Uq332802 Oa

Accepted Manuscript
Isolation of lactic acid bacteria with antifungal activity against the common cheese
spoilage mould Penicillium commune and their potential as biopreservatives in
cheese
Elsie Y.L. Cheong , Amrita Sandhu , Jayaram Jayabalan , Thu Thi Kieu Le , Nguyen
Thi Nhiep , Huong Thi My Ho , Jutta Zwielehner , Nidhi Bansal , Mark S. Turner
PII: S0956-7135(14)00257-6
DOI: 10.1016/j.foodcont.2014.05.011
Reference: JFCO 3844
To appear in: Food Control
Received Date: 9 July 2013

Revised Date: 6 May 2014
Accepted Date: 10 May 2014
Please cite this article as: CheongE.Y.L., SandhuA., JayabalanJ., Kieu LeT.T., NhiepN.T., My HoH.T.,
ZwielehnerJ., BansalN. & TurnerM.S., Isolation of lactic acid bacteria with antifungal activity against the
common cheese spoilage mould Penicillium commune and their potential as biopreservatives in cheese,
Food Control (2014), doi: 10.1016/j.foodcont.2014.05.011.
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1 Isolation of lactic acid bacteria with antifungal activity against the common cheese spoilage
2 mould Penicillium commune and their potential as biopreservatives in cheese.
4 Elsie Y. L. Cheong1, Amrita Sandhu1, Jayaram Jayabalan1, Thu Thi Kieu Le1,2, Nguyen Thi
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5 Nhiep 1, Huong Thi My Ho1,3, Jutta Zwielehner1, Nidhi Bansal1 and Mark S. Turner1,4*
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6 School of Agriculture and Food Sciences, University of Queensland, Brisbane, Australia;
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7 Department of Human Nutrition, Faculty of Food Science and Technology, Nong Lam
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8 University, Ho Chi Minh City, Vietnam; 3Department of Food Safety and Quality Assurance,
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9 Faculty of Food Technology, Ho Chi Minh City University of Food Industry, Ho Chi Minh City,
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10 Vietnam; 4Queensland Alliance for Agriculture and Food Innovation, University of Queensland,
11 Brisbane, Australia.
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*
13 Corresponding author:
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14 Mark S. Turner
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15 Tel: +61-7 33651171, Fax: +61- 7 33651177

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16 E mail. m.turner2@uq.edu.au
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18 Running head: Antifungal activity of lactic acid bacteria on cheese spoilage moulds.
19 Key words: Antifungal, lactic acid bacteria, Penicillium, cheese spoilage, biopreservative
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20 Abstract
21 Moulds are the most common cheese spoilage organisms which can lead to economic loss as
22 well as raising public health concerns due to the production of mycotoxins. In this study, 897
23 lactic acid bacteria (LAB) isolated from different herbs, fruits and vegetables were screened
24 for their antifungal activity in an agar plate overlay assay. Thirty-six isolates had weak
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25 activity, 11 had moderate activity and 12 were confirmed as having strong activity. The
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26 strong antifungal isolates were obtained from a range of different sources but were all
27 identified by 16S rDNA sequencing as being Lactobacillus plantarum. The antifungal spectra
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28 for these 12 isolates were determined against eight other moulds commonly associated with
29 cheese spoilage and all isolates were found to possess inhibition against Penicillium solitum,
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Aspergillus versicolour and Cladosporium herbarum, but not against Penicillium roqueforti,
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31 Penicillium glabrum, Mucor circinelloides, Geotricum candidum or Byssochlamys nivea. The
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32 absence of sodium acetate from MRS agar resulted in no inhibition of P. commune,
33 suggesting the synergistic effect of acetic acid with the antifungal LAB, similarly to that
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34 previously reported. To determine their potential as biopreservatives in cheese, LAB isolates

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35 were inoculated into cottage cheese prior to the addition of P. commune. All Lb. plantarum
36 isolates were found to prevent the visible growth of P. commune on cottage cheese by
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37 between 14 to >25 days longer than cottage cheese that contained either no added LAB or
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38 LAB that did not have antifungal activity (Lactococcus lactis, Weisella soli, Leuconostoc
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39 inhae and Leuconostoc mesenteroides isolates). The results of this study shows that LAB
40 isolated from various herbs, fruits and vegetables possess antifungal activity and have
41 potential for use as biopreservatives in cheese.
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43 1. Introduction
44 Fungal food spoilage is one of the main causes of food and economical loss all over the
45 world. Despite the dry climate and advanced technology, food loss due to fungal spoilage in
46 Australia is estimated to be more than $10,000,000 per annum, and it would be expected that
47 this level would be significantly higher in more humid countries with less developed
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48 technology (Pitt & Hocking, 2009). Although fungi such as yeast and moulds have been used
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49 in the processing of many cheese and fermented products (Marth & Yousef, 1991) they are
50 also responsible for the spoilage of many processed dairy foods (Fente-Sampayo et al., 1995).
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51
52 According to Pitt and Hocking (2009), cheese is very susceptible to the growth of mould,
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which makes it unsuitable for sale and consumption. Some of these spoilage moulds in cheese
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54 may also produce mycotoxins, such as Sterigmatocystin produced by Aspergillus versicolor,
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55 which could have serious consequences on consumers’ health (Northolt, van Egmond,
56 Soentoro & Deijll, 1980). The growth of mould in cheese is largely contributed by mould’s
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57 ability to grow at refrigeration temperature, low oxygen concentrations, low pH and low water
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58 activity. These moulds are also often resistant to the preservative action of free fatty acids and
59 have lipolytic activity that allows them to cause spoilage in cheese.

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60
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61 Various techniques have been applied to inhibit mould growth in retail cheese. Since most
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62 mould spores are killed by pasteurization of milk (Doyle & Marth, 1975), it is important to
63 prevent the recontamination and growth of mould. Modified atmosphere packaging (MAP)
64 has been used successfully to retard or prevent mould growth, and other treatments such as
65 the use of chemical preservatives: sorbates, propionate and natamycin have also been applied
66 as mould inhibitors (Ledenbach & Marshall, 2009). However, these techniques do not show
67 complete effectiveness as the ideal gas composition in MAP may vary from different varieties
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68 of cheese and the use of sorbates may lead to a “kerosene” flavour defect after being
69 decaboxylated by some fungi (specifically Penicillium species) (Liewen, 1992; Marth &
70 Yousef, 1991). Besides the negative effects on taste and flavour, consumers are also
71 becoming more concerned over the use of preservatives and chemicals in food, driving
72 demand towards natural and organic products. Therefore, there is a significant interest to
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73 develop natural preservatives to enhance or replace chemical treatments.
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75 Biopreservation or biocontrol refers to the use of natural or controlled microflora, or its
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76 antibacterial products to extend the shelf life and enhance the safety of foods (Stiles, 1996).
77 Since lactic acid bacteria (LAB) occur naturally in many food systems and have a long history
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of safe use in fermented foods, thus classed as Generally Regarded As Safe (GRAS), they
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79 have a great potential for extended use in biopreservation. Multiple publications have
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80 identified the antibacterial (O’Sullivan, Ross & Hill, 2002) and antifungal (Crowley, Mahony,
81 & van Sinderen, 2013b; Dalié, Deschamps & Richard-Forgot, 2010; Magnusson, Ström,
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82 Roos, Sjögren, & Schnürer, 2003; Schnürer & Magnusson, 2005) activities of LAB. The
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83 antimicrobial activity of LAB has been credited to the production of several antimicrobial
84 substances such as lactic acid, which lowers the pH in food and helps inhibit the growth of
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85 other microorganisms (Brul & Coote, 1999). Other compounds such as hydrogen peroxide,
86 acetic acid, reuterin, diacetyl and bacteriocins also contribute to its preserving capabilities
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87 (Caplice & Fitzgerald, 1999; Lindgren & Dobrogosz, 1990). LAB are commonly found on
88 fresh fruits and vegetables and these sources provide a potential source of new antimicrobial
89 strains. LAB from these sources have been investigated for anti-Listeria (Allende et al., 2007;
90 Trias, Babosa, Montesinos & Bañeras, 2008) and anti-mould activity (Sathe, Nawani,
91 Dhakephalkar & Kapadnis, 2007; Trias, Bañeras, Montesinos & Badosa, 2008) however only
92 limited work has been carried out specifically investigating the usefulness of LAB in
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93 controlling fungal growth in cheese (Garcha, & Natt, 2012; Muhialdin, Hassan, & Sadon,
94 2011; Schwenninger & Meile, 2004; Zhao, 2011)
95 This study aims to identify and investigate antifungal activity of LAB isolated from herbs,
96 fruits and vegetables against moulds commonly associated with cheese spoilage, with a focus
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97 on Penicillium commune which is a major cheese spoilage mould (Hocking, 1994). Those
98 LAB with anti-mould activity were then tested for their abilities to prevent mould growth
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99 when applied to cottage cheese, with the future perspective of using them in cheese
preservation.
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100
101
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102 2. Materials and Methods
103 2.1. Bacteria and mould strains and media.

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104 Eight hundred and ninety seven LAB isolated from fresh herbs, fruits and vegetables (see
105 Supplementary data) purchased from local supermarkets, grocers, markets and farms were
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106 isolated using de Mann Rogosa Sharpe (MRS; Oxoid Ltd) agar and incubation at 30°C for 3
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107 days under anaerobic conditions (AnaeroGen system; Oxoid Ltd.). They were then stored at -
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108 80°C in MRS broth supplemented with 40% glycerol. For the overlay assay (see below), two
109 different media were evaluated: MRS and modified MRS without sodium acetate (mMRS).
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110 The bacteria were inoculated on agar plates and incubated for 48 hours at 30°C under
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111 anaerobic conditions.
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113 Nine mould species, P. commune (FRR no. 4117), Penicillium roqueforti (FRR no. 0058),
114 Penicillium glabrum (FRR no. 4190), Penicillium solitum (FRR no. 4195), Geotricum
115 candidum (FRR no. 4204), A. versicolor (FRR no. 0038), Mucor circinelloides (FRR no.
116 4846), Byssochlamys nivea (FRR no. 4376), Cladosporium herbarum (FRR no. 4199) were
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117 obtained from CSIRO FRR culture collection (CSIRO, North Ryde, Australia). Fungal
118 inocula were prepared by resuspending the freeze dried lyophilized mould cultures in distilled
119 water and growing them on malt extract agar (MEA, Difco™) slants at 25°C for 7 days (or
120 until sporulation). Spores were collected by vigorously shaking slants after adding sterile
121 peptone water (0.2% w/v). The concentration of the P. commune inocula were adjusted with
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122 sterile peptone water (0.2% w/v) to an absorbance of 0.5 at 600nm using a spectrophotometer
which resulted in a spore number of ~1 x 106 spores/ml.
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123
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124 2.2. Screening of LAB for antifungal activity.
125 Antifungal activity assay was carried out using the overlay method described by Rouse et al.
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(2008) with slight modification. All 897 LAB isolates were tested for their antifungal activity
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127 against P. commune as an initial screening step. P. commune was selected as the indicator
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128 mould due to its common occurrence as spoilage mould on cheese (Hocking, 1994). Initial
129 screening was carried out by spotting 2µl of 18 LAB isolates from the frozen stock on one
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130 MRS agar plate. The plates were incubated anaerobically at 30°C for 48 hours. The plates
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131 were then overlaid with 6ml of malt extract soft agar (1.5% malt extract, 0.7% agar; Difco™)
132 containing 0.1ml (1 x 106 spores/ml) of P. commune and incubated aerobically at room
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133 temperature (~25°C) for 3-4 days or until a uniform layer of mould growth was observed.
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134 Zones of inhibition around the LAB spots were recorded according to the following scale: (-)
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135 no inhibition, colonies are entirely covered by mould, (+) weak inhibition seen on the LAB
136 colony but no distinct clearing zone near the LAB colony, (++) moderate inhibition with
137 small clearing zone near the LAB colony and (+++) strong inhibition with a large zone of
138 clearing around the LAB colony.
139
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140 LAB isolates that displayed strong inhibition in the preliminary screening were selected for
141 confirmation using the same overlay method. Two LAB isolates with antifungal activity and
142 one LAB isolate that displayed no antifungal activity (negative control) were spotted as above
143 but on a single MRS agar plates and zones of inhibition were recorded according to that
144 above.
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145 2.3. Screening of LAB antifungal activity on modified MRS.
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146 LAB isolates selected for confirmation screening were also tested using the same overlay
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147 method on modified MRS (mMRS) agar to determine the effect of sodium acetate on
148 antifungal activity. The mMRS agar was prepared according to MRS (Oxoid) formula without
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sodium acetate (10 g/l peptone, 8g/l “Lab-Lemco” powder, 4g/l yeast extract, 20g/l glucose,
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150 1ml/l C24H46O6, 2g/l K2HPO4, 2g/l C6H8O7×2NH3, 0.2g/l MgSO4×7H2O and 0.05g/l
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151 MnSO4×4H2O).
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152 2.4. Antifungal activity spectrum.

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153 LAB isolates that possessed strong antifungal activity were screened against eight other
moulds (P. roqueforti, P. glabrum, P. solitum, G. candidum, A. versicolor, M. circinelloides,

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155 B. nivea, C. herbarum) using the same overlay method described above with one LAB on
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156 each plate to determine their antifungal activity spectrum. These moulds were selected
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157 because of their common occurrence in cheese spoilage and ability to produce mycotoxins (A.
158 versicolor).
159 2.5 Antifungal activity of LAB against P. commune in cottage cheese.
160 LAB that displayed strong antifungal activity on MRS agar were selected for testing against
161 P. commune on cottage cheese. Some LAB with no antifungal activity were also selected as
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162 negative controls. LAB isolates were grown anaerobically in MRS broth for 48 hours at 30°C.
163 After incubation, the optical density of each LAB in broth was measured and recorded, and
164 2ml was centrifuged at 16,873 x g (Eppendorf Centrifuge, Model 5418) for 1 minute to obtain
165 the bacterial cell pellet. The spent MRS broth was discarded and bacterial cells were
166 resuspended with peptone water (0.1% w/v) to obtain a standard number of cells per ml using
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167 the OD600nm as a reference. Each LAB suspension was estimated to contain 1 x 109 CFU/ml
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168 and the exact concentration of each LAB suspension was determined by plating dilutions on
169 MRS agar and incubation.
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170 Twelve grams of commercially obtained cottage cheese which contained 11.3g protein, 5.4g
171 fat and 2.6g sugar per 100g (Dairy Farmers, Queensland, Australia) were weighed out on each
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petri dish (9cm in diameter; Labtek) and inoculated with 0.1ml of LAB suspension. The
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173 cottage cheese was mixed on the petri dish using a sterile spreader for at least 1 minute to
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174 ensure even distribution of bacterial cells. Controls were prepared by inoculating cottage
175 cheese with 0.1ml of peptone water not containing LAB. The plates were then incubated at
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176 room temperature (~24°C) for 2 days. P. commune fungal inoculum was prepared according
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177 to the method described earlier and 0.1ml (1 x 106 spores/ml) was inoculated onto each
178 cottage cheese plate (final concentration was ~1 x 104 spores/g of cottage cheese), including
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179 control plates without LAB, and mixed again as above to ensure even distribution. Plates were
180 incubated at room temperature and examined every few days for mould growth: (-) no mould
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181 growth; (+) small mould spots; (++) moderate sized mould spots or patches; (+++) mostly or
182 completely covered by mould. Each strain was tested at least twice and consistent results
183 were obtained. The data presented is from the replicate experiment which showed the least
184 antifungal activity.
185 2.6. Identification of LAB.

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186 LAB that possessed strong antifungal activity were selected for identification by sequence
187 analysis of 16S rDNA (Ström, Sjögren, Broberg & Schnürer, 2002). The LAB were grown
188 overnight in MRS broth anaerobically at 30°C and subsequently centrifuged to obtain the
189 bacteria cell pellet. Bacteria DNA was extracted using the previously described method
190 (Prasad & Turner, 2011). Amplification of the 16S rDNA gene was done by polymerase chain
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191 reaction (PCR) (94°C for 2 min, and 30 cycles of 94°C/20s, 53°C/30s, 72°C/1.5min) using
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192 primers 16S-S Forward (5’-AGAGTTTGATCCTGGCTC-3’) and 16S-R Reverse (5’-
193 CGGGAACGTATTCACCG-3’). The resulting PCR products were sent for purification and
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194 sequencing at Macrogen (South Korea). The partial 16S rDNA sequences of approximately
195 250-500bp were used to search public databases (Genbank using BLAST and the Ribosomal
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Database Project) for the identification of the LAB with the closest species match being
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197 reported.
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198
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200
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201 3. Results
202 3.1. Screening of LAB for antifungal activity.

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203 Out of the 897 LAB screened for antifungal activity against the indicator mould P. commune,
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204 approximately 7% showed antifungal activity during the initial screening. Figure 1 shows the
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205 percentage of antifungal LAB isolates out of the total number of LAB isolated from particular
206 herbs, fruits and vegetables which were sources of antifungal LAB. A variety of sources
207 yielded LAB with antifungal activity with thirty-six isolates showed weak inhibition (+), 11
208 isolates showed moderate inhibition (++), while 15 isolates showed strong (+++) inhibition
209 (Figure 1). After re-testing, 12 out of 15 of the strong inhibiting isolates generated
210 reproducible large zones of inhibition (Figure 2) and 4 isolates with no activity were also
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211 selected for further study as controls. LAB isolates that displayed antifungal activity on MRS
212 agar did not produce the same antifungal activity when tested on modified MRS agar made
213 without sodium acetate (data not shown).
214 3.2. Antifungal activity spectrum of LAB.
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215 The 12 LAB selected after confirmation of antifungal activity on MRS were tested against a
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216 selection of moulds to determine the range of their inhibitory activity. Eight other mould
217 species were chosen based on their involvement in cheese spoilage. It was found that all 12
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218 isolates had the same inhibitory spectrum (activity against P. solitum, A. versicolour and C.
219 herbarum, but not against P. roqueforti, P. glabrum, G. candidum, M. circinelloides and B.
220 nivea).
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221 3.3. Identification of anti-fungal LAB.
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222 LAB species were identified via partial 16S rDNA sequencing (Table 1). Out of the 12
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223 isolates that displayed antifungal activity, all isolates were identified as Lactobacillus
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224 plantarum. Four LAB that previously displayed no antifungal activity on MRS agar were
225 identified as Weissella soli, Lactococcus lactis, Leuconostoc inhae and Leuconostoc
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226 mesenteroides.
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227 3.4. Antifungal activity of LAB against P. commune on cottage cheese.

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228 The results of the antifungal activity of different species of LAB against P. commune that was
229 inoculated (~104 spores/g) on cottage cheese are shown in Table 1. The first control (control
230 1) was cheese that was inoculated with P. commune but without LAB, was found to display
231 moderate mould growth from day 4 and was completely covered in dark green mould on day
232 12 (Figure 3). Cottage cheese inoculated with antifungal LAB Lb plantarum isolates did not
233 show signs of mould growth until at least day 18 with some cheese not showing any visible
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234 mould at day 29 (Table 1 and Figure 3). Of note are two Lb. plantarum strains (#170 and
235 377) which were able to prevent visible mould growth beyond day 29, the last time-point of
236 the experiment.
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238 Four LAB isolates that did not display antifungal activity on MRS agars were also used as
239 negative controls. Cheese containing W. soli #33, Lc. lactis #49, Le. inhae #402 and Le.
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240 mesenteroides #844 all had visible mould growth at day 4 and were completely covered in
mould by day 12 (Table 1 and Figure 3).
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241
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243 4. Discussion
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244 LAB have a long history of use in fermented food products and are generally regarded as safe
245 organisms. Due to the production of lactic acid and several antimicrobial compounds,
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246 extensive studies have been conducted on their preservative potential, both against
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247 pathogenic bacteria (O’Sullivan, Ross & Hill, 2002) and fungi (Schnürer & Magnusson,
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248 2005). Multiple publications have highlighted the ability of some LAB strains to repress
249 mould growth (Dalié, Deschamps & Richard-Forgot, 2010) however, to the best of our
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250 knowledge, no studies were conducted on the antifungal activity of LAB isolated from a wide
251 range of herbs, fruits and vegetables.

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252
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253 In the present study, 897 LAB previously isolated from herbs, fruits and vegetables were
254 screened against an indicator mould P. commune on MRS agar using an overlay method to
255 identify their antifungal properties. Twelve LAB isolates with strong or moderate antifungal
256 activity were identified to species level and were all found to be Lb. plantarum. In our
257 previous work, the same large collection of LAB isolates were tested for their antimicrobial
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258 activity against pathogens Listeria monocytogenes and Salmonella Typhimurium and the
259 majority of the antimicrobial LAB were identified as species from Lactococcus, Leuconostoc
260 and Weissella, including strains of Le. mesenteroides, W. cibaria and Lc. lactis (data not
261 shown). Interestingly however, no Lb. plantarum were identified in this screening of LAB
262 against L. monocytogenes and S. Typhimurium. This shows that the Lb. plantarum identified
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263 in this study may possess antifungal but not antibacterial activity and that the screening
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264 methods used are highly specific for the target organism i.e. mould. Likewise a recent similar
265 large scale screening of around 7000 isolates of presumptive LAB from a variety of sources
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266 against Penicillium expansum identified Lb. plantarum strains as the most common antifungal
267 LAB (Crowley, Mahony, & van Sinderen, 2013a). Results of our current study also showed
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that the antifungal activity of LAB is likely strain dependent. Lb. plantarum isolated from
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269 different sources displayed varying degrees of antifungal activity (e.g. isolates #170 and
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270 #892). This suggests that not all LAB of the same genera and species may be used as
271 biopreservatives and further steps are likely needed to be taken to differentiate strains of
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272 bacteria using genotyping methods.

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273
274 In order to investigate their effectiveness as cheese preservatives, these antifungal LAB were
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275 screened against a variety of moulds commonly associated with cheese to evaluate their
276 antifungal activity spectrum. According to ICMSF (1998) and Taniwaki et al. (2001),
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277 commonly isolated spoilage fungi from cheese include Penicillium, Aspergillus,
278 Cladosporium, Geotrichum, Mucor and Trichoderma, with Penicillium being the most
279 predominant flora associated with cheese spoilage. Among the Penicillium sp., P. commune
280 and P. roqueforti, both of which have been involved in cheese production, are the two most
281 common spoilage species found in Australian and New Zealand retail cheeses (Hocking,
282 1994). Although the antifungal activity spectrum of the antifungal LAB identified in this
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283 study is not very wide, the selected LAB isolates were found to be inhibitory against several
284 important spoilage moulds (P. solitum, C. herbarum and A. versicolour) in cheese. According
285 to Oyugi and Buys (2007) and Hocking (1994), P. solitum is the main species of mould found
286 in shredded cheeses in South Africa and Australia while C. herbarum is commonly associated
287 with cheese with thread mould defects in Australian cheese factories (Hocking, 1994). A.
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288 versicolour, though not as commonly isolated from cheese spoilage, is an important mould
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289 that produces mycotoxins that could potentially harm consumers. None of the LAB were
290 found to be inhibitory against P. roqueforti, which makes it applicable as a biopreservative on
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291 blue cheese as P. roqueforti is often inoculated as secondary starter to achieve desired
292 properties in blue cheese. Similarly, antifungal LAB have also shown an activity spectrum
293
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which excluded P. roqueforti (Magnusson, Ström, Roos, Sjögren, & Schnürer, 2003).
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294
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295 The effect of sodium acetate on the antifungal activity of LAB was also investigated and
296 results showed that antifungal LAB requires sodium acetate in the MRS medium to exhibit
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297 antifungal activity. This finding is consistent with that of Schillinger and Villarreal (2010),
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298 who studied the influence of medium composition on the antifungal activity of LAB and
299 observed that LAB strains, previously shown to inhibit mould on MRS agar, were unable to
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300 produce inhibition zones when grown on MRS medium without sodium acetate or with
301 reduced glucose content. The authors also observed antifungal activity only when normal
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302 MRS broth containing 61mmol/l sodium acetate was used. Similarly, Cabo et al. (2002) and
303 Stiles et al. (2002) also reported a synergistic effect between acetate present in the growth
304 medium with lactic acid and other antifungal compounds produced by the LAB, and is likely
305 to be the main factor responsible for the antifungal properties of the selected strains. Our
306 finding further confirms the role of sodium acetate on the antifungal activity of LAB and
307 should be taken into consideration when evaluating the antifungal activity of LAB on MRS
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308 medium. However, it should be noted that the absence of sodium acetate from MRS agar did
309 not affect the antifungal activity of propionibacteria on P. roqueforti and Aspergillus
310 fumigatus (Lind, Jonsson & Schnürer, 2005).
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312 In order to evaluate the potential of these antifungal LAB as food biopreservatives, we tested
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313 them against P. commune on cottage cheese. Few papers reported the use of antimicrobial
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314 LAB in cheese, especially for their use in preventing mould spoilage. Neugebauer and
315 Gilliland (2005) studied the antagonistic action of Lb. delbrueckii RMZ-5 against
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316 Pseudomonas fluorescens on cottage cheese and found that the number of spoilage organisms
317 did not increase over 21 days with a treatment of 1 x 109 CFU/g Lb. delbrueckii RMZ-5.
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Strains of Bifidobacterium infantis and Bifidobacterium breve were also found to reduce the
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319 levels of Pseudomonas on cottage cheese (O’Riordan & Fitzgerald, 1998). Along with these
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320 studies using cottage cheese as a model, we also found this product to be a quick and simple
321 matrix for evaluation of the biopreservative potential of LAB on cheese. In our study,
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322 antifungal isolates of Lb. plantarum, were all able to prevent the growth of P. commune on
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323 cottage cheese for up to 14 to >25 days more than the no LAB control, however future work
324 looking at testing these LAB in other cheese will be of interest.

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325
326 The isolation of antifungal Lb. plantarum in this study supports the findings of several
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327 publications on the antifungal activity of this species. Lb. plantarum strains have been
328 extensively investigated as mould controlling agents in bread where they have shown positive
329 results (Coda et al., 2011; Dal Bello et al., 2007; Moore, Dal Bello and Arendt, 2008). The
330 use of Lb. plantarum in combination with calcium propionate was also found to inhibit mould
331 growth better than using calcium propionate alone (Ryan, Dal Bello & Arendt, 2008).
332 Besides the application on baked products, Sathe, Nawani, Dhakephalkar and Kapadnis
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333 (2007) studied the potential of LAB to prolong shelf life of fresh vegetables and found that
334 cell-free supernatant of Lb. plantarum inoculated into vegetables were able to significantly
335 delay fungal spoilage when challenged by Aspergillus flavus, Fusarium graminearum,
336 Rhizopus stolonifer and Botrytis cinerea. Application in of Lb. plantarum to control mould in
337 fruits, including apples and kumquats has also been demonstrated (Trias, Bañeras, Montesinos
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338 & Badosa, 2008; Wang, et al., 2013).
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340 Besides lactic acid, several compounds have been identified as the mechanism of action of Lb.
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341 plantarum. Phenyllactic acid and two cyclic dipeptides cyclo (L-Leu-L-Pro) and cyclo (L-
342 Phe-L-Pro) were identified in the cell-free supernatant of antifungal Lb. plantarum FST 1.7
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(Dal Bello et al., 2007), while cyclic dipeptides cyclo(L-Phe–L-Pro) and cyclo(L-Phe–trans-4-
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344 OH-L-Pro), were identified as the mechanism of action of antifungal Lb. plantarum
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345 MiLAB393 (Ström, Sjögren, Broberg & Schnürer, 2002). Lavermicocca et al. (2000) also
346 found that the production of phenyllactic and 4-hydroxy-phenyllactic acids contributed to the
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347 antifungal activity of Lb. plantarum 21B and in a later study, the same authors discovered that
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348 less than 7.5 mg/ml of phenyllactic acid were required to obtain full inhibition of mould
349 (Lavermicocca, Valerio & Visconti, 2003). Niku-Paavola, Laitila, Mattila-Sandholm and
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350 Haikara (1999) also isolated and identified benzoic acid, methylhydantoin, mevalonolactone
351 and cyclo (Gly-L-Leu) in the culture filtrate of Lb. plantarum VTT E-78076, and found it to
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352 be active against Fusarium avenaceum VTT D-80147. Recent work has identified antifungal
353 active compounds from isolates of Lb. plantarum from kimchi as 5-oxododecanoic acid, 3-
354 hydroxy decanoic acid and 3-hydroxy-5-dodecenoic acid (Ryu, Yang, Woo, & Chang, 2014)
355 or 3,6-bis(2-methylpropyl)-2,5-piperazinedion (Yang & Chang, 2010). A genome shuffling
356 approach has been used to enhance the antifungal activity of Lb. plantarum IMAU10014
357 (Wang, et al., 2013) and genome sequencing of antifungal Lb. plantarum strain 16 (Crowley,
15
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358 Bottacini, Mahony & van Sinderen, 2013) will provide a better understanding of the
359 antifungal mechanisms of Lb. plantarum.
360
361 The current study shows that LAB from different fruits and vegetables and from different
362 genera and species can exhibit antifungal activity against a number of common cheese
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363 spoilage moulds, particularly P. commune. The antifungal activity is observed not only on
364 MRS agar, but on cottage cheese as well, indicating potential for the control of spoilage
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365 moulds in cheese products. Further investigations to identify the minimum inhibitory
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366 concentration (MIC) of each species, as well as the characterization of the LAB antifungal
367 compounds may help in understanding the antifungal activity of these LAB. In order for LAB
368
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to be successfully applied as biopreservatives in cheese, more studies need to be done to
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369 explore the effectiveness on different variety of cheeses as well as the methods of application.
370 Sensory analysis would also be required to determine whether the LAB and/or their antifungal
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371 compounds would impart undesirable flavours to the cheese and cheese products.
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372
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373 Acknowledgements
374 Part of this project was funded by Horticulture Australia Limited (Grant No. VG09075). We
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375 thank Prascilla Prasad for her technical support in this project.
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376
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377 References
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498 OH-L-Pro) and 3-phenyllactic acid. Applied and Environmental Microbiology, 68(9), 4322-
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510 Genome shuffling of Lactobacillus plantarum for improving antifungal activity. Food
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516 Use to Inhibit Mold Growth in Cheese. (Masters Thesis). California Polytechnic State
517 University San Luis Obispo. California, USA.
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533 Figure legends.
534 Figure 1. Sources of LAB isolates with different levels of antifungal activity against P.
535 commune on MRS agar. Numbers on the bars indicate number of LAB in each group, while
536 the percentage of isolates from each food source is shown on the X-axis. Note: only foods are
537 shown which contained antifungal LAB. Antifungal activity scale (-, +, ++ and +++) is
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538 mentioned in section 2.2.
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539
540 Figure 2. Antifungal activity of LAB isolates 49 (antifungal score of -), 897 and 892
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541 (antifungal scores of +++) on MRS agar overlaid with P. commune. A and B are images from
542 the bottom and top of the same agar plate, respectively.
543
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544 Figure 3. Images showing growth of P. commune on cottage cheese with or without
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545 inoculated LAB.
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Table 1. Antifungal activity of selected LAB against P. commune on cottage cheese.
P. commune growth on cottage cheese*

No. Species Day
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Source LAB
LAB with antifungal activity on MRS agar CFU/g 0 4 8 12 16 18 20 25 29
170 Lb. plantarum Stevia (sweet leaf) 107 - - - - - - - - -
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377 Lb. plantarum Baby endive 107 - - - - - - - - -
845 Lb. plantarum Parsnips 107 - - - - - - + + +
108
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880 Lb. plantarum Asian Vegetables - - - - - + + + +
883 Lb. plantarum Spinach 107 - - - - - - - + +
884 Lb. plantarum Cos Lettuce 107 - - - - - + + ++ ++
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885 Lb. plantarum Broccoli 107 - - - - - + + + +
871 Lb. plantarum Red capsicum 107 - - - - - + + + +
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891 Lb. plantarum Cos lettuce 107 - - - - - + + + ++
892 Lb. plantarum Broccoli 107 - - - - - + + ++ +++
107
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895 Lb. plantarum Spinach - - - - - + + + +
897 Lb. plantarum Green bean 107 - - - - - - + + +
Control LAB with no antifungal activity on MRS agar
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33 W. soli Mixed salad 107 - ++ ++ +++ +++ +++ +++ +++ +++
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49 Lc. lactis Flat parsley 107 - + + +++ +++ +++ +++ +++ +++
402 Le. inhae Baby Rocket 108 - + +++ +++ +++ +++ +++ +++ +++
844 Le. mesenteroides Rocket leaves 107 - + + +++ +++ +++ +++ +++ +++
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Controls
1 no LAB 0 - ++ +++ +++ +++ +++ +++ +++ +++
2 no LAB and no P. commune 0 - - - - - - - - -
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550 *Unless stated all cheese samples were inoculated with P. commune. Scoring was as follows: (-) no mould growth; (+) small mould spots; (++) moderate sized mould spots
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551 or patches; (+++) mostly or completely covered by mould.

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Figure 1.
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Watermelon 8 1 2
Sprouts 18 1 1
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Spinach 53 12 2
Rocket leaves 34 1
Rock Melon 5 2
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Pear 6 2
Pawpaw 12 3
Parsnips 1 1 1
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Mixed vegetables 27 2 1 1
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Lettuce 111 9 32
(-)
Source
Herbs 135 21
(+)
Green Bean 18 1 1
(++)
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Cucumber 8 1
(+++)
Cherry tomatoes 9 1
Celery 2 1
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Capsicum 5 1 1
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Cabbage 31 2 1
Broccoli 17 2 1 2
Beetroot 15 3
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Asian Vegetable 9 2
Apple 25 1
0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
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Percentage of isolates
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Figure 2.
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Figure 3.
Control Day 4 Day 8 Day 12
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no LAB
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LAB with no
antifungal activity
Day 4 Day 8 Day 16
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W. soli
#33
Lc. lactis
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#49
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Le. mesenteroides
#844
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LAB with
antifungal activity Day 4 Day 8 Day 20
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Lb. plantarum
#170
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Lb. plantarum
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#845
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Lb. plantarum
#895
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Highlights
► Antifungal activity of 897 lactic acid bacteria was tested against Penicilium commune.
► All 12 strong antifungal lactic acid bacteria were Lactobacillus plantarum.
► Inhibition of P. solitum, Aspergillus versicolour & Cladosporium herbarum was seen.
► Antifungal isolates significantly inhibited P. commune growth in cottage cheese.
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LAB strains which
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possessed different
levels of antifungal
activity (scored as +,
++ or +++) on MRS
agar
Activity (+)
Strain No. Source of LAB
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46 Assorted lettuce (organic lettuce)
96 Cos lettuce
89 Fresh herb
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127 Fancy lettuce
147 Beetroot
151 Cos lettuce
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186 Green cabbage
212 Coriander
229 Chinese Broc
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238 Baby leaves with beetroot
245 Traditional stir fry vegetables
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276 Mixed salad: green lettuce, red lettuce, spinach, rocket lettuce.
280 Aromatic spinach blend
285 Baby leaves with beetroot
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336 Baby red capsicums

356 Snow pea sprouts
365 Parsley
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391 Summer lettuce

400 Summer lettuce
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401 Baby rocket

499 Iceberg lettuce
513 Baby cos leaf
526 Iceberg lettuce
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543 Cucumber
548 Nashi
552 Apple (Pink lady)
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685 Chinese cabbage

735 Pawpaw
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741 Persimmous pear

744 Rock melon
749 Rock melon
762 Watermelon
782 Pawpaw
789 Pawpaw
817 Green Bean
827 Parsnips
Activity (++)
97 Cos Iceberg lettuce
99 Iceberg lettuceACCEPTED MANUSCRIPT
181 Chinese Broc
217 Baby spinach
269 Traditional stir fry vegetables
275 Cherry tomatoes
289 English spinach
505 Iceberg lettuce
758 Watermelon
759 Watermelon
804 Organically Spouts
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Activity (+++)
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845 Parsnips
883 Vietnamese spinach (mong toi)
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884 Cos lettuce
885 Brocolli
890 Asian vegetable (Cai ngot)
891 Cos lettuce
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897 Green Bean
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895 Vietnamese spinach (mong toi)
170 Sweet leaves
871 Capsicum red
880 Asian Vegetable (Rau den)
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892 Brocolli
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436
437
438
439
440
442
443
444
445
446
447
448
449
450
PT
451
452
453
RI
454
455
456
SC
457
458
459
U
460
461
AN
462
463
464
M
465
466
467
D
468
469
TE
470
471
472
473
EP
474
475
476
C
477
478
AC
479
480
481
482
483
484
485
486
487
488
489
490
492
493
494
495
496
497
498
499
500
PT
501
502
503
RI
504
505
506
SC
507
508
509
U
510
511
AN
512
513
514
M
515
516
517
D
518
519
TE
520
521
522
523
EP
524
525
526
C
527
528
AC
529
530
531
532
533
534
535
536
537
538
539
540
542
543
544
545
546
547
548
549
550
PT
551
552
553
RI
554
555
556
SC
557
558
559
U
560
561
AN
562
563
564
M
565
566
567
D
568
569
TE
570
571
572
573
EP
574
575
576
C
577
578
AC
579
580
581
582
583
584
585
586
587
588
589
590
592
593
594
595
596
597
598
599
600
PT
601
602
603
RI
604
605
606
SC
607
608
609
U
610
611
AN
612
613
614
M
615
616
617
D
618
619
TE
620
621
622
623
EP
624
625
626
C
627
628
AC
629
630
631
632
633
634
635
636
637
638
639
640
642
643
644
645
646
647
648
649
650
PT
651
652
653
RI
654
655
656
SC
657
658
659
U
660
661
AN
662
663
664
M
665
666
667
D
668
669
TE
670
671
672
673
EP
674
675
676
C
677
678
AC
679
680
681
682
683
684
685
686
687
688
689
690
692
693
694
695
696
697
698
699
700
PT
701
702
703
RI
704
705
706
SC
707
708
709
U
710
711
AN
712
713
714
M
715
716
717
D
718
719
TE
720
721
722
723
EP
724
725
726
C
727
728
AC
729
730
731
732
733
734
735
736
737
738
739
740
742
743
744
745
746
747
748
749
750
PT
751
752
753
RI
754
755
756
SC
757
758
759
U
760
761
AN
762
763
764
M
765
766
767
D
768
769
TE
770
771
772
773
EP
774
775
776
C
777
778
AC
779
780
781
782
783
784
785
786
787
788
789
790
792
793
794
795
796
797
798
799
800
PT
801
802
803
RI
804
805
806
SC
807
808
809
U
810
811
AN
812
813
814
M
815
816
817
D
818
819
TE
820
821
822
823
EP
824
825
826
C
827
828
AC
829
830
831
832
833
834
835
836
837
838
839
840
842
843
844
845
846
847
848
849
850
PT
851
852
853
RI
854
855
856
SC
857
858
859
U
860
861
AN
862
863
864
M
865
866
867
D
868
869
TE
870
871
872
873
EP
874
875
876
C
877
878
AC
879
880
881
882
883
884
885
886
887
888
889
890
892
893
894
895
896
897
PT
RI
U SC
AN
M
D
TE
EP
C
AC
ACCEPTED MANUSCRIPT
Source of LAB
Mesculin leaves (salad mix)
PT
Coriander
Salad mix
Midi cos
RI
Rocket leaves
Green oak lettuce
SC
Coriander
Coriander
Salad mix
U
Spinach leaves
Green oak lettuce
AN
Rocket leaves
Rocket leaves
M
Flat parsley
Baby spinach
Red and green lettuce
D
Baby spinach
Iceberg lettuce
TE
Fancy lettuce
Mixed salad: green lettuce, red lettuce, spinach, rocket lettuce.
Green lettuce
Iceberg lettuce
EP

Baby spinach
C
Baby spinach
AC
Thai basil
Thai basil
Parley
Parley
Thai basil
Cos lettuce
Cos lettuce
Baby spinach
Baby spinach
Parley
Basil
ACCEPTED
Mixed salad: green lettuce, red lettuce, MANUSCRIPT
spinach, rocket lettuce.
Flat parley
Cos lettuce
Iceberg lettuce
Assorted lettuce (organic lettuce)
Iceberg lettuce
Iceberg lettuce
Flat parley
Flat parley
PT
Fancy lettuce
RI
Parley
Iceberg lettuce
Baby spinach
SC
Iceberg lettuce
Basil
Thai basil
U
Flat parley
AN
Parley
Parley
Sweet basil
M
Parley
Baby spinach
Sweet basil
D
Parley
English spinach
TE
Coriander
Coriander
Coriander
EP

English spinach
C
Iceberg lettuce
Parley
AC
Fancy lettuce
Coriander
Cos lettuce
Coriander
English spinach
Baby spinach
Coriander
Fancy lettuce
Celery
Fresh herb
Coriander
Parley ACCEPTED MANUSCRIPT
Parley
Fancy lettuce
Coriander
Celery
Cos lettuce
Cos Iceberg lettuce
Celery
Iceberg lettuce
Cos lettuce
PT
Coriander
Parley
RI
Celery
Celery
Iceberg lettuce
SC
Cos lettuce
Coriander
Iceberg lettuce
U
Iceberg lettuce
Coriander
AN
Iceberg lettuce
Cos Iceberg lettuce
Cos Iceberg lettuce
M
Cos Iceberg lettuce

Fancy lettuce
D
Celery
Fancy lettuce
TE
Fancy lettuce
Fancy lettuce
Iceberg lettuce
Cos Iceberg lettuce
EP
Fancy lettuce
Fancy lettuce
Iceberg lettuce
C
Fancy lettuce
Iceberg lettuce
AC
Parley
Iceberg lettuce
Spinach leaves
Cos lettuce
Fancy lettuce
Fresh herb
Parley
Organically sprouted
Fresh herb
sweet potato leaves
Red and green lettuce ACCEPTED MANUSCRIPT
Green bean leaves
Fresh herb
Mint
Mint
Beetroot
Beetroot
Beetroot
Fresh herb
Beetroot
PT
Cos lettuce
Chinese cabbage
Cos lettuce
RI
Mint
Pumpkin leaves
sweet potato leaves
SC
Chinese mint
Chinese cabbage
Coriander
U
Mint
Chinese mint
AN
Fancy lettuce
Chinese cabbage
Coriander
M
Sweet leaves
Pumpkin leaves
Coriander
D
Chinese mint
Chinese mint
TE
Sweet leaves
sweet potato leaves
Chinese mint
sweet leaves
EP
Pumpkin leaves
Green cabbage
Green cabbage
C
Coriander
Baby Choy sum
AC
Green cabbage
Pak choy
Chinese Broc
Celery
Broccoli
Celery
Broccoli
Green cabbage
Celery
Iceberg lettuce
Pak choy
Green bean leaves
sweet potato leaves ACCEPTED MANUSCRIPT
Pumpkin leaves
Pumpkin leaves
sweet potato leaves
Mint
Baby Choy sum
Celery
Coriander
Celery
Baby Choy sum
PT
Fancy lettuce
Pumpkin leaves
Broccoli
RI
Beetroot
Mint
Chinese Broc
SC
Celery
Sweet leaves
Beetroot
U
Mint
Fresh herb
AN
Coriander
Beetroot
Pumpkin leaves
M
Pumpkin leaves
Beetroot
Baby spinach
D
Iceberg lettuce
Celery
TE
Mint
Mint
Baby spinach
Celery
EP
Celery
Green bean leaves
Fresh herb
C
Mint
Broccoli
AC
Chinese Broc
Green bean leaves
Fresh herb
Salad mix
Baby spinach
Fancy lettuce
Fresh herb
Mint
Fresh herb
Baby leaves with beetroot
Baby Asian greens
Aromatic spinach blend
Aromatic spinach blend ACCEPTED MANUSCRIPT
Rainbow salad
Just broccoli
Traditional stir fry vegetables
Thyme
Thyme
Baby Asian greens
Thyme
Snow pea sprouts
PT
Baby brussels sprouts
Coleslaw
RI
Coleslaw
Cherry tomatoes
SC
Rainbow salad
U
Baby rocket
AN
Tuscan cabbage
M
Mint
Baby red capsicums
Coleslaw
D
Rainbow salad
TE
Rainbow salad
Spinach, rocket and kale
Rainbow salad
EP
Rainbow salad
Cherry tomatoes
C
Coleslaw
Baby Asian greens
AC
Mint
Baby rocket
Mint
English spinach
Iceberg lettuce
Baby leaves with beetroot ACCEPTED MANUSCRIPT
Rocket salad mix
Rocket salad mix
Cherry tomatoes
Cherry tomatoes
English spinach
Spinach leaves
English spinach
PT
Continental parley
Coleslaw
Tuscan cabbage
RI
Salanova red coral lettuce
Sweet raspberries
Spinach leaves
SC
Rocket salad mix
Baby Endive
U
Tuscan cabbage
Iceberg lettuce
AN
English spinach
Mint
Spinach leaves
M
Mint
English spinach
Strawberries
D
Butter lettuce
Tarragon leaves
TE
Oregano
English spinach
Rocket salad mix
Baby Asian greens
EP
Rocket leaves
Strawberries
Iceberg lettuce
C
Tuscan cabbage
English spinach
AC
Spinach leaves
English spinach
Baby Endive
Mint
Strawberries
Spinach leaves
Oz Berries
Baby red capsicums
Strawberries
Oregano
Coleslaw
Tuscan cabbage
Snow pea sprouts ACCEPTED MANUSCRIPT
Rocket leaves
Tuscan cabbage
Thyme
Treviso radicchio
Baby red capsicums
Tarragon leaves
Treviso radicchio
Baby Endive
Butter lettuce
PT
Iceberg lettuce
Sweet raspberries
Thyme
RI
Treviso radicchio
Baby red capsicums
Snow pea sprouts
SC
Salanova red coral lettuce
Thyme
U
Thyme
Rocket leaves
AN
Tuscan cabbage
Treviso radicchio
Cherry tomatoes
M
Parsley
Sweet raspberries
Treviso radicchio
D
Bistro salad
Tarragon leaves
TE
Butter lettuce
Sweet raspberries
Sweet raspberries
Oz Berries
EP
English spinach
Butter lettuce
Treviso radicchio
C
Baby Endive
Thyme
AC
Baby rocket
Oregano
Tarragon leaves
Baby red capsicums
Iceberg lettuce
Four leaf salad mix
Spinach leaves
Wild rocket
Spinach leaves
Baby mesculin
Summer lettuce
Baby mesculin
Summer lettuce ACCEPTED MANUSCRIPT
Summer lettuce
Spinach leaves
Fresh Italian Parsley
Baby rocket
Spinach leaves
Basil
Summer lettuce
Summer lettuce
PT
Baby rocket
Baby rocket
Summer lettuce
RI
Spinach leaves
Spinach leaves
Coriander
SC
Spinach leaves
Spinach leaves
baby mescullin salad
U
Baby spinach
Baby spinach
AN
Baby spinach
Red oak lettuce
M

Wild rocket
Baby spinach
D
Four leaf salad mix

Baby spinach
TE
Wild rocket
Baby spinach
Rocket leaves
Rocket leaves
EP
Leafy mix
Leafy mix
Green oak lettuce
C
Leafy mix
Leafy mix
AC
Leafy mix
Red oak lettuce
Red oak lettuce
Red oak lettuce
Red oak lettuce
Butter lettuce
Baby mesculin salad
Summer lettuce
Baby rocket
Red oak lettuce
Baby mesculin salad
Summer lettuce
Wild rocket ACCEPTED MANUSCRIPT
Baby mesculin salad
Four leaf salad mix
Baby spinach
Rocket leaves
Butter lettuce
Mint leaves
Baby rocket
Parsley
Mint leaves
PT
Coriander
Baby rocket
Coriander
RI
Mint leaves
Leafy mix
Mint leaves
SC
Baby coslettuce
Coriander
Mint leaves
U
Red oak lettuce
AN
Basil
Rosemary
Basil
M
Rocket leaves
Rocket leaves
D
Basil
Rocket leaves
TE
Baby coslettuce
Parsley
Green oak lettuce
Basil
EP
Parsley
Coriander
Parsley
C
Coriander
Red oak lettuce
AC
Leafy mix
Mint leaves
Mint leaves
Parsley
Butter lettuce
Green oak lettuce
Parsley
Parsley
Rosemary
Rocket leaves
Leafy mix
Mint leaves
Basil ACCEPTED MANUSCRIPT
Mixed florets (Broccoli)
Baby cos leaf
Gourmet salad
Fancy lettuce
Iceberg lettuce
Gourmet salad
Iceberg lettuce
PT
Celery
Iceberg lettuce
Fancy lettuce
RI
Gourmet salad
Iceberg lettuce
Celery
SC
Gourmet salad
Fancy lettuce
U
Baby cos leaf
AN
Gourmet salad
Baby cos leaf
Gourmet salad
M
Fancy lettuce
Iceberg lettuce
Fancy lettuce
D
Gourmet salad
Baby cos leaf
TE
Iceberg lettuce
Baby cos leaf
Celery
EP
Gourmet salad
Baby cos leaf
Iceberg lettuce
C
Baby rocket
Baby rocket
AC
Baby rocket
Coriander
Fresh herb
Fresh herb
Onion
Eggplant
Mint
Green bean
Fresh herb
Cucumber
Eggplant
Onion
Dragon bean ACCEPTED MANUSCRIPT
Cucumber
Cucumber
Dragon bean
Mint
Eggplant
Tomato
Nashi
Nashi
Nashi
PT
Eggplant
Apple (Pink lady)
Dragon bean
RI
Green bean
Green bean
Onion
SC
Nashi
Roma tomato
Onion
U
Fresh herb
Tomato
AN
Onion
Green bean
Apple (Pink lady)
M
Green bean
Green bean
Onion
D
Mint
Dragon bean
TE
Nashi
Apple (Pink lady)
Fresh herb
Cucumber
EP
Fresh herb
Nashi
Green bean
C
Dragon bean
Green bean
AC
Dragon bean
Roma tomato
Green bean
Roma tomato
Tomato
Nashi
Green bean
Tomato
Cucumber
Tomato
Onion
Dragon bean
Green bean ACCEPTED MANUSCRIPT
Onion
Dragon bean
Green bean
Zaccuchi
Nashi
Red grape
Cherry tomatoes
Banana
Nashi
PT
Nashi
Tomato
Mint
RI
Zaccuchi
Zaccuchi
Zaccuchi
SC
Green bean
Cherry tomatoes
Banana
U
Zaccuchi
Red grape
AN
Cherry tomatoes
Cherry tomatoes
Roma tomato
M
Starfruit
Nashi
Cherry tomatoes
D
Tomato
Endive
TE
Basil
Mint
Red grape
Purple perilla
EP
Purple perilla
Strawberries
Strawberries
C
Fuji apple
Red grape
AC
Fuji apple
Eggplant
Starfruit
Starfruit
Banana
Pawpaw
Fuji apple
Strawberries
Pawpaw
Tomato
Fresh herb
Apple (Granny Smith)
Apple (Granny Smith) ACCEPTED MANUSCRIPT
Red grape
Mint
Tomato
Starfruit
Fuji apple
Cucumber
Parsley
PT
Fenugreek
Red mustard
Purple perilla
RI
Starfruit
Red grape
SC
Starfruit
Red grape
Cucumber
U
Apple (Pink lady)
AN
Cucumber
Red grape
Kiwi
M
Royal gala (apple)

Kiwi
Chinese cabbage
D
Kiwi
Pineapple
TE
Susu
Roma tomato
Pineapple
Pineapple
EP
Royal gala (apple)

Susu
Kiwi
C
Chinese cabbage
Pineapple
AC
Green chilli
Chinese cabbage
Chinese cabbage
Kiwi
Kiwi
Green chilli
Chinese cabbage
Royal gala (apple)
Kiwi
Chinese cabbage
Susu
Green chilli
Green chilli ACCEPTED MANUSCRIPT
Kiwi
Sugar loaf cabbage
Susu
Royal gala (apple)
Chinese cabbage
Royal gala (apple)
Susu
Susu
Kiwi
PT
Royal gala (apple)
Kiwi
Royal gala (apple)
RI
Kiwi
Kiwi
Susu
SC
Chinese cabbage
Kiwi
Green chilli
U
Susu
Royal gala (apple)
AN
Kiwi
Sugar loaf cabbage
Sugar loaf cabbage
M
Sugar loaf cabbage

Sugar loaf cabbage
Sugar loaf cabbage
D
Pawpaw
Pawpaw
TE
Pawpaw
Mint
Mint
Spearmint
EP
Fenugreek
Mint
Chervil
C
Endive
Endive
AC
Endive
Fenugreek
Parsley
Parsley
Honeydew melon
Persimmous pear
Pawpaw
Pawpaw
Rock melon
Rock melon
Watermelon
Honeydew melon
Persimmous pear ACCEPTED MANUSCRIPT
Honeydew melon
Watermelon
Rock melon
Honeydew melon
Honeydew melon
Persimmous pear
Pawpaw
Rock melon
Rock melon
PT
Watermelon
Watermelon
Rock melon
RI
Watermelon
Persimmous pear
Rock melon
SC
Watermelon
Watermelon
Watermelon
U
Watermelon
Honeydew melon
AN
Watermelon
Chives
Chervil
M
Basil
Spearmint
Watermelon
D
Bosc pear
Red delicious apple
TE
Spearmint
Fuji apple
Pawpaw
Pear
EP
Pawpaw
Pink lady apple
Pawpaw
C
Pink lady apple

Pink lady apple
AC
Pawpaw
Sugar loaf cabbage
Sugar loaf cabbage
Pawpaw
Sugar loaf cabbage
Sugar loaf cabbage
Tomatoes Gourmet
Witlof
Witlof
Tomatoes Gourmet
Pawpaw
Witlof
Organically Spouts ACCEPTED MANUSCRIPT
Witlof
Brussel Sprouts Loose
Choy sum
Swiss Brown Mushrooms
Choy sum
Tomatoes Gourmet
PT
Pawpaw
Bosc Pear
RI
Organically Spouts
Tomatoes Gourmet
SC
Coriander
Mushrooms Button
Mushrooms Flat
U
Mushrooms Button
Strawberries
AN
Italian Parsley
Chives
Bean Sprouts
M
Okara
Asian Mix
Green Bean
D
String Bean
Baby Brocolli
TE
Sugarsnap Peas
Turinps
Beetroot
Organically Spouted
EP
Beetroot
Organically Sprouted
Zucchini
C
Parsnips
Yellow squash
AC
Yellow squash
Potatoes
Zucchini
Snow pea
Potatoes
Parsnips
Kipfler Potatoes
Radish Bunch
Snow pea ACCEPTED MANUSCRIPT
Potatoes
Baby spinach
Rocket Leaves
Parsnips
Mesculin Leaves
Snow pea
Radish Bunch
Rocket Leaves
Kipfler Potatoes
PT
Baby spinach
Tomatoes Gourmet
Tomatoes Brushed
RI
Zucchini Green
Mangoes
Cabbage
SC
Beetroot
Kiwifruit Green
Potatoes Kipfler
U
Zucchini Green
Tomatoes Brushed
AN
Capsicum red
Chokoe
Mangoes
M
Pears
Zucchini Green
Cucumber Lebanese
D
Potatoes Kipfler
Tomatoes Gourmet
TE
Cabbage
Capsicum red
Asian Vegetable (Rau den)
Green Bean
EP
Chieves
Banana (lady finger)
Eggplant
C
Eggplant
Nectarine Yellow
AC
Asian Vegetable (Cai ro)

Chieves
Vietnamese spinach (mong toi)
Cos lettuce
Brocolli
Glory Morning
Beetroot
Asian vegetable (Cai ngot)
Mint Leaves
Asian vegetable (Cai ngot)
Cos lettuce ACCEPTED MANUSCRIPT
Brocolli
Banana (lady finger)
Glory Morning
Vietnamese spinach (mong toi)
Brocolli
Green Bean
PT
RI
U SC
AN
M
D
TE
C EP
AC

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To appear in: Food Control

Received Date: 9 July 2013

2 mould Penicillium commune and their potential as biopreservatives in cheese.

15 Tel: +61-7 33651171, Fax: +61- 7 33651177

32 absence of sodium acetate from MRS agar resulted in no inhibition of P. commune,

34 previously reported. To determine their potential as biopreservatives in cheese, LAB isolates

41 potential for use as biopreservatives in cheese.

59 have lipolytic activity that allows them to cause spoilage in cheese.

75 Biopreservation or biocontrol refers to the use of natural or controlled microflora, or its

94 2011; Schwenninger & Meile, 2004; Zhao, 2011)

103 2.1. Bacteria and mould strains and media.

111 anaerobic conditions.

which resulted in a spore number of ~1 x 106 spores/ml.

138 clearing around the LAB colony.

152 2.4. Antifungal activity spectrum.

moulds (P. roqueforti, P. glabrum, P. solitum, G. candidum, A. versicolor, M. circinelloides,

159 2.5 Antifungal activity of LAB against P. commune in cottage cheese.

169 MRS agar and incubation.

184 antifungal activity.

185 2.6. Identification of LAB.

202 3.1. Screening of LAB for antifungal activity.

213 without sodium acetate (data not shown).

214 3.2. Antifungal activity spectrum of LAB.

227 3.4. Antifungal activity of LAB against P. commune on cottage cheese.

236 the experiment.

mould by day 12 (Table 1 and Figure 3).

251 range of herbs, fruits and vegetables.

272 bacteria using genotyping methods.

290 found to be inhibitory against P. roqueforti, which makes it applicable as a biopreservative on

310 fumigatus (Lind, Jonsson & Schnürer, 2005).

324 looking at testing these LAB in other cheese will be of interest.

355 or 3,6-bis(2-methylpropyl)-2,5-piperazinedion (Yang & Chang, 2010). A genome shuffling

359 antifungal mechanisms of Lb. plantarum.

381 Microbiology, 24(7), 759-766.

383 resistance mechanisms. International Journal of Food Microbiology, 50(1-2), 1-17.

394 sequence of Lactobacillus plantarum strain 16, a broad-spectrum antifungal-producing lactic

395 acid bacterium. Genome Announcements, 1(4), e00533-13.

417 International Commission on Microbiological Specifications of Foods (ICMSF), 1998.

434 Dekker, Inc.

441 Letters, 219(1), 129-135.

444 York: Marcel Dekker, Inc.

447 Research and Technology, 226(6), 1309-1316.

450 paracasi D5 on selected foods. Journal of Food Science, 76(7), M493-M499.

453 Dairy Science, 88(4), 1335-1341.

455 antimicrobial compounds produced by Lactobacillus plantarum. Journal of Applied

456 Microbiology, 86(1), 29-35.

458 presence of sterigmatocystin in hard cheese. Journal-Association of Official Analytical

459 Chemists, 63(1), 115.

461 antimicrobial compounds and assessment of performance in cottage cheese at refrigeration

462 temperature. Journal of Applied Microbiology, 85(1), 103-114.

466 in modified atmospheres. International Journal of Dairy Technology, 60(2), 89-95.

477 Microbiology. Food Microbiology 41 (In press) 19e26

483 Microbiology , 103(6), 2622-2628.

486 Applied Microbiology, 27(2), 229-237.

490 biopreservatives. Trends in Food Science & Technology, 16(1), 70-78.

492 International Journal of General and Molecular Microbiology, 70 (2-4), 331-345.

501 production on cheese under modified atmospheres. International Journal of Food

502 Microbiology, 68(1-2), 125–133.

505 Microbiology, 11(4), 231-236.

508 Food Microbiology, 127(1), 91-98.

511 Control, 32(2013), 341-347.

514 Microbiology, 139(1), 56-63.