Cold-adapted biocontrol yeast was selected from four yeast isolates from Tibet against gray mold of cherry tomato in cold storage. The strain numbered LB2 showed the best biocontrol activity and identified as Cryptococcus laurentii. Competition for nutrient, space, and induced fruit resistance was also its antagonistic mechanism. Compared with C. laurentii from sea-level place, the reason why LB2 had a better biocontrol activity was studied. More trehalose and proline in cell of LB2 made it exhibit a better cellular activity at low temperature, such as higher population dynamics in the wounds of cherry tomato and more biocontrol-related enzyme secretion, chitinase and β-glucanase. The better oxidative stress tolerance was another characteristic of LB2. Maybe because of the ideal culture condition, there was no obvious difference between these two yeasts in the growth in vitro test at low temperature. Although the same phenomenon existed in the low pH stress test, LB2 still had higher cell concentration under this stress. Comparative transcriptomics method was also applied to analyze the cell activity of LB2 and C. laurentii at different temperatures. The results showed that more active response in the intracellular structure and intracellular metabolic process to cold temperature made LB2 had a better activity. The present study indicated a possibility to select cold-adapted biocontrol yeast from Tibet and also showed its primary action mechanism.

This study conducts a systematic review and meta-analysis to assess the association between dairy consumption and hepatocellular carcinoma (HCC) risk.The association between consumption of overall dairy, specific dairy products (such as milk, cheese, butter, yoghurt, condensed milk, whey, casein, lactose) and HCC risk has not been assessed before. This association between dairy consumption and HCC risk has been reported in several epidemiological studies, but results were controversial and inconsistent. The PubMed, EMBASE and Cochrane databases were searched for relevant studies published up to September 20, 2015. The relative risks (RRs) and odds ratios (ORs) with 95% confidence intervals (CIs) for dairy consumption associated with HCC risk were extracted from each included cohort as well as case-control study. In our study, the pooled RR was obtained using the random-effects model. Heterogeneity was evaluated by Q and I2 statistics.A total number of 1,084,666 participants and 2041 HCC patients from three cohort studies and five case-control studies were included in this meta-analysis. The pooled RR of dairy was 1.38 (95% CI: 1.00–1.91, p = 0.00) in all studies. The pooled RR of milk, yoghurt, and cheese was 1.13 (95% CI: 0.67–1.88, p = 0.00), 0.40 (95% CI: 0.14–1.14, p = 0.00) and 1.45 (95% CI: 1.02–2.07, p = 0.80), respectively.This meta-analysis indicates that consumption of overall dairy may be associated with increased HCC risk and a risk factor involved in the etiology of HCC.

Highly specific monoclonal and polyclonal antibodies are the key components in a diverse set of immunoassay applications, from research work to routine monitoring and analysis. In the current manuscript, combinatorial strategies for a single mixture immunization, screening and rabbit hybridoma cell technology were described. Fluoroquinolones (FQs) drugs were chosen as representative analytes. Six FQs were conjugated with bovine serum albumin and used as immunogens for subsequent immunization, while a mixture of all was injected for coimmunization. The hybridomas obtained against the individual and multiple FQs were used for the production of diverse varieties of rabbit monoclonal antibodies (RabMAbs) against the target analytes. As was proven by indirect competitive ELISA and quantitative lateral flow immunoassay, this approach opens a new way for simultaneously obtaining functional monoclonal antibodies which are capable of recognizing both individual and multiple analytes in a single preparation circle. This addresses various needs of different monitoring regulations as analytical methodology advances.

Six lactic acid bacterial (LAB) strains were isolated from traditionally fermented Xinjiang cheese and evaluated for functional and probiotic properties and potentials as starter cultures. The isolated six LAB strains comprised Lactobacillus rhamnosus (one strain), Lactobacillus helveticus (one strain), and Enterococcus hirae (four strains). All of the six strains were tolerant to acidic and bile salt conditions. Among which, the L. rhamnosus R4 strain showed more desirable antimicrobial, auto-aggregation, and hydrophobic activity. In addition, the strain L. rhamnosus R4 exhibited the highest level of free radical scavenging activity (53.78% of 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals and 45.79% of hydroxyl radicals). L. rhamnosus R4 also demonstrated cholesterol and triglyceride degradation by 50.97% and 28.92%, respectively. To further examine the health-promoting effects of these LAB strains on host lifespan, Caenorhabditis elegans was used as an in vivo model. Worms fed LAB as a food source had significant differences in lifespan compared to those fed Escherichia coli OP50 (as a negative control). Feeding of L. rhamnosus R4 extended the mean lifespan of C. elegans by up to 36.1% compared to that of the control. The results suggest that the strains isolated from Xinjiang fermented dairy products have high potential as starter cultures in the cheese industry.分离鉴定新疆牧民家庭自制的4 份奶酪样品中的6 株乳酸菌,研究其益生特性。通过对新疆传统发酵乳制品中乳酸菌的分离及其抗氧化和降脂特性的测定,筛选到益生特性良好同时也能延长模式生物秀丽线虫寿命的菌株。对分离自传统发酵乳制品中的乳酸菌进行生理生化分析和16S rDNA 分子生物学鉴定,筛选出6株乳酸菌,并测定其耐酸、耐胆盐、抑菌、亲水性和共聚集能力;测定其抗氧化、降解胆固醇和甘油三酯的能力;测定乳酸菌对秀丽线虫寿命的影响。实验表明,该6 株菌具有较好的耐酸性,在胆盐环境中也有较好的存活率。同时,这6 株菌均有清除自由基和降解脂肪的能力。此外,其中3 株具有延长秀丽线虫寿命的能力。综上所述,这6株菌具有较好的益生特性,可作为优良的潜在益生菌株。

American ginseng (Panax quinquefolius L.) and Chinese jujube (Zizyphus jujuba Mill.) are commonly used in traditional Chinese medicine to enhance immune function.The present study aimed to develop one Chinese prescription, Shenzao Cha (SZC), consisting of American ginseng and Chinese jujube, and systematically investigate its immunomodulation in healthy ICR mice.Normal ICR mice received intragastric administration of SZC (1.3, 2.6, and 5.2 g raw material/kg body weight) once daily for four weeks, while a control group received the same amount of sterile water.SZC significantly increased the spleen and thymus indices and T-lymphocyte proliferation, while the T-lymphocyte proliferation in the 5.2 g/kg group was 1.4-fold higher than that in the control. Further, 1.3 g/kg SZC could markedly improve hemolytic activity by 25.2%, and 2.6 g/kg SZC increased the NK cell activity by 78.6% relative to the control. In addition, the activities of antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase), that participated in modulating oxidative stress, were significantly increased in the liver, spleen, thymus, and serum, while the contents of malondialdehyde were dramatically decreased.SZC exhibited potent immunomodulatory effects on innate and adaptive immunity in healthy ICR mice, as well as potential antioxidant activity for prevention of oxidative stress, which was suggested to partly contribute to the immune enhancement.综合评价复方西洋参和红枣制剂对于小鼠非特异 性免疫和特异性免疫的影响, 为开发新产品奠定 基础。本文首次以西洋参提取物和红枣提取物为原料制 成复方制剂, 并通过动物实验发现该制剂具有增 强免疫功能, 为开发功能性食品提供新思路。将西洋参与红枣提取物按一定比例制备复方制 剂, 以1.3、2.6 和5.2 g/kg 为低、中和高剂量给 健康ICR 小鼠连续灌胃此复方制剂4 周, 对照组 灌胃同等剂量的无菌水, 最后通过测定免疫相关 指标对该复方制剂的免疫调节功能进行综合评 价。此款复方制剂可以显著提高小鼠的胸腺和脾脏指 数(表1), 同时低剂量组小鼠的血清溶血素水 平显著高于对照组, 高剂量组小鼠的脾淋巴细胞 增值能力约是对照组的2.4 倍, 自然杀伤细胞的 活性也得到显著增强, 中剂量组小鼠的NK 细胞 活性与对照组相比提高了78.6%(图2)。另外, 此复方制剂能有效提高小鼠组织(肝脏、脾脏和 胸腺)和血清中超氧化物歧化酶(SOD)、谷胱 甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT) 的活性, 同时降低丙二醛(MDA) 的含量 (图3~6)。综上所述, 此复方西洋参和红枣制 剂可有效提高健康ICR 小鼠的非特异性和特异 性免疫功能, 而其抗氧化功能有可能是其免疫调 节活性的作用机制之一。

•The mechanism by which R. paludigenum induces resistance was global analysed.•Plant hormone and signalling transduction contributed to yeast–fruit interaction.•R. paludigenum increased phenolic acids & lignin production in mandarin orange.•R. paludigenum elicited energy shift from primary metabolism to immunity.To investigate the basis of inducible resistance response in postharvest mandarin orange, cDNA microarray and high-performance liquid chromatography were performed to study transcriptional and metabolic changes in Rhodosporidium paludigenum strain treated fruit. The microarray data mining revealed that R. paludigenum activated transcription of genes important for plant hormones, signalling transduction, stress and defensive responses in orange peel tissue. Moreover, up-regulation of phenylalanine and tyrosine metabolism, phenylpropanoids biosynthesis, and alkaloid biosynthesis I, were observed at the transcription level. Conversely, large amounts of genes involved in starch metabolism, oligosaccharide and glycoside metabolism were markedly repressed by R. paludigenum treatment. Activation of phenylpropanoids biosynthesis pathway was correlated with the increasing production of phenolic acids and their subsequent metabolite lignin, indicating antifungal metabolites indeed contributed to biocontrol yeast enhanced fruit protection. Our findings provide an important basis for understanding the mechanisms of resistance induction in mandarin orange, as well as for reducing postharvest losses.

A total of 20 strains of yeast isolated from Tibetan fermented products were screened for antagonism against blue mold of pear caused by Penicillium expansum. Six isolates that inhibited incidence of postharvest decay by 35% or more were selected for further screening. Among them, the most effective was Rhodotorula mucilaginosa. The results showed that washed cell suspensions of R. mucilaginosa yielded better antagonistic efficacy than unwashed cell-culture mixtures, cell-free culture filtrates, and autoclaved cell cultures. Biocontrol activity improved with increasing concentrations of incubated cells. The best concentration was 1×108 cells/ml, at which the incidence of decay was only 16.7% after 6 d of incubation. The germination of conidia of P. expansum in vitro was significantly inhibited by both washed cell-suspensions and unwashed cell-culture mixtures. Rapid colonization by yeast at different concentrations showed a relationship between yeast-cell concentration and biocontrol activity. Although the titratable acidity of pear fruits increased after treatment, R. mucilaginosa did not affect the total soluble solids or ascorbic acid content. This is the first study to report that the yeast R. mucilaginosa from Tibet Autonomous Region of China may have potential as an antagonist to control the postharvest decay of pear fruits.从西藏发酵制品中分离筛选出对水晶梨采后青霉病具有较好防治效果的拮抗酵母, 并对其作用机理进行研究。首次从西藏发酵制品中筛选得到一株对水晶梨采后青霉病具有较好防治效果的拮抗酵母Rhodotorula mucilaginosa, 并对其作用机理进行了初步研究。采用水晶梨果实体内筛选的方法, 经过初筛与复筛, 确定具有较好生防效果的拮抗酵母; 通过对筛选所得拮抗酵母不同浓度、不同处理液的生防效果, 果实伤口处生长动态, 以及对梨果实采后品质影响的研究, 初步探讨西藏发酵制品中拮抗酵母的生防作用机理。从西藏发酵制品中筛选得到一株拮抗酵母R. mucilaginosa, 其对水晶梨采后青霉病具有较好的生物防治效果。研究结果发现其主要作用机理为在果实伤口处与病原菌的营养与空间竞争。通过进一步研究, 可开发为梨果实采后病害防治的新型生物保鲜剂。

Tomato yellow leaf curl virus (TYLCV) resistant cultivar T07-4 and susceptible cultivar T07-1 were inoculated with the root endophytic fungus, Piriformospora indica in greenhouse to study the effects of P. indica inoculation on the tomato growth, early yield, fruit quality and resistance to TYCLV. The results indicated that P. indica stimulated root growth, promoted the growth of tomato plants between 2–6 weeks for T07-1 and 2–4 weeks for T07-4 cultivar after inoculation. The early fruit yield was improved by 12.8 % for susceptible cultivar T07-1, but no significant difference for resistant cultivar T07-4. The taste of fruits are even better because of higher ratio of TSS to TA for two cultivars and P. indica increased TSS and firmness for cultivar T07-1. P. indica enhanced more pathogensis-related genes expressions in inoculated susceptible cultivar T07-1 than in resistant cultivar T07-4 at 2 weeks after inoculation. P. indica induced resistance against TYCLV for susceptible cultivar, reduced TYCLV incidence and decreased disease index by 26 % and 1.25 in natural TYCLV infection. One may draw an inference that P. indica inoculation can lead to better vegetative growth, higher early yield and induced resistance for TYLCV-susceptiable cultivar T07-1 in practical greenhouse condition.

Cold-adapted yeasts were isolated from soil samples collected in Tibet and evaluated as potential biocontrol agents against blue mold (Penicillium expansum) of pear fruit in cold storage. YC1, an isolate identified as Rhodotorula mucilaginosa, was found to exhibit the greatest biocontrol activity among the different isolates that were screened. A washed cell suspension of YC1 exhibited the best biocontrol activity among three different preparations that were used in the current study. A concentration of 108 cells/ml reduced the incidence of decay to 35 %, compared to the control where decay incidence was 100 %. A higher intracellular level of trehalose and a higher proportion of polyunsaturated acids present in YC1, was associated with increased the tolerance of this strain to low temperatures, relative to the other strains that were evaluated. The increased tolerance to low temperature allowed the YC1 strain of yeast to more effectively compete for nutrients and space in wounded pear fruit that had been inoculated with spores of P. expansum and placed in cold storage. The present study demonstrated the ability to select cold-adapted yeasts from cold climates and use them as biocontrol agents of postharvest diseases of fruit placed in cold storage.

•Chitosan efficiently inhibited green mold rot in satsuma orange.•Chitosan reduced germination of P. digitatum on PDA medium.•Chitosan significantly improved biocontrol efficiency of antagonistic yeast.•Increased resistance dominates biocontrol efficiency of chitosan in fruit.•Chitosan had no adverse influence on the edible quality of oranges.This study investigated the capacity of chitosan oligomers (COS), applied before harvest singly or in combination with antagonists, in controlling postharvest green mold caused by Penicillium digitatum in satsuma orange. Oranges treated with COS or Rhodosporidium paludigenum were observed having a delay in onset and progression of disease symptoms relative to wounded controls. Preharvest application of COS at different concentrations achieved similar biocontrol efficiency rates in green mold control after 4 days storage. However, the combination of pre-COS (1%, w/v) and R. paludigenum showed a more effective decay control than any other treatments. COS (1%, w/v) alone did not negatively affect R. paludigenum growth in wounds, but severely inhibited P. digitatum spore germination than lower dose treatments in vitro. The expression levels of the defense-related genes chitinase and phenylalanine ammonia lyase increased with decreased disease symptoms. Moreover, this phenomenon was more prominent in the integrated treatments than in the individual ones.

The objectives of this work were to assess the optimum conditions for induction of acid tolerance in the marine yeast Rhodosporidium paludigenum and evaluate the biocontrol activity of non-adapted and acid-adapted yeasts in controlling apple blue mold caused by Penicillium expansum. R. paludigenum grown in malic and lactic acid treatments were stimulated after 12 h incubation. Moreover, medium modified with malic and lactic acid significantly enhanced the acid tolerance of R. paludigenum (p < 0.05). In acid tolerance response test, the highest viability of R. paludigenum was obtained at initial pH of 5.5 in the NYDB medium modified with malic acid (91.6 %). In addition, all R. paludigenum treatments significantly reduced the disease incidences and lesion diameters of blue mold in apples. Furthermore, there was no significant difference between acid-adapted and unadapted yeasts in the apple wounds after 48 h dynamics. Acid stress improved R. paludigenum viability under acidic conditions. However, there was no significant difference between acid-adapted and unadapted yeasts in controlling P. expansum on apple fruit (p < 0.05). These results indicate the potential for maintaining the survival level of biocontrol agents by physiological inducement strategy.

We present our experiment about adding anthocyanins to the daily food of mice. Three kinds of anthocyanins (cyanidin-3-glucoside, cyanidin-3-rutinoside and pelargonidin-3-glucoside) purified from Chinese mulberry (Morus australis Poir) were evaluated for suppressing body weight gain of the male C57BL/6 mice fed with high-fat diet (HFD). The results from a 12-week experiment show that consumption of purified mulberry anthocyanins (MACN) of 40 or 200 mg/kg can significantly inhibit body weight gain, reduce the resistance to insulin, lower the size of adipocytes, attenuate lipid accumulation and decrease the leptin secretion. Thus, dietary supplementation with MACN can protect against body weight gain of the diet-induced obese mice.•Purified mulberry anthocyanins exhibit anti-obesity effect.•Cyanidin-3-glucoside and cyanidin-3-rutinoside reduced the resistance to insulin.•Consumption mulberry anthocyanins attenuated lipid accumulation.

•Myricitrin inhibits peroxynitrite-induced DNA damage and cytotoxicity in astrocytes.•Myricitrin significantly scavenges hydroxyl radicals from peroxynitrite.•Myricitrin prevents peroxynitrite-mediated GSH depletion in astrocytes.Peroxynitrite, a potent oxidising and nitrating species, has been implicated in the pathogenesis of neurodegenerative diseases. This study was undertaken to investigate the protective effect of myricitrin on peroxynitrite-mediated toxicity and the underlying mechanism. Our results showed that the presence of myricitrin was found to significantly inhibit peroxynitrite-mediated DNA damage. EPR spectroscopy demonstrated that myricitrin potently diminished the DMPO-hydroxyl radical adduct signal from peroxynitrite. Further study showed that glutathione (GSH) depletion caused by peroxynitrite can be effectively prevented by pre-incubation of astrocytes with myricitrin. Moreover, co-incubation of astrocytes with myricitrin and buthionine sulfoximine (BSO) eliminated the myricitrin-induced GSH increase. In contrast, co-incubation of myricitrin with BSO slightly protected astrocytes against cytotoxicity and DNA damage mediated by peroxynitrite. These results revealed that myricitrin can protect against peroxynitrite-induced DNA damage and cytotoxicity, which might have implications for myricitrin-mediated neuroprotection.

This study investigated the anti-obesity effects of honeysuckle anthocyanins (HA) in a high fat diet-induced mouse model. The mice were initially fed with a low-fat diet (LFD) or high-fat diet (HFD) for 8 weeks. After that, the HFD-fed mice were divided into five groups, with 12 mice in each group, including a HFD group, a HFD plus Orlistat group, and three HFD plus HA (at a dose of 50, 100, or 200 mg kg−1) groups, for another 8-week experiment. HA at 100 or 200 mg kg−1 can suppress body weight gain, reduce serum and liver lipid profiles, ameliorate impaired hepatic function, and significantly increase serum adiponectin concentration while decreasing serum insulin and leptin levels. These results suggest that the anti-obesity effect of HA might be through the blockage of lipid accumulation.

Cecropin A gene was cloned into the expression vector pPIC9k and was successfully expressed in methylotrophic yeast, Pichia pastoris GS115. The yeast had effective antimicrobial activity on Geotrichum citri-aurantii spores by the thiazolyl blue (MTT) assay. There was no large growth difference between nontransformed strain GS115 and recombinant strain GS115/CEC in citrus fruits wounds. Yeast transformants could significantly inhibit growth of germinated G. citri-aurantii spores and inhibited decay development caused by G. citri-aurantii in citrus fruits compared to the yeast strain GS115/pPIC. This study demonstrates the potential of expression of an antifungal peptide in yeast for enhancing suppression of postharvest diseases and represents a new approach for the biological control of postharvest diseases.Highlights► The study showed that cecropin A can inhibit G. citri-aurantii spores effectively. ► Recombinant strain GS115/CEC can express antifungal cecropin A successfully. ► GS115/CEC yeast is suitable for biocontrol tests on citrus fruit. ► GS115/CEC yeast can inhibit growth of G. citri-aurantiiin vitro and in vivo.

Recombinant Pichia pastoris yeasts expressing cecropin A (GS115/CEC), was evaluated for the control of the blue mold of apple caused by Penicillium expansum due to cecropin A peptide’s effective antimicrobial effects on P. expansum spores by the thiazolyl blue (MTT) assay. Then, the protein concentration was determined and it was expressed at high levels up to 14.2 mg/L in the culture medium. Meanwhile, the population growth was assayed in vivo. The population growth of recombinant strain GS115/CEC was higher than that of non-transformed strain GS115 in red Fuji apples wounds. Recombinant yeast strains GS115/CEC significantly inhibited growth of germinated P. expansum spores in vitro and inhibited decay development caused by P. expansum in apple fruits in vivo when compared with apple fruits inoculated with sterile water or the yeast strain GS115/pPIC (plasmid pPIC9k transformed in GS115). This study demonstrated the potential of expression of the antifungal peptide in yeast for the control of postharvest blue mold infections on pome fruits.

We investigated the effects of marine yeast Rhodosporidium paludigenum in combination with a food additive, carboxymethylcellulose sodium (CMC-Na), on prevention of postharvest decay and food quality of Chinese winter jujubes. R. paludigenum (1 × 108 cells/ml) combined with CMC-Na (0.3%) significantly increased the inhibition of black rot on jujubes at 25 °C when compared with R. paludigenum-alone treatment (5.8% vs. 20%, p < 0.05). The combination also reduced natural rot from 86% (control) to 56%. The combination caused transient changes in enzyme activities or contents of some oxidation reactive markers such as peroxidase (POD), superoxide dismutase (SOD), and malondialdehyde (MDA) of jujubes. The combination had no significant effect on the food qualities such as colour (chroma and hue angle), total soluble solid (TSS) and titratable acidity (TA) of the fruit. While enhancing these effects, CMC-Na did not affect the survival of R. paludigenum in nutrient yeast dextrose agar (NYDA) culture. Thus, we conclude that the combination of R. paludigenum and CMC-Na is a promising formulation to control postharvest decay of Chinese winter jujubes.

LePR-5, a putative PR5 like protein gene was amplified from a cherry tomato (Lycopersicon esculentum), which encodes a precursor protein of 250 amino acid residues, and shares high degrees of homology with a number of other PR5 genes. Expression of LePR-5 in different tomato organs was analyzed with Semi-quantitative RT–PCR, showing that LePR-5 expressed at different levels in leaves, stems, roots, flowers and fruits. In addition, expression of LePR-5 under different abiotic stresses was carried out at different time points. Three of the four tested abiotic stimuli, ethophen, salicylic acid and methyl jasmonate, triggered a significant induction of LePR-5 after treatment. However, LePR-5 was weaker induced by abscisic acid than by others. The positive responses of LePR-5 to the three abiotic stimuli suggested that LePR-5 may play an important role in response to abiotic stresses, and it may also be involved in plant defense system against pathogens. In addition, different expression patterns between tomato fruit and seedling suggested that LePR-5 may play a distinctive role in the defensive system protecting tomato fruit and seedling.

Culture medium for keratinase production from hair substrate by a new Bacillus subtilis strain, KD-N2, was optimized. Effects of culture conditions on keratinase production were tested, and optimal results were obtained with 10% inocula (v/v), 16 g/L hair substrate, an initial pH value of 6.5 and a culture volume of 20 mL. Several carbon sources (sucrose, cornflour) and nitrogen sources (yeast extract, tryptone and peptone) had positive effects on keratinase production, with sucrose giving optimal results. To improve keratinase yield, statistically based experimental designs were applied to optimize the culture medium. Fractional factorial design (FFD) experiments showed that MgSO4 and K2HPO4 were the most significant factors affecting keratinase production. Further central composite design (CCD) experiments indicated that the optimal MgSO4 and K2HPO4 concentrations were 0.91 and 2.38 g/L, respectively. Using an optimized fermentation medium (g/L: NaCl 1.0, CaCl2 0.05, KH2PO4 0.7, sucrose 3, MgSO4 0.91, K2HPO4 2.38), keratinase activity increased to 125 U/mL, an approximate 1.7-fold increase over the previous activity (75 U/mL). Human hair was degraded during the submerged cultivation.

The aim of this study was to purify and characterize a keratinase produced by a new isolated Bacillus subtilis KD-N2 strain. The keratinase produced by the isolate was purified using ammonium sulphate precipitation, Sephadex G-75 and DEAE (diethylaminoethyl)-Sepharose chromatographic techniques. The purified enzyme was shown to have a molecular mass of 30.5 kDa, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The optimum pH at 50 °C was 8.5 and the optimum temperature at pH 8.5 was 55 °C. The keratinase was partially inactivated by some metal ions, organic solvents and serine protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetic acid (EDTA) had positive effect on the keratinase activity. Reducing agents including dithiothreitol (DTT), mercaptoethanol, L-cysteine, sodium sulphite, as well as chemicals of SDS, ammonium sulfamate and dimethylsulfoxide (DMSO) stimulated the enzyme activity upon a feather meal substrate. Besides feather keratin, the enzyme is active upon the soluble proteins ovalbumin, bovine serum albumin (BSA), casein and insoluble ones as sheep wool and human hair. Calf hair, silk and collagen could not be hydrolyzed by the keratinase.

Postharvest decay of fruit may be controlled by the use of a variety of diverse microorganisms acting as biocontrol agents, but the mechanisms associated with control are not fully understood. In order to gain insight into the action of antagonistic microorganisms on fruit, a forward subtractive suppression hybridization (SSH) cDNA library was constructed. SSH was performed with cDNA from cherry tomato fruit (Lycopersicon esculentum) inoculated with water as the “driver” and cDNA from tomato fruit inoculated by Cryptococcus laurentii as the “tester”. A total of 150 clones in the SSH library were sequenced and found to represent 50 unigenes. BLASTX results reveal that 35 cDNAs had significant sequence homologies with known sequences in the NCBI database. The identified cDNAs encode proteins involved in cellular processes such as primary metabolism, signal transduction, defense and responses to pathogens, stress-related, cell wall assembly, and photosynthesis and transcription related sequences. Six cDNA clones were selected for temporal expression analysis using RT-PCR. The results show that a number of transcripts encoding proteins/enzymes which are known to be up-regulated under some biotic and abiotic stresses are also up-regulated after the application of biological control yeast to cherry tomato fruit. The expression of these proteins may play a role in increasing fruit resistance to postharvest pathogen infection.

•R. paludigenum induces resistance against Penicillium digitatum in citrus.•Efficacy of induced resistance is dependent on the time of the yeast application.•The inhibition rate is influenced by the concentration of yeast used.•R. paludigenum stimulates the activities of GLU, PAL, POD and PPO in citrus.Induced disease resistance against plant pathogens is a promising non-fungicidal decay control strategy. In this study, a potential biocontrol yeast, Rhodosporidium paludigenum, was investigated for its induction of disease resistance against Penicillium digitatum in citrus fruit. The results showed that R. paludigenum is the most effective yeast among three selected yeasts in stimulating the resistance of citrus fruit to green mold. When R. paludigenum was applied 48–72 h before inoculation with P. digitatum, disease incidence and disease severity in citrus fruit significantly decreased. Application of R. paludigenum at concentrations of 1 × 108 and 1 × 109 cells mL−1 respectively resulted in 49.6% and 52.5% reductions in the percentage of infections. Induction of resistance to P. digitatum by R. paludigenum treatment significantly enhanced the activities of defense-related enzymes, including β-1,3-glucanase, phenylalanine ammonia-lyase, peroxidase, and polyphenoloxidase, which may be an important mechanism by which the biocontrol yeast reduces the fungal disease of citrus fruit caused by P. digitatum.

•Preventive activity of MeJA combined with C. laurentii in inhibiting green mold in citrus fruit inoculated with Penicillium digitatum was better than MeJA or antagonistic yeast applied alone.•MeJA increased the population growth of C. laurentii in citrus fruit wounds.•MeJA and C. laurentii activated the PPO, POD and CAT activities of citrus fruit.•MeJA and C. laurentii induced a rise in the mRNA expression level of PR5 of citrus fruit peel.The objective of this study was to evaluate the preventive activity of methyl jasmonate (MeJA) alone and in combination with antagonistic yeast in suppressing green mold decay in citrus fruit, and to explore the mechanisms involved. At 100 μmol/L, MeJA inhibited disease incidence and lesion diameter of mold decay compared with the control (P < 0.05) The preventive application of Cryptococcus laurentii at 1 × 108 cells/mL combined with 100 μmol/L MeJA reduced green mold incidence compared to the control and the other treatment groups (P < 0.05) when tested in wounded citrus fruit inoculated with Penicillium digitatum. MeJA and C. laurentii induced higher activity of polyphenol oxidase, peroxidase and catalase than control. Moreover, treatment with MeJA and C. laurentii induced a rise in the mRNA expression level of PR5 (pathogenesis-related protein family 5), which was stronger than in the single-treatment groups and the control. In addition, 100 μmol/L MeJA improved the rapid proliferation of C. laurentii in citrus fruit wounds. This combined treatment can induce natural resistance and stimulate the proliferation of antagonistic yeast on the fruit surface.

•A stable relationship between pigment secretion and Val-A production was found.•An equation with SA value of pigment to determine Val-A was fitted.•Accuracy and applicability of Spectrophotometry was proved in determining Val-A productivity and screening high-yield mutants.•Spectrophotometry assay is reported first as a rapid method in antibiotic fermentation.Validamycin A (Val-A), produced by Streptomyces hygroscopicus 5008 in industrial fermentation, is one of the most widely used anti-fungal agro-antibiotics in Asia and high performance liquid chromatography (HPLC) assay is usually used to determine the production of Val-A. A new approach to determine Val-A by spectrophotometer is developed. During the fermentation of S. hygroscopicus 5008, a pigment secretion was found along with the Val-A biosynthesis. There was a stable relationship between the concentration of Val-A and spectral absorption (SA) value of this pigment at 450 nm, even in different fermentation cultures or conditions. Using SA value as interior label, a rapid spectrophotometric method for determining Val-A production was established. In comparing Val-A productivity by HPLC method with that by SA method, the relative standard deviation (R.S.D.) was 0.007 (less than 0.05, no variation) and the conditional probability [Pr(T < t)] was 0.3491 (greater than 0.05, no difference) at the optimal time point of Val-A fermentation, which demonstrated SA method was as stable and accurate as standard HPLC method. It was applied successfully to finding positive strains with high Val-A productivity and short fermentation time. SA assay is an accurate and cost-effective method for measuring Val-A and screening high-producing strains, and this work provides a new insight for rapid quantitative analysis of antibiotics in fermentation of pigment-producing strains.

The potential of using Rhodotorula glutinis alone or in combination with hot water for the control of postharvest blue mold decay of pear fruit, and their effects on postharvest quality of fruit was investigated. Spore germination of Penicillium expansum was inhibited by hot (46 °C) water. Both hot water for 10–20 min and R. glutinis, as stand-alone treatments, reduced the incidence of blue mold decay, but complete control was not achieved by either treatment. However, a combination of hot water treatment and R. glutinis completely controlled decay of inoculated fruit. In addition, the combination of hot water treatment with R. glutinis on naturally infected, intact fruit, reduced decay from 66.7% in the control fruit to 13.3% after 15 days at 20 °C, and from 46.7 to 6.7% after 4 °C for 60 days followed by 20 °C for 15 days. None of the treatments impaired fruit quality. The combination of hot water and R. glutinis could be an alternative to synthetic fungicides for the control of postharvest blue mold decay on pears.

The effectiveness of the cytokinin N6-benzyladenine (6-BA), alone or in combination with the biocontrol yeast Cryptococcus laurentii, in controlling blue mold on pear fruit was assessed. The application of 6-BA (500–2000 μg mL−1) or C. laurentii was effective in reducing Penicillium expansum infection in pear fruit wounds, but its efficacy declined rapidly as the incubation time increased. Integrated application of C. laurentii and 6-BA at 500–2000 μg mL−1, especially at the optimal concentration (1000 μg mL−1), resulted in a more effective and stable inhibition of the mold rots than that of the 6-BA or C. laurentii alone. Treatments of pears with 6-BA at 1000 μg mL−1, alone or with C. laurentii also led to an increase in catalase activity and an inhibition of the activities of both peroxidase and lipoxygenase as well as ethylene production. In addition, 6-BA from 20 to 2000 μg mL−1 did not influence the population growth of C. laurentii in pear fruit wounds. These data suggested that a combination of 6-BA and C. laurentii could integrate the dual biological activities from 6-BA and C. laurentii and might be developed into a novel protection strategy for reduction of the blue mold rot of pear fruit.

This study was conducted to evaluate the efficacy of the biocontrol yeast Cryptococcus laurentii and salicylic acid (SA) in suppressing the blue and gray mould rots in pear fruit and to explore possible mode of action involved. Our results showed that the combined treatment of pear fruit with C. laurentii with SA at 100 μg ml− 1 resulted in a remarkably improved control of Penicillium expansum and Botrytis cinerea infections, including the pre-inoculated P. expansum, in comparison with the application of C. laurentii or SA alone. The biocontrol yeast C. laurentii proliferated rapidly within the first 24 h of incubation in pear fruit wounds. Although SA at 100 μg ml− 1 neither affected the population growth of C. laurentii nor directly inhibited the blue mold when the inoculation concentrations of P. expansum were above 5 × 102 spore per ml in vivo, it induced the fruit resistance to the blue and gray mold rots when the time interval between SA treatment and pathogens inoculation was more than 48 h, being associated with a rapid and strong activation of the peroxidase activity in pear fruit. Thus we assume that SA may be regarded as a secondary defense line in a combination of C. laurentii and SA, which could reinforce the biocontrol efficacy of C. laurentii by induction of the fruit natural resistance.

This study was conducted to determine the efficacy of chitosan at different concentrations with various intrinsic viscosities alone, and in its combination with a yeast antagonist Cryptococcus laurentii in reducing the blue mold rot caused by Penicillium expansum in apple fruit. The results indicated that application of chitosan alone was effective in inhibiting the blue mold rot in apple fruit wounds, especially with the high concentrations and low viscosities. But its efficacy was declining with the incubation time so that chitosan alone could not provide enduring protection of apple fruit from P. expansum infections. When applied at the concentration range from 0.001 to 0.1% (wt/vol), chitosan did not influence the population growth of C. laurentii in vivo, whereas it markedly repressed the yeast growth as its concentrations were increased up to 0.25% (wt/vol) or higher. Moreover, combination of chitosan and C. laurentii resulted in a synergistic inhibition of the blue mold rot, being the most effective at the optimal concentration of 0.1% of chitosan with the lowest viscosity (12 cP). The possible mode of action of the combination of chitosan and C. laurentii was discussed.

•Pyrimethanil (Pyr) at 400 μg/mL efficiently inhibited blue mold rot.•Pyr at 40 μg/mL failed to reduce P. expansum infections in vivo•C. laurentii was insensitive to pyr in vitro and in vivo.•C. laurentii with pyr at 40 μg/mL significantly enhanced mold inhibition.The effect of biocontrol yeasts and pyrimethanil at low concentration on inhibition of blue mold rot caused by Penicillium expansum in pear fruit was investigated. Pyrimethanil at low concentration (40 μg/mL) alone had little inhibitory activity against the P. expansum infection in pear fruit wounds although it was effective in inhibiting the survival of P. expansum on Asp-agar medium. Pyrimethanil at this low concentration significantly enhanced the efficacy of Cryptococcus laurentii at 1 × 107 CFU/mL in reducing blue mold rot in vivo compared with C. laurentii at 1 × 107 CFU/mL alone. However, there was no additive inhibitory activity when pyrimethanil was combined for application with biocontrol yeasts Rhodosporidium paludigenum or Rhodotorula glutinis. Combination of pyrimethanil and C. laurentii at low concentration also inhibited blue mold rot when P. expansum was inoculated into fruit wounds 12 h before treatment and fruit was stored at low temperature (4 °C). Pyrimethanil at 0.04 to 400 μg/mL did not influence the survival of C. laurentii in vitro, and it only slightly reduced the population growth of C. laurentii after 48 h of incubation in the pear fruit wounds. There was no significant difference in quality parameters including total soluble solids, titratable acidity and ascorbic acid of pear fruit wounds among all treatments after 5 days of treatment at 25 °C. Integration of C. laurentii and pyrimethanil at low concentration might be an effective and safe strategy to control P. expansum infection in pear fruit, especially in an integrated postharvest disease management strategy.

The potential of using heat treatment alone or in combination with an antagonistic yeast for the control of blue mold decay and Rhizopus decay of peaches caused by Penicillium expansum and Rhizopus stolonifer respectively, and in reducing natural decay development of peach fruits, as well as its effects on postharvest quality of fruit was investigated. In vitro tests, spore germination of pathogens in PDB was greatly controlled by the heat treatment of 37 °C for 2 d. In vivo test to control blue mold decay of peaches, heat treatment and antagonist yeast, as stand-alone treatments, were capable of reducing the percentage of infected wounds from 92.5% to 52.5% and 62.5%, respectively, when peach fruits stored at 25 °C for 6 d. However, in fruit treated with combination of heat treatment and Cryptococcus laurentii, the percentage of infected wounds of blue mold decay was only 22.5%. The test of using heat treatment alone or in combination with C. laurentii to control Rhizopus decay of peaches gave a similar result. The application of heat treatment and C. laurentii resulted in low average natural decay incidences on peaches after storage at 4 °C for 30 days and 20 °C for 7 days ranging from 40% to 30%, compared with 20% in the control fruit. The combination of heat treatment and C. laurentii was the most effective treatment, and the percentage of decayed fruits was 20%. Heat treatment in combination with C. laurentii had no significant effect on firmness, TSS, ascorbic acid or titratable acidity compared to control fruit. Thus, the combination of heat treatment and C. laurentii could be an alternative to chemicals for the control of postharvest decay on peach fruits.