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.     

Combination of salicylic acid and ultrasound to control postharvest blue mold caused by Penicillium expansum in peach fruit

The effect of ultrasound (40 kHz, 10 min) and salicylic acid (SA, 0.05 mM) either separately, or combined on blue mold caused by Penicillium expansum in peach fruit was investigated. The results showed that the application of SA alone could reduce blue mold, while the use of ultrasound had no effect. Our results also revealed that SA combined with ultrasound treatment was more effective in inhibiting fungal decay during storage than the SA treatment alone. The combined treatment increased the activities of defense enzymes such as chitinase, β-1,3-glucanase, phenylalanine ammonia-lyase, polyphenol oxidase and peroxidase, which were associated with higher disease resistance induced by the combined treatment. Furthermore, the combined treatment did not impair the quality parameters of peach fruit after 6 days of storage at 20 °C. These results suggested that the combination of ultrasound and SA treatment may be a useful technique to reduce blue mold in peach fruit.

Industrial relevance

This paper investigates the effect of ultrasound combined with SA on decay incidence of peach fruit. The results presented demonstrate that the effect of the combined treatment on the disease resistance and fruit quality should be considered by processors prior to its adoption as a preservation technique.     

Evaluation of Penicillium expansum isolates for aggressiveness, growth and patulin accumulation in usual and less common fruit hosts

Experiments were carried out in vivo and in vitro with four isolates of Penicillium expansum (I 1, E 11, C 28 and I 12) to evaluate their aggressiveness, growth and patulin accumulation in both usual (pears and apples) and less common hosts (apricots, peaches, strawberries and kiwifruits) of the pathogen. The 75% of isolates showed the ability to cause blue mould in all tested hosts. In particular, C 28 and I 1 were the most and the least aggressive isolates, respectively (52.9 and 10.6% infection and 20.7 and 15.4 mm lesion diameters). ‘Candonga’ strawberries and ‘Pinkcot’ apricots showed the largest lesion diameters (29.8 and 25.3 mm), followed by ‘Conference’ pears, ‘Spring Crest’ peaches and ‘Abate Fetel’ pears. With the exception of ‘Candonga’ strawberries, the formation of colonies and mycelial growth of P. expansum isolates on fruit puree agar media (PAMs) was stimulated in comparison to a standard growth medium (malt extract agar, MEA). Two of the most aggressive isolates in our assays (I 12 and C 28) showed the greatest accumulation of patulin both in vitro and in vivo, while the least aggressive isolate (I 1) produced patulin only in a few growth media and cvs. Patulin concentration on fruit PAMs was higher than patulin detected in infected fruit tissues. Apple PAMs were the more favorable substrates for patulin accumulation in vitro (maximum concentration 173.1 and 74.1 μg/mL in ‘Pink Lady and ‘Golden Delicious’ PAMs, respectively) and ‘Pink Lady’ apples inoculated with the isolate E 11 showed the greatest accumulation of patulin in the whole in vivo assay (33.9 μg/mL). However, infected tissue of cv Golden Delicious showed lower average accumulation of patulin (1.7 μg/mL) than that of cv Pink Lady (19.1 μg/mL), and no significant differences in patulin concentrations were found among ‘Golden Delicious’ apples and tested cvs of pears, kiwifruits and strawberries. Peaches were highly susceptible to patulin accumulation, showing average concentrations of 27.4 and 18.6 μg/mL in vitro and in vivo, respectively. Apricots were also consistently positive for patulin accumulation, both in vitro (average values of 20.1 μg/mL) and in vivo (average values of 9.4 μg/mL). Our study showed the potential of some less common hosts of P. expansum (in particular peaches and apricots) to support patulin production, indicating that a steady monitoring of patulin contamination should be carried out in fruit substrates other than apples and pears.     

Inhibition of Penicillium expansum by an oxidative treatment

Several oxidizing compounds such as sodium hypochlorite (NaClO) and hydrogen peroxide (H₂O₂) are used to control postharvest decay in fresh fruit due to their antimicrobial effects. Here, we applied these compounds in vitro, in the presence of CuSO₄, against Penicillium expansum, causal agent of apple blue mold. MICs were 50 mg L⁻¹ and 400 mmol L⁻¹ for NaClO and H₂O₂, respectively, when these compounds were individually applied to conidia suspensions during 2 min. A combined oxidative treatment (OT) consisting on an incubation with 1 mg L⁻¹ NaClO and 200 mmol L⁻¹ H₂O₂, in the presence of 6 mmol L⁻¹ CuSO₄, inhibited growth, conidial germination and fungal infectivity on apple. The fractional inhibitory concentration index for the interaction between NaClO and H₂O₂ in the OT was 0.52 indicating a synergistic effect of the oxidizing compounds. These results suggest that the OT could be an interesting alternative for apple diseases postharvest control.     

Biocontrol of fungal decay of citrus fruit by Pichia pastoris recombinant strains expressing cecropin A

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.     

Influencing cocoa flavour using Pichia kluyveri and Kluyveromyces marxianus in a defined mixed starter culture for cocoa fermentation

The potential impact of aromatic and pectinolytic yeasts on cocoa flavour was investigated using two defined mixed starter cultures encompassing strains of Pichia kluyveri and Kluyveromyces marxianus for inoculating cocoa beans in small scale tray fermentations. Samples for microbial and metabolite analysis were collected at 12–24 hour intervals during 120 h of fermentation. Yeast isolates were grouped by (GTG)₅-based rep-PCR fingerprinting and identified by sequencing of the D1/D2 region of the 26S rRNA gene and the actin gene. Pulsed Field Gel Electrophoresis (PFGE) was conducted on isolates belonging to the species P. kluyveri and K. marxianus to verify strain level identity with the inoculated strains. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) was performed to follow yeast and bacterial dynamics over time including the presence of the bacterial inoculum consisting of Lactobacillus fermentum and Acetobacter pasteurianus. Yeast cell counts peaked after 12 h of fermentation with the predominant species being identified as Hanseniaspora opuntiae and Hanseniaspora thailandica. P. kluyveri and K. marxianus were found to compose 9.3% and 13.5% of the yeast population, respectively, after 12 h of fermentation whilst PFGE showed that ~ 88% of all P. kluyveri isolates and 100% of all K. marxianus isolates were identical to the inoculated strains. Despite never being the dominant yeast species at any stage of fermentation, the un-conched chocolates produced from the two inoculated fermentations were judged by sensory analysis to differ in flavour profile compared to the spontaneously fermented control. This could indicate that yeasts have a greater impact on the sensory qualities of cocoa than previously assumed.     

Study of natural mango juice spoilage and microbial contamination with Penicillium expansum by high resolution H NMR spectroscopy

High resolution ¹H NMR has been applied to monitor the changes in the composition of natural mango juice subjected to spoilage and to microbial contamination with Penicillium expansum. A vast number of compounds undergoing changes upon these processes have been identified and their variations followed throughout time (132 h). Besides the formation of typical fermentation products (e.g. acetate, lactic acid, acetoin and isopropanol/2,3-butanediol) and the utilization of the major sugars (sucrose, glucose and fructose), there were changes in organic acids (e.g. decreases of quinic and shikimic acids with formation of 3,4,5-trihydroxycyclohexane acid in spoiled juice, and decreases of citric and malic acids in contaminated juice), amino acids (decreases of alanine, leucine, isoleucine and valine), and less abundant components such as oligosaccharides and aromatic compounds. The possibility of using these changes as early indicators of natural spoilage or P. expansum contamination is discussed.     

Cloning and characterization of a novel phytase from wastewater treatment yeast Hansenula fabianii J640 and expression in Pichia pastoris

Phosphohydrolysis of organic phosphorus compounds by acid phosphatases (EC 3.1.3.1 and EC 3.1.3.2) is an important method for efficient removal of phosphorus from high concentration organic wastewater. Another important method is supplementation of animal feed with phytase (EC 3.1.3.8 and EC 3.1.3.26), which improves the availability of phytate-phosphates (phosphate that are hydrolyzed by phytases), making it possible to add less phosphate to animal feed and resulting in the excretion of less phosphorus by the animals. In the present study, we purified a novel phytase from the wastewater treatment yeast Hansenula fabianii J640 (Hfphytase), cloned the 1456 bp open reading frame (ORF) encoding Hfphytase, and characterized Hfphytase. The molecular weight of Hfphytase after deglycosylation by PNGaseF was 49 kDa. The optimal pH and temperature for enzyme activity were 4.5 and 50 °C, respectively. Hfphytase exhibits 40% identity with Debaryomyces castellii phytase, 37% identity with Aspergillus niger PhyB, and 34% identity with Saccharomyces cerevisiae Pho5p. Recombinant Hfphytase was transformed and expressed in Pichia pastoris. The yield was 23 g/l by jar fermenter cultivation. The marked phosphohydrolysis activity exhibited by Hfphytase on six substrates (pNP-P, sodium phytate, glucose-1 phosphate, glucose-6 phosphate, α-glycerophosphate and β-glycerophosphate) indicated that it is a non-specific acid phosphatase.     

Control of specific growth rate to enhance the production of a novel disintegrin,saxatilin, in recombinant Pichia pastoris

The methylotrophic yeast Pichia pastoris is one of the best hosts for the production of foreign proteins because of the presence of a strong alcohol oxidase 1 (AOX1) promoter that can be induced by methanol. Feeding the yeast, methanol induces protein production and provides an energy source for the host cells. However, excessive levels of methanol inhibit the growth of host cells, and insufficient methanol levels lead to poor growth and protein production. We have used various methanol feeding strategies to enhance the production of saxatilin. Saxatilin is a novel snake venom-derived disintegrin that inhibits tumor angiogenesis and metastasis and has been shown to suppress ovarian cancer cell invasion. A two-step increase feeding strategy to control the specific growth rate led to the best results in terms of specific protein production rates and final saxatilin amounts within the limited fermentation time.     

A peach (Prunus persica)-specific gene, Lhcb2, used as an endogenous reference gene for qualitative and real-time quantitative PCR to detect fruit products

Among the methods used to detect food adulteration, the amplification of endogenous reference genes is particularly important. Endogenous reference genes for many different species, such as cotton, papaya, maize and others, have been reported, yet an endogenous reference gene for the peach is still lacking. In this paper, the chlorophyll a/b-binding protein (Lhcb2) gene was identified as a species-specific gene for the peach. Lhcb2 was assayed in 4 species of peaches and 8 non-peach species by both qualitative and quantitative PCR. No amplification was observed in other species. The detection limit of quantitative PCR was as low as 5 pg of DNA, equal to 9 copies, and Southern blot analysis confirmed that the Lhcb2 gene was present in a single copy in the peach genome. All of these experiments indicated that the Lhcb2 gene is a useful endogenous reference gene for the detection of peach material via both qualitative and quantitative PCR assays, even in the processed food samples such as juices containing peach.     

Combination of Kluyveromyces marxianus and sodium bicarbonate for controlling green mold of citrus fruit

Biocontrol efficacy of an antagonistic yeast Kluyveromyces marxianus was evaluated individually or in combination with sodium bicarbonate (SBC) against green mold of citrus fruit caused by Penicillium digitatum. Their effects on postharvest quality of citrus fruit were also investigated. The results indicated that the antagonistic activity of K. marxianus at 1 × 10⁸ CFU/mL on green mold of citrus fruit was enhanced by 2% SBC treatment. In artificial inoculation trials, disease control after 3 and 6 days, respectively, with the mixture of K. marxianus and 2% SBC (18.33%, 58.33%) was significantly improved over that obtained with K. marxianus (41.67%, 70.00%) or SBC (43.33%, 81.67%) alone. The combination of K. marxianus with SBC was as effective as the imazalil treatment in natural infection trials, which gave about 90% control of green mold. Addition of 2% SBC significantly stimulated the growth of K. marxianus in citrus fruit wounds after 72 h. Moreover, K. marxianus, SBC and their combination did not impair quality parameters including weight loss, fruit firmness, total soluble solids, titratable acidity and ascorbic acid at 4 °C for 30 days followed by 20 °C for 15 days. These results suggested that the use of SBC is a useful approach to improve the efficacy of K. marxianus for the postharvest green mold of citrus fruit.     

Optimization of conditions for secretion of dengue virus type 2 envelope domain III using Pichia pastoris

We have developed a recombinant clone of the methylotrophic yeast Pichia pastoris capable of secreting dengue virus type 2 envelope domain III (sEDIII-2). We explored various induction parameters including media composition, temperature, pH, and methanol concentration, to optimize conditions for sEDIII-2 expression in shake flask culture. Induction at 20 °C in the presence of 2% (v/v) methanol in a medium buffered to pH 5.8 resulted in highest secretion of sEDIII-2. This yield could be further enhanced up to 70% by repeated induction of the same initial biomass. Using a fed-batch cultivation strategy, we observed that shake-flask yields can be scaled up ∼ 8-fold in a bioreactor. We obtained ∼ 94% purity with > 70% recovery after purification. This study, which demonstrates for the first time the feasibility of secreting envelope domain III using the P. pastoris host, will be relevant to sub-unit approaches to dengue vaccine development.     

Formation of vinylphenolic pyranoanthocyanins by Saccharomyces cerevisiae and Pichia guillermondii in red wines produced following different fermentation strategies

The strains of two species of yeast, Saccharomyces cerevisiae and Pichia guillermondii, both with high hydroxycinnamate decarboxylase (HCDC) activity (56% and 90% respectively), were used in the fermentation of musts enriched with grape anthocyanins, to favour the formation of highly stable vinylphenolic pyranoanthocyanin pigments. The different strains were used to ferment the must separately, simultaneously, or sequentially, the latter involving an initial period using the yeast with the greatest HCDC activity (P. guillermondii). The must was made from concentrated grape juice diluted to 220 g/l of sugar, and enriched with grape anthocyanins to 100 mg/l and with p-coumaric acid to 120 mg/l. The pH was fixed to 3.5. All 50 ml micro-fermentations were done in triplicate. The development of anthocyanin-3-O-glucoside precursors, the decarboxylation of p-coumaric acid, and the formation of 4-vinylphenol and vinylphenolic pyranoanthocyanin derivatives were studied during the fermentation. The fermentation strategy used and the yeast HCDC activity significantly influenced the formation of vinylphenolic pyranoanthocyanins. The latter molecules were separated, identified, and quantified using high performance liquid chromatograph with diode array detection and electrospray-mass spectrometry (HPLC-DAD-ESI/MS). The volatile compounds profile was screened during fermentation using gas cromatogrphy-flame ionisation detection (GC-FID), in order to detect and quantify the main molecules. The best results were reached with the sequential fermentation (3.74 mg/l of malvidin-3-O-glucoside-4-vinylphenol). This work shows that during mixed or sequential fermentations carried out with non-Saccharomyces or highly fermentative Saccharomyces strains, with high HCDC activity, the content of stable pigments can be increased without sensorial modifications.     

采后茉莉酸甲酯处理对桃果实青霉病及细胞壁降解酶的影响

以桃果实为试材,研究了采后不同浓度茉莉酸甲酯（Me JA）处理对损伤接种桃果实扩展青霉（Penicillium expansum）病斑扩展的影响,并分析了最佳浓度Me JA处理后桃果实细胞壁降解酶活性的变化。同时研究离体条件下Me JA对P.expansum菌丝生长和孢子萌发的影响。结果表明,100μmol/L Me JA对桃果实P.expansum的病斑抑制效果最好,并且显著（p≤0.05）抑制了菌丝生长和孢子萌发。100μmol/L Me JA处理明显抑制了桃果实多聚半乳糖醛酸酶（PG）、果胶甲酯酶（PME）、果胶甲基反式消除酶（PMTE）、多聚半乳糖醛酸反式消除酶（PGTE）和β-葡萄糖苷酶活性。由此表明,Me JA抑制桃果实青霉病的发生与延缓果实软化有关。      

Bioethanol production from ball milled bagasse using an on-site produced fungal enzyme cocktail and xylose-fermenting Pichia stipitis

Sugarcane bagasse is one of the most promising agricultural by-products for conversion to biofuels. Here, ethanol fermentation from bagasse has been achieved using an integrated process combining mechanical pretreatment by ball milling, with enzymatic hydrolysis and fermentation. Ball milling for 2 h was sufficient for nearly complete cellulose structural transformation to an accessible amorphous form. The pretreated cellulosic residues were hydrolyzed by a crude enzyme preparation from Penicillium chrysogenum BCC4504 containing cellulase activity combined with Aspergillus flavus BCC7179 preparation containing complementary β-glucosidase activity. Saccharification yields of 84.0% and 70.4% for glucose and xylose, respectively, were obtained after hydrolysis at 45 °C, pH 5 for 72 h, which were slightly higher than those obtained with a commercial enzyme mixture containing Acremonium cellulase and Optimash BG. A high conversion yield of undetoxified pretreated bagasse (5%, w/v) hydrolysate to ethanol was attained by separate hydrolysis and fermentation processes using Pichia stipitis BCC15191, at pH 5.5, 30 °C for 24 h resulting in an ethanol concentration of 8.4 g/l, corresponding to a conversion yield of 0.29 g ethanol/g available fermentable sugars. Comparable ethanol conversion efficiency was obtained by a simultaneous saccharification and fermentation process which led to production of 8.0 g/l ethanol after 72 h fermentation under the same conditions. This study thus demonstrated the potential use of a simple integrated process with minimal environmental impact with the use of promising alternative on-site enzymes and yeast for the production of ethanol from this potent lignocellulosic biomass.     

Citrus paradisi and Citrus sinensis flavonoids: Their influence in the defence mechanism against Penicillium digitatum

Citrus peel is rich in flavanone glycosides and polymethoxyflavones. In view of their importance for industrial application as well as for their pharmacological properties, their content was analyzed in the mature fruits of several Citrus paradisi (grapefruit) and Citrus sinensis (orange) varieties, with a view to select the most interesting for isolation. The results shows that the Star Ruby grapefruit and the Sanguinelli orange stand out for their high contents of naringin and hesperidin, respectively. The presence of the polymethoxyflavones nobiletin, heptamethoxyflavone and tangeretin, could be ascertained in all the grapefruit varieties analysed. Higher polymethoxyflavone levels were recorded in orange, with Valencia Late showing the greatest nobiletin, sinensetin and tangeretin contents and Navelate the highest heptamethoxyflavone levels. An in vitro study revealed that these compounds acted as antifungal agents against Penicillium digitatum, the polymethoxyflavones being more active than the flavanones in this respect. The possible participation of these phenolic compounds in the defence mechanism of Citrus against P. digitatum is discussed.