combating fungi in the food industry by applying antifungal lactic acid bacteria

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Title “Green preservatives” – combating fungi in the food industry by
applying antifungal lactic acid bacteria
Author(s) Pawlowska, Agata
Publication date 2013
Original citation Pawlowska, A. 2013. “Green preservatives” – combating fungi in the
food industry by applying antifungal lactic acid bacteria. PhD Thesis,
University College Cork.
Type of publication Doctoral thesis
Rights © 2013, Agata Pawlowska
http://creativecommons.org/licenses/by-nc-nd/3.0/
Embargo information No embargo required
Item downloaded http://hdl.handle.net/10468/1510
from
Downloaded on 2023-01-07T00:56:28Z
Ollscoil na hÉireann

The National University of Ireland

Coláiste na hOllscoile, Corcaigh

University College Cork

School of Food and Nutritional Sciences

“Green preservatives” – combating fungi in the food
industry by applying antifungal lactic acid bacteria

Thesis presented by
Agata Maria Pawlowska

For the degree of
Doctor of Philosophy
(PhD, Food Science and Technology)

Under the supervision of
Prof. DSc. Dr. Elke K. Arendt

July 2013
1
Table of Contents
Table of Contents ....................................................................................................... 2
List of Figures ............................................................................................................. 6
List of Tables .............................................................................................................. 8
Declaration .................................................................................................................. 9
Abstract ..................................................................................................................... 10
Chapter 1 Introduction ............................................................................................ 11
1.1 Introduction ...................................................................................................... 12
1.2 References ........................................................................................................ 15
Chapter 2 “Green Preservatives”: Combating fungi in the food and feed
industry by applying antifungal lactic acid bacteria – a review .......................... 20
2.1 Abstract ............................................................................................................ 21
2.2 Introduction ...................................................................................................... 21
2.3 Food quality and safety .................................................................................... 29
2.4 LAB as biopreservatives .................................................................................. 32
2.4.1 Application of antifungal LAB in bread ................................................... 40
2.4.2 Application of antifungal LAB in dairy products ..................................... 42
2.4.3 Application of antifungal LAB in fresh fruits and vegetables .................. 44
2.4.4 Application of antifungal LAB in cereal- based beverages ...................... 45
2.4.5 Application of antifungal LAB in feed ..................................................... 46
2.5 Conclusions ...................................................................................................... 49
2.6 References ........................................................................................................ 51
Chapter 3 Potential of lactic acid bacteria to inhibit common toxigenic food
spoilage fungi ............................................................................................................ 71
3.1 Abstract ............................................................................................................ 72
3.2 Introduction ...................................................................................................... 72
3.3 Materials and Methods ..................................................................................... 74
3.3.1 Bacterial cultures ....................................................................................... 74
3.3.2 Fungal cultures .......................................................................................... 74
3.3.3 In vitro antimicrobial activity.................................................................... 75
3.3.4 LCMS analyses of antifungal compounds ................................................ 75
3.3.5 HPLC analysis of weak organic acids ....................................................... 77
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3.3.6 Determinantion of Minimum Inhibitory Concentrations (MIC) ............... 77
3.4 Results and Discussion ..................................................................................... 78
3.4.1 Antifungal activity of lactic acid bacteria ................................................. 78
3.4.2 Antifungal potential of L. reuteri ee1p ..................................................... 89
3.4.3 Production of antifungal substances by L. reuteri ee1p ............................ 89
3.5 Conclusions ...................................................................................................... 92
3.6 References ........................................................................................................ 93
Chapter 4 Characterisation of Lactobacillus reuteri R29 as a new antifungal
strain improving the quality and shelf life of wheat bread .................................. 99
4.1 Abstract .......................................................................................................... 100
4.2 Introduction .................................................................................................... 100
4.3 Materials and Methods ................................................................................... 103
4.3.1 Bacterial cultures ..................................................................................... 103
4.3.2 Fungal cultures ........................................................................................ 103
4.3.3 In vitro antimicrobial activity.................................................................. 103
4.3.4 LCMS analyses of antifungal compounds .............................................. 104
4.3.5 HPLC analysis of weak organic acids ..................................................... 104
4.3.6 Sourdough fermentation .......................................................................... 104
4.3.7 Fundamental rheology ............................................................................. 105
4.3.8 Bread making .......................................................................................... 105
4.3.9 Antifungal activity tests .......................................................................... 106
4.4 Results ............................................................................................................ 107
4.4.1 Antifungal activity in vitro ...................................................................... 107
4.4.2 Identification and quantification of antifungal components ................... 108
4.4.3 Fundamental rheology ............................................................................. 110
4.4.4 Bread quality evaluation ......................................................................... 113
4.4.5 Antifungal activity tests .......................................................................... 113
4.5 Discussion ...................................................................................................... 115
4.6 Conclusions .................................................................................................... 117
4.7 References ...................................................................................................... 118
Chapter 5 Application of Lactobacillus amylovorus as an antifungal adjunct to
extend the shelf-life of Cheddar cheese ................................................................ 122
5.1 Abstract .......................................................................................................... 123
5.2 Introduction .................................................................................................... 123
5.3 Materials and Methods ................................................................................... 126
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5.3.1 Cultures and growth conditions .............................................................. 126
5.3.2 In vitro antifungal activity assay ............................................................. 127
5.3.3 LC-FTMS analyses of antifungal compounds ........................................ 127
5.3.4 HPLC analysis of weak organic acids ..................................................... 129
5.3.5 Miniature Cheddar cheese manufacture .................................................. 129
5.3.6 Microbiological analysis of cheeses ....................................................... 130
5.3.7 Gross composition of cheeses ................................................................. 131
5.3.8 Assessment of proteolysis ....................................................................... 131
5.3.9 Antifungal activity of L. amylovorus in cheese ...................................... 132
5.4 Results and Discussion ................................................................................... 133
5.4.1 In vitro antifungal activity of L. amylovorus strains ............................... 133
5.4.2 Identification and quantification of antifungal components ................... 134
5.4.3 Microbiological analyses and cheese composition ................................. 138
5.4.4 Antifungal activity of L. amylovorus DSM 19280 in cheese .................. 140
5.4.5 Proteolysis during cheese ripening ......................................................... 143
5.5 Conclusions .................................................................................................... 145
5.6 References ...................................................................................................... 146
Chapter 6 Lactic acid bacteria with antifungal activity and their application in
a novel wort-based beverage ................................................................................. 153
6.1 Abstract .......................................................................................................... 154
6.2 Introduction .................................................................................................... 154
6.3 Material and Methods .................................................................................... 158
6.3.1 Cultures and growth conditions .............................................................. 158
6.3.2 In vitro antifungal activity ....................................................................... 158
6.3.3 LCMS analyses of antifungal compounds .............................................. 159
6.3.4 HPLC analysis of weak organic acids and ethanol ................................. 159
6.3.5 Brewing materials ................................................................................... 159
6.3.6 Milling ..................................................................................................... 160
6.3.7 Wort preparation ..................................................................................... 160
6.3.8 Standard wort analysis ............................................................................ 161
6.3.9 Beverage preparation .............................................................................. 161
6.3.10 Antifungal activity assay of fermented wort ......................................... 162
6.4 Results and Discussion ................................................................................... 162
6.5 Conclusions .................................................................................................... 172
6.6 References ...................................................................................................... 173
Chapter 7 Overall discussion ................................................................................ 181
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7.1 Overall discussion .......................................................................................... 182
7.2 References ...................................................................................................... 189
Appendix ................................................................................................................. 195
Appendix 1 List of Publications ........................................................................... 196
Appendix 2 Lactobacillus amylovorus DSM 19280 as a novel food-grade
antifungal agent for bakery products ................................................................... 196

5
List of Figures
Figure 2.1 Distribution of the patents within the food and feed fields ...................... 48
Figure 2.2 Distribution of the patents within LAB species ........................................ 49
Figure 3.1 LAB species screened for antifungal activity ........................................... 80
Figure 3.2 Percentage of each LAB species showing antifungal activity ................. 80
Figure 3.3 Percentage of LAB showing antifungal activity against four common
fungi species ........................................................................................................... 81
Figure 3.4 LCMS chromatogram of antifungal compounds found in MRS fermented
by (A) L. reuteri ee1p and (B) L. reuteri M13·10-5 3 .............................................. 91
Figure 4.1. LCMS chromatogram of antifungal compounds found in MRS fermented
by (A) L. reuteri R29 and (B) L. reuteri hhIp ....................................................... 109
Figure 4.2 Effect of sourdough on the complex modulus (A) and phase angle (B) of
wheat batters. (♦) sourdough fermented with L. reuteri R29, (●) sourdough
fermented with L. reuteri hhIp, (▲) control, without sourdough addition .......... 112
Figure 4.3 Shelf life of wheat bread challenged against F. culmorum FST 4.05 (A),
A. niger FST 4.21 (B), P. expansum FST 4.22 (C), P. roqueforti FST 4.11 (D), and
environmental moulds from a pilot scale industrial bakery (E) ........................... 114
Figure 5.1 Antifungal activity of L amylovorus DSM 19280 (left) and antifungal-
negative control L. amylovorus DSM 20531 (right) against P. expansum on milk
agar, as differentiated by measuring the zone of clearance surrounding the LAB
spot ....................................................................................................................... 134
Figure 5.2 LC-FTMS chromatograms of standard antifungal compounds and those
identified in MRS fermented by L. amylovorus DSM 19280 ................................ 136
Figure 5.3 Microbial counts of non-starter adjunct lactobacilli in MRS agar in
miniature Cheddar-type cheeses after 7, 14, 30, 60, and 90 d of ripening. Cheeses
made using antifungal adjunct L. amylovorus DSM 19280 (●) and L. amylovorus
DSM 20531 (▲). .................................................................................................. 139
Figure 5.4 Growth of P. expansum (A) and airborne mould (B) on the surface of
miniature Cheddar cheeses. Cheese produced using antifungal adjunct
L. amylovorus DSM 19280 (●), L. amylovorus DSM 20531 (▲), control adjunct-
free cheese ((cid:1)). .................................................................................................... 142
Figue 6.1 Antifungal activity against F. culmorum of L. brevis PS1 (1) and non-
inhibiting control L. brevis NS (2) by overlay method on mMRS5 agar plate 163
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Figure 6.2 LCMS chromatogram of antifungal compounds found in MRS fermented
by L. brevis PS1 .................................................................................................... 164
Figure 6.3 Growth of L. brevis PS1 (●) and L. brevis NS (■) in wort (A). Changes in
pH (L. brevis PS1 (●), L. brevis NS (■)) and TTA (L. brevis PS1 (○), L. brevis NS
(□)) over 48h wort fermentation period by L. brevis strains (B).......................... 168
Figure 6.4 Growth of F. culmorum in wort fermented by the antifungal strain
L. brevis PS1 (●), control strain showing no inhibitory activity L. brevis NS (■),
uninoculated wort (▲). (A) sterile filtered sample, (B) pasteurized sample ....... 170
Figure 6.5 Inhibitory effect against F. culmorum of wort fermented by the antifungal
strain L. brevis PS1 (1) and showing no antifungal activity strain L. brevis NS (2).
(3) is the sample of uninoculated wort contaminated by F. culmorum, (4) is
uninoculated wort with no spores added. Each sample is performed in triplicate.
.............................................................................................................................. 171

7
List of Tables
Table 2.1 Mycotoxins commonly found in food and feed .......................................... 27
Table 2.2 Lactic acid bacteria with the ability to inhibit mould growth ................... 35
Table 3.1 Antifungal activity of selected LAB against spoilage moulds. Halo
measured in mm. ..................................................................................................... 82
Table 3.2 Chromatographic, spectrometric and quantitative data (µg L-1) of
antifungal components produced by L. reuteri ee1p and M13·10-5 3 strains. Values
are means (n = 2): the relative standard deviations for all compounds were <6%
................................................................................................................................ 91
Table 4.1 Wheat dough formulations ...................................................................... 106
Table 4.2 Inhibitory activity of L. reuteri R29 and L. reuteri hhIp strains against a
range of reference fungi as determined by the overlay method ........................... 107
Table 4.3 Quantitave amounts (mg L-1) of antifungal components produced by
L. reuteri strains ................................................................................................... 110
Table 4.4 Characteristics of bread fermented with L. reuteri strains and control
bread ..................................................................................................................... 113
Table 5.1 Chromatographic, spectrometric and quantitative data (mg L-1) of
antifungal components produced by L. amylovorus DSM 19280......................... 137
Table 5.2 Biochemical analysis of 90-day-old cheeses ........................................... 140
Table 5.3 Shelf-life of cheeses made with antifungal adjunct L. amylovorus DSM
19280 and L. amylovorus DSM 20531 compared with adjunct-free control cheeses
.............................................................................................................................. 141
Table 6.1 Chromatographic, spectrometric and quantitative data (mg L-1) of
antifungal components produced by L. brevis PS1 and NS strain ....................... 165
Table 6.2 Concentrations of organic acids, ethanol, pH and TTA values and viable
cell counts in the supernatant of wort inoculated with 108 bacteria mL-1 and
incubated at 30 °C for 48 h .................................................................................. 169
8
Declaration
I hereby declare that this thesis is my own work and effort and that it has not been
submitted anywhere for any award. Where other sources of information have been
used, they have been acknowledged.
Signature:

Date: 05/07/2013

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