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Exploring the microbiomeinvolved in the control of mycotoxigenic fungi in
sorghumsilage
Silvana Vero
Microbiology Area
Biosciences Department
Facultad de Química – UdeLaR Montevideo, Uruguay
Introduction
Ensilage
The process of preserving grains (such as corn, rye, sorghum, etc.) compacted and
stored in airtight conditions, typically in a silo, without first being dried, and used
as animal feed in the winter.
This process has different steps, concluding in 30 to 40 days (Choisonne, 2011)
Aerobic phase Fermentation Stabilisation Aerobic deterioration
O2
pH
Lactic acid
Grain sorghum silage: Grain is harvested
with a moisture content between 23 y 40%
and ensiled without drying in a anaerobic
conditions (Scarpitta, 2008).
Feed-out
Aspergillus spp.
Penicillium spp.Fusarium spp.
Aspergillus flavus
Aflatoxins
Carcinogenic – Group I (IARC)
Variables affecting A. flavus growth in sorghum silage
Factors/Levels -1.68 -1 0 1 +1.68
Moisture content 13.2 20 30 40 46.8
Temperature 3 10 20 30 37
Time (hours) 63.36 96 144 192 224.64
A. flavusconcentration
2.09E+02 1.00E+03 1.00E+04 1.00E+05 4.79E+05
Central Composite Designα=1.68
Design Expert 7.0.0
RunMoisture
content (%)Temperature (°C) Time (days)
Starter inoculum 10x
(conidias/g SAT)
1 40 10 8 3
2 40 10 4 3
3 40 10 4 5
4 30 36.8 6 4
5 46.8 20 6 4
6 40 30 4 5
7 30 20 9.36 4
8 30 20 6 4
9 20 30 4 5
10 30 20 2.64 4
11 20 10 4 5
12 13.2 20 6 4
13 20 10 8 5
14 40 30 4 3
15 20 10 8 3
16 40 30 8 3
17 20 30 8 3
18 30 20 6 5.68
19 20 30 4 3
20 20 30 8 5
21 20 10 4 3
22 40 10 8 5
23 30 3.2 6 4
24 30 20 6 4
25 40 30 8 5
26 30 20 6 4
27 30 20 6 2.32
Incubation temperatures:• 3.2°C• 10°C• 20°C• 30°C• 36.8°C
Lyophilization
Grinding
Stomacher1 min
DNA extraction(ZR Fungal/Bacterial
DNA MiniPrep – ZymoResearch-)
Real time PCR quantification
(Shweta et al., 2013)
Optimized method
2 mL
Shweta, S., Madhavan, S.; Paranidharan, V.; Velazhahan, R. (2013). Detection of Aspergillus flavus in maize kernels by conventional and real-time PCR assays. International
Food Research Journal 20(6): 3329-3335
Growth curves
Growth of A. flavus in sorghum silage at 25C y 30% moisture(aerobic phase)
After 24 h aflatoxin B1 concentration is higher than theadmitted value
34 ppm aflatoxin B1
252 ppm aflatoxin B1
Adapted from de la Roza-Delgado, B. (2005). El ensilado en zonas húmedas y sus indicadores de calidad. IV Jornadas de Alimentación Animal. Laboratorio de Mouriscade. Lalín (Pontevedra). Disponible en: ww.mouriscade.com/doc.../ensilado_zonas_humedas_e_indicadores_calidad.pdf
Additives used to improve ensilage
Preservatives Inoculants Enzymes Substrates
Acids:
Ac. Sulfuric
Ac. phosphoric
Ac. formic
Ac. acetic
Ac. lactic
Ac. propionic
Ac. benzoic
Ac. caproic
Lactic acid bacteria:
Lactobacillus
Pediococcus
Streptococcus
Amylases
Cellulases
Hemicellulases
Pectinases
Molasses
Glucose
Saccharose
Whey
Cereal grains
Pulps (beet)
Biological Control: control of the growth of a
population by the action of one or more
antagonistic organisms
Yeast as biocontrol agents in ensiled grains
Disadvantages
Competition for fermentable substrates with lactic acid bacteria
Lactic acid metabolization leading to a higher pH
Undesirable fermentations: ethanol production
Advantages
• Fungal growth inhibition
• Increment of feed nutritional value
• Oxygen consumption in aerobic phase
• No mycotoxins production
Pichia anomala J 121 (Schnurer, Sweeden)
Mixed inoculum
Olstorpe, M.; Borling , J.; Schnürer, J.; Passoth, V. (2010b). Pichia anomala yeast improves feed hygiene during storage ofmoistcrimped barley grain under Swedish farm conditions. Animal Feed Science and Technology, 156: 47–56
Water kefir
Water kefir is a drink obtained by the fermentation of sugary solutions with microorganismspresent in kefir grains
Kefir grains: complex association of bacteria and yeast bound within a dextran matrix
• Yeast• Lactic acid bacteria (BAL)• Acetic acid bacteria (BAA)
Caro Vélez, C.; León Peláez, A. (2014). Inhibición del crecimiento de Aspergillus ochraceus mediante “panela” fermentada con gránulos de kefir de agua. Vitae, 21 (3):223-232.
AttributesSource of probiotics with potential health benefits Antioxidant and anti-inflammatory propertiesAntifungal and antibacterial activity
Sterilizedpanela
solution 4.5%pH=5.5
Kefir granules CMUNLP1
10% Fermentation
24 h, 28°C pH=4
Filter sterilization(0.45 µm)
Methodology
Sorghum bicolor
High tannin
Cracked grain. sterilized by moist
heat (SAT)
Control
1:1 SAT: sterile water
Treatment 1:
1:1 SAT:WK Treatment 2:
1:1 SAT:SWK
A. flavus
inoculation
1x104 conidia/g
Moisture level 30%
Granules remotion
Water kefir (WK)
Sterilized waterkefir (SWK)
Mini silos wereprepared and incubated 7 daysat 25C
Methodology
Stomacher1 min
10 mL
Bacteria and yeast plate count• Lactic acid bacteria (MRS + Cicloheximide)• Yeast (PCA +Cloranfenicol)(T1 before and after incubation)
DNA extraction(ZR Fungal/Bacterial
DNA MiniPrep – ZymoResearch-)
Quantification of A. flavus growth by qPCR)
Control, T1 and T2(Shweta et al., 2013with modifications
Shweta, S., Madhavan, S.; Paranidharan, V.; Velazhahan, R. (2013). Detection of Aspergillus flavus in maize kernels by conventional and real-time
PCR assays. International Food Research Journal 20(6): 3329-3335
Massive sequencing16S rDNA
ITS(T1 before and after
incubation)
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
180.00
200.00
C aire KA KA estéril
ng
AD
N A
. fla
vus/
g d
e so
rgo
93.89a
0.90b
189.12c
A. flavus growth during aerobic phase in mini silos
Treatment 1: 1:1 SAT:WK
Treatment 2: 1:1 SAT:SWK
Control1:1 SAT: sterilewater
Fungalgrowth
Bacteria and yeast growth in minisilos amended with water kefir
Lactic acid bacteria Yeasts
t=0 t=7 days
AirPlate count in:
Lactobacillus spp. isolates
Yeast isolates
NGS results 16S rDNA
A- Before incubation
B- After incubation (7 días)
Sample Sequences OTUs
A 66571 41
B 109365 51
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
KA t=0 (KA+Af) t=7días
Ab
un
dan
cia
filo
gen
étic
a a
niv
el d
e Fi
lo (
%)
Proteobacteria
Firmicutes
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
KA t=0 (KA+Af) t=7 díasAb
un
dan
cia
filo
gen
étic
a a
niv
el
de
gén
ero
(%
)
Lactobacillus Acetobacter Gluconobacter
Minisilos amendedwith water kefir
Percentage of sequences assigned to OTUs in the total of Lactobacillus sequences in
mini-silos before and after incubation.
Lactobacillusdiversity
Lactobacillus parakefiri JCM 8573 T
Lactobacillus buchneri JCM 1115 T
Lactobacillus hilgardii JCM 1155 T
Lactobacillus farraginis NRIC 0676 T
Lactobacillus diolivorans JCM 12183 T
OTU 14
Lactobacillus kefiri JCM 5818 T
OTU 19
Lactobacillus odoratitofui JCM 15043 T
Lactobacillus similis JCM 2765 T
Lactobacillus silagei IWT126 T
Lactobacillus brevis JCM 1059 T
OTU 11
OTU 16
Lactobacillus wasatchensis WDC04 T
OTU 13
OTU 28
OTU 29
Lactobacillus paracasei ATCC 25302 T
Lactobacillus casei ATCC 393 T
Lactobacillus perolens L532 T
OTU 9
Lactobacillus harbinensis NBRC 100982 T
Lactobacillus shenzhenensis LY-73 T
OTU 10
Lactobacillus capillatus JCM 15044 T
Lactobacillus sucicola NRIC 0736 T
OTU 15
OTU 20
OTU 22
Lactobacillus ghanensis L489 T
OTU 17
OTU 18
OTU 25
Lactobacillus satsumensis JCM 12392 T
Lactobacillus nagelii NRIC 0559 T
OTU 12
OTU 21
OTU 24
77
86
66
77
66
68
79
91
74
7354
64
55
56
69
67
62
65
56
Isolated fromminisilos
Increased afterincubation
Decreased afterincubation
NGS Results ITS
0
20
40
60
80
100
120
KA t=0 (KA +Af) t= 7 días
Rel
ativ
eab
un
dan
ce(%
)
Dekkera bruxellensisSaccharomyces cerevisiaePichia membranifaciens
Minisilos amended with water kefir
Other: Aspergillus, Dekkera, Candida,Malassezia, Mortierella, Rhodotorula, Penicillium, Alternaria, Hypocrea
Conclusions
• A. flavus is a problem in sorghum silage during the aerobic phase of silage process
• Microorganisms from kefir could prevent the growth of A. flavus in sorghum silage
• A consortium of Lactobacillus spp., Sacharomyces cerevisiae and Pichia membranifaciens are involve in A. flavus control
• Lactobacillus nagelli and Lactobacillus harbinensis seem to have animportant role in the biocontrol
• Massive sequencing approach helped to see the fate of differentmicroorganisms after incubation in sorghum silos
Thank you!