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!