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Els Debonne, Ghent UniversityEde, 28 January 2020
The use of growth/no-growth models as a tool
to predict bread shelf-life
TABLE OF CONTENTS
01OBJECTIVESBread moulds – clean label
02METHODOLOGYScreening of antifungal activity of natural antifungal compounds
03RESULTS ANALYSISValidation of growth/no-growth models with bread shelf-life
04CONCLUSIONSModels as a tool to predict bread shelf-life
2
Penicillium spp. Cladosporium spp. Aspergillus spp. …
INTRODUCTION Is it safe to eat mouldy bread?
visible mycelia natural repellant
chemical preservatives
invisible network breathing problems andallergic reactions
mycotoxins diseases and death
3
“Any food that requires enhancing by the use of chemical substances should in no way be considered as food.”— JOHN H. TOBE
“Old people shouldn’t eat healthy foods. They need all the preservatives they can get.”— ROBERT ORBEN
4
5
Traditional bread2 – 5 daysretrogradation
Par-baked bread, toast breadClean label, MAP 3 weeksWith preservatives, MAP 6 - 8 weeks
Antifungal compounds (chemicals): organic acids; acetic acid, lactic acid,
phenyllactic acid, …pH dependent antifungal effect
CTOT and pH
LITERATURE REVIEW
Micro-organisms are only active in the aqueous phase.Migration of antifungal compounds (water versus oil phase)
.Protonated form of organic acid (undissociated concentration). Undissociated acid (mmol) / L aqueous phase CHA (mM)
Natural characterChemical (volatile) compoundsStrong sensorial and physico-chemical adverse effectsAntifungal = anti – fungi (moulds AND yeasts)
Active concentration expressed on the aqueous phase
Sourdough Essential oils & plant extracts
6
Natural antifungal compounds
Screening Shelf-lifeG/NG models
Data analysisBread baking trials
& storage
STUDY OBJECTIVES
7
Micro versus macro dilution
Selection of growth medium
Screening of antifungal activity requires either
working with standardized amounts of pure chemicals OR
requires detection methods of chemicals in food products
Development of models
Validation with bread shelf-life
METHODOLOGY
In-vitro screening
Chemicals
G/NG models + validation in
8
METHODOLOGY
chemicals
HA
A-
H+
H+
H+H+
H+
HA H+
H+
pH effect on growth of moulds?
Weak organic acids Acetic acid Lactic acid Phenyllactic acid
Undissociated acid (CHA)
Henderson- Hasselbalch equation
Sourdough – organic acids
𝑝𝐻 = 𝑝𝐾𝑎 + log10[𝐴−]
[𝐻𝐴]
9
METHODOLOGY
chemicals
pH effect on growth of moulds?
Weak organic acids Acetic acid Lactic acid Phenyllactic acid
Undissociated acid (CHA)
Henderson- Hasselbalch equation
CHA in mmole / L aqueous phase
Sourdough – organic acids
Example: 33 % moistureactive concentration = 3 x conc
𝑝𝐻 = 𝑝𝐾𝑎 + log10[𝐴−]
[𝐻𝐴]
10
METHODOLOGY
chemicals
Lipophilic behavior of EOs/ components
Partitioning to oil – water phase Kp: partitioning coefficient
Modified Henderson- Hasselbalchequation
e.g. thyme essential oil (thymol)
Caqua in mmole / L aqueous phase
Essential oils – terpenes, terpenoids, phenylpropenes & others
Example thyme EO (~ thymol): 33 % moistureKp (thymol) = 3,34 (103,34/1: parts oil/water)Oil in bread: 57% (free) of 1,2% lipids in flour
𝐶𝑡ℎ𝑦𝑚𝑜𝑙 , 𝑎𝑞𝑢𝑎 =𝑛𝑇𝑂𝑇,𝑡ℎ𝑦𝑚𝑜𝑙
𝑚𝑇𝑂𝑇 ∗ 𝐾𝑝 ∗𝑟𝜌𝑜𝑖𝑙
+1 − 𝑟𝜌𝑎𝑞𝑢𝑎
11
METHODOLOGY
In-vitro screening
Screening method can vary.
Important to know the mode of action of the chemical compound, e.g. volatilebehavior of EOs.
Essential oilsThe chemical variability of EOs due tovariations in geographical conditions, age of the plants, time of harvesting andthe method of extraction, complicatesthe use of EOs as natural preservativesin food products.
Therefore in-vitro screening requiresstandardization of the chemicals.
Organic acidsMicro-and macro dilution methods
Growth of fungi
12
METHODOLOGY
In-vitro screening
Screening method can vary.
Important to know the mode of action of the chemical compound, e.g. volatilebehavior of EOs.
Essential oilsThe chemical variability of EOs due tovariations in geographical conditions, age of the plants, time of harvesting andthe method of extraction, complicatesthe use of EOs as natural preservativesin food products.
Therefore in-vitro screening requiresstandardization of the chemicals.
Organic acidsMicro-and macro dilution methods
Growth of fungi Macro-dilution
Micro-dilution
12
fungibacteriaGrowth kinetics is less important.Maximal quality levels are defined.
0
0,5
1
1,5
2
2,5
0 50 100 150
O.D.
Time (h)
G/NG
0
0,5
1
1,5
2
2,5
0 50 100 150
O.D.
Time (h)
lag
log
stationary
decay
Bread at the end of shelf-life
METHODOLOGY
G/NG models
+
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G/NG modelsScreening method: macro-dilutionMould: Penicillium paneumIncubation temperature: 22 °C
RESULTS ANALYSIS CHA acetic acid CHA acetic & lactic acid
Antifungal activity of acetic acid >> lactic acid
CHA acetic acid ≥ 150 – 200 mmole/L
14
0
10
20
30
40
50
0 - 100 100 - 150 150 - 200 > 200s
he
lf-l
ife
(d
ays
)
CHA,AA (mmole/L)
Shelf-lifePackaging: air packagedBaking: par-baked and full-bakedContamination: airborne mouldsIncubation temperature: 22 °C
RESULTS ANALYSIS
CHA acetic acid in sourdough bread & in chemically acidified bread
CHA acetic acid ≥ 150 – 200 mmole/L
No significant difference between SD bread & chem. acid. wheat bread
PB/SD PB/chemical
FB/SD FB/chemical
Sourdough with ≥ 150 mM acetic acid:L. sanfranciscensisS. cereviseae
15
RESULTS ANALYSIS
Par-baked bread shelf-lifeIn-vitro screeningThyme EO added to bread doughof thyme essential oil
G/NG modelsScreening method: micro-dilutionMould: Penicillium paneumpH: 6 – aw: 0.97Incubation temperature: 22 °C
Bread shelf-lifeScreening method: shelf-life Moulds: airborne post-baking contaminationpH: 6 – aw: 0.97Incubation temperature: 22 °C
16
RESULTS ANALYSIS
Par-baked bread shelf-lifeIn-vitro screeningThyme EO added to bread doughof thyme essential oil
0.2 – 0.3 mL / 100 g dough5 – 7 µL / mL aqeous phase in bread
(modified HH equation + moisture content of bread)
Further optimization needed
C (thyme EO) : ± 1 µL / mL medium
16
CONCLUSIONS
Take-home messages:
1. Benefits of chemical preservatives (& E-numbers)2. G/NG models as a tool to screen antifungal compounds3. Role of expressing undissociated acid concentrations4. Antifungal effect of sourdough is more than pH alone
And the most important thing: validation of in-vitro G/NG models results in bread products is essential to obtain safe & qualitative food products!
17
● DEBONNE (2019). Growth/no-growth models of in-vitro growth of Penicillium paneum as a function of thyme essential oil, pH, aw, temperature. Food Microbiology (Elsevier).
● DEBONNE (2020). Validation of in-vitro antifungal activity of the fermentation quotient on bread spoilage moulds through growth/no-growth modelling and bread baking trials. LWT (Elsevier).
● DEBONNE (xxxx). Comparison of the antifungal effect of undissociated lactic and acetic acid in sourdough bread and in chemically acidified wheat bread.. Unpublished results
BIBLIOGRAPHY
18
THANKSDoes anyone have any questions?
Els.Debonne@ugent.be +32 9 243 24 94Ghent University
ir. Els DebonnePhD student/Teaching Assistant
DEPARTMENT OF FOOD TECHNOLOGY, SAFETY & HEALTHFACULTY OF BIOSCIENCE ENGINEERINGGHENT UNIVERSITY, BELGIUM
CREDITS
Presentation template & features:
● Presentation template by Slidesgo● Author introduction slide photo created by Freepik
Ph.D promotors
● Prof. dr. ir. Frank Devlieghere (Ghent University)● Prof. dr. ir. Mia Eeckhout (Ghent University)● Prof. dr. ir. Filip Van Bockstaele (Ghent University)
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