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Induction and Quantification of Patulin Production in Penicillium Species
青霉菌展青霉素生成的诱导和定量   

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Abstract

Patulin, a worldwide regulated mycotoxin, is primarily produced by Penicillium and Aspergillus species during fruit spoilage. Patulin contamination is a great concern with regard to human health because exposure of the mycotoxin can result in severe acute and chronic toxicity, including neurotoxic, mutagenic, and immunotoxic effects. Penicillium expansum is known as the main producer of patulin. This protocol addresses the cultivation procedure of P. expansum under patulin permissive conditions and describes the method of collection and detection of patulin.

Keywords: Penicillium expansum(扩展青霉), Patulin induction(展青霉素诱导), HPLC(HPLC), Quantification(定量)

Background

Patulin is a polyketide lactone mycotoxin and is produced by several species of fungi including Penicillium, Aspergillus and other species. Among them, Penicillium expansum, which is a well-known postharvest pathogen causing decay of pomaceous fruits during storage, is the main producer. Patulin levels in apple products are of great concern because of the severe acute and chronic effects caused by the toxin. Therefore the patulin level in food is limited in many countries around the world. The European Commission (2006) has set maximum permitted levels in apple juices (50 μg/kg), solid apple products (25 μg/kg) and, above all, fruit-derived baby foods (10 μg/kg), as children are major consumers of apple derived products.

Studies on patulin in recent years have focused on environmental factors regulating patulin production, molecular basis of patulin biosynthesis and biodegradation of patulin. The methods of induction and quantification of patulin production are important in these studies. Patulin analysis in fruits usually follows the AOAC method 995.10 (Brause et al., 1996). After treatment with pectinase, patulin is extracted with ethyl acetate from the puree of decayed portion of fruits. Many methods have been developed for measuring patulin such as TLC, mass spectrometry and gas chromatography/mass spectrometry. Now, high performance liquid chromatography with ultra violet light detection (HPLC-UV) is the most frequently used method (Baert et al., 2007).

In this protocol, we address two methods of patulin induction in vitro and describe the specific parameters appropriate for HPLC-UV analysis of patulin.

Materials and Reagents

  1. 1,000 µl pipette tips (Corning, Axygen®, catalog number: TF-1000-R-S )
  2. 200 µl pipette tips (Corning, Axygen®, catalog number: TF-200-R-S )
  3. 10 µl pipette tips (Corning, Axygen®, catalog number: TF-300-R-S )
  4. Cheesecloth (Aladdin, catalog number: G6902 )
  5. 90 x 15 mm Petri dish (any brand will suffice)
  6. 10 ml centrifuge tubes (Sangon Biotech, catalog number: F601889 )
  7. 1.5 ml centrifuge tubes (Corning, Axygen®, catalog number: MCT-150-C )
  8. Filter (pore size 0.45 μm) (EMD Millipore, catalog number: SLHV033RB )
  9. Cellophane sheets (Bio-Rad Laboratories, catalog number: 1650963 )
  10. 24-well culture plates (Corning, Costar®, catalog number: 3524 )
  11. Penicillium expansum T01: was isolated by our laboratory and whole-genome sequenced (Li et al., 2015)
  12. Glycerol (AMRESCO, catalog number: M152 )
  13. Tween 20 (Sigma-Aldrich, catalog number: T2700 )
  14. Liquid nitrogen
  15. Sterile distilled water
  16. Water (HPLC grade) (Alfa Aesar, catalog number: 19391 )
  17. Acetonitrile (HPLC grade) (Alfa Aesar, catalog number: 22927 )
  18. Potato
  19. Dextrose (Macklin, catalog number: D823520 )
  20. Agar (HUAAOBIO, catalog number: HA0552 )
  21. Sodium nitrate (NaNO3) (Beijing Chemical Works, GB/T 647-1993)
  22. Potassium phosphate dibasic trihydrate (K2HPO4·3H2O) (Beijing Chemical Works, HG/T 3487-2000)
  23. Potassium chloride (KCl) (Aladdin, catalog number: P112133 )
  24. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Macklin, catalog number: M813599 )
  25. Iron(II) sulfate heptahydrate (FeSO4·7H2O) (Aladdin, catalog number: F116341 )
  26. Sucrose (Beijing Chemical Works, HG/T 3462-1999)
  27. Yeast extract (Oxoid, catalog number: LP0021 )
  28. Hydrochloric acid (HCl) (AMRESCO, catalog number: 0369 )
  29. PDA medium (see Recipes)
  30. CY medium (see Recipes)
  31. Acidified distilled water (pH 4.0) (see Recipes)

Equipment

  1. Glass spreading rod (any brand will suffice)
  2. Hemacytometer (QIUJING, model: XB-K-25 )
  3. 100-1,000 µl pipette (Eppendorf, catalog number: 3120000267 )
  4. 10-100 µl pipette (Eppendorf, catalog number: 3120000240 )
  5. 0.5-10 µl pipette (Eppendorf, catalog number: 3120000224 )
  6. Centrifuge (Beckman Coulter, model: Microfuge 16 , catalog number: A46473)
  7. Tweezer (Thermo Fisher Scientific, catalog number: 402011 )
  8. High-performance liquid chromatography (WATERS Corp., MA, USA)
  9. Auto sampler (WATERS, catalog number: 2498 )
  10. Binary HPLC pump (WATERS, catalog number: 1525 )
  11. UV/Visible detector (WATERS, catalog number: 2487 )
  12. C18 column (5 μm, 250 x 4.6 mm) (GL Sciences, model: Inertsil® ODS-3 )
  13. Vortexer (Select BioProducts, catalog number: SBS100-2 )
  14. Optical microscope (Chongqing Optec Instrument, model: B Series Biological Microscope , catalog number: B203LED)
  15. Clean bench (Donglian Electronic & Technology Development, model: SCB-1520 )
  16. Constant temperature incubator (TAICANG, model: THZ-C )

Software

  1. Microsoft Excel
  2. Empower 3.0
  3. SPSS 13.0 (SPSS Inc., Chicago, IL, USA)

Procedure

  1. Patulin induction with PDA (solid) medium (Zong et al., 2015)
    1. Five-microliter spore suspension (5 x 106/ml in 16% glycerol, stored at -80 °C) of Penicillium expansum is inoculated on PDA plate (Figure 1A) and cultured for 2 weeks at 25 °C in the dark.
      Conidia are harvested with a glass spreading rod with 3-5 ml 0.05% Tween 20 (Figure 1B) and filtered through four layers of sterile cheesecloth (Figure 1C). Conidia are counted with a hemocytometer and diluted to a concentration of 1 x 105 conidia/ml with sterile distilled water.


      Figure 1. Preparation of P. expansum spore suspension. A. Activation of P. expansum on PDA plate; B. Conidia harvesting with 0.05% Tween 20; C. Filtration of spore suspension with four layers of sterile cheesecloth.

    2. Five-microliter spore suspension is inoculated at the center of 9-cm PDA plates and incubated at 25 °C in the dark for 10 days (Figure 2A).
    3. Each of the Petri dish is washed with a glass spreading rod with 5 ml of acidified distilled water (pH 4.0) and the spore suspension is transferred to a 10 ml centrifuge tube with a 100-1,000 µl pipette (Figure 2B). Then, the spore suspension is centrifuged at 10,000 x g for 10 min and the supernatant is filtered through a 0.45 μm filter for patulin detection.


      Figure 2. Patulin induction with PDA medium. A. Asexual sporulation (top) and mycelial pigmentation (bottom) patterns of P. expansum after 10 days of cultivation on PDA plate; B. The spore suspension washed with 5 ml of acidified distilled water (pH 4.0).

  2. Patulin induction with CY (liquid) medium
    1. P. expansum spore suspension is prepared as described above and diluted to a concentration of 1 x 106 conidia/ml with sterile distilled water.
    2. Aliquots of 1 μl of spore suspension are spread on cellophane sheets (1 x 1 cm) placed on PDA plates and cultured at 25 °C for 36 h (Figure 3A).
    3. Then the cellophane sheets covered with P. expansum mycelia are transferred to CY liquid medium. Each cellophane sheet is floated on 1 ml CY liquid medium in a 24-well culture plate (Figure 3B). The whole plate is covered and sealed with Parafilm, then cultured under static conditions at 25 °C in the dark.
    4. At 48 h after transfer, mycelium and spores are collected with a tweezer and quickly frozen with liquid nitrogen for some other use (RNA/DNA/protein extraction) and the medium (Figure 3C) is filtered through a 0.45 μm filter for patulin detection.


      Figure 3. Patulin induction with CY medium. A. P. expansum mycelium incubated on cellophane sheets (1 x 1 cm) placed on PDA plates; B. Cellophane sheets covered with mycelium incubated on CY liquid medium in a 24-well culture plate; C. CY medium after removal of mycelium.

  3. HPLC analysis
    1. High-performance liquid chromatography analysis is performed for determining patulin production.
    2. 10 μl of the filtrate extract is injected into a liquid chromatography equipped with an auto sampler (WATERS 2498), a binary HPLC pump (WATERS 1525), and a UV/Visible detector (WATERS 2487). A C18 column (5 μm, 250 x 4.6 mm, GL Sciences, Japan) is used for patulin detection.
    3. The mobile phase is a mixture of water and acetonitrile (90:10, v/v) at a flow rate of 1 ml/min in isocratic elution mode.
    4. The detection wavelength of patulin is 276 nm and the column oven is set at 25 °C.

Data analysis

  1. To construct a standard curve
    The solutions of patulin were prepared at concentrations of 20, 50, 100, 200 and 250 μg/ml. The volume injected into the column was 10 μl. The retention time of patulin is about 9.28 min in the HPLC analysis with above experimental conditions (Figure 4A). Linear regression was used to prepare the standard curve by using the mean values of peak areas of five injections of the five solutions (Figure 4B). Equation of the standard curve was:

        E = 50990C + 39869 R² = 1

    where, E is the peak area of patulin injected (10 μl) and C is the concentration (μg/ml) of the standard patulin injected.
    The concentrations of experimental samples can be calculated with the corresponding peak area on the retention time of patulin (9.28 min) read by HPLC.


    Figure 4. HPLC analysis (A) and standard curve of patulin (B)

  2. Statistical analysis
    Data from three independent experiments, each with five replicates, can then be analyzed with the statistic software SPSS version 13.0. ANOVA test was performed using Duncan’s multiple range test; P < 0.05.

Recipes

  1. PDA medium (1 L)
    200 g potato
    20 g dextrose
    15 g agar
    Boiling 200 g of sliced potatoes in 1 L distilled water for 30 min, then decanting the broth through cheesecloth and adding 20 g dextrose and 15 g agar powder in the broth. Add distilled water to make up 1 L, and the medium is sterilized by autoclaving at 121 °C for 20 min
  2. CY medium (1 L)
    3 g NaNO3
    1 g K2HPO4·3H2O
    0.5 g KCl
    0.5 g MgSO4·7H2O
    0.01 g FeSO4·7H2O
    30 g sucrose
    5 g yeast extract
    dH2O up to 1 L
    Adjust to pH 5.2 by adding HCl, autoclave at 121 °C for 20 min
  3. Acidified distilled water (pH 4.0, 100 ml)
    Prepare 100 ml distilled water, adjust to pH 4.0 with HCl, filter sterile through a 0.22 μm filter

Acknowledgments

This protocol was adapted from Li et al. (2015) and Zong et al. (2015). The work was supported by Chinese Ministry of Science and Technology (grant number 2016YFD0400902).

References

  1. Baert, K., Meulenaer, B., Kasase, C., Huyghebaert, A., Ooghe, W. B. and Devlieghere, F. (2007). Free and bound patulin in cloudy apple juice. Food Chem 100: 1278-1282.
  2. Brause, A. R., Trucksess, M. W., Thomas, F. S. and Page, W. S. (1996). Determination of patulin in apple juice by liquid chromatography: collaborative study. J AOAC Int 79: 451-455.
  3. European Commission Regulation (2006). Commission Regulation EC 1881/2006 setting maximum levels for certain contaminants in foodstuffs. Off J Eur Commun L364: 5-24.
  4. Li, B., Zong, Y., Du, Z., Chen, Y., Zhang, Z., Qin, G., Zhao, W. and Tian, S. (2015). Genomic characterization reveals insights into patulin biosynthesis and pathogenicity in Penicillium species. Mol Plant Microbe Interact 28(6): 635-647.
  5. Zong, Y., Li, B. and Tian, S. (2015). Effects of carbon, nitrogen and ambient pH on patulin production and related gene expression in Penicillium expansum. Int J Food Microbiol 206: 102-108.

简介

Patulin是世界范围内调节的真菌毒素,主要是在水果腐败期间由青霉菌和曲霉菌种生产的。蛋白污染是人类健康的重大问题,因为霉菌毒素的暴露会导致严重的急性和慢性毒性,包括神经毒性,诱变性和免疫毒性作用。青霉素被称为patulin的主要生产者。该协议涉及到P的培育程序。膨化条件下的膨胀,并描述了patulin的收集和检测方法。

背景 丙氨酸是一种聚酮内酯霉菌毒素,是由几种真菌,包括青霉菌,曲霉菌和其他物种产生的。其中,广西青霉菌是一种着名的收获后病原体,在贮存期间引起了果皮果实的腐烂,是主要的生产者。由于毒素引起的严重急性和慢性作用,苹果产品中的蛋白酶水平受到极大的关注。因此,世界上许多国家的食品中的蛋白质水平有限。欧盟委员会(2006年)确定了苹果汁(50μg/ kg),固体苹果产品(25μg/ kg),最重要的是果实婴儿食品(10μg/ kg)的最高允许水平,因为儿童苹果衍生产品的主要消费者。
&NBSP;近年来对青霉素的研究主要集中在调节蛋白质生产的环境因素,patulin生物合成的分子基础和patulin的生物降解。在这些研究中,嘌呤产生的诱导和定量的方法是重要的。水果中的蛋白质分析通常遵循AOAC方法995.10(Brause等人,1996)。用果胶酶处理后,用醋酸乙酯从果实的腐烂部分提取白蛋白。已经开发了许多用于测量patulin的方法,例如TLC,质谱和气相色谱/质谱。现在,使用紫外光检测(HPLC-UV)的高效液相色谱法是最常用的方法(Baert等人,2007)。
&NBSP; &nbsp;在本协议中,我们讨论了两种在体外进行patulin诱导的方法,并描述了适用于patulin的HPLC-UV分析的具体参数。

关键字:扩展青霉, 展青霉素诱导, HPLC, 定量

材料和试剂

  1. 1,000μl移液器吸头(Corning,Axygen ®,目录号:TF-1000-R-S)
  2. 200μl移液器吸头(Corning,Axygen ®,目录号:TF-200-R-S)
  3. 10μl移液器吸头(Corning,Axygen ®,目录号:TF-300-R-S)
  4. Cheesecloth(阿拉丁,目录号:G6902)
  5. 90 x 15毫米培养皿(任何品牌都可以满足)
  6. 10ml离心管(Sangon Biotech,目录号:F601889)
  7. 1.5ml离心管(Corning,Axygen ,目录号:MCT-150-C)
  8. 过滤器(孔径0.45μm)(EMD Millipore,目录号:SLHV033RB)
  9. 玻璃纸(Bio-Rad Laboratories,目录号:1650963)
  10. 24孔培养板(Corning,Costar ®,目录号:3524)
  11. 通过我们的实验室和全基因组测序(Li et al。,2015),分离出青霉菌 T01
  12. 甘油(AMRESCO,目录号:M152)
  13. 吐温20(Sigma-Aldrich,目录号:T2700)
  14. 液氮
  15. 无菌蒸馏水
  16. 水(HPLC级)(Alfa Aesar,目录号:19391)
  17. 乙腈(HPLC级)(Alfa Aesar,目录号:22927)
  18. 土豆
  19. 葡萄糖(Macklin,目录号:D823520)
  20. 琼脂(HUAAOBIO,目录号:HA0552)
  21. 硝酸钠(NaNO 3)(北京化工,GB / T 647-1993)
  22. 磷酸氢二钾三水合物(K 2 O 3 HPO 4·3H 2 O)(北京化工,HG / T3487-2000)
  23. 氯化钾(KCl)(阿拉丁,目录号:P112133)
  24. 硫酸镁七水合物(MgSO 4·7H 2 O)(Macklin,目录号:M813599)
  25. 硫酸铁(II)七水合物(FeSO 4·7H 2 O)(阿拉丁,目录号:F116341)
  26. 蔗糖(北京化工,HG / T 3462-1999)
  27. 酵母提取物(Oxoid,目录号:LP0021)
  28. 盐酸(HCl)(AMRESCO,目录号:0369)
  29. PDA媒体(见食谱)
  30. CY培养基(参见食谱)
  31. 酸化蒸馏水(pH 4.0)(参见食谱)

设备

  1. 玻璃传播杆(任何品牌都可以满足)
  2. 血细胞计数器(QIUJING,型号:XB-K-25)
  3. 100-1,000μl移液器(Eppendorf,目录号:3120000267)
  4. 10-100μl移液器(Eppendorf,目录号:3120000240)
  5. 0.5-10μl移液器(Eppendorf,目录号:3120000224)
  6. 离心机(Beckman Coulter,型号:Microfuge 16,目录号:A46473)
  7. 镊子(Thermo Fisher Scientific,目录号:402011)
  8. 高效液相色谱(WATERS Corp.,MA,USA)
  9. 自动取样器(WATERS,目录号:2498)
  10. 二元HPLC泵(WATERS,目录号:1525)
  11. UV /可见检测器(WATERS,目录号:2487)
  12. C18柱(5μm,250 x 4.6 mm)(GL Sciences,型号:Inertsil ODS-3)
  13. Vortexer(选择BioProducts,目录号:SBS100-2)
  14. 光学显微镜(重庆光学仪器,型号:B系列生物显微镜,目录号:B203LED)
  15. 洁净台(东联电子科技开发,型号:SCB-1520)
  16. 恒温培养箱(TAICANG,型号:THZ-C)

软件

  1. Microsoft Excel
  2. Empower 3.0
  3. SPSS 13.0(SPSS Inc.,Chicago,IL,USA)

程序

  1. 用PDA(固体)培养基进行的Patulin诱导(Zong等人,2015)
    1. 接种在PDA平板上的5微升孢子悬浮液(储存于-80℃的16%甘油中的5×10 6个/ ml)接种在PDA平板上(图1A)并在25℃下在黑暗中培养2周。
      用具有3-5ml 0.05%Tween 20的玻璃铺展杆(图1B)收获分生孢子,并通过四层无菌粗纱布过滤(图1C)。用血细胞计数器计数分生孢子,用无菌蒸馏水稀释至浓度为1×10 5 /分钟/ ml的分生孢子。


      图1.准备。膨胀孢子悬浮液。 :一种。激活P。 PDA板上的膨胀 B.用0.05%吐温20收获分生孢子; C.用四层无菌的干酪包布过滤孢子悬浮液
    2. 将5微升孢子悬浮液接种在9cm PDA平板的中心,并在25℃下在黑暗中孵育10天(图2A)。
    3. 每个培养皿用玻璃传播杆用5ml的酸化蒸馏水(pH4.0)洗涤,孢子悬浮液用100-1000μl移液管转移到10ml离心管中(图2B)。然后,将孢子悬浮液以10,000×g离心10分钟,并通过0.45μm过滤器过滤上清液以进行patulin检测。


      图2.使用PDA培养基进行的Patulin诱导。 A.A。在PDA板上培养10天后,膨胀 B.用5ml酸化蒸馏水(pH 4.0)洗涤孢子悬浮液。

  2. 丙氨酸诱导用CY(液体)培养基
    1. P上。膨胀孢子悬浮液如上所述制备,并用无菌蒸馏水稀释至浓度为1×10 -6分配孢子/ ml。
    2. 将1μl孢子悬浮液的等分试样铺在置于PDA平板上的玻璃纸(1×1cm)上并在25℃培养36小时(图3A)。
    3. 然后玻璃纸被

      P覆盖。菌丝体将菌丝体转移至CY液体培养基。每个玻璃纸在24孔培养板中漂浮在1ml CY液体培养基上(图3B)。整个板用Parafilm覆盖并密封,然后在25℃在黑暗中的静态条件下培养。

    4. 转移后48h,用镊子收集菌丝体和孢子,并用液氮快速冷冻以进行其他一些用途(RNA / DNA /蛋白质提取),并通过0.45μm过滤器过滤培养基(图3C)进行蛋白质检测。


      图3.具有CY培养基的Patulin诱导 A.放置在PDA平板上的玻璃纸片(1×1cm)上孵育的膨胀菌菌丝体; B.用在24孔培养板中的CY液体培养基上培养的菌丝体覆盖的玻璃纸; C.清除菌丝体后的CY培养基

  3. HPLC分析
    1. 执行高效液相色谱分析以确定patulin生产
    2. 将10μl滤液萃取液注入配有自动进样器(WATERS 2498),二元HPLC泵(WATERS 1525)和UV /可见检测器(WATERS 2487)的液相色谱仪中。使用C18柱(5μm,250×4.6mm,GL Sciences,Japan)进行patulin检测
    3. 流动相是在等度洗脱模式下,流速为1ml / min的水和乙腈(90:10,v / v)的混合物。
    4. patulin的检测波长为276nm,柱温箱设定在25℃

数据分析

  1. 构建标准曲线
    以20,50,100,200和250μg/ ml的浓度制备patulin溶液。注入柱中的体积为10μl。在上述实验条件下,HPLC分析中patulin的保留时间约为9.28分钟(图4A)。使用线性回归法通过使用五种溶液的五次注射的峰面积的平均值来制备标准曲线(图4B)。标准曲线的方程为:

    &NBSP;&NBSP; &NBSP; E = 50990C + 39869R²= 1

    其中E是注射的patulin的峰面积(10μl),C是注射的标准patulin的浓度(μg/ ml)。
    实验样品的浓度可以通过HPLC读取的patulin保留时间(9.28分钟)的相应峰面积计算。


    图4. HPLC分析(A)和patulin标准曲线(B)

  2. 统计分析
    然后可以用SPSS 13.0统计软件分析来自三个独立实验的数据,每个实验有五个重复。使用Duncan的多范围测试进行ANOVA测试; 0.05。

食谱

  1. PDA媒体(1升)
    200克土豆
    20克右旋糖
    15克琼脂
    将200克切片的土豆在1升蒸馏水中煮沸30分钟,然后通过干酪布滗析肉汤,并在肉汤中加入20克葡萄糖和15克琼脂粉末。加入蒸馏水组成1升,培养基通过121℃高压灭菌20分钟灭菌
  2. CY培养基(1L)
    3g NaNO 3
    1 g K 2 2 HPO 4·3H 2 O - / - 0.5克KCl
    0.5g硫酸镁·7H 2 O 2·
    0.01g FeSO 4·7H 2 O
    30克蔗糖
    5克酵母提取物
    dH <2> O至1 L
    加入HCl调节至pH 5.2,121℃高压灭菌20分钟
  3. 酸化蒸馏水(pH 4.0,100 ml)
    准备100ml蒸馏水,用HCl调节至pH 4.0,通过0.22μm过滤器无菌过滤

致谢

该协议由Li等人(2015)和Zong等人改编。 (2015)。这项工作得到了中国科技部的支持(授权号码2016YFD0400902)。

参考

  1. Baert,K.,Meulenaer,B.,Kasase,C.,Huyghebaert,A.,Ooghe,WB and Devlieghere,F。(2007)。&nbsp; 多云苹果汁中的自由和结合的蛋白酶。 食品化学 100:1278-1282。 />
  2. Brause,AR,Trucksess,MW,Thomas,FS and Page,WS(1996)。&nbsp; 通过液相色谱法测定苹果汁中的patulin:合作研究。 J AOAC Int 79:451-455。
  3. 欧洲委员会条例(2006)。委员会条例EC 1881/2006设定食品中某些污染物的最高水平。 Eur Commun L364:5-24。
  4. Li,B.,Zong,Y.,Du,Z.,Chen,Y.,Zhang,Z.,Qin,G.,Zhao,W。和Tian,S。(2015)。基因组表征揭示了青霉属物种中patulin生物合成和致病性的见解。 。Mol Plant Microbe Interact 28(6):635-647。
  5. Zong,Y.,Li,B.and Tian,S。(2015)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/26001378”目标=“_ blank”>碳,氮和环境pH对青霉素生产和相关基因表达的影响 Int J Food Microbiol 206:102 -108。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Chen, Y., Li, B., Zhang, Z. and Tian, S. (2017). Induction and Quantification of Patulin Production in Penicillium Species. Bio-protocol 7(11): e2324. DOI: 10.21769/BioProtoc.2324.
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