natural antifungal systems for prevention of mould spoilage in

263 Pages·2012·25.85 MB·English

Cranfield University
MARIONA ARROYO
NATURAL ANTIFUNGAL SYSTEMS
FOR PREVENTION OF MOULD
SPOILAGE IN BAKERY PRODUCTS
APPLIED MYCOLOGY GROUP
INSTITUTE OF BIOSCIENCE AND TECHNOLOGY
Ph.D. THESIS
Cranfield University
Applied Mycology Group
Institute of BioScience and Technology
Ph.D. Thesis
Academic year 2002-03
MARIONA ARROYO
NATURAL ANTIFUNGAL SYSTEMS
FOR PREVENTION OF MOULD
SPOILAGE IN BAKERY PRODUCTS
Supervisor:
Professor Naresh Magan
Apri12003
This thesis is submitted in partial fulfilment of the requirements for the
degree of Doctor of Philosophy
ABSTRACT
Growth of spoilage fungi in bread and other bakery products is currently controlled
with the addition of weak acid preservatives. Consumers demand more natural
products and thus there is a need to reduce the amount of chemical preservatives
added to foods, or to identify alternative, more 'natural' antifungal compounds with
strong biological activity. This thesis reports on several areas of research undertaken
in the project: evaluation of sub-optimal concentrations of existing preservatives,
under different environmental conditions, on growth and ochratoxin A (OT A)
production by six bread spoilage moulds; efficacy of new/natural antifungal
compounds for possible use in bread preservation; evaluation of the impact of
preservation hurdles on ecophysiology of the spoilage fungi, including niche overlap;
and mechanisms of action of preservatives on hydrolytic enzymes.
It was found that the use of currently applied levels of the existing preservatives
potassium sorbate, calcium propionate and sodium benzoate were effective, under low
pH environments (pH 4.5) at completely controlling growth of spoilage moulds
(Aspergillus ochraceus, Eurotium repens, Cladosporium herbarum, Penicillium
corylophilum and Penicillium verrucosum) on wheat flour-based substrates over a
period of 30 days. At higher pH levels, the efficacy decreased being almost nil at pH
7.5. The use of sub-optimal concentrations of weak acid preservatives led in most
cases to reductions in lag times and/or stimulation of mould growth and ochratoxin A
productionb y P. verrucosum strains.
Four differeht antioxidants and twenty plant essential oils were screened for in vitro
antifungal activity. Antioxidants butylated hydroxyanisole (BRA) and p
hydroxybenzoate (propyl paraben) and essential oils of clove, cinnamon, bay and
thyme showed the strongest antifungal activity, and were therefore considered as
possible alternative compounds for bread preservation. Under different environmental
conditions MIC (Minimum Inhibitory Concentration) values ranged from 200-500
ppm (w/w) for antioxidants and from 200-1000 ppm for essential oils. In contrast to
weak organic acids, the antifungal efficacy of these alternative preservatives was less
dependent on the pH of the media.
The addition of antioxidants and essential oils to bread analogues resulted in a
significant decrease in their biological activity, probably due to preservative
molecules binding to food components such as lipids, proteins or polysaccharides.
Maximum doses studied (1000 ppm) had no effect on mould growth of the species
tested.
On wheat flour agar A.ochraceus and three strains of P. verrucosum did not produce
any OTA at 25°C on a range of a x temperature conditions and 54 days incubation.
w
However, on bread analogues, P.verrucosum strains produced up to 75 ppm after 28
days. A.ochraceus produced no OT A on bread analogues. Stimulation of OT A
production was observed on both substrates by the use of sub-optimal concentrations
of existing preservatives. Conversely, although growth of P. verrucosum on bread
analogues was not inhibited by the addition of 1000 ppm of antioxidants and essential
oils, a significant reduction in inhibition on OT A was found.
Nutritional assimilation by spoilage moulds of 32 key bread carbon sources, under
different environmental factors and in the presence of existing preservatives, was
determined for the first time. Niche sizes and niche overlap indices (NOl) varied with
species, environmental factors (a pH and temperature) and with the presence of
w,
preservatives. Overall, the addition of potassium sorbate increased the inter-specific
competition for nutrients.
Lastly, the impact of environmental conditions and existing/alternative preservatives
on hydrolytic enzyme production by moulds colonising wheat flour-based substrates
was studied. Of seven different enzymes assayed, only (3 -D-glucosidase, ex -D
galactosidase and N-acetyl-(3 -D-glucosaminidase were detected. The type and level
of enzyme produced varied with species and was significantly affected by time of
incubation, a and by the presence of preservatives.
w
From the results presented in this thesis it can be concluded that antioxidants and
essential oils are promising alternative compounds for controlling growth and toxin
production of common bread spoilage moulds. The effectiveness of these compounds
will be dependent on other factors such as the mode in which they are applied and the
11
A fa mareta,
ACKNOWLEDGMENTS
First and most importantly, I would like to thank my supervisor Professor Naresh
Magan, the 'tarzan' of the Fungal Jungle, for his constant advice and support. Thank
you for the unforgettable experience that it has been working for you and for your
contagious enthusiasm and optimism throughout the project.
I would also like to thank my second supervisor, Dave Aldred for being always ready
to listen (if spoken loud enough) and help me in my crusade against the HPLC. Hola
Guapo! !. It has been a privilege knowing you.
A huge "thank you" to my wonderful Spaniards: Olinda 'la tonta', my personal bench
warrior, Belen, Laura, Esther, and Nievinos, the pottery Queen, and to my favourite
Spaniard in training, Ioannis, filakia.
I wish also to thank Russ and Mike for the great companionship, to Silvia "beijinhos,
beijinhos" and to the always smiley Stella. You will be sorely missed. Grazie mile! to
Iva and Manuela, the best house mates, for their patience with me and my Italian.
To everyone at mST and to all those whom I may have forgotten, a big thank you.
Last but not least, I thank my family, "Mizu" and friends for their encouragement and
love.
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ABREVIATION LIST
pres Preservative
eso Essential oil
ant Antioxidant
WFA Wheat Flour Agar
MEA Malt Extract Agar
e.r.h. Equilibrium Relative Humidity
Water Activity
BHT Butylated Hydroxytoluene
BRA Butylated Hydroxyanisole
PP Propyl p-Hydroxybenzoate (Propyl Paraben)
OTA Ochratoxin A
OTB Ochratoxin B
HPLC High Performance Liquid Chromatography
CS Carbon Sources
NOI Niche Overlap Index
Spp Species
CP Calcium Propionate
SB Sodium Benzoate
PS Potassium Sorbate
AO Aspergillus ochraceus
CH Cladosporium herbarum
ERE E urotium repens
PC Penicillium corylophilum
PR Penicillium roquefortii
M450 Penicillium verrucosum strain M450
M453 Penicillium verrucosum strain M453
PV3 Penicillium verrucosum strain PV3
IV
Abstract
Acknowledgments
111
Abbreviation list
IV
Contents
v
List of Figures
x
List of Tables xvu
List of Plates
XXUI
CONTENTS
1 LITERATURE REVIEW
1.1 FOOD SPOILAGE 1
1.1.1 Parameters affecting fungal growth 1
1.1.2 Mycotoxin production in foodstuffs 5
1.2 FOOD PRESERVATION 7
1.2.1 Preservation techniques 8
1.2.2 Antimicrobial agents: an overview 9
1.2.3 Existing chemical antimicrobials 14
1.2.4 Natural antimicrobials 20
1.2.5 Combined methods in food preservation 29
1.3 BREAD 29
1.3.1 Situation of the bakery sector 29
1.3.2 Mould spoilage 30
1.3.3 Preservation 32
1.3.4 Future 34
v
2 AIMS AND OBJECTIVES
2.1 AIMS OF THE PROJECT 35
2.2 GENERAL OBJECTIVES 36
3 MATERIALS AND METHODOLOGY
3.1 FUNGAL SPECIES AND CULTURE MAINTENANCE 39
3.2 SUBSTRA TES FOR FUNGAL GROWTH 39
3.2.1 Basic nutritional media 39
3.2.2 Preparation of bread analogues 42
3.2.3 Control of water activity and pH levels 45
3.3 SUBSTRATE INOCULATION AND INCUBATION 45
3.4 PARAMETERS FOR MEASUREMENT OF FUNGAL GROWTH 47
3.5 EFFECT OF EXISTING PRESERVATIVES ON GROWTH OF
SPOILAGE FUNGI UNDER DIFFERENT ENVRIONEMTAL
CONDITIONS 48
3.5.1 In vitro studies on 2% wheat flour agar 48
3.5.2 In situ studies on bread analogues 48
3.6 EFFECT OF ANTIOXIDANTS AND ESSENTIAL OILS ON GROWTH
OF SPOILAGE FUNGI UNDER DIFFERENT ENVIRONMENTAL
CONDITIONS 49
3.6.1 In vitro studies on 2% wheat flour agar 49
3.6.2 In situ studies on bread analogues 51
3.7 EFFECT OF PRESERVATIVES AND ENVIRONMENT ON
OCHRATOXIN A PRODUCTION BY A.OCHRACEUS AND
P. VERRUCOSUM 51
3.7.1 Sampling 51
52
3.7.2 Toxin extraction
3.7.3 Quantification 57
Vi
3.8 CARBON SOURCES UTILISATION PROFILE AND NICHE
OVERLAP BETWEEN SPOILAGE FUNGI IN THE PRESENCE
OF SUB-OPTIMAL CONCENTRATIONS OF POTASSIUM SORBATE 60
3.8.1 Media preparation 60
3.8.2 Spore suspension preparation and inoculation 62
3.8.3 Measurement of Niche Overlap Index (NOI) 66
3.9 EFFECT OF EXSISTING AND ALTERNATIVE PRESERVATIVES ON
HYDROLYTIC ENZYME ACTIVITY OF SPOILAGE FUNGI 68
3.9.1 Sampling and inoculation 68
3.9.2 Enzyme extraction 69
3.9.3 Measurement of enzyme activity 69
3.10 STATISTICAL TOOLS 73
4 RESULTS
4.1 EFFECT OF EXISTING PRESERVATIVES ON GROWTH OF
SPOILAGE FUNGI ON 2% WHEAT FLOUR AGAR AT DIFFERENT
ENVIRONMENTAL CONDITIONS 75
4.1. 1 Effect of environmental factors on fungal growth 75
4.1.2 Effect of existing chemical preservatives 82
4.2 EFFECT OF EXISTING PRESERVATIVES ON GROWTH OF
SPOILAGE FUNGI ON BREAD ANALOGUES AT DIFFERENT
ENVIRONMENTAL CONDTIONS 90
4.2.1 Effect of environmental conditions 90
4.2.2 Effect of existing preservatives 91
4.3 EFFECT OF ANTIOXIDANTS AND ESSENTIAL OILS ON GROWTH
OF SPOILAGE FUNGI 96
4.3.1 Screening for essential oils and antioxidants with antifungal activity on
2% wheat flour agar 96
4.3.2 Effect of dose and environmental conditions on the in vitro antifungal
efficacy of best antioxidants and essential oils 101
4.3.3 Efficacy of best antioxidants and essential oils on bread analogues 111
Vll