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Bamboo leaf flavonoid enhances the control effect of Pichia caribbica against Penicillium expansum growth and patulin accumulation in apples
Postharvest Biology and Technology 141 (2018) 1–7
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Bamboo leaf flavonoid enhances the control effect of Pichia caribbica against Penicillium expansum growth and patulin accumulation in apples
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Gustav Komla Mahunu, Hongyin Zhang , Maurice Tibiru Apaliya, Qiya Yang, Xiaoyun Zhang, Lina Zhao School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 12013, Jiangsu, People’s Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Pichia caribbica Bamboo leaf flavonoid Blue mold Biocontrol agent Patulin Postharvest storage
Patulin (PAT) mainly produced by Penicillium expansum is a very problematic mycotoxin, and its presence is often reported in different foods and food products. The prevention of these products from initial contamination and/or degradation is therefore of foremost importance in protecting consumer health. In this report, Pichia caribbica (1 × 108 cells mL−1) antagonistic yeasts incorporated with bamboo leaf flavonoid (0.01% w/v) were assessed for PAT degradation capabilities in in-vitro and in-vivo. Also the effects of treatments on wound lesion diameter, lignification and pH level were investigated. Clearly, the combined biocontrol agent (P. caribbica and bamboo leaf flavonoid) had the ability to degrade PAT in vitro with undetected level at 96 h of incubation in NYDB media. In apple fruits, PAT accumulation was significantly lower at 20 d (at 20 °C) of storage. Wound lignification was significantly enhanced from 0.31 to 0.27 after 15 d of storage, whereas pH was modified (> 4.0) in host; which might lead to an increase in PAT breakdown. P. caribbica combined with bamboo leaf flavonoid could be a promising biocontrol agent for PAT degradation processes in apple fruits.
1. Introduction Patulin (PAT) is among the main mycotoxins, which is considered an unwanted natural contaminant of fruits. It is toxic secondary metabolite biosynthesized by several species of filamentous fungi of the genera Penicillium, Aspergillus and Byssochlamys (Ritieni, 2003). PAT has been detected mostly in fresh pome fruits (such as apples, pears and peaches) and products derived from these fruits, however, it could also be found in vegetables, cereals and other foods (Prieta et al., 1993). P. expansum is the postharvest fungi considered the predominant producer of patulin and its usually known as blue mold decayed apples in storage (Andersen and Thrane, 2006; Pitt, 2000). Patulin has been detected in raw and processed products such as juices and puree, making its occurrence a global health issue (Organization W.H., 2012). The risk associated with PAT is that often surfaces of fruits may appear healthy but contaminated with PAT, thus damaged fruits or fruits previously infected by pathogens during pre-harvest and postharvest conditions (Laidou et al., 2001). Prolong intake of patulin contaminated products has been reported to cause immunotoxicity and genotoxicity (Bürger et al., 1988), mutagenicity and neurotoxicity (Devaraj et al., 1982), cytotoxicity and teratogenicity (Bonerba et al., 2010). Therefore, the prevention of initial infection of fruits by P. expansum and then possible inhibition of PAT production is important step to
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protect the health of consumers, particularly infants. Several methods (physical and chemical) have been proposed for the elimination of PAT. Some of these methods could be promising, capable of destroying or reducing the spores and mycelia of P. expansum but will not be able to destroy the PAT completely (Salas et al., 2012). According to Chen et al. (2012), treatment of apples with fungicides will not completely prevent P. expansum infections and neither will it ensure absence of PAT in the final products. The most preferred strategy is preventing fresh fruits from PAT contamination, since PAT is not confined to the decayed tissues and may be found in fruits that appear healthy and also one infected fruit can cause contamination of final product. Over the last 30 years significant progress has been made in the use of microbial antagonists (as postharvest biocontrol agents) and controlled atmosphere (CA) for the control of PAT-producing fungi and its consequence on reduction of PAT accumulation in apples (Lima et al., 2011; Spadaro et al., 2013). However, beyond the use of single treatment microbial antagonists, in recent times emphasis is being placed on the use of two or more combined biocontrol agents (BCAs) to enhance their performance and reliability; and expand the alternatives for postharvest disease control (Wisniewski et al., 2016). From previous studies, synergistic effect of chitosan and Cryptococcus laurentii were found to inhibit infections caused by Penicillium expansum (Yu et al., 2007). Coelho et al., 2007 indicated the effectiveness of Pichia ohmeri
Corresponding author. E-mail address: [email protected] (H. Zhang).
https://doi.org/10.1016/j.postharvbio.2018.03.005 Received 3 October 2017; Received in revised form 16 February 2018; Accepted 7 March 2018 0925-5214/ © 2018 Elsevier B.V. All rights reserved.
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determine the concentrations of the spores and adjusted as required with sterile distilled water.
and Saccharomyces cerevisiae in the biodegradation of patulin. Similarly, Lentinula edodes was reported to enhance the biocontrol activity of C. laurentii against P. expansum infection contamination and patulin production in apple fruits (Tolaini et al., 2010). To continue the search for alternative approaches to manage PATproducing fungi, our study opted to treat P. caribbica yeast with bamboo leaf flavonoid (powder form) in order to understand the mechanisms of action that control PAT accumulation in apple fruit. The bamboo leaf extract powder is rich in flavonoids with protective function of inducing antioxidant responses in the host under stress conditions. It is highly underutilized natural bioactive compound in management of postharvest diseases, despite its function as food additive and medicine (Vastano et al., 2000). The Ministry of Health of the People’s Republic of China has approved bamboo leaves extract as an innovative natural antioxidant and has been proven to be safe and virtually non-toxic for use as a food additive, after both rats and mice tests were conducted (Mao et al., 2015). To the best of our knowledge, there is no information available on the integration of P. caribbica with bamboo leaf flavonoid (BLF) with the aim of enhancing biocontrol efficacy of P. caribbica against apple decay caused by P. expansum. In addition, we evaluated the treatments effect on lignin content and pH level. Lastly, the effect of treatments on the biocontrol of PAT accumulation was investigated.
2.3. Determination of pH in vitro The pH of cultures containing patulin and various treatments (0.01 %0.01% Bamboo leaf flavonoid or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid + P. caribbica) and patulin alone in (without yeast or Bamboo leaf flavonoid) as control were determined. By this method, the pH electrode InLab 427 (Mettler Toledo) connected to a SG2-SevenGo pH meter (Mettler Toledo) was used to determine the pH of the treatments at time points as follows 0 (1 h), 12 h, 1 d, 2 d, 3 d, 4 d and 5 d) during incubation at 20 °C. This experiment constituted three replicates of 12 flask containing medium for each treatment, and the experiment was conducted thrice. 2.4. Determination of the effect of P. caribbica combined with BLF on controlling patulin in vitro In this experiment, the method of sample preparation according to Coelho et al. (2007) was used with some modifications. In each 250 mL Erlenmeyer flasks containing 50 mL NYDB with 1 mL of patulin (0.00445 μg L−1), the following suspensions were added; bamboo leaf flavonoid (0.01%) or P. caribbica alone, bamboo leaf flavonoid (0.01%) + P. caribbica and then patulin alone (without yeast or bamboo leaf flavonoid) as control. Afterwards, all Erlenmeyer flasks were incubated using a rotary shaker at 180 × g at 28 °C. At each time point of 0 (1 h), 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, and 144 h, samples were collected and centrifuged at 7000 × g for 10 min. One milliliter of each supernatant was collected and filtered through a 45 μm filter, then retained for HPLC analysis of patulin.
2. Material and methods 2.1. Apple fruit samples Apples (Malus domestica Borkh, cv. Fuji) were harvested at commercial maturity from an orchard in Yantai, Shandong Province, and selected based on absence of physical injury uniform ripeness and size. Selected apples were disinfected with (0.1% sodium hypochlorite) for 1 min, washed using tap water before used.
2.5. Efficacy of P. caribbica combined with BLF in controlling patulin in vivo
2.2. Antagonistic yeast and pathogen inoculum Fruit treatment: To determine the effect of BLF on the efficacy of P. caribbica in preventing blue mold, the selected apples were wounded, inoculated and stored. Here, three uniform wounds with dimensions of 5 mm diameter and approximately 3 mm deep were made at the equator of each apple fruit using the tip of a sterile cork borer. Each of the wounds was inoculated with 30 μl of P. caribbica suspension (1 × 108 cells mL−1) combined with bamboo leaf flavonoid at concentration of 0.01% w/v or sterile distilled water as a control. Each of the wounds was inoculated with 30 μL of P. expansum (1 × 105 spores mL−1) suspension, 2 h later. Then, treated apples were air-dried and kept in plastic trays protected with polythene films to maintain a favorable relative humidity (95%) for 15 d at 20 °C. There were three replicates of 12 fruit for each treatment, and the experiment was conducted thrice.
Antagonistic yeast: The yeast antagonist P. caribbica was isolated from soils of peach orchard (the central shoal of Yangtze River, Zhenjiang, Jiangsu Province) for its reliable antagonistic efficacy against P. expansum (Zhao et al., 2012). The classical methods based on colony and cell morphologies were used for a preliminary characterization of the yeast (Kurtzman et al., 2011). Subsequently, sequence analysis of the 5.8S internal transcribed spacer (ITS) ribosomal DNA (rDNA) region was used to identify the yeast (Li et al., 2014). P. caribbica has been shown to be safe in animal testing, including physiology experiments, acute toxicity studies, and the Ames test (unpublished data). Then P. caribbica isolates were maintained at 4 °C on nutrient yeast dextrose agar medium (NYDA-0.8% nutrient broth, 0.5% yeast extract, 1% glucose and 2% agar). Liquid cultures of the yeast were grown in 250-ml Erlenmeyer flasks containing 50 ml of nutrient yeast dextrose broth (NYDB) which had been inoculated with a loop of the culture and incubated on a rotary shaker (180 rpm) at 28 °C for 24 h. Following incubation, cells were centrifuged (6000 × g for 10 min) at 4 °C and then washed twice with sterile distilled water in order to remove the growth medium (Li et al., 2014). Afterwards the cell pellets were re-suspended in sterile distilled water and adjusted to an initial concentration before being adjusted to the concentration required for the each experiment. Pathogen inoculum: P. expansum was isolated from infected apples and cultured as described by Zheng et al. (2017) with slight modifications. This culture was maintained on potato dextrose agar (PDA, extract of boiled potatoes, 200 ml; dextrose, 20 g; agar, 20 g and distilled water, 800 ml) at 4 °C, and fresh cultures were grown on PDA plates before use. Spore suspensions of P. expansum were prepared by removing the conidia of 7 d old culture with a bacteriological loop, and suspended in sterile distilled water. Hemocytometer was used to
2.6. Determination of lignin content The lignin content of apples was determined at different time points after 15 d of storage at 20 °C (relative humidity of 95%). First, wounds were made at the equator of each apple as described above. Later each wound was inoculated with 30 μl of P. caribbica (1 × 108 cells mL−1) containing BLF (0.01%) or each of them used as stand-alone treatment with sterile distilled water as control. After application of treatments, fruits were allowed to air dry for 2 h at room temperature. The fruits samples were packaged in trays and covered with plastic film for storage and observation. The assessment of the lignin content was conducted according to the method suggested by Vilanova et al. (2014) with minor modification. Here, the apple disks were lyophilized for 4 d and then ground to a fine powder. In sequence, each sample was washed with water, ethanol, acetone and diethyl ether through Whatman 1 filter paper until the 2
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washed tissue was colourless. After the final powder was dried for 1 h at 70 °C, 20 mg samples were digested with a solution of 25% (w/w) acetyl bromide in acetic acid (2.5 mL) and HClO4 (70%, 0.12 mL) and heated in a bath for 30 min at 70 °C with shaking. After cooling with ice, 10 mL of 2 M NaOH and 12 mL of acetic acid were added to the reaction tubes and 1.5 mL of the resulting solution was centrifuged at 10,000 × g for 30 min at 4 °C to be sure that the resulting sample was completely clear. Five times dilution was made for each solution with acetic acid and absorbance was determined at 280 nm. Samples were collected at each time point and each treatment was replicated three times. 2.7. Determination of pH in fruit wounded site The pH of each fruit mesocarp was measured according to the method described by Neri et al. (2010). In brief, the pH electrode In Lab 427 (Mettler Toledo) was inserted at 15 mm depth through the wounds (decayed). Similarly, the pH of non-decayed (control) tissue were also measured. The pH was measured at 15 d of storage at 20 °C (relative humidity 95%). Three replicates of 12 fruit for each treatment were used, and the experiment was conducted thrice. Fig. 1. pH of culture of patulin in 0.01% Bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc), or untreated control in medium. pH was measured at time points as follows 0 (1 h), 12 h, 1 d, 2 d, 3 d, 4 d and 5 d after incubation at 20 °C. Bars represent standard error of the means according to the Tukey test at P = 0.05.
2.8. Determination of extent of lesions Lesion diameters of the equatorial sections of each apple was measured. Two perpendicular diameters from each of the lesions were measured using a pair of calipers (Morales et al., 2007). The tissues were removed and weighed for patulin content analysis.
incubation at 28 °C (Fig. 1). The combined treatment of BLF and Pc (P. caribbica) retained the highest pH, reaching values of about 4.8 after 4 d of incubation, followed by a slight increase of pH after a day of incubation. Actually, very little changes in pH were observed in Pc compared with BLF, except at 4 d and 5 d. The final difference in pH after 5 d of incubation were BLF and Pc (4. 9 ± 0.127), Pc (3.8 ± 0.114), BLF (3.6 ± 0.021) and control (3.4 ± 0.064). However, the control medium showed the greatest decrease of pH during the 5 d of incubation.
2.9. Extraction and purification Patulin accumulation in decayed tissue was determined by removing from each apple wound using a cork borer (at least 1 cm wider than lesion diameter), and juice extracted. Then apple juice was clarified using pectinase (Boonzaaijer et al., 2005). Ethyl acetate (25 mL) was added to 10 mL of the juice and vortexed for 60 s. Then the supernatant was transferred into a separatory funnel (250 mL). This step was repeated thrice and then 10 mL of 1.4% w/v Na2CO3 was added to the ethyl acetate and mixed vigorously for 2 min. The ethyl acetate layer was vacuum dried at 40 °C in a rotary evaporator (Germany). Immediately, 1 mL acetate buffer made of 0.2 mol L−1 and pH 4 was added and vortexed until it completely dissolved. This mixture was then transferred into a vial and inserted into the auto sampler for HPLC analysis.
3.2. Effect of treatments on patulin retention in medium Efficacies of the different treatments varied when medium were inoculated with Patulin (Fig. 2). In particular, when the medium was
2.10. HPLC conditions An Agilent Technologies 1100 series system equipped with a quaternary pump and variable wavelength detector (Switzerland) was used. The analytical column was from Zorbax, SB-C18 250 × 4.6 mm 5 μm (US). The mobile phase 90 – 10% water/acetonitrile was used at 1 mL min−1. The UV detection was conducted at 276 nm. 2.11. Statistical analysis Generally, each treatment consisted of three replicates of 12 fruit, and the experiment was performed thrice. The data were analyzed for significant differences by analysis of variance (ANOVA) with OriginPro version 9.0 (OriginLab, Northampton, USA) statistical package. The significance among the data was assessed by Tukey test at P < 0.05. 3. Results Fig. 2. Effect of treatment with 0.01% Bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc) and untreated fruits as control on patulin concentration in vitro. Data were the means of three experiments. Bars represent standard error of the means according to the Tukey test at P = 0.05.
3.1. pH in medium after incubation In general, the decrease of pH began after 1 h during the period of 3
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Fig. 4. Effects of treatments on lesion diameter of apple wounds were determined. After initial treatment with 0.01% Bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc) or untreated control in medium was done. After drying the inoculated wounds for 2 h, same wounds were inoculated with P. expansum before storage for 15 d at 20 °C (RH 95%). The pH values were pooled from three wound along the equator of the fruit. Bars represent standard error of the means according to the Tukey test at P = 0.05.
Fig. 3. Determination of pH in decayed fruit wounds. Each wound was initially inoculated with different treatments of 0.01% bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc), and untreated control before infected by P. expansum. pH of fruits was measured at time points as follows 0 (1 h), 3 h, 6 d, 9 d, 12 d, 15 d and 18 d after storage at 20 °C. Bars represent standard error of the means according to the Tukey test at P = 0.05.
inoculated with BLF combined with Pc, it yielded a significant reduction in the patulin concentration, compared with yeast alone or BLF alone. The untreated control gave the highest patulin residue, which implies that it was the least effective against patulin accumulation. At the final time of 144 h after incubation the following patulin concentration were observed: BLF + Pc (undetected), Pc (undetected), BLF (0.001 μg L−1), and untreated control (0.003 μg L−1). However, the combined treatment showed undetected patulin earlier during incubation at 96 h compared to antagonistic yeast, which reached undetectable limit at 120 h.
3.4. Effect of treatment on lignin content of fruit wound Table 1 shows lignin content measured in the tissues of the wound. The results showed higher levels of lignin contents in treatments compared with the control. The highest lignin content was found in the combined treatment of BLF and Pc (0.345 ± 0.011) followed by Pc (0.304 ± 0.011) and BLF (0.279 ± 0.015), each as individual treatment, and the control was the lowest (0.203 ± 0.017). 3.5. Lesion diameter and incidence of patulin accumulation Lesion diameter: When wounded apple fruits were treated with BLF + Pc, rot inhibition was significantly (P < 0.05) increased, achieving the highest inhibition in controlling blue mold. The lesion diameter were BLF + Pc (5.74 mm), Pc (8.33 mm), BLF (17.60 mm) and control (69.00 mm). The results suggest that P. expansum growth on apple wounds could be significantly inhibited by this treatment under the existing experimental conditions (Fig. 4). Patulin accumulation: The present data in relation to patulin content in wounded apples contaminated with P. expansum and treated or untreated with biocontrol agent Pc and with BLF have been are showed in Fig. 5 It was evident that after 20 d of incubation at 20 °C, BLF + Pc significantly inhibited patulin production in comparison with the
3.3. pH of wound area of fruits Fig. 3 indicates the pH measured after 18 d storage of fruits at 20 °C. There was a significant difference (P > 0.05) between all the samples tested particularly on the 18 d after incubation. On the 18 d after incubation, the pH of BLF combined with Pc inoculated tissues was higher (pH 4.41 ± 0.022), followed by Pc alone (pH 4.10 ± 0.081), BLF alone (pH 3.87 ± 0.053) and control was the least (pH 3.63 ± 0.055) fruit. This implied that pH values of BLF + Pc > Pc > BLF > control.
Table 1 Lignin content (absorbance at 280 nm) of apple wound inoculated withP. expansum and stored at 20 °C for 15 d. Each wound was initially inoculated with different treatments of 0.01% Bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc), and untreated control before infected by P. expansum. Days after incubation
Treatments Ck
0 3 6 9 12 15
0.2134 0.2318 0.2326 0.2365 2.0243 0.1920
0.01% BLF ± ± ± ± ± ±
0.004ae 0.013a 0.002a 0.002a 0.0024a 0.002a
0.2140 0.2439 0.2456 0.2472 0.2705 0.2837
± ± ± ± ± ±
Pc 0.002be 0.004b 0.004b 0.004b 0.003b 0.005a
0.2369 0.2310 0.2384 0.2472 0.2571 0.2718
0.01% BLF + Pc ± ± ± ± ± ±
0.007c 0.006c 0.004c 0.002c 0.006c 0.006a
0.2712 0.2954 0.2981 0.2986 0.3049 0.3058
± ± ± ± ± ±
0.003d 0.003d 0.005d 0.007d 0.004d 0.009a
Means are pooled values of three trials ± the standard error. Bars represents standard errors and dissimilar alphabets are statistically different based on the Tukey test at P = 0.05.
4
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at finial incubation time. Similar to a study by Moss (1991), at pH 4.0 yeast biomass in apple juice was highest (12.4 g L−1), In agreement with Prusky et al. (2004), in apples, they observed pH of 4–6 was detected in non-decayed tissues and lower pH of 3.6–4.1 in decayed tissue (caused by P. expansum) between 4 and 6 d after inoculation. Neri et al. (2010) also observed a decline of pH (kiwifruits and pears tissues) in infected sites to be less than 4.0. According to these authors (Prusky et al., 2014), Penicillium spp. is known to be influenced by the decrease of pH to support pathogen attack and that change in pH around the host infection sites can modulate the expression of factors of pathogenicity. For instance, pH range of 3.2 – 3.8 enables fungi to produce patulin vigorously supports (Moss, 1991), whiles pH increase to 6 in a solution, might lead to an increase in PAT breakdown (Collin et al., 2008). From our results, probably the presence of bamboo leaf flavonoid during yeast activity limited the rapid growth of the pathogen, contributing to the trend of pH observed in the combined treated sample. Indeed, as suggested, the aggressive growth of P. expansum will significantly reduce the pH of potato dextrose broth, while less aggressive growth will limit dropping of pH in the substrate (Neri et al., 2010). Clearly, our results indicate connection between increased lignin content and decreased wound lesion extension. As such wounds treated with BLF combined with Pc, gave the highest lignin content and the lowest lesion diameter. Our study, which reports the beneficial effect of yeast amended with BLF in apple fruit, supports other findings that lignin may perhaps impede penetration of pathogen and increase disease resistance in fruit (Schirra et al., 2000). With regards to similar findings, higher induction of disease resistance is linked with higher phenolic content and lignin in fruit tissue as suggested by other studies (Valentines et al., 2005). We also confirmed in our previous trials that BLF combined with Pc increased the phenolic compound in wounds of apple fruits (unpublished data); thus, both phenolic and lignin contents play a significant role in the mechanism of defense against P. expansum in vivo. It was reported earlier that stimulation of lignification of plant cells by polymerization of phenolic phenylpropanoid precursors such as hydroxylated and methoxylated cinnamoyl alcohols, plays an essential role in disease resistance expression (Hahlbrock and Scheel, 1989). Valentines et al. (2005) confirmed that lignification, which can also be connected to H2O2 through the action of POX enzyme, is a universal process implicated in plant defence reactions. In many plants, it was observed that lignification is a reaction to pathogenic infection, in order to enhance mechanical strength of cell walls and deters fungal invasion (Walter et al., 2010). In addition, it was earlier reported that the production of wound periderm is dependent on the wound healing response (Vilanova et al., 2014), which was found to be interrelated to a local accumulation of lignin and phenolics, controlling the cell wall thickening around wounds (Spotts et al., 1998). In support of these findings, different authors reported on the correlation between wound healing processes in apples with lignin accumulation (Vilanova et al., 2012). Indeed, lignification or lignin deposition is known as one of the mechanisms of disease resistance in fruit (Valentines et al., 2005), and in citrus fruit it appeared to influence wound healing and prevention of green mold (Nafussi et al., 2001). From our previous review report, P. expansum is often involved in spoilage of apple fruits and apple juice, and because of public health concerns, various biocontrol methods that are comparatively safe have been confirmed as possible agents to control P. expansum (Mahunu et al., 2015). In addition, P. caribbica as a single treatment, decreased patulin accumulation in vitro (Cao et al., 2013). However, the enhancement of BCAs through combined treatment approaches, have been proposed as most effective way of reducing PAT (Spadaro et al., 2013b), and besides it also improves their persistence and colonization of BCAs wounded and unwounded fruit surfaces (Castoria et al., 2008). According to the results of our study, non-detectable of patulin production was achieved using 0.01% (w/v) bamboo leaf flavonoid. To the best of our knowledge, no previous reports have found on the effect of the combination action of bamboo leaf flavonoid and P. caribbica on
Fig. 5. Patulin accumulation after 20 d at 20 °C of storage condition. Treatment include 0.01% Bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc), or untreated control. Each value is the mean of two experiments. Bars represent standard error of the means according to the Tukey test at P = 0.05.
untreated control (0.029 μg kg−1 ± 0.373) inoculated with P. expansum. Furthermore, when treated separately, Pc (0.002 μg kg−1 ± 0.28) followed by BLF (0.002 μg kg−1 ± 0.23) controlled patulin production. All samples treated with the biocontrol agents or the bioactive compound showed a large inhibiting effect on patulin biosynthesis when fruits were incubated at 20 °C for 20 d.
4. Discussion Our initial investigations using P. caribbica combined with different concentrations of bamboo leaf flavonoid as amendment yielded promising results against P. expansum. In the same experiment, we observed that P. caribbica combined with low concentration (0.01% w/v) of bamboo leaf flavonoid was able to reduce natural decay and maintained physiochemical parameters of apple fruits (data unpublished). Furthermore, the treatment combination was able to inhibit effectively both mycelial growth and germination of P. expansum, which according to literature was mainly due to competition and production of secondary metabolites with antifungal action (Trias et al., 2010). We therefore decided to investigate further, whether P. caribbica amended with bamboo leaf extract will also able to reduce patulin, and also influence pH levels, lignin content and lesion diameter. As mentioned earlier, the combination of more treatments produced a complex mechanism of action, which disrupts the possible development of pathogen resistance (Castoria et al., 2003; Paster and Barkai-Golan, 2008) and the potential inhibition of PAT production. Again, combined treatments have the potential to enhance the antifungal activity of yeast in broth cultures and decrease in PAT production (Coelho et al., 2007), though some factors could influence their performance response during the process. According to Yiannikouris et al. (2007), pH is one of the parameters that controls patulin breakdown. The change in the incidence and level of patulin could be altered according to various factors such as pH of fruit and vegetable (Drusch et al., 2007). In our trial, we found similar trends in pH both in vitro and in vivo. When we conducted a long-term incubation of samples for 5 d at 28 °C, where P. caribbica cells combined with bamboo leaf flavonoid had been exposed to patulin containing media, we discovered the higher pH value. In general, though pH showed a decline in all the samples, P. caribbica cells + bamboo leaf flavonoid inoculated sample maintained a higher curve with > pH 4.0 5
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mycotoxin patulin production. The minimum quantity of patulin was favored by the yeast activity in the presence of bamboo leaf flavonoid. This combined treatment, according to earlier investigation, has inhibitory effect on the growth of P. expansum, probably preventing subsequent effects. According to Hasan (2000), patulin production is amplified when the growth of P. expansum reaches the late phase and the energy source in the mycological medium is nearly exhausted and sufficient intermediates might have accumulated. On the other hand, they also observed that the decrease in patulin production associated with length of incubation period, possibly maybe due to its degradation by certain substances as organic acids depleting from the vacuole and by mycelium of P. expansum itself. In conclusion, although the use of P. caribbica alone was found to inhibit PAT accumulation in stored apple fruits, however, its integration with BLF has shown better effect. Thus, the enhancement of lignification and the extension of storage time of apple fruits by the yeast combined with natural bioactive compound demonstrated here suggests that the use of P. caribbica integrated with BLF is a potential agent for postharvest diseases management. Notes The authors declare no competing financial interest. Funding This work was supported by the National Natural Science Foundation of China (31571899; 31772037), and the National Key Research and Development Program of China (2016YFD0400902). References Andersen, B., Thrane, U., 2006. Food-borne fungi in fruit and cereals and their production of mycotoxins. Adv. Food Mycol., vol. 572. Springer, pp. 137–152. http://dx.doi.org/ 10.1007/0-387-28391 9_812. Bonerba, E., Ceci, E., Conte, R., Tantillo, G., 2010. Survey of the presence of patulin in fruit juices. Food Addit. Contam. 3, 114–119. http://dx.doi.org/10.1080/19393210. 2010.490882. Boonzaaijer, G., Bobeldijk, I., van Osenbruggen, W.A., 2005. Analysis of patulin in dutch food, an evaluation of a SPE based method. Food Control 16, 587–591. http://dx.doi. org/10.1016/j.foodcont.2004.06.020. Bürger, M., Brakhage, A., Creppy, E., Dirheimer, G., Röschenthaler, R., 1988. Toxicity and mutagenicity of patulin in different test systems. The Target Organ and the Toxic Process, vol. 12. Springer, pp. 347–351. http://dx.doi.org/10.1007/978-3-64273113-6_62. Cao, J., Zhang, H., Yang, Q., Ren, R., 2013. Efficacy of Pichia caribbica in controlling blue mold rot and patulin degradation in apples. Int. J. Food Microbiol. 162, 167–173. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.01.007. Castoria, R., Caputo, L., De Curtis, F., De Cicco, V., 2003. Resistance of postharvest biocontrol yeasts to oxidative stress: a possible new mechanism of action. Phytopathology 93, 564–572. http://dx.doi.org/10.1094/PHYTO.2003.93.5.564. Castoria, R., Wright, S.A., Droby, S., 2008. Biological control of mycotoxigenic fungi in fruits. Mycotoxins Fruits Vegetables 311–333. Chen, X., Li, J., Zhang, L., Xu, X., Wang, A., Yang, Y., 2012. Control of postharvest radish decay using a Cryptococcus albidus yeast coating formulation. Crop Prot. 41, 88–95. http://dx.doi.org/10.1016/j.cropro.2012.05.015. Coelho, A.R., Celli, M.G., Ono, E.Y.S., Wosiacki, G., Hoffmann, F.L., Pagnocca, F.C., Hirooka, E.Y., 2007. Penicillium expansum versus antagonist yeasts and patulin degradation in vitro. Braz. Arch. Biol. Technol. 50, 725–733. http://dx.doi.org/10. 1590/S1516-89132007000400019. Collin, S., Bodart, E., Badot, C., Bouseta, A., Nizet, S., 2008. Identification of the main degradation products of patulin generated through heat detoxication treatments. J. Inst. Brew. 114, 167–171. http://dx.doi.org/10.1002/j.2050-0416.2008.tb00322.x. Devaraj, H., Radha, S.K., Shanmugasundaram, E., 1982. Neurotoxic effect of patulin. Indian J. Exp. Biol. 20, 230–231. Drusch, S., Kopka, S., Kaeding, J., 2007. Stability of patulin in a juice-like aqueous model system in the presence of ascorbic acid. Food Chem. 100, 192–197. http://dx.doi. org/10.1016/j.foodchem.2005.09.043. Hahlbrock, K., Scheel, D., 1989. Physiology and molecular biology of phenylpropanoid metabolism. Annu. Rev. Plant Biol. 40, 347–369. http://dx.doi.org/10.1146/ annurev.pp.40.060189.002023. Hasan, H., 2000. Patulin and aflatoxin in brown rot lesion of apple fruits and their regulation. World J. Microbiol. Biotechnol. 16, 607–612. http://dx.doi.org/10.1023/ A:1008982511653. Kurtzman, C., Fell, J.W., Boekhout, T., 2011. The Yeasts: a Taxonomic Study. Elsevier. Laidou, I., Thanassoulopoulos, C., Liakopoulou‐Kyriakides, M., 2001. Diffusion of patulin
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efficacy of Pichia carribbica to postharvest diseases of strawberries by addition of trehalose to the growth medium. Int. J. Mol. Sci. 13, 3916–3932. http://dx.doi.org/ 10.3390/ijms13033916. Zheng, X., Yang, Q., Zhang, X., Apaliya, M.T., Ianiri, G., Zhang, H., Castoria, R., 2017. Biocontrol agents increase the specific rate of patulin production by Penicillium expansum but decrease the disease and total patulin contamination of apples. Front. Microbiol. 8. http://dx.doi.org/10.3389/fmicb.2017.01240.
Yiannikouris, A., Jouany, J.-P., Bertin, G., Lyons, T., Jacques, K., Hower, J., 2007. Counteracting mycotoxin contamination: the effectiveness of Saccharomyces cerevisiae cell wall glucans in Mycosorb for sequestering mycotoxins. Nutritional Biotechnology in the Feed and Food industries: Proceedings of Alltech’s 23rd Annual Symposium. The New Energy crisis: Food, Feed or Fuel. pp. 11–19. Yu, T., Li, H.Y., Zheng, X.D., 2007. Synergistic effect of chitosan and Cryptococcus laurentii on inhibition of Penicillium expansum infections. Int. J. Food Microbiol. 114, 261–266. Zhao, L., Zhang, H., Li, J., Cui, J., Zhang, X., Ren, X., 2012. Enhancement of biocontrol
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Postharvest Biology and Technology journal homepage: www.elsevier.com/locate/postharvbio
Bamboo leaf flavonoid enhances the control effect of Pichia caribbica against Penicillium expansum growth and patulin accumulation in apples
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Gustav Komla Mahunu, Hongyin Zhang , Maurice Tibiru Apaliya, Qiya Yang, Xiaoyun Zhang, Lina Zhao School of Food and Biological Engineering, Jiangsu University, Zhenjiang, 12013, Jiangsu, People’s Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Pichia caribbica Bamboo leaf flavonoid Blue mold Biocontrol agent Patulin Postharvest storage
Patulin (PAT) mainly produced by Penicillium expansum is a very problematic mycotoxin, and its presence is often reported in different foods and food products. The prevention of these products from initial contamination and/or degradation is therefore of foremost importance in protecting consumer health. In this report, Pichia caribbica (1 × 108 cells mL−1) antagonistic yeasts incorporated with bamboo leaf flavonoid (0.01% w/v) were assessed for PAT degradation capabilities in in-vitro and in-vivo. Also the effects of treatments on wound lesion diameter, lignification and pH level were investigated. Clearly, the combined biocontrol agent (P. caribbica and bamboo leaf flavonoid) had the ability to degrade PAT in vitro with undetected level at 96 h of incubation in NYDB media. In apple fruits, PAT accumulation was significantly lower at 20 d (at 20 °C) of storage. Wound lignification was significantly enhanced from 0.31 to 0.27 after 15 d of storage, whereas pH was modified (> 4.0) in host; which might lead to an increase in PAT breakdown. P. caribbica combined with bamboo leaf flavonoid could be a promising biocontrol agent for PAT degradation processes in apple fruits.
1. Introduction Patulin (PAT) is among the main mycotoxins, which is considered an unwanted natural contaminant of fruits. It is toxic secondary metabolite biosynthesized by several species of filamentous fungi of the genera Penicillium, Aspergillus and Byssochlamys (Ritieni, 2003). PAT has been detected mostly in fresh pome fruits (such as apples, pears and peaches) and products derived from these fruits, however, it could also be found in vegetables, cereals and other foods (Prieta et al., 1993). P. expansum is the postharvest fungi considered the predominant producer of patulin and its usually known as blue mold decayed apples in storage (Andersen and Thrane, 2006; Pitt, 2000). Patulin has been detected in raw and processed products such as juices and puree, making its occurrence a global health issue (Organization W.H., 2012). The risk associated with PAT is that often surfaces of fruits may appear healthy but contaminated with PAT, thus damaged fruits or fruits previously infected by pathogens during pre-harvest and postharvest conditions (Laidou et al., 2001). Prolong intake of patulin contaminated products has been reported to cause immunotoxicity and genotoxicity (Bürger et al., 1988), mutagenicity and neurotoxicity (Devaraj et al., 1982), cytotoxicity and teratogenicity (Bonerba et al., 2010). Therefore, the prevention of initial infection of fruits by P. expansum and then possible inhibition of PAT production is important step to
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protect the health of consumers, particularly infants. Several methods (physical and chemical) have been proposed for the elimination of PAT. Some of these methods could be promising, capable of destroying or reducing the spores and mycelia of P. expansum but will not be able to destroy the PAT completely (Salas et al., 2012). According to Chen et al. (2012), treatment of apples with fungicides will not completely prevent P. expansum infections and neither will it ensure absence of PAT in the final products. The most preferred strategy is preventing fresh fruits from PAT contamination, since PAT is not confined to the decayed tissues and may be found in fruits that appear healthy and also one infected fruit can cause contamination of final product. Over the last 30 years significant progress has been made in the use of microbial antagonists (as postharvest biocontrol agents) and controlled atmosphere (CA) for the control of PAT-producing fungi and its consequence on reduction of PAT accumulation in apples (Lima et al., 2011; Spadaro et al., 2013). However, beyond the use of single treatment microbial antagonists, in recent times emphasis is being placed on the use of two or more combined biocontrol agents (BCAs) to enhance their performance and reliability; and expand the alternatives for postharvest disease control (Wisniewski et al., 2016). From previous studies, synergistic effect of chitosan and Cryptococcus laurentii were found to inhibit infections caused by Penicillium expansum (Yu et al., 2007). Coelho et al., 2007 indicated the effectiveness of Pichia ohmeri
Corresponding author. E-mail address: [email protected] (H. Zhang).
https://doi.org/10.1016/j.postharvbio.2018.03.005 Received 3 October 2017; Received in revised form 16 February 2018; Accepted 7 March 2018 0925-5214/ © 2018 Elsevier B.V. All rights reserved.
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determine the concentrations of the spores and adjusted as required with sterile distilled water.
and Saccharomyces cerevisiae in the biodegradation of patulin. Similarly, Lentinula edodes was reported to enhance the biocontrol activity of C. laurentii against P. expansum infection contamination and patulin production in apple fruits (Tolaini et al., 2010). To continue the search for alternative approaches to manage PATproducing fungi, our study opted to treat P. caribbica yeast with bamboo leaf flavonoid (powder form) in order to understand the mechanisms of action that control PAT accumulation in apple fruit. The bamboo leaf extract powder is rich in flavonoids with protective function of inducing antioxidant responses in the host under stress conditions. It is highly underutilized natural bioactive compound in management of postharvest diseases, despite its function as food additive and medicine (Vastano et al., 2000). The Ministry of Health of the People’s Republic of China has approved bamboo leaves extract as an innovative natural antioxidant and has been proven to be safe and virtually non-toxic for use as a food additive, after both rats and mice tests were conducted (Mao et al., 2015). To the best of our knowledge, there is no information available on the integration of P. caribbica with bamboo leaf flavonoid (BLF) with the aim of enhancing biocontrol efficacy of P. caribbica against apple decay caused by P. expansum. In addition, we evaluated the treatments effect on lignin content and pH level. Lastly, the effect of treatments on the biocontrol of PAT accumulation was investigated.
2.3. Determination of pH in vitro The pH of cultures containing patulin and various treatments (0.01 %0.01% Bamboo leaf flavonoid or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid + P. caribbica) and patulin alone in (without yeast or Bamboo leaf flavonoid) as control were determined. By this method, the pH electrode InLab 427 (Mettler Toledo) connected to a SG2-SevenGo pH meter (Mettler Toledo) was used to determine the pH of the treatments at time points as follows 0 (1 h), 12 h, 1 d, 2 d, 3 d, 4 d and 5 d) during incubation at 20 °C. This experiment constituted three replicates of 12 flask containing medium for each treatment, and the experiment was conducted thrice. 2.4. Determination of the effect of P. caribbica combined with BLF on controlling patulin in vitro In this experiment, the method of sample preparation according to Coelho et al. (2007) was used with some modifications. In each 250 mL Erlenmeyer flasks containing 50 mL NYDB with 1 mL of patulin (0.00445 μg L−1), the following suspensions were added; bamboo leaf flavonoid (0.01%) or P. caribbica alone, bamboo leaf flavonoid (0.01%) + P. caribbica and then patulin alone (without yeast or bamboo leaf flavonoid) as control. Afterwards, all Erlenmeyer flasks were incubated using a rotary shaker at 180 × g at 28 °C. At each time point of 0 (1 h), 12, 24, 36, 48, 60, 72, 84, 96, 108, 120, 132, and 144 h, samples were collected and centrifuged at 7000 × g for 10 min. One milliliter of each supernatant was collected and filtered through a 45 μm filter, then retained for HPLC analysis of patulin.
2. Material and methods 2.1. Apple fruit samples Apples (Malus domestica Borkh, cv. Fuji) were harvested at commercial maturity from an orchard in Yantai, Shandong Province, and selected based on absence of physical injury uniform ripeness and size. Selected apples were disinfected with (0.1% sodium hypochlorite) for 1 min, washed using tap water before used.
2.5. Efficacy of P. caribbica combined with BLF in controlling patulin in vivo
2.2. Antagonistic yeast and pathogen inoculum Fruit treatment: To determine the effect of BLF on the efficacy of P. caribbica in preventing blue mold, the selected apples were wounded, inoculated and stored. Here, three uniform wounds with dimensions of 5 mm diameter and approximately 3 mm deep were made at the equator of each apple fruit using the tip of a sterile cork borer. Each of the wounds was inoculated with 30 μl of P. caribbica suspension (1 × 108 cells mL−1) combined with bamboo leaf flavonoid at concentration of 0.01% w/v or sterile distilled water as a control. Each of the wounds was inoculated with 30 μL of P. expansum (1 × 105 spores mL−1) suspension, 2 h later. Then, treated apples were air-dried and kept in plastic trays protected with polythene films to maintain a favorable relative humidity (95%) for 15 d at 20 °C. There were three replicates of 12 fruit for each treatment, and the experiment was conducted thrice.
Antagonistic yeast: The yeast antagonist P. caribbica was isolated from soils of peach orchard (the central shoal of Yangtze River, Zhenjiang, Jiangsu Province) for its reliable antagonistic efficacy against P. expansum (Zhao et al., 2012). The classical methods based on colony and cell morphologies were used for a preliminary characterization of the yeast (Kurtzman et al., 2011). Subsequently, sequence analysis of the 5.8S internal transcribed spacer (ITS) ribosomal DNA (rDNA) region was used to identify the yeast (Li et al., 2014). P. caribbica has been shown to be safe in animal testing, including physiology experiments, acute toxicity studies, and the Ames test (unpublished data). Then P. caribbica isolates were maintained at 4 °C on nutrient yeast dextrose agar medium (NYDA-0.8% nutrient broth, 0.5% yeast extract, 1% glucose and 2% agar). Liquid cultures of the yeast were grown in 250-ml Erlenmeyer flasks containing 50 ml of nutrient yeast dextrose broth (NYDB) which had been inoculated with a loop of the culture and incubated on a rotary shaker (180 rpm) at 28 °C for 24 h. Following incubation, cells were centrifuged (6000 × g for 10 min) at 4 °C and then washed twice with sterile distilled water in order to remove the growth medium (Li et al., 2014). Afterwards the cell pellets were re-suspended in sterile distilled water and adjusted to an initial concentration before being adjusted to the concentration required for the each experiment. Pathogen inoculum: P. expansum was isolated from infected apples and cultured as described by Zheng et al. (2017) with slight modifications. This culture was maintained on potato dextrose agar (PDA, extract of boiled potatoes, 200 ml; dextrose, 20 g; agar, 20 g and distilled water, 800 ml) at 4 °C, and fresh cultures were grown on PDA plates before use. Spore suspensions of P. expansum were prepared by removing the conidia of 7 d old culture with a bacteriological loop, and suspended in sterile distilled water. Hemocytometer was used to
2.6. Determination of lignin content The lignin content of apples was determined at different time points after 15 d of storage at 20 °C (relative humidity of 95%). First, wounds were made at the equator of each apple as described above. Later each wound was inoculated with 30 μl of P. caribbica (1 × 108 cells mL−1) containing BLF (0.01%) or each of them used as stand-alone treatment with sterile distilled water as control. After application of treatments, fruits were allowed to air dry for 2 h at room temperature. The fruits samples were packaged in trays and covered with plastic film for storage and observation. The assessment of the lignin content was conducted according to the method suggested by Vilanova et al. (2014) with minor modification. Here, the apple disks were lyophilized for 4 d and then ground to a fine powder. In sequence, each sample was washed with water, ethanol, acetone and diethyl ether through Whatman 1 filter paper until the 2
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washed tissue was colourless. After the final powder was dried for 1 h at 70 °C, 20 mg samples were digested with a solution of 25% (w/w) acetyl bromide in acetic acid (2.5 mL) and HClO4 (70%, 0.12 mL) and heated in a bath for 30 min at 70 °C with shaking. After cooling with ice, 10 mL of 2 M NaOH and 12 mL of acetic acid were added to the reaction tubes and 1.5 mL of the resulting solution was centrifuged at 10,000 × g for 30 min at 4 °C to be sure that the resulting sample was completely clear. Five times dilution was made for each solution with acetic acid and absorbance was determined at 280 nm. Samples were collected at each time point and each treatment was replicated three times. 2.7. Determination of pH in fruit wounded site The pH of each fruit mesocarp was measured according to the method described by Neri et al. (2010). In brief, the pH electrode In Lab 427 (Mettler Toledo) was inserted at 15 mm depth through the wounds (decayed). Similarly, the pH of non-decayed (control) tissue were also measured. The pH was measured at 15 d of storage at 20 °C (relative humidity 95%). Three replicates of 12 fruit for each treatment were used, and the experiment was conducted thrice. Fig. 1. pH of culture of patulin in 0.01% Bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc), or untreated control in medium. pH was measured at time points as follows 0 (1 h), 12 h, 1 d, 2 d, 3 d, 4 d and 5 d after incubation at 20 °C. Bars represent standard error of the means according to the Tukey test at P = 0.05.
2.8. Determination of extent of lesions Lesion diameters of the equatorial sections of each apple was measured. Two perpendicular diameters from each of the lesions were measured using a pair of calipers (Morales et al., 2007). The tissues were removed and weighed for patulin content analysis.
incubation at 28 °C (Fig. 1). The combined treatment of BLF and Pc (P. caribbica) retained the highest pH, reaching values of about 4.8 after 4 d of incubation, followed by a slight increase of pH after a day of incubation. Actually, very little changes in pH were observed in Pc compared with BLF, except at 4 d and 5 d. The final difference in pH after 5 d of incubation were BLF and Pc (4. 9 ± 0.127), Pc (3.8 ± 0.114), BLF (3.6 ± 0.021) and control (3.4 ± 0.064). However, the control medium showed the greatest decrease of pH during the 5 d of incubation.
2.9. Extraction and purification Patulin accumulation in decayed tissue was determined by removing from each apple wound using a cork borer (at least 1 cm wider than lesion diameter), and juice extracted. Then apple juice was clarified using pectinase (Boonzaaijer et al., 2005). Ethyl acetate (25 mL) was added to 10 mL of the juice and vortexed for 60 s. Then the supernatant was transferred into a separatory funnel (250 mL). This step was repeated thrice and then 10 mL of 1.4% w/v Na2CO3 was added to the ethyl acetate and mixed vigorously for 2 min. The ethyl acetate layer was vacuum dried at 40 °C in a rotary evaporator (Germany). Immediately, 1 mL acetate buffer made of 0.2 mol L−1 and pH 4 was added and vortexed until it completely dissolved. This mixture was then transferred into a vial and inserted into the auto sampler for HPLC analysis.
3.2. Effect of treatments on patulin retention in medium Efficacies of the different treatments varied when medium were inoculated with Patulin (Fig. 2). In particular, when the medium was
2.10. HPLC conditions An Agilent Technologies 1100 series system equipped with a quaternary pump and variable wavelength detector (Switzerland) was used. The analytical column was from Zorbax, SB-C18 250 × 4.6 mm 5 μm (US). The mobile phase 90 – 10% water/acetonitrile was used at 1 mL min−1. The UV detection was conducted at 276 nm. 2.11. Statistical analysis Generally, each treatment consisted of three replicates of 12 fruit, and the experiment was performed thrice. The data were analyzed for significant differences by analysis of variance (ANOVA) with OriginPro version 9.0 (OriginLab, Northampton, USA) statistical package. The significance among the data was assessed by Tukey test at P < 0.05. 3. Results Fig. 2. Effect of treatment with 0.01% Bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc) and untreated fruits as control on patulin concentration in vitro. Data were the means of three experiments. Bars represent standard error of the means according to the Tukey test at P = 0.05.
3.1. pH in medium after incubation In general, the decrease of pH began after 1 h during the period of 3
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Fig. 4. Effects of treatments on lesion diameter of apple wounds were determined. After initial treatment with 0.01% Bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc) or untreated control in medium was done. After drying the inoculated wounds for 2 h, same wounds were inoculated with P. expansum before storage for 15 d at 20 °C (RH 95%). The pH values were pooled from three wound along the equator of the fruit. Bars represent standard error of the means according to the Tukey test at P = 0.05.
Fig. 3. Determination of pH in decayed fruit wounds. Each wound was initially inoculated with different treatments of 0.01% bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc), and untreated control before infected by P. expansum. pH of fruits was measured at time points as follows 0 (1 h), 3 h, 6 d, 9 d, 12 d, 15 d and 18 d after storage at 20 °C. Bars represent standard error of the means according to the Tukey test at P = 0.05.
inoculated with BLF combined with Pc, it yielded a significant reduction in the patulin concentration, compared with yeast alone or BLF alone. The untreated control gave the highest patulin residue, which implies that it was the least effective against patulin accumulation. At the final time of 144 h after incubation the following patulin concentration were observed: BLF + Pc (undetected), Pc (undetected), BLF (0.001 μg L−1), and untreated control (0.003 μg L−1). However, the combined treatment showed undetected patulin earlier during incubation at 96 h compared to antagonistic yeast, which reached undetectable limit at 120 h.
3.4. Effect of treatment on lignin content of fruit wound Table 1 shows lignin content measured in the tissues of the wound. The results showed higher levels of lignin contents in treatments compared with the control. The highest lignin content was found in the combined treatment of BLF and Pc (0.345 ± 0.011) followed by Pc (0.304 ± 0.011) and BLF (0.279 ± 0.015), each as individual treatment, and the control was the lowest (0.203 ± 0.017). 3.5. Lesion diameter and incidence of patulin accumulation Lesion diameter: When wounded apple fruits were treated with BLF + Pc, rot inhibition was significantly (P < 0.05) increased, achieving the highest inhibition in controlling blue mold. The lesion diameter were BLF + Pc (5.74 mm), Pc (8.33 mm), BLF (17.60 mm) and control (69.00 mm). The results suggest that P. expansum growth on apple wounds could be significantly inhibited by this treatment under the existing experimental conditions (Fig. 4). Patulin accumulation: The present data in relation to patulin content in wounded apples contaminated with P. expansum and treated or untreated with biocontrol agent Pc and with BLF have been are showed in Fig. 5 It was evident that after 20 d of incubation at 20 °C, BLF + Pc significantly inhibited patulin production in comparison with the
3.3. pH of wound area of fruits Fig. 3 indicates the pH measured after 18 d storage of fruits at 20 °C. There was a significant difference (P > 0.05) between all the samples tested particularly on the 18 d after incubation. On the 18 d after incubation, the pH of BLF combined with Pc inoculated tissues was higher (pH 4.41 ± 0.022), followed by Pc alone (pH 4.10 ± 0.081), BLF alone (pH 3.87 ± 0.053) and control was the least (pH 3.63 ± 0.055) fruit. This implied that pH values of BLF + Pc > Pc > BLF > control.
Table 1 Lignin content (absorbance at 280 nm) of apple wound inoculated withP. expansum and stored at 20 °C for 15 d. Each wound was initially inoculated with different treatments of 0.01% Bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc), and untreated control before infected by P. expansum. Days after incubation
Treatments Ck
0 3 6 9 12 15
0.2134 0.2318 0.2326 0.2365 2.0243 0.1920
0.01% BLF ± ± ± ± ± ±
0.004ae 0.013a 0.002a 0.002a 0.0024a 0.002a
0.2140 0.2439 0.2456 0.2472 0.2705 0.2837
± ± ± ± ± ±
Pc 0.002be 0.004b 0.004b 0.004b 0.003b 0.005a
0.2369 0.2310 0.2384 0.2472 0.2571 0.2718
0.01% BLF + Pc ± ± ± ± ± ±
0.007c 0.006c 0.004c 0.002c 0.006c 0.006a
0.2712 0.2954 0.2981 0.2986 0.3049 0.3058
± ± ± ± ± ±
0.003d 0.003d 0.005d 0.007d 0.004d 0.009a
Means are pooled values of three trials ± the standard error. Bars represents standard errors and dissimilar alphabets are statistically different based on the Tukey test at P = 0.05.
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at finial incubation time. Similar to a study by Moss (1991), at pH 4.0 yeast biomass in apple juice was highest (12.4 g L−1), In agreement with Prusky et al. (2004), in apples, they observed pH of 4–6 was detected in non-decayed tissues and lower pH of 3.6–4.1 in decayed tissue (caused by P. expansum) between 4 and 6 d after inoculation. Neri et al. (2010) also observed a decline of pH (kiwifruits and pears tissues) in infected sites to be less than 4.0. According to these authors (Prusky et al., 2014), Penicillium spp. is known to be influenced by the decrease of pH to support pathogen attack and that change in pH around the host infection sites can modulate the expression of factors of pathogenicity. For instance, pH range of 3.2 – 3.8 enables fungi to produce patulin vigorously supports (Moss, 1991), whiles pH increase to 6 in a solution, might lead to an increase in PAT breakdown (Collin et al., 2008). From our results, probably the presence of bamboo leaf flavonoid during yeast activity limited the rapid growth of the pathogen, contributing to the trend of pH observed in the combined treated sample. Indeed, as suggested, the aggressive growth of P. expansum will significantly reduce the pH of potato dextrose broth, while less aggressive growth will limit dropping of pH in the substrate (Neri et al., 2010). Clearly, our results indicate connection between increased lignin content and decreased wound lesion extension. As such wounds treated with BLF combined with Pc, gave the highest lignin content and the lowest lesion diameter. Our study, which reports the beneficial effect of yeast amended with BLF in apple fruit, supports other findings that lignin may perhaps impede penetration of pathogen and increase disease resistance in fruit (Schirra et al., 2000). With regards to similar findings, higher induction of disease resistance is linked with higher phenolic content and lignin in fruit tissue as suggested by other studies (Valentines et al., 2005). We also confirmed in our previous trials that BLF combined with Pc increased the phenolic compound in wounds of apple fruits (unpublished data); thus, both phenolic and lignin contents play a significant role in the mechanism of defense against P. expansum in vivo. It was reported earlier that stimulation of lignification of plant cells by polymerization of phenolic phenylpropanoid precursors such as hydroxylated and methoxylated cinnamoyl alcohols, plays an essential role in disease resistance expression (Hahlbrock and Scheel, 1989). Valentines et al. (2005) confirmed that lignification, which can also be connected to H2O2 through the action of POX enzyme, is a universal process implicated in plant defence reactions. In many plants, it was observed that lignification is a reaction to pathogenic infection, in order to enhance mechanical strength of cell walls and deters fungal invasion (Walter et al., 2010). In addition, it was earlier reported that the production of wound periderm is dependent on the wound healing response (Vilanova et al., 2014), which was found to be interrelated to a local accumulation of lignin and phenolics, controlling the cell wall thickening around wounds (Spotts et al., 1998). In support of these findings, different authors reported on the correlation between wound healing processes in apples with lignin accumulation (Vilanova et al., 2012). Indeed, lignification or lignin deposition is known as one of the mechanisms of disease resistance in fruit (Valentines et al., 2005), and in citrus fruit it appeared to influence wound healing and prevention of green mold (Nafussi et al., 2001). From our previous review report, P. expansum is often involved in spoilage of apple fruits and apple juice, and because of public health concerns, various biocontrol methods that are comparatively safe have been confirmed as possible agents to control P. expansum (Mahunu et al., 2015). In addition, P. caribbica as a single treatment, decreased patulin accumulation in vitro (Cao et al., 2013). However, the enhancement of BCAs through combined treatment approaches, have been proposed as most effective way of reducing PAT (Spadaro et al., 2013b), and besides it also improves their persistence and colonization of BCAs wounded and unwounded fruit surfaces (Castoria et al., 2008). According to the results of our study, non-detectable of patulin production was achieved using 0.01% (w/v) bamboo leaf flavonoid. To the best of our knowledge, no previous reports have found on the effect of the combination action of bamboo leaf flavonoid and P. caribbica on
Fig. 5. Patulin accumulation after 20 d at 20 °C of storage condition. Treatment include 0.01% Bamboo leaf flavonoid (BLF) or P. caribbica (Pc), 0.01% Bamboo leaf flavonoid (BLF) + P. caribbica (Pc), or untreated control. Each value is the mean of two experiments. Bars represent standard error of the means according to the Tukey test at P = 0.05.
untreated control (0.029 μg kg−1 ± 0.373) inoculated with P. expansum. Furthermore, when treated separately, Pc (0.002 μg kg−1 ± 0.28) followed by BLF (0.002 μg kg−1 ± 0.23) controlled patulin production. All samples treated with the biocontrol agents or the bioactive compound showed a large inhibiting effect on patulin biosynthesis when fruits were incubated at 20 °C for 20 d.
4. Discussion Our initial investigations using P. caribbica combined with different concentrations of bamboo leaf flavonoid as amendment yielded promising results against P. expansum. In the same experiment, we observed that P. caribbica combined with low concentration (0.01% w/v) of bamboo leaf flavonoid was able to reduce natural decay and maintained physiochemical parameters of apple fruits (data unpublished). Furthermore, the treatment combination was able to inhibit effectively both mycelial growth and germination of P. expansum, which according to literature was mainly due to competition and production of secondary metabolites with antifungal action (Trias et al., 2010). We therefore decided to investigate further, whether P. caribbica amended with bamboo leaf extract will also able to reduce patulin, and also influence pH levels, lignin content and lesion diameter. As mentioned earlier, the combination of more treatments produced a complex mechanism of action, which disrupts the possible development of pathogen resistance (Castoria et al., 2003; Paster and Barkai-Golan, 2008) and the potential inhibition of PAT production. Again, combined treatments have the potential to enhance the antifungal activity of yeast in broth cultures and decrease in PAT production (Coelho et al., 2007), though some factors could influence their performance response during the process. According to Yiannikouris et al. (2007), pH is one of the parameters that controls patulin breakdown. The change in the incidence and level of patulin could be altered according to various factors such as pH of fruit and vegetable (Drusch et al., 2007). In our trial, we found similar trends in pH both in vitro and in vivo. When we conducted a long-term incubation of samples for 5 d at 28 °C, where P. caribbica cells combined with bamboo leaf flavonoid had been exposed to patulin containing media, we discovered the higher pH value. In general, though pH showed a decline in all the samples, P. caribbica cells + bamboo leaf flavonoid inoculated sample maintained a higher curve with > pH 4.0 5
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mycotoxin patulin production. The minimum quantity of patulin was favored by the yeast activity in the presence of bamboo leaf flavonoid. This combined treatment, according to earlier investigation, has inhibitory effect on the growth of P. expansum, probably preventing subsequent effects. According to Hasan (2000), patulin production is amplified when the growth of P. expansum reaches the late phase and the energy source in the mycological medium is nearly exhausted and sufficient intermediates might have accumulated. On the other hand, they also observed that the decrease in patulin production associated with length of incubation period, possibly maybe due to its degradation by certain substances as organic acids depleting from the vacuole and by mycelium of P. expansum itself. In conclusion, although the use of P. caribbica alone was found to inhibit PAT accumulation in stored apple fruits, however, its integration with BLF has shown better effect. Thus, the enhancement of lignification and the extension of storage time of apple fruits by the yeast combined with natural bioactive compound demonstrated here suggests that the use of P. caribbica integrated with BLF is a potential agent for postharvest diseases management. Notes The authors declare no competing financial interest. Funding This work was supported by the National Natural Science Foundation of China (31571899; 31772037), and the National Key Research and Development Program of China (2016YFD0400902). References Andersen, B., Thrane, U., 2006. Food-borne fungi in fruit and cereals and their production of mycotoxins. Adv. Food Mycol., vol. 572. Springer, pp. 137–152. http://dx.doi.org/ 10.1007/0-387-28391 9_812. Bonerba, E., Ceci, E., Conte, R., Tantillo, G., 2010. Survey of the presence of patulin in fruit juices. Food Addit. Contam. 3, 114–119. http://dx.doi.org/10.1080/19393210. 2010.490882. Boonzaaijer, G., Bobeldijk, I., van Osenbruggen, W.A., 2005. Analysis of patulin in dutch food, an evaluation of a SPE based method. Food Control 16, 587–591. http://dx.doi. org/10.1016/j.foodcont.2004.06.020. Bürger, M., Brakhage, A., Creppy, E., Dirheimer, G., Röschenthaler, R., 1988. Toxicity and mutagenicity of patulin in different test systems. The Target Organ and the Toxic Process, vol. 12. Springer, pp. 347–351. http://dx.doi.org/10.1007/978-3-64273113-6_62. Cao, J., Zhang, H., Yang, Q., Ren, R., 2013. Efficacy of Pichia caribbica in controlling blue mold rot and patulin degradation in apples. Int. J. Food Microbiol. 162, 167–173. http://dx.doi.org/10.1016/j.ijfoodmicro.2013.01.007. Castoria, R., Caputo, L., De Curtis, F., De Cicco, V., 2003. Resistance of postharvest biocontrol yeasts to oxidative stress: a possible new mechanism of action. Phytopathology 93, 564–572. http://dx.doi.org/10.1094/PHYTO.2003.93.5.564. Castoria, R., Wright, S.A., Droby, S., 2008. Biological control of mycotoxigenic fungi in fruits. Mycotoxins Fruits Vegetables 311–333. Chen, X., Li, J., Zhang, L., Xu, X., Wang, A., Yang, Y., 2012. Control of postharvest radish decay using a Cryptococcus albidus yeast coating formulation. Crop Prot. 41, 88–95. http://dx.doi.org/10.1016/j.cropro.2012.05.015. Coelho, A.R., Celli, M.G., Ono, E.Y.S., Wosiacki, G., Hoffmann, F.L., Pagnocca, F.C., Hirooka, E.Y., 2007. Penicillium expansum versus antagonist yeasts and patulin degradation in vitro. Braz. Arch. Biol. Technol. 50, 725–733. http://dx.doi.org/10. 1590/S1516-89132007000400019. Collin, S., Bodart, E., Badot, C., Bouseta, A., Nizet, S., 2008. Identification of the main degradation products of patulin generated through heat detoxication treatments. J. Inst. Brew. 114, 167–171. http://dx.doi.org/10.1002/j.2050-0416.2008.tb00322.x. Devaraj, H., Radha, S.K., Shanmugasundaram, E., 1982. Neurotoxic effect of patulin. Indian J. Exp. Biol. 20, 230–231. Drusch, S., Kopka, S., Kaeding, J., 2007. Stability of patulin in a juice-like aqueous model system in the presence of ascorbic acid. Food Chem. 100, 192–197. http://dx.doi. org/10.1016/j.foodchem.2005.09.043. Hahlbrock, K., Scheel, D., 1989. Physiology and molecular biology of phenylpropanoid metabolism. Annu. Rev. Plant Biol. 40, 347–369. http://dx.doi.org/10.1146/ annurev.pp.40.060189.002023. Hasan, H., 2000. Patulin and aflatoxin in brown rot lesion of apple fruits and their regulation. World J. Microbiol. Biotechnol. 16, 607–612. http://dx.doi.org/10.1023/ A:1008982511653. Kurtzman, C., Fell, J.W., Boekhout, T., 2011. The Yeasts: a Taxonomic Study. Elsevier. Laidou, I., Thanassoulopoulos, C., Liakopoulou‐Kyriakides, M., 2001. Diffusion of patulin
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