Introduction
Recently, the research of microbial antimicrobial
substance development has become a hot field of microbiological research. Since
1945, Johnson reported Bacillus
subtilis antimirobial substances, various
antimicrobial substances produced by microorganisms caused widespread concern (Maget and Peypoux 1994). Bacillus is a non-pathogenic bacteria
existing widely in nature, can produce a variety of antimicrobial substances,
most of which are polypeptide, mainly against gram positive bacteria and some
gram negative bacteria, fungi and yeast (Magnusson and Schnürer 2001). B. amyloliquefaciens
and B. subtilis
affinity was high, in recent years, it has been reported that a series of
metabolic products can be produced in the growth process itself, these
metabolites with active inhibition of fungi and bacteria (Wright et al. 2001). Many scholars have studied
on B. amyloliquefaciens,
the bacteria have the advantages of easy separation, easy culture.
Identification methods consist of the inhibitory effect of screening, the
individual morphology, physiological and biochemical characteristics,
determination of fatty acid, sequence analysis of 16S rDNA homology, HPLC-MS
analysis and others. Extraction and separation method of antimicrobial
substances are mainly macro porous resin method, acid-alkaline
precipitation, ammonium sulfate precipitation, chromatography etc.
A kind of oligopeptide
was found in B. amyloliquefaciens
23
Antimicrobial substances produced
by microorganisms including antibiotics,
bacteriocin, antimicrobial protein, hydrogen peroxide and organic acids etc. Therefore, it was necessary to
exclude the interference factors for the screening of bacteriocin producing
strains and inhibition of bacteriocin to be determined. There were some methods
such as eliminating acid,
eliminating hydrogen peroxide,
and protease detection (Leelasuphakul et al. 2008). Bacteriocins were protein
or polypeptide produced by the bacteria metabolic process, there was some
inhibition effect of certain microbial growth activity, and some degradation by
protease. Bacteriocins have some advantages of
non-drug-resistant, no residue, and inhibition of pathogenic bacteria (Garneau et al. 2002; Wang et al. 2013). So, it has a broad
application prospect in food, medical, health and veterinary etc. (Neville and O’Toole 2010; Maldonado et
al. 2010).
The strain
HRH317 of B. amyloliquefaciens was isolated from corn fields after harvest around
Shanxi Agricultural University by professor Hao Lin's team of Shanxi
Agricultural University in 2011. The genomic DNA of strain H317 was extracted
by the kits method, then bacterial universal primers were used as amplification
primer, 16S rDNA sequence was amplified by PCR, and the sequence and homology
were compared. The results showed that 16S rDNA sequence of HRH317 were same as
homology of B. amyloliquefaciens, reach
to 99–100%. At the same time the phylogenetic tree of 16S rDNA sequences were
drawn. Comprehensive comparison, HRH317 was identified as B. amyloliquefaciens. The study found
that the strain has excellent antimicrobial activity, its antimicrobial
spectrum was widely, not only can inhibit the gram negative and gram positive
bacteria, but also can inhibit the yeast and mold, it was a good kind of
resource with the development in the microorganism. At present, the antimicrobial spectrum and antimicrobial substances component of HRH317 were studied, to lay a good
foundation and theoretical support for the further development and utilization.
Materials
and Methods
Microorganisms
and medium
Strain HRH317 of B. amyloliquefaciens, Penicillium:
provided by biology engineering experiment of Shanxi Agricultural University.
Other indicator strains and number are given in Table 1.
Beef extract peptone medium (used
by the tested bacteria and indicator bacteria) (Hao 2001): beef extract 0.5 g, peptone
1.0 g, NaCl 0.5 g, agar 1.5 g, water 100 mL, pH 7.2, 0.1 MPa, sterilization 20 min.
Mould medium (Hao
2001): potato 200 g, were peeled and cut into the water 1000 mL and boiled for
30 min, filtrated by double-layer gauze, glucose 20 g, agar 20 g, 0.075 MPa, sterilization
20 min.
Yeast medium (Hao 2001): peptone 10
g, maltose 20 g, yeast extract 5 g, agar16 g, distilled water 1000 mL.
Liquid activation medium (Hao 2001): beef extract 0.5 g peptone 1.0 g, NaCl
0.5 g, water 100 mL, 0.1 MPa, sterilization 20 min.
Medicine and reagent: 1 mol/L HClˏ 1 mol/L NaOH ˏ catalaseˏ
protease Kˏ ammonium sulphateˏ Tris buffer solutionˏ DEAE
Sepharose Fast Flowˏ sephadex G-200ˏ Coomassie brilliant blue G-250.
Antimicrobial activity detection
Oxford-cup
tests
(Hao 2001; Schnürer and Magnusson 2005), The solid medium sterilized 15 mL
were added into the culture dish sterilized, the spore suspension of indicator bacteria
0.2 mL were added into the culture dish by micro pipette after solidification,
and were speeded evenly with glass spreading rod. The oxford-cup were put into
the culture dish equidistantly when the suspension was absorbed, the fermentation supernatant
of strain HRH317 0.2 mL were injected into each
oxford-cup and put in the refrigerator at 4℃ for 24 h and then put in the
incubator at 30℃ for 3–5 days. Observe the inhibition circle.
Antimicrobial spectrum detection
Gram positive
bacteria, gram negative bacteria, yeast and mould
used as indicator bacteria were detected by the antimicrobial substance of the
strain HRH317 of B. amyloliquefaciens.
The antimicrobial spectrum could be determined through the inhibition effect (Quiroga
et al. 2001; Flynn et al. 2002; Gong et al. 2006).
Preparation method of bacteria suspension: the inclined bacteria were
picked by sterilized inoculating loop into the liquid medium 30 mL in the
shaker incubator at 37℃, 160 r/min for 24 h.
Preparation method of yeast suspension: the same to bacteria, at
30℃, 150r/min for 48 h.
Preparation method of mould spore’s suspension:
the inclined mould were picked by sterilized
inoculating loop put into the normal saline 5 mL, concentration was made into
1.5×106 per mL.
Every kind of indicator bacteria experiment repeated 3 times measured the
diameter of inhibition circle and calculated the average value.
Antimicrobial substance screening
and analysis
Eliminating acid
The inhibition
effectiveness of fermentation
supernatant to the indicator bacteria may be due to the acid produced by strain
HRH317. Fermentation liquid was centrifuged under the conditions of 5000 r/min, 20 min, added 1 mol/L HCl and 1 mol/L NaOH into fermentation supernatant to adjust the pH 7 and operated the inhibition experiment by oxford-cup
tests (Joshi et al. 2008).
Eliminating hydrogen peroxide
Antagonism could inhibit the microorganism
by the hydrogen peroxide produced by it.
Fermentation liquid were centrifuged under the conditions of 5000 r/min,
20 min, added the catalase into fermentation
supernatant in the bain-marie at 37℃ for 4 h and operated the
inhibition experiment by oxford-cup tests after eliminating hydrogen peroxide
(Li et al. 2006).
Protease
detection
Fermentation liquid was centrifuged
under the conditions of 5000 r/min, 20 min, added 1 mol/L HCl and 1 mol/L
NaOH into fermentation supernatant to adjust
the pH 7 and added catalase and protease K in the bain-marie at 37℃ for 2
h, bacteria was used as indicator bacteria and operated the inhibition
experiment by oxford-cup tests after eliminating hydrogen peroxide (Kamel et al. 2012).
SDS-PAGE
detection
Precipitation of ammonium sulphate (Matynia et al.
2005): The fermentation supernatant was
filtrated by 0.22 µm
%20685-690_files/image002.png)
Fig. 1: Inhibition activity after eliminating acid
Left tube: the control, right tube: the treatment
bacteriological filter, the cell-free cultural filtrate was gained. The
ammonium sulphate were added into the filtrate to the saturation of 50% at 4℃
left to stand 2 h, the sediment was collected under the centrifuge conditions
of l0000 r/min at 4℃ for 30 min. The sediment was dissolved by pH 6.8
Tris buffer solution of 1/10 of sediment volume and put into the bag filter of
the same concentration of Tris buffer solution one night. The crude protein
from sediment were re-dissolved and treated by DEAE ion exchange chromatography
and Sephadex G-200 column in sequence (Queiroz 2004).
SDS-PAGE: According to literature (Idriss et al.
2002). spacer gel 4%, separation gel 12%, standard
protein produced by Shanghai Biology Chemical Institute. The antimicrobial
protein component was detected by oxford-cup tests with three repeated
experiments.
Results
Antimicrobial
spectrum of fermentation supernatant
Antimicrobial spectrum of the fermentation supernatant of strain HRH317 of B. amyloliquefaciens to common microorganisms were showed in Table 1.
Table 1 indicated that the antimicrobial substances of fermentation supernatant
consist of three kinds of microorganisms, such as bacteria,
yeast and mould, so the strain HRH317 of B. amyloliquefaciens
has widely antimicrobial spectrum.
Antimicrobial substance screening and analysis
Eliminating acid
The strain HRH317 of B. amyloliquefaciens in the shaker incubator were cultured at 37℃, 160 r/min for 24 h,
fermentation liquid pH was 6.8. Acid
substances were eliminated still have antimicrobial activity, and the diameters
of inhibition bacteria circle were nearly the same (Fig. 1), the results showed that inhibition effective were
not the acid substances come from the strain HRH317.
Eliminating hydrogen
peroxide
Adding catalase treatment was compared to the
non-catalase treatment, the results showed that inhibition activity of CK and
treatment group were the same nearly
for B. coagulans
63501-2aq (Fig. 2a). For Penicillium (Fig. 2b) and yeast (Fig. 2c), the
inhibition activity of treatment group was disappeared completely. The results
showed that antimicrobial substances of strain HRH317 were not only consisting
of hydrogen peroxide that inhibit the bacteria, mould and yeast, but also included non-hydrogen peroxide
substances that inhibit bacteria.
Protease detection
The protease treatment could decide
the antimicrobial substance were protein or polypeptide. The inhibition
bacteria circle disappeared mostly when fermentation liquid of strain HRH317 was combined with protease K
and the CK had an obvious inhibition activity (Fig. 3). The results showed that non hydrogen peroxide
substances produced by the strain HRH317 of B. amyloliquefaciens belonged to protein or polypeptide.
Shown experiments, the strain HRH317 of
B. amyloliquefaciens
when fermented could produce two
kind of Table 1: The antimicrobial
spectrum of substance from HRH317
|
Indicator bacteria |
Source |
Diameter of
inhibition bacteria circle (average value:mm) |
|
Bacillus spp. H108 |
Microbiology
Laboratory of SXUCM (ML of SXUCM) |
26.5 ± 1.20 |
|
B. coagulans 10144 |
CICC |
18.0 ± 0.51 |
|
B. coagulans 63501-2aq |
CMCC |
14.5 ± 0.23 |
|
Escherichia coli 44102 |
CMCC |
13.0 ± 1.30 |
|
Staphlococcus aureus 26003 |
CMCC |
16.0 ± 1.06 |
|
Saccharomy cescerevisiae H226 |
(ML of SXUCM) |
14.0 ± 0.92 |
|
Rhodotorula H215 |
(ML of SXUCM) |
15.5 ± 0.86 |
|
Saccharomyces
ellipsoideus H222 |
(ML of SXUCM) |
14.5 ± 1.12 |
|
Alternaria kikuchiana |
Isolate from pear,
(ML of SXUCM) |
21.5 ± 0.98 |
|
Plasmopara
uiticola R6 |
Isolate from grape,
(ML of SXUCM) |
18.5 ± 1.35 |
|
Aspergillus niger |
(ML of SXUCM) |
20.0 ± 1.28 |
|
Penicillium |
(ML of SXUCM) |
19.5 ± 1.03 |
|
Fusarium moniliforme |
(ML of SXUCM) |
18.5 ± 0.98 |
|
F. graminearum |
(ML of SXUCM) |
16.5 ± 0.75 |
%20685-690_files/image004.png)
Fig.
2: Inhibition activity
after adding catalase
Left
tube: the control, right tube: adding catalase
%20685-690_files/image006.jpg)
Fig. 3: Inhibitory activity after adding protease K
Left tube: the control, right tube: adding protease K
antimicrobial
substances, as follows hydrogen peroxide that inhibition spectrum widely
and protein and polypeptide that antibacterial selectively.
SDS-PAGE
purity detection
From Fig. 4 the results showed that the crude proteins that
inhibition activity was gained when the fermentation
supernatant was precipitated fractionally by ammonium sulphate, and
consist of different kind of protein components (column 1). Meanwhile,
indicated that strain HRH317 include many kinds of different molecular weight
proteins except for antimicrobial protein. The crude
protein from sediment were re-dissolved and treated by DEAE ion exchange
chromatography, there were many different protein components of peak 2 (column
2). Furthermore, the peak 2 from DEAE were purified by Sephadex G-200 column,
an obvious single band were obtained and determined as antimicrobial protein
that molecular weight was 36 KD (column 3). Finally, the
antimicrobial protein component was detected by oxford-cup tests, the
antimicrobial activity effect of three repeated experiments were good to the
indicator strain in Fig. 5.
The
bacterium B. amyloliquefaciens
is a kind of biological control resource, it has strong reproductive
capacity, easy industrialization production, harmless to human and animal, it
also causes no pollution to the environment so on, has become a hot point of
research and application after the application of the B. subtilis in recent years (Sowanpreecha et al. 2018). The small molecular weight of
antibiotics and antimicrobial proteins or peptides and
other active substances were produced by B. amyloliquefaciens
which can inhibit various plant pathogenic bacteria, play an important role in
the control of food preservation, plant disease. In biological control, B. amyloliquefaciens
can be produced as biological pesticide or fertilizer, and has wide application
prospect. In this experiment,1 strain of antagonistic bacteria strain HRH317
were isolated from maize fields after harvest, through
the detection, it was found that B. amyloliquefaciens HRH317 fermentation
liquid with broad antimicrobial spectrum characteristics, can inhibit a
variety of bacteria, yeasts and molds, especially has good inhibition for the
mold caused fruit spoilage.
%20685-690_files/image008.jpg)
Fig. 4: SDS-PAGE spectrum of antimicrobial protein
Note:1. Ammonium sulfate-precipitated
protein;2. DEAE peak 2;
3. Sephadex G-200 peak 2
M.Marker
%20685-690_files/image010.jpg)
Fig. 5: Antimicrobial activity detection of antimicrobial protein component
The antimicrobial
substances were
studied by the experiment of identification method of producing bacteriocin, the results showed that the antimicrobial
substances include hydrogen peroxide and proteins or polypeptides. The
fermentation liquid still has antimicrobial activity after eliminating acid and hydrogen peroxide, and then the antimicrobial
activity disappeared by
the protease treatment, therefore, the antimicrobial substances were determined
as protein, furthermore, the B. amyloliquefaciens HRH317 were determined as bacteriocin
producing strains. The antimicrobial activity was significantly enhanced
through the ammonium sulfate precipitation, the dialysis and concentration.
Furthermore, the crude proteins were purified by sequential column
chromatography using DEAE Sepharose Fast Flow and Sephadex G-200 as column
filling matrix．The
antimicrobial protein of molecular weight 36 KD were gained through SDS-PAGE
detection. At present, the usually methods of isolation and purification of
antimicrobial protein were ammonium sulfate precipitation and gradually
chromatography purification. But there were small amounts of heavy metal ions,
a sensitive effect on protein thiol, and this method
often requires a longer time and more equipment. In order to reduce the
extraction of protein loss, HCl precipitate can be used to extract the
antimicrobial crude protein. Hang get stable relatively antimicrobial protein
by above methods (Zhang and Zhang 2010). The molecular weight of antimicrobial
protein 40 kD were extracted
by B. amyloliquefaciens
MET0908 to control anthracnose (Kim and Chung 2004).
The antimicrobial protein of separation
and purification has advantages as follows good safety, moderate molecular
weight, good stability, a widely antimicrobial spectrum and etc.
There will be good prospect in food preservative and new antimicrobial
agent development. The mechanism of antimicrobial protein and gene expression
of B. amyloliquefaciens HRH317 needs further study.
Conclusion
The results of this study indicate that antimicrobial
spectrum was widely by detecting the antimicrobial substance activity with
oxford-cup tests and especially inhibition effect was good for the fruit
spoilage mould. The antimicrobial substance was determined as bacteriocin by
eliminating acid, H2O2 and enzyme treatment experiment,
meanwhile, the crude proteins were gained from the fermentation supernatant by
fractional precipitation of ammonium sulphate, and purified by sequential
column chromatography using DEAE Sepharose Fast Flow and Sephadex G-200 as
column filling matrix. Finally the antimicrobial subtance was a kind of
antimicrobial proteins found that molecular weight was 36 kilo Dalton by
SDS-PAGE.
Acknowledgments
This study was supported by Project of Natural Science Foundation of Shanxi
Province (201801D121256) & Doctoral Research Foundation Project of Shanxi
University of Chinese Medicine (2016BK010) & Shanxi Provincial Planning
Commission Research Project(201601115).
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