Fungal contamination
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fungal contamination. chemical presevatives. biological presevatives. Antifungal peptide 抗真菌肽. A bacterial strain isolated from a honey sample showed antifungal activity against mold. What is this strain? The essence of this antifungal material?. ?. 芽胞杆菌 BH072 产生的抗真菌肽研究

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Fungal contamination

fungal contamination


Fungal contamination

chemical presevatives

biological presevatives

Antifungal peptide抗真菌肽


Fungal contamination

A bacterial strain isolated from a honey sample showed antifungal activity against mold.

What is this strain?

The essence of this antifungal material?


Fungal contamination

芽胞杆菌BH072产生的抗真菌肽研究

The Antifungal Peptides Produced by Bacillus BH072

2012.9


Fungal contamination

The Antifungal Peptides Produced by Bacillus BH072

outline

①Identification and characterization of Bacillus BH072

② The antifungal activity of Bacillus BH072

③ PCR analysis

④ Purification and characterization of this peptide

⑤ Discussion


Fungal contamination

Part 1 Identification and characterization

of Bacillus BH072


Fungal contamination

Technical route


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Morphological,Biochemical and Physiological tests

注:+表示实验结果为阳性;﹣表示实验结果为阴性


Fungal contamination

pH

temperature


16s ribosomal dna sequence analysis

16S Ribosomal DNA sequence analysis

  • 8F (5’-AGAGTTTGATCATGGCTCAG-3’)

  • 1492R(5’-ACGGTTACCTTGTTACGACTT-3’)

  • The PCR amplification system :25µ L system,

  • 0.2 µ L Taq enzyme ( 0.5 U/mL )

  • 2.5 µ L 10× Buffer

  • 1.8 µ L Mg2+

  • 1 µ L dNTPs Mixture

  • 1 µ L template DNA

  • 0.5 µ L forward primer ( 10 µ mol/L )

  • 0.5 µ L reverse primer ( 10 µ mol/L )

  • 17.5µ L ddH2O


16s ribosomal dna sequence analysis1

16S Ribosomal DNA sequence analysis

  • Amplification factor:

  • 95 ° C 3 min;

  • 95° C 30 s,

  • 55 °C 60 s, 30 cycles

  • 72 ° C 60s,

  • 72 ° C 5 min,

  • 4 ° C termination reaction.

The 16S rDNA sequence of the amplified PCR product was determined.

Sequences of 1120 bp fragments showed similarity (minimum identity 98%) with the Bacillus subtilis.


Fungal contamination

Results


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细菌DNA的制备

PCR引物的设计

PCR扩增

结合常规检测

枯草芽胞杆菌

琼脂糖凝胶电泳

芽胞杆菌BH072

PCR产物16S rDNA测序

Blast分析


Fungal contamination

Part 2 The antifungal activity of

Bacillus BH072


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agar well diffusion assay

注:++表示抑菌作用显著;+表示抑菌作用一般;﹣表示无抑菌作用

Antifungal activity


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Antifungal activity graph


Fungal contamination

Part 3 PCR analysis


Primer design

Primer design

  • iturinA gene

  • 上游引物:5’- atgaaaatttacggagtatatatg - 3’

  • 下游引物:5’- ttataacagctcttcatacgtt - 3’

  • 扩增长度:675 bp

  • tasA gene

  • 上游引物:5’- atgggtatgaaaaagaaattaag - 3’

  • 下游引物:5’- ttagtttttatcctcactgtga - 3’

  • 扩增长度:786 bp


Fungal contamination

  • Amplification factor:

  • 95 ° C 3 min;

  • 95° C 30 s,

  • 51 °C 60 s, 30 cycles

  • 72 ° C 60s,

  • 72 ° C 5 min,

  • 4 ° C termination reaction.

  • The PCR amplification system :25µ L system,

  • 0.2 µ L Taq enzyme ( 0.5 U/mL )

  • 2.5 µ L 10× Buffer

  • 1.8 µ L Mg2+

  • 1 µ L dNTPs Mixture

  • 1 µ L template DNA

  • 0.5 µ L forward primer ( 10 µ mol/L )

  • 0.5 µ L reverse primer ( 10 µ mol/L )

  • 17.5µ L ddH2O

The amplified sequences were purified, connected to the pUCm-T carrier, transformated, cultivated, extracted of plasmid, and then sequenced in both directions by Sangon Biotech Co., Ltd. (Shanghai, China). Sequencing data were collected. The BLAST algorithm was used to retrieve for homologous sequences in GenBank (National Center for Biotechnology Information [http://www.ncbi.nlm. nih.gov])

tasA

iturin


Fungal contamination

  • Sequence of the amplified PCR product was determined. Sequences of 675 bp fragments showed elevated similarity (99%) with the gene-encoding iturinA, and only 10 point mutations were observed.

  • Sequences of 786 bp fragments showed elevated similarity (99%) with the gene-encoding tasA, and only 10 point mutations were observed.


Fungal contamination

Part 4 Purification and characterization

of this peptide


Separation and purification

Separation and purification

  • Crude extraction stage

    • ammonium sulphate precipitation

    • 硫酸铵沉淀法。该法是细菌素粗提阶段最常用的一种有效方法,其优点在于成本低,操作简便, 条件温和, 不改变蛋白质活性。

    • organic solvent precipitation

    • 有机溶剂沉淀法。该法的优点在于有机溶剂不会残留在细菌素中,容易蒸发除去。有机溶剂沉淀法比硫酸铵沉淀法容易使细菌素变性,所以很少使用。

    • pH absorption

    • 在细菌素粗提阶段, 如果对细菌素的性质不了解时, 可以首先采用硫酸铵分级沉淀法, 如果细菌素具有吸附性,可采用吸附法提取细菌素。也可根据实验要求,采用几种方法相结合对细菌素进行粗提。

  • Purification stage

    • gel chromatography

    • 是利用蛋白质分子量大小的差异来得到目的蛋白的一种方法。

    • ion-exchange chromatography

    • 根据蛋白质所带电荷的性质与离子交换剂的结合力大小的差别而进行分离的一种层析方法。大多细菌素带正电荷,在细菌素纯化时常常选择阳离子交换剂。

    • HPLC

    • 纯度达到99 %以上


  • Fungal contamination

    New discovery:

    Bac 14B is the first antimicrobial protein to show a spectrum of action that is particularly inhibitory against Agrobacterium spp. Strains.

    It also revealed that this bacteriocin contained a unique sequence, namely M-L-K-A-N-L-Q-N-P-L-N-A, suggesting the identification of a novel compound.


    Methods and results

    Methods and Results

    • Bacterial strains and growth conditions

    • Bac 14B from B. subtilis 14B strain

      • pH7.2 and 37°C

      • LB (peptone, 10 g/L; yeast extract, 5 g/L; NaCl, 5 g/L; pH 7.4) supplemented with 10 g/L glucose, 15 g/L K2HPO4, 5 g/L MgSO4·7H2O.

  • Agrobacterium tumefaciens C58, which is routinely used as an indicator strain, was grown on mannitol glutamate agar medium

  • Antimicrobial activity determination

    • agar well diffusion assay


  • Methods and results1

    Methods and Results

    • Bac 14B purification procedure

    supernatant

    Centrifuged

    30min

    9000*g

    pH 7

    ammonium

    sulfate saturation

    Centrifuged

    30min

    9000*g

    pH 7

    Precipitation

    2.5 × 25 cm

    Sephadex G-50

    column

    Fraction I

    10 mM Tris-HCl

    pH 9 (buffer A)

    Elution

    Elution

    Fraction III

    Fraction II

    Mono Q column (2.5 × 20 cm)

    attached to a fast protein

    liquid chromatography system


    Methods and results2

    Methods and Results

    Molecular mass determination

    mass spectrometry

    amino acid sequencing


    Fungal contamination

    • N-Terminal sequence of the purified Bac 14B

      • N-terminal sequencing of the blotted purified Bac 14B led to the identification of 12 residues, M-L-K-A-N-L-Q-N-P-L-N-A. This sequence was submitted to the GenBank nonredundant nucleotide database for comparison with protein sequences using the BLASTP and tBlastn search programs. It was further submitted to the Swiss-Prot database for comparison using BLASTP search software. No similar sequence was found in either database, indicating that Bac 14B has unique N-terminal amino acids.


    Effect of ph and temperature

    Effect of pH and temperature

    • 100°C for 2 h

    • 90°C for 45 min~loss 20% activity

    • 120°C for 20 min~complete loss activity

    • Low temperature storage (−20 and 4°C for 1 month)~ loss activity

    • pH 4-8 retained itsbiological activity, reduced at pH 9

    • Triton X-100 , Tween 80~strongly inhibited

    • Completely inactivation after treatment with different proteolytic enzymes


    Inhibitory spectrum mode of action

    Inhibitoryspectrummode of action


    Fungal contamination

    Part 5 Discussion


    Fungal contamination

    Early achievements

    A bacterial strain isolated from a honey sample showed antifungal activity against mold. Based on the morphological, biochemical and physiological tests, the strain was like Bacillus sp. The 16S rDNA sequence of the Bacillus sp. was amplified by PCR and sequenced. By Blast assay, the strain was identified to be Bacillus subtilis.

    The antibacterial spectrum against different microorganisms was detected by agar diffusion method, the results indicated that it had antibacterial effects against Aspergillus, Pythium, Botrytis cinerea, but not E. coli, Staphylococcusaureus and Saccharomyces cerevisiae.

    The Bacillus subtilis culture was centrifuged and the supernatant was used to conduct antibacterial test. The antibacterial substances were found in the supernatant. The antibacterial substances could remain inhibitory effect at pH between 5 and 9, and at temperature between 40 and 100℃. The antibacterial activity was found lost after proteinase K treatment. It seemed to be a proteinous material and had a stable physical and chemical properties.。


    Fungal contamination

    Current experiments

    Purification of the antifungal peptide

    1 ammonium sulfate saturation (70%)

    2 gel chromatograph (seperdex G75)

    3 HPLC

    4 mass spectrometry

    5 amino acid sequencing

    Molecular characterization (DNA)

    1 NCBI→ Bacillus subtilis antifungal peptide gene

    2 primer design

    3 PCR amplify aimed sequence

    4 clone and express

    5 antifungal assay→if “+”

    6 sequencing

    correspondence


    Fungal contamination

    Thank You !


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