillium expansum and/or a combination of P. expansum and B. cinerea strains. B. cinerea strains were isolated from different (a) vineyards in Spain and Portugal, (b) grape varieties (c) bunches (i.e., sound and botrytized) and (d) positions in the botrytized bunch (i.e., interior or exterior). A novel Headspace-Phase Microextraction (SPME) followed by Gas Chromatrography/Mass Spectrometry (GC-MS) dedicated to analyze geosmin, methylisoborneol (MIB), 1-octen-3-ol, fenchone and fenchol in grape broth medium was used. Approximately 50% of the B. cinerea strains induced detectable geosmin. One strain accumulated significant amounts of anisoles, demonstrating that this contamination might already occur in the vineyard. Strains from the interior of Cainho grape bunches induced more geosmin and hence it may be possible to reduce this volatile in wine by avoiding using these grapes in case of B. cinerea attack.     

不同地区来源藏曲对西藏传统酿造青稞酒风味特征的影响

通过模拟酿造实验比较了不同地区来源藏曲对西藏传统酿造青稞酒理化指标和风味的影响。结果显示不同来源藏曲均能顺利完成青稞酒发酵,所酿造的青稞酒酒度、残糖、游离氨基酸含量没有明显差异,但总酸含量差异显著。酿造的青稞酒中主要有机酸为乳酸、乙酸和琥珀酸,拉萨和日喀则地区藏曲发酵青稞酒中总有机酸显著高于其他地区藏曲发酵酒青稞酒。采用顶空固相微萃取(headspace solid phase micro-extraction,HSSPME)结合气相色谱-质谱(GC-MS)的方法分析了不同青稞酒中挥发性香气组分种类含量特征,共检测到66种挥发性香气组分,包括酯类物质19种、芳香族化合物9种,醇类物质11种、酸类物质9种、醛酮类物质10种、其他类8种。不同来源藏曲酿造青稞酒香气组分含量差异显著,通过藏曲的选择可以针对性地调控青稞酒的风味特征。     

The effects of the combination of Pichia membranefaciens and BTH on controlling of blue mould decay caused by Penicillium expansum in peach fruit

The feasibility of 0.2 g l⁻¹ benzo-thiadiazole-7-carbothioic acid S-methyl ester (BTH) to improve the efficacy of Pichia membranefaciens in controlling postharvest blue mould decay in peach fruit was investigated. Our results showed that biocontrol activity of P. membranefaciens against blue mould caused by Penicillium expansum in peach fruit could be enhanced by addition of 0.2 g l⁻¹ BTH. The combination of P. membranefaciens and BTH resulted in a more effective control of blue mould than individual treatment of P. membranefaciens or BTH alone. The combined treatment had a synergistic effect on the induction of superoxide dismutase, catalase, ascorbate peroxidase, chitinase and β-1,3-glucanase activities, which induced stronger disease resistance in fruit than BTH or yeast alone, and resulted in a lower lesion diameter and disease incidence of blue mould decay in peaches. Furthermore, the combined treatment did not impair the quality parameters including fruit firmness and contents of total soluble solids, titratable acidity and vitamin C of peach fruit after 6 days of storage at 20 °C. These results suggested that the use of BTH may be an effective method to improve the biological activity of P. membranefaciens.     

Multi-element analysis for determining the geographical origin of mutton from different regions of China

Elemental fingerprints were investigated for their potential to classify mutton samples according to their geographical origin. The concentration of 25 element contents in 99 mutton samples from three pastoral regions and two agricultural regions of China were analysed by ICP-MS. Multivariate statistical analysis including principal component analysis (PCA) and linear discriminate analysis (LDA) were used for this purpose. Twenty-one elements (Be, Na, Al, Ca, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Ag, Sb, Ba, Tl, Pb, Th and U) in de-fatted mutton showed significant differences (p < 0.05). LDA gave an overall correct classification rate of 93.9% and cross-validation rate of 88.9%. Furthermore, mutton samples from agricultural regions and pastoral regions were differentiated with 100% accuracy. These results demonstrate the usefulness of multi-element fingerprints as indicators for authenticating the geographical origin of mutton in China.     

Comparative chemical composition,free radical-scavenging and cytotoxic properties of essential oils of six Stachys species from different regions of the Mediterranean Area

The chemical composition of essential oils of six Stachys species, S. cretica L. ssp. vacillans Rech. fil., S. germanica L., S. hydrophila Boiss., S. nivea Labill., S. palustris L. and S. spinosa L., obtained by hydrodistillation, was studied by GC and GC–MS. All the oils have in common a great percentage of fatty acids and esters (24.2–58.5%) and a high amount of sesquiterpenes (16–35.9%), with the exception of the oil from S. palustris, which consisted mainly of carbonylic compounds (25.4%). The antioxidant activity by DPPH test and the antiproliferative activity on a series of human cancer cell lines (C32, amelanotic melanoma and ACHN, renal cell adenocarcinoma) were investigated for all the oils. S. palustris,S. cretica and S. hydrophila showed the highest antiradical effect, with IC₅₀ values of 0.482, 0.652 and 0.664 mg/ml, respectively. The most antiproliferative essential oil against C32 cell line was the oil of S. germanica with a 77% of inhibition at a concentration of 100 μg/ml. S. germanica, S. palustris and S. spinosa showed the most antiproliferative activity on ACHN cell line, at a concentration of 100 μg/ml,with 81%, 77% and 73% inhibition, respectively.     

A CAPS test allowing a rapid distinction of Penicillium expansum among fungal species collected on grape berries, inferred from the sequence and secondary structure of the mitochondrial SSU-rRNA

Penicillium expansum is a fungal species highly damageable for the postharvest conservation of numerous fruits. In vineyards, this fungus is sometimes isolated from grape berries where its presence may lead to the production of geosmin, a powerful earthy odorant, which can impair grapes and wines aromas. However, the discrimination of P. expansum from related fungi is difficult because it is based on ambiguous phenotypic characters and/or expensive and time-consuming molecular tests. In this context, the complete sequences and secondary structures of Penicillium expansum and Penicillium thomii mitochondrial SSU-rRNAs were achieved and compared with those of two other phylogenetically related Ascomycota: Penicillium chrysogenum and Emericella nidulans. The comparison has shown a high conservation in size and sequence of the core and of the variable domains (more than 80% of nt identity) of the four SSU-rRNAs, arguing for a close phylogenetic relationship between these four species of the Trichocomaceae family. Large (from 10 to 18 nt) inserted/deleted (indel) sequences were evidenced in the V1, V5 and V6 variable domains. The size variations (10 to 18 nt) of the V1 indel sequence allowed the distinction of the four species; the V5 indel (15 nt) was specifically recovered in E. nidulans; the V6 indel (16 nt), shared by the three Penicillium species, was lacking in E. nidulans. A couple of conserved primers (UI/R2) were defined to generate a PCR product containing the V1 to V5 variable domains. This product contained the two regions of the four SSU-rRNAs showing the highest rates of nt substitutions, namely the V2 variable domain and, surprisingly, a helix (H17) of the core. The H17 sequence was shown to specifically possess in P. expansum a recognition site for the ClaI restriction endonuclease. Hence, this enzyme generates a digestion pattern of the PCR product with two bands (350 bp+500 bp), specific to P. expansum and easily separable by agarose gel electrophoresis. This leads to a CAPS test usable for P. expansum discrimination among grape berries isolated filamentous fungi. The CAPS test was validated by a comparative analysis involving 29 strains belonging to 17 species currently isolated from grape berries in the Bordeaux vineyards.     

几种不同产地棉籽及其混合油精炼工艺的对比研究

对比了新疆克拉玛依、新疆喀什、甘肃和河北棉籽的各项指标,试验结果显示,新疆棉籽粗蛋白含量和粗脂肪含量均高于甘肃和河北棉籽。用气相色谱检测了四地区棉籽油脂肪酸组成,亚油酸含量最高,在58%以上,油酸和亚油酸含量达到75%左右。探讨了四地区棉籽在棉籽油混合油精炼过程中的工艺控制,得出不同地区棉籽适宜的混合油精炼工艺条件。     

Evaluation of different methods for the production of juice concentrates and fruit powders from cactus pear

The present work aimed both at improvement and extension of a previously developed process for the production of cactus pear juice. The total yield was increased by 10% through processing whole instead of peeled fruits and by further optimisation of pulp enzymation. As an alternative to HTST pasteurisation, cross-flow microfiltration was applied for non-thermal cactus pear juice preservation. Juice concentrates and fruit powders were produced by rotary evaporation and freeze drying at laboratory scale and compared to products obtained at pilot plant-scale applying a three-stage column evaporator and spray drying, respectively. To monitor process-related quality changes, the resulting products were characterised in terms of colour and selected quality parameters. For both juice concentrates and fruit powders, initial colour characteristics were retained. In addition to betanin isomerisation, C₁₁-epimerisation of proline-betaxanthin was demonstrated to be a valuable indicator of the respective heat treatment applied. Whereas no adverse reactions were observed during juice production, non-enzymatic browning and HMF formation were found after concentration at pilot plant-scale and freeze drying, respectively.

Industrial relevance

Although cactus pear meets all requirements for food colouring purposes, no attempt has been made so far to exploit this potential. The feasibility of processing cactus pear juice into concentrates and fruit powders was demonstrated in this study. In contrast to single-strength juice, concentrates and powders are easier to handle during transportation and storage and also open further fields of application that may promote cactus pear fruit processing at industrial scale in the future.     

Pulsed-field gel electrophoresis for the discrimination of Oenococcus oeni isolates from different wine-growing regions in Germany

Reliable techniques are needed for the identification individual Oenococcus oeni strains with desirable flavor characteristics and to monitor the survival and contribution of inoculated and indigenous bacteria. Therefore, we investigated the suitability of pulsed-field gel electrophoresis (PFGE) for the discrimination of 65 O. oeni isolates from six different wine-producing regions in Germany. Among the restriction enzymes tested, genomic DNA digestions with Sfi I were most effective by displaying 56 (86%) different banding profiles. Our results underline the high capacity of PFGE for strain identification and differentiation. Cluster analysis of the DNA restriction patterns revealed no distinct region specificity of O. oeni populations.     

Aflatoxin B1 contamination in wheat grain samples collected from different geographical regions of India: A multicenter study

In a multicenter study conducted by the Indian Council of Medical Research, 1,646 samples of wheat grain collected from rural and urban areas of 10 states representing different geographical regions of India were analyzed for aflatoxin B1 (AFB1). AFB1 concentrations of > or = 5 microg kg(-1) were recorded in 40.3% of the samples, and concentrations above the Indian permissible regulatory limit of 30 microg kg(-1) were found in 16% of the samples. The proportion of samples with AFB1 concentrations above the Indian regulatory limit ranged from 1.7 to 55.8% in different states, with the minimum in Haryana and the maximum in Orissa. The variation in wheat contamination among states seems to be mainly the result of unsatisfactory storage conditions. Median AFB1 concentrations of 11, 18, and 32 microg kg(-1) were observed in samples from Uttar Pradesh, Assam, and Orissa, respectively; concentrations in other states were <5 microg kg(-1). The maximum AFB1 concentration of 606 microg kg(-1) was observed in a sample from the state of Uttar Pradesh. The calculated probable daily intakes of AFB1 through consumption of contaminated wheat for the population in some states were much higher than the suggested provisional maximum tolerable daily intake. Human health hazards associated with such AFB1 exposure over time cannot be ruled out.     

Aflatoxin B1 in betel nuts (Areca catechu L.) imported to Pakistan from different regions of South Asia

Aflatoxin B₁ (AFB₁) levels were evaluated in betel nuts (Areca catechu L.) being imported to Pakistan during 2010–2011. In total, 278 betel nut samples (India = 21, Indonesia = 51, Sri-Lanka = 34 and Thailand = 172) were received from the Department of Customs and were analysed by thin layer chromatography (TLC). All Indian origin betel nuts showed AFB₁ contamination ranging from 11.7–262.0 µg kg^?1 with a mean of 92.5 µg kg^?1. Among Indonesian and Sri Lankan shipments, 80.4% and 73.5% betel nuts were contaminated with AFB₁ ranging between 3.3–39.2 and 6.5–103.4 µg kg^?1 with a mean of 11.6 and 35.0 µg kg^?1, respectively. However, only 30.2% of Thailand origin samples showed AFB₁ contamination ranging 3.3–77.0 µg kg^?1 with a mean of 6.6 µg kg^?1. The widespread occurrence of AFB₁ increases the hazard associated with betel nuts. Thus, strict control is a pre-requisite for the production and import/export of psychoactive substances as betel nuts.     

基于超高效液相色谱串联三重四级杆质谱的不同产区赤霞珠葡萄酒花色苷特征性研究

利用超高效液相色谱结合三重4级杆质谱(ultra-performance liquid chromatography-mass spectrometry/-mass spectrometry,UPLC-MS/MS)对我国4个主要产区(新疆石河子、宁夏贺兰山东麓、陕西泾阳、山东烟台)赤霞珠葡萄新酒中游离花色苷的种类、含量及特征性进行探讨。结果显示,从新鲜赤霞珠葡萄酒中共检测出16种游离花色苷,其中二甲花翠素葡萄糖苷是赤霞珠葡萄酒中含量最高的花色苷,二甲花翠素乙酰化葡萄糖苷次之。此外,宁夏产区葡萄酒花色苷含量显著高于其他产区。利用XLSTAT 2014建立不同产区葡萄酒基于花色苷种类及含量的判别模型,可将葡萄酒按产地明显区分开,判别正确率达100%。