bioproductionof antifungal and antimicrobial peptides...
TRANSCRIPT
Bioproduction of antifungal and antimicrobial peptides and their applications in biocontrol
Pascal DHULSTER
1
Charles VIOLLETTE InstituteLaboratory research in agri‐food and biotechnology
ICV EA 7394 USC INRA 1411 AFP
USC ANSES
FR CNRS no 3417
2
« Charles VIOLLETTE Institute »FR CNRS no
3417
2 Associated International Laboratories
CRIBLAGE, CARACTERISATION ET PRODUCTION DE METABOLITES D’ORIGINE MICROBIENNE
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ContextProBioGEM‐ Lille MiPI‐ Gembloux
Collaboration from 2003
Production of lipopeptides by microorganisms
Improvement of strains by genetic engineering
Development of production and separation and
purification processes
Mechanisms of action
Applications biopesticides
Discovery of new molecules
▪ start‐up Lipofabrik
Biocontrol
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ContextICV
TEAM ProBioGEMand QSA
INAF TEAMS: Laurent Bazinet and Ismail Fliss
Collaboration 10 years ago
Production of antimicrobial peptides and peptide fractions
Lactic acid bacteriabacteriocins
Development of production and
purification processes
Mechanisms of actionMicrobiote Interactions
Animals HealthPackaging
Inert surfacesEmployees
Hydrolysis of proteins
Volet Qualité‐santé‐innocuitéProcesses for the production ofActive fractions
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• Peptidic sequenceL-Leu- D-Leu- L-Asp-L-Val- D-Leu- L-Leu- L-Glu
• Acide gras : C13 to C17• bacterial strain
BBG21, BBG131
• Peptidic sequenceL-Glu- L-Orn- D-Tyr- D-alloThr- L-Glu- D-Ala- L-Pro- L-Gln- L-Tyr- L-Ile
• Acide gras : C14 to C18• bacterial strain
BBG21
Surfactins FengycinsIturins(mycosubtilin)
• Peptidic sequenceL-Asn- D-Tyr- D-Asn-L-Gln- L-Pro- D-Ser- L-Asn
• Fatty acids: C14 to C17• bacterial strain
BBG101
Lipopeptides produced by Bacillus subtilis
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• Lipopeptides properties
surfactants:Surfactins, iturins, fengycins
antifungal activity:Fengycins, iturinsPotential biopesticides
eliciting activity (plant resistance induction)Surfactins
• Bacterial strainB. subtilis , model strain BBG21 (surfactin and fengycin producing strain)aerobic metabolismproduces up to around 1g/L of lipopeptidic biosurfactants
Background
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Role of mycosubtilin in the protection of tomatoes against Pythium aphanidermatum
A B C
(Leclère et al.,2005, appl. environ. microbiol., 71, 4577-4584)
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Laitue/ Bremia lactucae
0
10
20
30
40
50
60
70
Témoin Mycosubtiline
nom
bre
de s
alad
es
classe 4classe 3classe 2classe 1 classe 0
Mycosubtilin reduces the severity of the disease
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lettuce/ Bremia lactucae
0102030405060708090
100
Témoin DMSO (0,1%) surf 50 mg/L myco 50 mg/L myco+surf 50/50 mg/LPour
cent
age
de la
itues
infe
ctée
s (%
)
Traitements
Pourcentage de laitues infestées par B. lactucae
a
ab
bb
ab
The surfactin / mycosubtline mixture has a synergistic effect on the protection of lettuce
15
Raisin/Botrytis cynerea
Traitement avec un mélange surfactine/fengycine
Preventive treatment with a surfactin / fengycin mixture reduces the percentage of contaminated berries
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Production issue
• The production of bacterial surfactants requires an important supply of oxygen• Aeration and conjugate agitation generate foam
T 6h: Agitation + aeration + surfactant= foamT 0h: inoculation T 12h: Loss of culture
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Production issue• The production of bacterial surfactants requires an important supply of oxygen• Aeration and conjugate agitation generate foam
• Consequences: Uncontrolled losses of cells and substrateDifficulty controlling fermentationImpossibility of coupling a purification step
T 6h: Agitation + Aeration + Surfactant= FoamT 0h: Inoculation T 12h: Loos of culture
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Control by anti‐foam?Chemical anti‐foam agent
makes difficult biosurfactant purification (BS)can alter biological properties of biosurfactantsconsumption increases with amount of BS produced
Mechanical anti‐foam system: not efficient enough
Solutionsto monitor foam formation and to use foam as an extraction process• Overflowing exponential fed bioreactor
to change air sparging aeration to bubbleless aeration• Three phase inverse fluidized bed bioreactor• Membrane contactor aerated bioreactor• Rotating disk bioreactor
Foam formation control
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• Schéma du bioréacteur O‐EFBC
Feed vessel
Air Inlet
TemperaturepH, pO2 , Feed rate Control
Pump 2 Cooling device 1
Cooling device 2
Pump 1
Pump 3
Base Acid
Balance
Air Outlet
UPPER LEVEL
Cooling agent
fout
Foam collector
Foam
fin
• Capacité moussante = volume de mousse stabilisée/volume d’air injecté
• CM de l’iturin A: 0,99 (Razafindralambo et al., 1998)
• CM de la surfactin: 0,98 (Razafindralambo et al., 1996)
Foam formation control
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A specific productivity of 1.1 mg mycosubtiline of biomass. g‐1h‐1 is obtained for an average growth rate of 0.071 h‐1
The estimation of the quantity of biomass produced over time allows to calculate the evolution of μ and therefore a mean μ
The analyses of the quantities of mycosubtiline produced over time allow to calculate the specific productivity in mycosubtiline qP
Foam formation control
Problem for the scale up.Difficulty of coupling to separation techniques (foam stability)
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Objectives
To reach at least the same growth level and the same lipopeptide concentration than in a conventional air sparged bioreactor (foamingbioreactor)
Foam formation control
to change air sparging aeration to bubbleless aeration:
Three phase inverse fluidized bed bioreactorMembrane contactor aerated bioreactorRotating disk bioreactor
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• Three phase inverse fluidized bed bioreactor (TPIFBB)(Fahim et al., 2013)
culture volume: 2.5L
down‐flow circulation of the culture medium
partial foam control by a falling film in the upperpart of the bioreator
increase of gas hold‐up in the culture medium
aeration‐ with air sparging (periodic feed)‐ with falling film: gaz‐film interfacial transfer
Limited air-sparging aeration
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• TPIFBB’s performancesOxygen transfer coefficient (Kla): between 0.006 s‐1 and 0.06 s‐1
Lipopeptide production in batch culture with B. subtilis BBG21
surfactins = 1232 mg L‐1 fengycins= 50 mg L‐1
Modelisation of oxygen transfer coefficient: correlation to gas and liquidvelocities and surface tension
Limited air-sparging aeration
To conclude
Well aerated bioreactorThe complexicity of design limits up‐scaling
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Bioreactor withexternal aerationmodule
Bioreactor withimersed aerationmodule
O2 O2 O2
AirCulture
Oxygen transfer through the porous hollow fiber membrane
Air‐liquid contactor aerated bioreactor(Coutte et al. , 2010; Coutte et al. 2013)
Choice of the membrane
Aeration without mixing air-culture medium
Applications :‐ Blood oxygenation (Tsugi et al., 1981, Khoshbinet al., 2005)‐ Waste water treatment (Brindle et al., 1996; Pankhania et al., 1999)‐ Animal cell cultures (Schneider et al., 1995)
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1 All the values presented are means value of duplicates (standards deviations were essentially within 1‐12%). After two washings with water at 30°C and two washings with NaOH pH 10.0 at 50°C.
Lipopeptides Foaming Bioreactor
Externalmodule
bioreactor
Immersedmembranebioreactor
Surfactin production analysisAmount of surfactin produced in the broth (mg)
Amount of surfactin produced in the foam (mg)
Amount of surfactin desorbed from the hollowfiber after washing* (mg)Total amount of surfactin produced (mg)
Fengycin production analysisAmount of fengycin produced in the broth (mg)
Amount of fengycin produced in the foam (mg)
Amount of fengycin desorbed from the hollowfiber after washing* (mg)Total amount of fengycin produced (mg)
60
714
‐
774
‐
45
‐
45
168
‐
604
772
35
‐
112
147
319
‐
150
469
982
‐
0
982
Lipopeptide production results
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Influence of oxygen transfer on lipopeptide production
Oxygen transfer coefficient (Kla) in flasks and lipopeptide production
KLa = 6,67x10‐6N1,16VL‐0,83d00,38d1,92 (Fahim et al., Bioresource Technol., 2012)
High KLaMedium KLaLow KLa
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Oxygen transfer coefficient (Kla) (h-1)
Foaming Bioreactor
External module bioreactor
Immersed membrane bioreactor
Initial KlaKla during the fermentation aKla after fermentation Kla after washing*
27.29 (SD : 0.17)23.70 (SD : 9.83)
--
39.36 (SD : 0.09)22.21 (SD : 4.5)4.07 (SD : 0.01)15.47 (SD : 0.09)
28.24 (SD : 0.15)13.19 (SD : 3.48)1.75 (SD : 0.06)4.46 (SD : 0.51)
a Values are mean data from two experiments with dynamic measurement of KLa. SD: Standard deviation* After one washing with water at 30°C
Oxygen transfer evolution during culture
Aeration membrane fouling
Evolution of oxygen transfer coefficient a
External module bioreactor
Immersed bioreactor
Initial Kla (h‐1)Kla after surfactin adsorption (h‐1)
39.36 (SD : 0.09)25.53 (SD : 0.07)
28.24 (SD : 0.15)2.57 (SD : 0.02)
a Values are mean data from three experiments with dynamic measurement of KLa. SD: Standard deviation.
Influence of surfactin on oxygen transfer
Origin of membrane foulingSurfactin adsorption for immersed membrane bioreactorBiofilm formation for external module
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Results
Equivalent production of lipopeptides with the conventional air sparged bioreactor
Good stability of the performances. Easy up‐scaling because of membrane modularity.
Objectives
A model will be developped to describe oxygen transfer through the membrane for the bioreactor with an external aeration module (A. Berth’s ) using dimensionalanalysis. This model should help for up‐scaling.
Aeration without mixing air-culture medium
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Production
Extraction
Concentration
PurificationPurity (surfactine): 95%
Diafiltration
Fermentation and downstream processing coupled for lipopeptide production and purification
patented by the University of Lille 1 Sciences et Technologies
(Coutte et al., 2013)
Bioreactor integration
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• Rotating disc bioreactor (Chtioui et al., 2012)
Biomass‐ biofilm on the disks (< 1mm thickness)‐ plantonic biomass
surface oxygen transfer(repetitive biofilm emersion, medium mixing, no bubble)
Stainless steeldisks as biofilm substrate
Disk surface bacterial colonization
Aeration without mixing air-culture medium
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0
200
400
600
800
1000
7 disques sans agitateurs 14 disques sans agitateurs
Conc
entra
tion
(mg
L-1 )
Surfactine
Fengycine
KLa = 0,0011 s‐1
KLa = 0,0019 s‐1
Adhered cells= 0,5 g
Adhered cells = 0,8 g
Aeration without mixing air-culture medium
fg/sf 4.4 3.8
•Proportional increase of oxygen transfer rate and biomass and lipopeptide production with the disk number
• Limitation of disk rotation speed because of foam production
Influence of disk number on oxygen transfer rate
7 disks 14 disks
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Results
Highly heterogenous bioreactor with very low aeration of the culture medium but a highly aerated biofilm
Easy to run, quite low energy consumption, easy up‐scaling
Aeration without mixing air-culture medium
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Acknowledgements
Thanks to my colleagues of ProBioGEM Team at Institut Charles Viollette
Financial support :
Philippe JacquesDidier LecouturierFrédérique GancelKrasimir DimitrovPeggy VauchelFrançois CoutteIordan NikovJean‐Sébastien Guez Alice RochexValérie Leclère
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Protein sources: Blood (cruor, hemoglobin),Milk (Caseins and Alpha‐lactalbumin), Alfalfa (RuBisCO)
Fish coproducts
Food Coproducts
Digestive Enzymes (Pepsin and Chymotrypsin)
Peptidic hydrolysates
KineticModeling
Déterminant MinimalRelation structure/function
mechanism of action
Identification and characterization of peptides
Bioactives peptides research
peptide mappings Processdesign and control
Valorisation of peptides
Obtaining and characterization of active peptides from agro‐food co‐products
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major source of pollution is a real problem on ecological and environmental
co‐product
source of peptides with biological activities
antibacterial agents
antistress
antihypertensiveantioxidant opioid
analgesic
60% 40%
Thèse de LUDIVINE SIONThèse de Rémi PRZYBYLSKI
Large amount of proteins(albumin, fibrinogen…)
Pharmaceutical interest!
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Waste of meat industries:30000 T/year in France
Chaîne alpha
Chaîne bêta
Hème
T0 (t=0min)
DHc = 0%
T1 (t=15min)
DHc = 7,8%
T2 (t=30min)
DHc = 9,9%
T3 (t=1h)
DHc = 11,9%
T4 (t=2h)
DHc = 13,9%
T5 (t=4h30)
DHc = 14,2%
T6 (t=10h)
DHc = 14,9%
Chaîne alpha
Chaîne bêta
Hème
T0 (t=0min)
DHc = 0%
T1 (t=2,5min)
DHc = 3,2%
T2 (t=5min)
DHc = 4%
T3 (t=30min)
DHc = 7%
T4 (t=1h)
DHc = 8,5%
T5 (t=2h)
DHc = 11,4%
T6 (t=3h)
DHc = 13,9%
5 40
Thèse d’Estelle ADJE
Estelle Yaba Adje, Rafik Balti , Mostafa kouach, Didier Guillochon and Naïma Nedjar‐Arroume (2013), Probiotics Antimicrob. Prot. 5: 176–186.
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Hémoglobine bovine Pepsine porcine
55 Peptides antibactériens
1‐32 33‐98 107‐141
5 Familles Peptidiques5 Familles Peptidiques
Chaîne Chaîne Chaîne Chaîne
114‐145
1‐30
Cartographies peptidiques
Naïma Nedjar-Arroume, Véronique Dubois-Delval, Estelle Yaba Adje, Jonathan Traisnel, François Krier, Patrice Mary, Mostafa Kouach,Gilbert Briand, and Didier Guillochon (2008), Peptides, 29 : 969-977.
Thèse de Véronique DUBOIS
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Groupe 1 Groupe 2
Hélice αGrand nombre d’AA
Random coilPetit nombre d’AA
CMI = 80 M CMI = 5 M
55 Peptides antibactériens55 Peptides
antibactériens
Cartographies peptidiques
Cartographies peptidiques
Naïma Nedjar-Arroume, Véronique Dubois-Delval, Estelle Yaba Adje, Jonathan Traisnel, François Krier, Patrice Mary, Mostafa Kouach,Gilbert Briand, and Didier Guillochon (2008), Peptides, 29 : 969-977.
Thèse de Véronique DUBOIS
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Objectifs Compétences Bilan/projets Perspectives
107VTLASHLPSDFTPAVHASLDKFLANVSTVLTSKYR141
137TSKYR141
1 38SKYR141
139KYR141
140YR141
133TVLTSKYR141
Déterminant peptidique antimicrobien minimal KYR
CMI:80 M
CMI: 8 M
CMI: 6 MCMI: 4 M
CMI: 1 M
Pas d’activité
Déterminant peptidique antimicrobien minimal RYH
140LAHRYH145
143RYH145
114ARNFGKFFTPVLQADFQKVVAGVANALAHRYH145
121FTPVLQADFQKVVAGVANALAHRYH145
CMI:85 M
CMI: 80 M
CMI: 71 M
CMI: 18 M
CMI: 1 M
126QADFQKVVAGVANALAHRYH145
antibacterial peptidic sequence minimal
Lucie Catiau, Johnatan Traisnel, Véronique Delval-Dubois, Nour-Eddine Chihib, Didier Guillochon, and Naïma Nedjar-Arroume(2011), Peptides 32 : 633–638.
Lucie Catiau, Johnatan Traisnel, Nour-Eddine Chihib, Guillaume Le Flem, Annick Blanpain, Oleg Melnyk, Didier Guillochon, andNaïma Nedjar-Arroume (2011), Peptides 32: 1463–1468.
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The target peptide: the neokyotorphin
• α‐chain of bovine hemoglobin1VLSAADKGNV10KAAWGKVGGH20AAEYGAEALE30RMFLSFPTTK40TYEPHFDLSH50GSAQVKGHGA60KVAAALTKAV70EHLDDLPGAL80SELSDLHAHK90LRVDPVNFKL100LSHSLLVVTLA110SHLPSDFTPA120VHASLDKFLA130NVSTVLTSKY140R
Final peptide653 DapI = 10.5
BioactivitiesAnalgesic
ANTIMICROBIAL
Micrococcus luteusStaphylococcus aureusSalmonella enteritidis
Escherichia coliListeria innocua
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…
The challenge of the hydrolysate fractionationHemoglobin subunitsare rapidly digested…
…to give intermediate peptideswith high molecular weights…
…then to give more and more final peptides (low MW) !
…
Target peptide:
Neokyotorphin
The challenge of the hydrolysate fractionation
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Pilote d’ÉDUF
‐ +
MUF10 kDa
MEC MEA
10 g.L‐118 L.h‐1
2 g.L‐118 L.h‐1
20 g.L‐112 L.h‐1
20 g.L‐112 L.h‐1
+-Él
ectrolytes
Électrolytes
Récupérationcationique Hydrolysat
Thesis of Rémi PRZYBYLSKI
Developed a selective method of obtaining of the antimicrobial peptide α137-141 from the co-product of slaughterhouses by using the electrodialysis coupled with an ultrafiltration membrane (EDUF)
α137-141+
47
Study the effect of hydrolysis degree on the peptides migration during the EDUF treatment
Selection of the most appropriate hydrolysis degree for the selective separation of neokyotorphin
Improve the neokyotorphin purity by pH‐controlduring the EDUF treatment
Neokyotorphin fractionation strategy: overview
48
Conclusions about EDUF
The EDUF is a suitable method to fractionate the bovine hemoglobin hydrolysates and to separate selectively the NKT.
The most appropriate hydrolysis degree is 5% to separate the neokyotorphin.The enrichment factor in NKT was superior than 13‐fold comparedto the initial hydrolysate.
Selecting a pH of 9.0 increased the NKT purity about 75‐foldcompared to the initial hydrolysate.
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Du co‐produit à sa valorisation
Peptides enzyme production
Séparation of bioactive peptides from hydrolysat
valorization of enriched fractions
Valorisation of bioactive peptidesCoproducts from industrial waste
Peptides as antibacterial and antioxidant agents in meatThe rejection becomes an added value
Identifications et caractérisations
Thèse de Rémi PRZYBYLSKIR. Przybylski, L. Firdaous, G. Châtaigné, P. Dhulster, N. Nedjar (2016)Food Chemistry 211 (2016) 306–313
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Fraction d’ÉDUF : potentiel conservateur ?
0
2
4
6
8
0 2 4 7 9 11 14
Col
onie
s tot
ales
(log
UFC
/g)
Durée de conservation (jours)
0
2
4
6
8
0 2 4 7 9 11 14
Bac
étie
s col
iform
es (l
og U
FC/g
)
Durée de conservation (jours)
COLONIES TOTALES
COLIFORMES
Thèse de Rémi PRZYBYLSKI
R. Przybylski, L. Firdaous, G. Châtaigné, P. Dhulster, N. Nedjar (2016) Food Chemistry 211 (2016) 306–313
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Acknowledgements
Thanks to my colleagues of ProBioGEM Team at Institut Charles Viollette
Naïma NEDJARLoubna FIRDAOUSRozenn RAVALLECBenoit CUDENNECChristophe FLAHAUTFrançois KRIERKrasimir DimitrovPeggy Vauchel
Véronique DUBOISEstelle ADJEKarima HEDHILIRémi PRZYBYLSKI poster
Titre du poster 17: Électroséparation d’un peptide antimicrobien à partir d’un co‐produitdes abattoirs et son application comme potentiel conservateur de la viande.
… Special thanks to Jean‐François Goossens and Mostafa Kouach(C.U.M.A. – EA 4481) for technical assistance in mass spectrometry…
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