**4. Purification of antifungal metabolites from soil bacteria: A practical approach**

#### **4.1. Culture conditions for metabolite production**

The PCR mixtures were denatured at 95°C for 5 min, which was followed by 35 cycles of 94°C for 30s, 55°C for 30s, and 72°C for 90s and then a final extension at 72°C for 5 min. Am‐ plification was checked for purity by electrophoresis on a 1.0% agarose gel. The bands of in‐ terest were excised from the gel, and the DNA was purified using QIAquick PCR purification columns (Qiagen, Inc., Valencia, CA). Purified DNA fragments were sequenced using the same sets of primers that were used for amplification by an ABI Prism Big Dye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems). Bacteria were identified based on sequence similarities to homologous 16S rRNA gene fragments in the Ribosomal Data‐ base Project database (Cole et al., 2005) (accessed at http://rdp.cme.msu.edu/index.jsp).

Cell free culture supernatants of inhibitory bacteria were used in an antagonistic assay sys‐ tem. Table 2 represents the strongest antagonistic bacteria which were identified by a combi‐

> **% of growth inhibition**

 Maize 57.7 95.7 + Maize 48.9 78.2 – Peanuts 60.4 63.4 + Maize 64.5 85.3 + Maize 55.6 74.7 + Pistachio 55.7 73.6 + Maize 59.3 65.3 + Maize 59.0 87.6 + Peanuts 35.0 84.4 + Maize 62.6 96.9 + Maize 69.3 70.3 +

298 Pistachio 70.6 18.7 + 295 Pistachio 56.0 43.0 +

**% of AFB1 inhibition** **Surfactant production on blood agar**

nation of biochemical and molecular methods in relation to their source of isolation.

**3.4. Antagonistic activity against aflatoxigenic** *A. parasiticus* **NRRL 2999**

**Field**

*P. aeruginosa* 320 Maize 63.9 95.3 +

*P. chlororaphis* 236 Peanuts 15.3 65.9 – *P. fluorescens* 82 Pistachio 72.7 91.1 – *B. subtilis* 248 Maize 52.0 19.1 +

*B. amyloliquefaciens* 296 Maize 66.7 24.4 +

growth rate of 51.17 mg and an AFB1 amount of 697.78 ng/mg fungal dry weight.

**Table 2.** Inhibitory effects of the strongest antagonistic bacteria selected from screening plates of visual agar plate assay on *A. parasiticus* NRRL 2999 growth and AF production in Potato dextrose broth. Control fungal culture had a

**Strain number**

**Antagonistic bacteria**

30 Aflatoxins - Recent Advances and Future Prospects

As the first step for production of bioactive antifungals, different culture conditions includ‐ ing medium, incubation time and aeration should be optimized. In order to initial purifica‐ tion of inhibitory metabolites, the selected bacterium with strongest antifungal activity in initial screening was cultured on suitable liquid media such as GY (2% glucose, 0.5% yeast extract), SCD (2% bacto dextrose, 20% potato infusion), PDB (potato dextrose broth) or even KB (King´s B). The cultures were checked for optimal conditions of aeration (stationary cul‐ tures to shaking at different rpm from 100 to 250), incubation times (for at least 1 to maxi‐ mum 7 days) and temperature (from 20 to 40°C). After culturing the bacterium at optimized condition, the whole culture as the main source of secretory metabolites was centrifuged at 8,000 x *g* for 30 min at room temperature. The cell free culture filtrate was then sterilized by filtration through a 0.22-µm-pore-size Millipore membrane (Millex-GV; Millipore) and kept at -20°C before use. The heat stability of the inhibitory metabolites can be examined by incu‐ bating the bacterial culture filtrate at 60, 80 and 100°C for 120 min or autoclaving at 121°C for 15 min. The acid and alkaline stabilities of the inhibitory metabolites can be checked by changing the pH of the culture medium to 1.5 and 11 by adding 1 M HCl or 1 M NaOH and incubating the solution at room temperature for 3 h.

*4.2.1. Metabolite production at pre-optimized culture conditions*

natant was used for purification of the inhibitory metabolites.

*4.2.2. Ion exchange column chromatography*

for further purification (Fig. 5A).

*4.2.3. Preparative thin layer chromatography*

*4.2.4. High performance liquid chromatography (HPLC)*

The selected bacterium with strongest antifungal activity was cultured in 1000 ml capacity flasks contained 250 ml GY as selected medium from section 4.1. The cultures were incubat‐ ed at pre-optimized conditions (28°C for 5 days with shaking at 120 rpm). The whole culture (2 liters totally) was then centrifuged at 8,000 × g at room temperature for 30 min. The super‐

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33

A glass column (2.5 × 60.0 cm) was equilibrated with MeOH. Five hundred grams of Diaion HP20 resin was suspended in MeOH and then packed onto the glass column. After remov‐ ing of MeOH, the column was equilibrated with distilled water. The culture broth of select‐ ed bacterium (500 ml) was loaded onto the column. The resin was washed with 3 liters of distilled water, and the substances bound to the resin were then stepwise eluted by using 2 liters each of 40, 60, 80, and 100% methanol (MeOH) in water. Each elution was concentrated to dryness with a rotary evaporator and dissolves in desirable amounts of 100% MeOH. The 80% MeOH fraction which showed the highest growth and/or AF inhibitory activity against NA-mutant of *A. parasiticus* NRRL 2999 in microtiter agar plate assay (MPA), was selected

The 80% MeOH fraction from section 4.2.2 (an approximate of 250 mg dry weight) was ap‐ plied to Silica gel 60F254 TLC plate and then developed with a mixture of chloroform/metha‐ nol/water (65:25:4, v/v/v) as mobile phase. Total area developed on the TLC plate was divided into at least 5 regions under 365 nm UV light, and the silica gel was scraped sepa‐ rately from each region. The substances presented in the silica gel were extracted with tenfold amounts of 100% MeOH. Each fraction was concentrated to dryness, dissolves in a small amount of MeOH, and subjected to the MPA on 96-well microplates. The fraction "b" (75.6 mg dry weight) which contained the strongest inhibitory activity against fungal

The fraction "b" from section 4.2.3 was finally purified by HPLC equipped with a Cosmosil 5C18-AR column (4.6 × 150 mm; 5 µm). After injecting the sample, the column was washed with MeOH/water (50:50, v/v) for 80 min. The flow rate was adjusted at 1.0 ml/min, and elu‐ tion was monitored at 290 nm wavelength. The number of 6 separated peaks (P1 to P6) were collected from the ODS column as shown in Fig. 5. Based on the MPA results, two peaks i.e. P2 and P3 were able to inhibit fungal growth and pigment production by *A. parasiticus* NRRL 2999 (Fig. 5C). These peaks were selected for further characterization by LC-MS and MALDI-TOF.

growth and/or AF production was selected for further purification (Fig. 5B).

#### **4.2. Purification of antifungal metabolites**

Consecutive steps of purification of bioactive metabolites from bacterial culture filtrate are summarized in Fig. 5. As the first step, the inhibitory bacterium should be cultured at opti‐ mized culture conditions from section 4.1. The next steps are Ion exchange column chroma‐ tography on Diaion HP20 resin, preparative thin layer chromatography on silica gel 60F254 and finally HPLC purification of bioactive metabolites.

**Figure 5.** Sequential steps of purification of *A. parasiticus* growth inhibitory metabolites from bacterial culture fil‐ trate:A) Stepwise elution of culture broth from a Diaion HP20 resin column using 40-100% aqueous MeOH. Fungal growth inhibition was reported for only 80% MeOH elution in microtiter agar plate assay (MPA).B) Further purification of fungal growth inhibitory metabolites from active Diaion HP20 column fraction (80% MeOH from step A) by thin layer chromatography (TLC). According to MPA result, section "b" was scrapped from TLC gel contained inhibitory compounds and thus, it was selected for further study.C) Final purification of inhibitory metabolites from section "b" of TLC in step B by normal-phase HPLC. Among 6 separated peaks shown (P1 to P6), two peaks i.e. P2 and P3 showed fungal growth inhibition in MPA.

#### *4.2.1. Metabolite production at pre-optimized culture conditions*

The selected bacterium with strongest antifungal activity was cultured in 1000 ml capacity flasks contained 250 ml GY as selected medium from section 4.1. The cultures were incubat‐ ed at pre-optimized conditions (28°C for 5 days with shaking at 120 rpm). The whole culture (2 liters totally) was then centrifuged at 8,000 × g at room temperature for 30 min. The super‐ natant was used for purification of the inhibitory metabolites.

#### *4.2.2. Ion exchange column chromatography*

mum 7 days) and temperature (from 20 to 40°C). After culturing the bacterium at optimized condition, the whole culture as the main source of secretory metabolites was centrifuged at 8,000 x *g* for 30 min at room temperature. The cell free culture filtrate was then sterilized by filtration through a 0.22-µm-pore-size Millipore membrane (Millex-GV; Millipore) and kept at -20°C before use. The heat stability of the inhibitory metabolites can be examined by incu‐ bating the bacterial culture filtrate at 60, 80 and 100°C for 120 min or autoclaving at 121°C for 15 min. The acid and alkaline stabilities of the inhibitory metabolites can be checked by changing the pH of the culture medium to 1.5 and 11 by adding 1 M HCl or 1 M NaOH and

Consecutive steps of purification of bioactive metabolites from bacterial culture filtrate are summarized in Fig. 5. As the first step, the inhibitory bacterium should be cultured at opti‐ mized culture conditions from section 4.1. The next steps are Ion exchange column chroma‐ tography on Diaion HP20 resin, preparative thin layer chromatography on silica gel 60F254

**Figure 5.** Sequential steps of purification of *A. parasiticus* growth inhibitory metabolites from bacterial culture fil‐ trate:A) Stepwise elution of culture broth from a Diaion HP20 resin column using 40-100% aqueous MeOH. Fungal growth inhibition was reported for only 80% MeOH elution in microtiter agar plate assay (MPA).B) Further purification of fungal growth inhibitory metabolites from active Diaion HP20 column fraction (80% MeOH from step A) by thin layer chromatography (TLC). According to MPA result, section "b" was scrapped from TLC gel contained inhibitory compounds and thus, it was selected for further study.C) Final purification of inhibitory metabolites from section "b" of TLC in step B by normal-phase HPLC. Among 6 separated peaks shown (P1 to P6), two peaks i.e. P2 and P3 showed

incubating the solution at room temperature for 3 h.

and finally HPLC purification of bioactive metabolites.

**4.2. Purification of antifungal metabolites**

32 Aflatoxins - Recent Advances and Future Prospects

fungal growth inhibition in MPA.

A glass column (2.5 × 60.0 cm) was equilibrated with MeOH. Five hundred grams of Diaion HP20 resin was suspended in MeOH and then packed onto the glass column. After remov‐ ing of MeOH, the column was equilibrated with distilled water. The culture broth of select‐ ed bacterium (500 ml) was loaded onto the column. The resin was washed with 3 liters of distilled water, and the substances bound to the resin were then stepwise eluted by using 2 liters each of 40, 60, 80, and 100% methanol (MeOH) in water. Each elution was concentrated to dryness with a rotary evaporator and dissolves in desirable amounts of 100% MeOH. The 80% MeOH fraction which showed the highest growth and/or AF inhibitory activity against NA-mutant of *A. parasiticus* NRRL 2999 in microtiter agar plate assay (MPA), was selected for further purification (Fig. 5A).

#### *4.2.3. Preparative thin layer chromatography*

The 80% MeOH fraction from section 4.2.2 (an approximate of 250 mg dry weight) was ap‐ plied to Silica gel 60F254 TLC plate and then developed with a mixture of chloroform/metha‐ nol/water (65:25:4, v/v/v) as mobile phase. Total area developed on the TLC plate was divided into at least 5 regions under 365 nm UV light, and the silica gel was scraped sepa‐ rately from each region. The substances presented in the silica gel were extracted with tenfold amounts of 100% MeOH. Each fraction was concentrated to dryness, dissolves in a small amount of MeOH, and subjected to the MPA on 96-well microplates. The fraction "b" (75.6 mg dry weight) which contained the strongest inhibitory activity against fungal growth and/or AF production was selected for further purification (Fig. 5B).

#### *4.2.4. High performance liquid chromatography (HPLC)*

The fraction "b" from section 4.2.3 was finally purified by HPLC equipped with a Cosmosil 5C18-AR column (4.6 × 150 mm; 5 µm). After injecting the sample, the column was washed with MeOH/water (50:50, v/v) for 80 min. The flow rate was adjusted at 1.0 ml/min, and elu‐ tion was monitored at 290 nm wavelength. The number of 6 separated peaks (P1 to P6) were collected from the ODS column as shown in Fig. 5. Based on the MPA results, two peaks i.e. P2 and P3 were able to inhibit fungal growth and pigment production by *A. parasiticus* NRRL 2999 (Fig. 5C). These peaks were selected for further characterization by LC-MS and MALDI-TOF.

#### **4.3. Structural elucidation of antifungal metabolites**

With a combination of Liquid chromatography-Mass spectrometry (LC-MS) and Matrix-as‐ sisted laser desorption/ionization (MALDI-TOF), we will be able to elucidate the chemical structure of a protein or peptide in a best way. LC-MS spectrum determines retention time and an approximate mass of a purified compound, while complementary MALDI-TOF ena‐ ble us to explain chemical formula and precise mass of the compound as the final step of identification. LC-MS and MALDI-TOF spectra of purified antifungal are shown in Fig. 6.

*4.3.2. MALDI-TOF*

Matrix-assisted laser desorption ionization-time of flight spectrometer (MALDI-TOF) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (bio‐ polymers such as DNA, proteins, peptides and sugars) and large organic molecules (poly‐ mers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. The MALDI-TOF is a two step process. First, desorption is triggered by a UV laser beam. Matrix material heavily absorbs UV laser light, leading to the ablation of upper layer of the matrix material. The second step is ioniza‐ tion which takes place in the hot plume. Aside from peptide mass fingerprinting and useful application in identifying of microorganisms such as bacteria and fungi, MALDI-TOF is used for the rapid identification of proteins isolated by using gel electrophoresis: SDS-PAGE, size exclusion chromatography, affinity chromatography, strong/weak ion exchange, isotope cod‐ ed protein labeling (ICPL), and two-dimensional gel electrophoresis. MALDI-TOF analysis of inhibitory compounds with defined retention time and an approximate mass from LC-MS step reveals valuable data about chemical formula and exact mass and provides finally identifica‐ tion of the absolute configuration of the purified inhibitory bacterial metabolite (Fig. 6).

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AF contamination of food and feed remains a major risk for human and animal health all over the world. Despite the long history of our knowledge about AF, little has been documented on how we can virtually combat the global distress of AF contamination of crops and agricultur‐ al commodities. AF-producing fungi can infect grains from pre-harvest conditions in the field through to post-harvest stages in the stores. Several pre- and post-harvest strategies have be‐ ing tested to reduce risk of AF contamination. One of the management strategies being devel‐ oped is biological control using various antagonistic microorganisms such as fungi, bacteria, and actinomycetes by a competitive exclusion mechanism. Biological control in conjunction with other management practices has potential to dramatically reduce AF contamination. Nat‐ ural population of *A. flavus* consists of toxigenic strains that produce considerable amount of AF and atoxigenic strains that lack the capacity to produce AF. Nowadays, introducing atoxi‐ genic strains has been successfully used to compete and exclude toxigenic strains in the field thereby reducing AF production in contaminated crops. However, there are some important limitations from the type of vegetative compatibility groups which shows the progeny of the fungus for AF-producing ability to geographic limitations in selection of atoxigenic strains. Considerable tolerance of *B. subtilis* and *P. chlororaphis* to environmental stresses, their large ca‐ pacity for producing diverse array of beneficial antifungal metabolites and their readily pro‐ ducing by current fermentation technology make them promising tools for biocontrol of aflatoxigenic fungi in practice. Bacterial population from the genera *Bacillus* and *Pseudomonas* identified in pistachio, maize and peanut fields in the present study with potent antagonistic activity against aflatoxigenic *Aspergillus parasiticus* can potentially be developed into new bio‐ control agents for combating AF contamination of crops in the field. These bacteria must be evaluated for a set of selection criteria for further use in biocontrol field experiments. Inabili‐

**5. Concluding remarks and future prospective**

#### *4.3.1. Liquid chromatography-Mass spectrometry (LC-MS)*

The LC-MS system usually consists of a LC-10Avp separation module equipped with a SPD-M10Avp photodiode array detector and LC-MS2010A single quadruple mass spectrometer with atmospheric pressure photo ionization (APPI) source. The probe can be operated in the positive/negative mode under the condition of defined probe voltage, temperature of 300°C, CDL temperature of 200°C, nabulization gas (N2) flow 2.5 1/min, and scan range 900-1600 m/z (sec/scan). The amount of 2 µl of each inhibitory peak purified from HPLC separation was injected to an Ascentis C18 column (150 mm × 2.1 mm, 5 µm) and washed with MeOH (65% aqueous solution) acidified with 0.1% acetic acid in a flow rate of 0.2 ml/min. The col‐ umn temperature should be maintained at 40°C during the operation. Approximate mass and retention time of the compound were recorded at the end of analysis.

**Figure 6.** Liquid chromatography-Mass spectrometry (LC-MS) analysis of a HPLC purified inhibitory metabolite for *A. parasiticus* growth shows an approximate retention time of 17.0 min and a mass of 1042.0 m/z (A), while MALDI-TOF data indicates a structural formula of C48H76N12O14 and an exact mass of 1042.5447 m/z (B).

## *4.3.2. MALDI-TOF*

**4.3. Structural elucidation of antifungal metabolites**

34 Aflatoxins - Recent Advances and Future Prospects

*4.3.1. Liquid chromatography-Mass spectrometry (LC-MS)*

With a combination of Liquid chromatography-Mass spectrometry (LC-MS) and Matrix-as‐ sisted laser desorption/ionization (MALDI-TOF), we will be able to elucidate the chemical structure of a protein or peptide in a best way. LC-MS spectrum determines retention time and an approximate mass of a purified compound, while complementary MALDI-TOF ena‐ ble us to explain chemical formula and precise mass of the compound as the final step of identification. LC-MS and MALDI-TOF spectra of purified antifungal are shown in Fig. 6.

The LC-MS system usually consists of a LC-10Avp separation module equipped with a SPD-M10Avp photodiode array detector and LC-MS2010A single quadruple mass spectrometer with atmospheric pressure photo ionization (APPI) source. The probe can be operated in the positive/negative mode under the condition of defined probe voltage, temperature of 300°C, CDL temperature of 200°C, nabulization gas (N2) flow 2.5 1/min, and scan range 900-1600 m/z (sec/scan). The amount of 2 µl of each inhibitory peak purified from HPLC separation was injected to an Ascentis C18 column (150 mm × 2.1 mm, 5 µm) and washed with MeOH (65% aqueous solution) acidified with 0.1% acetic acid in a flow rate of 0.2 ml/min. The col‐ umn temperature should be maintained at 40°C during the operation. Approximate mass

**Figure 6.** Liquid chromatography-Mass spectrometry (LC-MS) analysis of a HPLC purified inhibitory metabolite for *A. parasiticus* growth shows an approximate retention time of 17.0 min and a mass of 1042.0 m/z (A), while MALDI-TOF

data indicates a structural formula of C48H76N12O14 and an exact mass of 1042.5447 m/z (B).

and retention time of the compound were recorded at the end of analysis.

Matrix-assisted laser desorption ionization-time of flight spectrometer (MALDI-TOF) is a soft ionization technique used in mass spectrometry, allowing the analysis of biomolecules (bio‐ polymers such as DNA, proteins, peptides and sugars) and large organic molecules (poly‐ mers, dendrimers and other macromolecules), which tend to be fragile and fragment when ionized by more conventional ionization methods. The MALDI-TOF is a two step process. First, desorption is triggered by a UV laser beam. Matrix material heavily absorbs UV laser light, leading to the ablation of upper layer of the matrix material. The second step is ioniza‐ tion which takes place in the hot plume. Aside from peptide mass fingerprinting and useful application in identifying of microorganisms such as bacteria and fungi, MALDI-TOF is used for the rapid identification of proteins isolated by using gel electrophoresis: SDS-PAGE, size exclusion chromatography, affinity chromatography, strong/weak ion exchange, isotope cod‐ ed protein labeling (ICPL), and two-dimensional gel electrophoresis. MALDI-TOF analysis of inhibitory compounds with defined retention time and an approximate mass from LC-MS step reveals valuable data about chemical formula and exact mass and provides finally identifica‐ tion of the absolute configuration of the purified inhibitory bacterial metabolite (Fig. 6).
