The use of growth/no- growth models as a tool to predict ... · The use of growth/no-growth models as a tool to predict bread shelf-life. TABLE OF CONTENTS 01 OBJECTIVES Bread moulds

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

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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

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“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

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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

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Natural antifungal compounds

Screening Shelf-lifeG/NG models

Data analysisBread baking trials

& storage

STUDY OBJECTIVES

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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

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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]

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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]

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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

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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

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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

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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

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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

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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

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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

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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!

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● 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

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THANKSDoes anyone have any questions?

[email protected] +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)