Culture-Independent Bacterial Community Profiling of Carbon Dioxide Treated Raw Milk

ÔØ ÅÒÙ×Ö ÔØ

Culture-independent bacterial community proﬁling of carbon dioxide treated raw milk

Raquel Lo, Mark S. Turner, Mike Weeks, Nidhi Bansal

PII: S0168-1605(16)30308-7 DOI: doi: 10.1016/j.ijfoodmicro.2016.06.015 Reference: FOOD 7262

To appear in: International Journal of Food Microbiology

Received date: 1 March 2016 Revised date: 30 May 2016 Accepted date: 14 June 2016

Please cite this article as: Lo, Raquel, Turner, Mark S., Weeks, Mike, Bansal, Nidhi, Culture-independent bacterial community proﬁling of carbon dioxide treated raw milk, International Journal of Food Microbiology (2016), doi: 10.1016/j.ijfoodmicro.2016.06.015

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT

Culture-independent bacterial community profiling of carbon dioxide treated raw milk

Raquel Lo a, Mark S. Turner a,c, Mike Weeksb, Nidhi Bansal a,*

a School of Agriculture and Food Sciences, The University of Queensland, St Lucia, QLD

4072, Australia b Dairy Innovation Australia, Werribee, VIC 3030, Australia c Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St

Lucia, QLD 4072, Australia

*Corresponding author. Tel.: +61 7 33651673.

E-mail address: [email protected] (N. Bansal)

Postal address: School of Agriculture and Food Sciences, Hartley Teakle Building (No. 83),

The University of Queensland, St Lucia, QLD 4072, Australia

ACCEPTED MANUSCRIPT

Abstract

Due to technical simplicity and strong inhibition against the growth of psychrotrophic bacteria in milk, CO2 treatment has emerged as an attractive processing aid to increase the storage time of raw milk before downstream processing. However, it is yet to be adopted by the industry. In order to further explore the suitability of CO2 treatment for raw milk processing, the bacterial populations of carbonated raw milk collected locally from five different sources in Australia were analysed with next-generation sequencing. Growth inhibition by CO2 was confirmed, with spoilage delayed by at least 7 days compared with non-carbonated controls. All non-carbonated controls were spoiled by Gammaproteobacteria, namely Pseudomonas fluorescens group bacteria, Serratia and Erwinia. Two out of the five carbonated samples shared the same spoilage bacteria as their corresponding controls. The rest of the three carbonated samples were spoiled by the lactic acid bacterium (LAB)

Leuconostoc. This is consistent with higher tolerance of LAB towards CO2 and selection of

LAB in meat products stored in CO2-enriched modified atmosphere packaging. No harmful bacteria were found to be selected by CO2. LAB are generally regarded as safe (GRAS), thus the selection for Leuconostoc by CO2 in some of the samples poses no safety concern. In addition, we have confirmed previous findings that 454 pyrosequencing and Illumina sequencing of 16SACCEPTED rRNA gene amplicons from MANUSCRIPT the same sample yield highly similar results. This supports comparison of results obtained with the two different sequencing platforms, which may be necessary considering the imminent discontinuation of 454 pyrosequencing.

Keywords

Raw milk spoilage; Raw milk microbiota; CO2 treatment; Bacterial community profiling;

Next-generation sequencing

ACCEPTED MANUSCRIPT

1. Introduction

The establishment of the cold chain has extended the storage time of raw milk before processing due to inhibition of mesophilic bacteria. However, low temperature favours the growth of psychrotrophic organisms, among which those belonging to the Pseudomonas fluorescens group are predominant spoilage bacteria of raw milk (Boor and Fromm, 2009;

Heyndrickx et al., 2010; Sørhaug and Stepaniak, 1997; Ternström et al., 1993). Although the majority of psychrotrophic bacteria can be effectively killed by pasteurisation, many species, particularly those of the P. fluorescens group, produce heat-stable lipases and proteinases which remain active post-pasteurisation (Fairbairn and Law, 1986; Sørhaug and Stepaniak,

1991, 1997; Stead, 1986). Therefore, controlling the proliferation of psychrotrophic bacteria during refrigerated storage of raw milk is crucial to maintaining the shelf-lives of the derived dairy products.

Carbon dioxide has emerged as an attractive preservative of raw milk as it is inhibitory against the growth of psychrotrophic bacteria including P. fluorescens (Hotchkiss et al., 2006;

Loss and Hotchkiss, 2003; Martin et al., 2003), it is generally regarded as safe (GRAS), its treatment method is non-thermal, simple and economical, and it does not have adverse effects on the nutritionalACCEPTED content of milk (Loss and Hotchkiss,MANUSCRIPT 2003; Ruas-Madiedo et al., 1996;

Sierra et al., 1996). The effectiveness of CO2 in extending the storage life of raw milk and cottage cheese has been well established (Amigo et al., 1995; Chen and Hotchkiss, 1991,

1993; Espie and Madden, 1997; King and Mabbitt, 1982; Kosikowski and Brown, 1973;

Maniar et al., 1994; Moir et al., 1993; Roberts and Torrey, 1988; Ruas-Madiedo et al., 1996;

Ruas-Madiedo, Bascarán, et al., 1998). CO2 treatment is already routinely used in commercial cottage cheese production in the US (Loss and Hotchkiss, 2003), and an unpublished scaled-up field trial of CO2 treatment of raw milk showed a 4 day increase in

ACCEPTED MANUSCRIPT

storage life of the treated sample (Hotchkiss et al., 2006). However, CO2 remains to be adopted by the industry for raw milk processing.

Some reasons for the hesitation of introducing CO2 into raw milk processing could be the possible selection of pathogens and sporeformers and increased risk of toxin production during the extended storage time before downstream processing. Studies to date on the effect of CO2 on the growth of Listeria monocytogenes, Bacillus cereus and Clostridium sporogenes in milk or cottage cheese and toxingenesis of Clostridium botulinum in milk have shown that CO2 treatment does not pose increased risk with regard to these factors (Chen and

Hotchkiss, 1993; Glass et al., 1999; Werner and Hotchkiss, 2002) . In addition, culture- independent studies on the native bacterial composition of carbonated raw milk during cold storage have found no evidence of selection of harmful bacteria (Rasolofo et al., 2011;

Rasolofo et al., 2010). The current study aims to further explore the bacterial profiles of carbonated raw milk using culture-independent next-generation sequencing of 16S rRNA gene amplicons. Compared to the 16S rRNA gene-based techniques used in previous studies, namely gene clone libraries, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) (Rasolofo et al., 2011; Rasolofo et al., 2010), next-generatACCEPTEDion amplicon sequencing MANUSCRIPT offers much greater sampling depth and requires less manual handling and subjective judgement, making results more representative and reproducible. Thus the current study will be a meaningful addition to the literature of CO2 treatment of dairy products.

Pyrosequencing has already been used in a large number of recent studies in which the microbial communities of dairy products were characterised, including raw and pasteurised milk and various types of cheese (Aldrete-Tapia et al., 2014; de Filippis et al., 2014; de

ACCEPTED MANUSCRIPT

Pasquale et al., 2014; Ercolini et al., 2012; Guidone et al., 2016; Masoud et al., 2011; Masoud et al., 2012; Quigley, McCarthy, et al., 2013; Quigley et al., 2012; Riquelme et al., 2015). We have recently analysed the bacterial populations of fresh and spoiled carbonated raw sweet whey using pyrosequencing of 16S rRNA gene amplicons (Lo et al., 2016). The carbonated sample was spoiled by the same bacteria as the non-carbonated control, namely Pseudomonas,

Serratia and other Enterobacteriaceae, which are common milk spoilage organisms. It would be of interest to determine whether similar organisms would be found in spoiled carbonated raw milk.

This study aims to confirm the effectiveness of CO2 in inhibiting bacterial growth in raw milk samples obtained from geographically distinct sites and to characterise the microbial populations of fresh and spoiled raw milk using culture-independent 16S rRNA community profiling. The findings will help determine the suitability of incorporating CO2 treatment into raw milk processing.

2. Materials and Methods

2.1. Sources of raw milk Raw milk was collectedACCEPTED from five different sourcesMANUSCRIPT located in two different states of Australia (Table 1) and transported to the University of Queensland (UQ), where carbonation and analyses were performed. Milk samples were transported in an insulated container with ice bricks. Samples collected in Queensland arrived at UQ within an hour. SF2, which was collected in Victoria, was first transported to Dairy Innovation Australia Limited (DIAL) where it was stored in a 4°C cold room before being packaged with ice bricks and transported by plane to Queensland. The SF2 sample arrived at UQ within the same day as sample collection. Upon arrival at UQ, all samples were held in a 4°C cold room before carbonation.

ACCEPTED MANUSCRIPT

The day of sample collection was designated as day 0. All samples were carbonated on day 0.

Henceforth, the samples are abbreviated by their source and treatment. For example, DP1- control and DP1-CO2 stand for the non-carbonated control and carbonated sample from DP1 respectively.

2.2. Carbonation, measurement of CO2 concentration and milk storage conditions

Carbonation and measurement of CO2 concentration was carried out as previously described

(Lo et al., 2016). Samples were carbonated to achieve CO2 saturation (2146-3540 ppm). The carbonated milk was divided into 30 mL aliquots in sterile sealed plastic containers and stored at 4°C. One aliquot was used for analysis at each time point. A non-carbonated control was included in the analysis of each sample.

2.3. Bacterial growth studies

Bacterial growth in the raw milk samples was monitored by a plate count method. One millilitre of milk was taken from a well mixed 30 mL sample aliquot and used to prepare serial tenfold dilutions in 0.1% peptone water. One hundred microlitres were spread plated onto nutrient agar (Oxoid, Basingstoke, UK) and incubated at 30°C for 3 days. Undiluted samples were alsoACCEPTED plated on day 0 to allow forMANUSCRIPT low bacterial counts. Analysis of a sample was terminated when it was spoiled. Spoilage was defined by bacterial counts on nutrient agar

6 reaching a threshold of 10 CFU/mL. CO2-treated samples were also plated onto MRS agar

(Oxoid, Basingstoke, UK) and incubated at 37°C anaerobically to monitor the growth of

Lactobacillus and Leuconostoc.

ACCEPTED MANUSCRIPT

2.4. DNA extraction for pyrosequencing

One mL aliquots of milk were collected on the same day as plating for treatment with propidium monoazide (PMA) (Biotium, Hayward, CA, USA) and stored at -80°C until ready for DNA extraction. The purpose of the PMA treatment was to prevent DNA from dead bacteria from being extracted and amplified in PCR (Nocker et al., 2006). All milk samples except those from DP1 were treated with PMA prior to DNA extraction. PMA treatment and

DNA extraction was performed using methods described previously (Lo et al., 2016).

2.5. Pyrosequencing

PCR was performed with GoTaq® DNA Polymerase (Promega, Madison, WI, USA) according to the manufacturer’s instructions. The primers, provided by the Australian Centre for Ecogenomics (ACE, UQ), were non-barcoded and targeted the V5-V8 region of the 16S rRNA gene (forward primer: a mixture of TTAGATACCCTGGTAGTC,

TTAGATACCCSGGTAGTC, TTAGATACCCYHGTAGTC and

TTAGAGACCCYGGTAGTC in a 2:1:1:1 ratio respectively; reverse primer:

ACGGGCGGTGWGTRC). The resulting PCR products were then submitted to ACE for a second PCR with barcoded primers and Roche 454 pyrosequencing (Margulies et al., 2005; Rothberg and Leamon,ACCEPTED 2008). The sequencing MANUSCRIPT data were analysed with QIIME (Caporaso et al., 2010). The default parameters of the scripts were used in most cases. Reads were excluded if their average quality score were below 25, if their length was outside 200 - 1000 bp, if they had more than 6 ambiguous bases, if they had a homopolymer run exceeding 6 bases or if there were any primer mismatches. Reverse primer sequences, if present, were removed from the reads. OTUs (operational taxonomic units) were picked de novo by grouping sequences with a similarity of at least 97% into OTUs by Uclust (Edgar, 2010). A representative set of sequences were then picked from the OTUs, and their taxonomy 7

ACCEPTED MANUSCRIPT

assigned by Uclust against the Greengenes 16S rRNA database (McDonald et al., 2012) with a sequence similarity threshold of 90%. The resulting OTU tables were modified by excluding singletons and OTUs with unassigned taxonomies. The modified OTU tables were then used for plotting rarefaction curves and calculating Good's coverage with the multiple_rarefactions, alpha_diversity and collate_alpha scripts in QIIME. For bacterial composition analysis, the OTU tables were further processed with the summarize_taxa_through_plots script in QIIME to collate OTUs with the same taxonomy assignment and for conversion to relative proportions. The most specific taxonomy assignment was often to the genus level only. Representative sequences of dominant OTUs belonging to the same genus were analysed by BLAST (Altschul et al., 1990) and species assignment was given where applicable.

2.6. Illumina sequencing

Due to anomalous pyrosequencing results from day 0 MF-CO2 and the lack of sequencing reads from day 0 SF1-control, these samples were resubmitted to ACE for Illumina sequencing (pyrosequencing services were no longer provided by ACE by that time). 16S rRNA gene amplicons were prepared with the same primers as for pyrosequencing (section 2.5). In order to ensureACCEPTED that results from pyrosequencing MANUSCRIPT and Illumina sequencing were comparable, additional samples with previous pyrosequencing results were included in the same Illumina sequencing run. Quality trimming as per ACE's method was done using

Trimmomatic (Bolger et al., 2014) in Galaxy (Afgan et al., 2016) as follows: (i) trimming the first 20 bases of the raw reads to remove primer sequence; (ii) quality trimming using a sliding window of 4 bases with an average base quality above 15; (iii) hard trimming to 250 bases; (iv) excluding reads shorter than 250 bases. OTU picking and rarefaction analysis were done using the same methods as for pyrosequencing data (section 2.5).

ACCEPTED MANUSCRIPT

2.7. Identification of bacterial isolates using Sanger sequencing

Bacterial isolates of interest from the spread plates in bacterial growth studies (section 2.3) were grown in nutrient or MRS broth. Two mL of stationary phase cultures were centrifuged to obtain cell pellets. The pellets were stored at -20°C until DNA extraction. DNA was extracted using chloroform-isoamyl alcohol as previously described (Prasad and Turner,

2011). The resulting DNA extract acted as template for PCR amplification of the 16S rRNA gene using primers designed against conserved regions upstream of V1 and downstream of

V8 in the 16S rRNA gene (forward primer: 5'-AGAGTTTGATCCTGGCTCA-3', reverse primer: 5'-CGGTGAATACGTTCCCG-3'). The PCR products were sent to Macrogen (Seoul,

Korea) for DNA purification and Sanger sequencing. The isolates were identified using

BLAST (Altschul et al., 1990).

2.8. Accession number of pyrosequencing and Illumina sequencing data

Raw sequencing data of the samples analysed in this study have been deposited into the

European Nucleotide Archive under the study number PRJEB11534. A key listing the sample

IDs in the sequence files with the corresponding sample descriptions is available in the supplemental data.ACCEPTED MANUSCRIPT

3. Results and discussion

3.1. Carbonation delays spoilage of raw milk

It has been well established by independent research groups in different countries that CO2 is inhibitory against bacterial growth in raw milk, particularly against Gram-negative bacteria such as coliforms and Pseudomonas (Amigo et al., 1995; Espie and Madden, 1997; Roberts and Torrey, 1988; Ruas-Madiedo et al., 1996). In order to confirm this inhibition locally, raw

ACCEPTED MANUSCRIPT

milk from five different sources with varying scales of production were collected (Table 1) and the effect of CO2 on bacterial growth was studied. The initial bacterial counts of the samples ranged from 3.3×103 to 4.4×104 CFU/mL (Fig. 1). Non-carbonated controls all spoiled between day 3 and 7 except MF which spoiled between day 7 and 14. The carbonated samples did not spoil until 1 to 4 weeks after their corresponding non-carbonated controls. The bacterial counts of the carbonated samples from DP1, MF and SF2 remained stable for 21 days of storage (Fig.1). Hence, CO2 was confirmed to be effective at delaying bacterial spoilage in raw milk from our local sources, thus justifying further investigation into its potential to be developed as a processing aid.

The average day 0 pH of the raw milk samples was 6.57, and CO2 decreased the pH to an average of 5.82, equivalent to an average reduction of 0.75 pH units. However, inhibition of bacterial growth could not be attributed to pH reduction alone, as has been previously documented (Hendricks and Hotchkiss, 1997; King and Mabbitt, 1982; Lo et al., 2016).

3.2. Characteristics of pyrosequencing and Illumina reads

Although CO2 was confirmed to be effective at inhibiting bacterial growth in raw milk, it was important to determineACCEPTED whether any undesirable MANUSCRIPT bacteria were selected by the CO2 treatment. A culture-independent approach was adopted in order to avoid bias due to selection of culture media. Bacterial compositions of day 0 and spoiled raw milk samples were analysed by 16S rRNA gene amplicon pyrosequencing. Two samples had to be resubmitted for sequencing due to unsatisfactory or anomalous results: (1) day 0 SF1-control, whose sequencing reaction failed and (2) day 0 MF-CO2, which contained 87.4% Bifidobacterium compared to 6.2% in day 0 MF-control. These two samples were analysed by Illumina sequencing as pyrosequencing had been discontinued by our sequencing service provider. In order to ensure

ACCEPTED MANUSCRIPT

that the results obtained with the two sequencing platforms were comparable, day 0 SF1-CO2 and day 0 MF-control were also included in the same Illumina sequencing run. The characteristics of the pyrosequencing and Illumina data are shown in Tables 2 and 3. The mean length of the pyrosequencing reads after quality trimming is 450 bp for samples from

DP1 and 503 bp for the other samples. Illumina reads were hard trimmed to 250 bp. The alpha rarefaction curves of the observed number of species of all samples show plateauing

(Fig. 2). Although only a small number of reads were obtained for spoiled DP1-control and spoiled DP2-control (539 and 361 respectively after quality filtering, Table 2), the Good's coverage at the largest number of sequences of both samples were above 0.995. Therefore, the low sequencing depths of these samples are acceptable.

Illumina sequencing was successful for all resubmitted samples. Pyrosequencing and

Illumina yielded highly similar results from day 0 MF-control and SF1-CO2 and (Fig. 3). This is consistent with findings in previous studies which showed pyrosequencing and Illumina sequencing produced similar results from the same sample (Caporaso et al., 2011; Nelson et al., 2014). There are only minor differences between the samples. In the MF sample,

Mycoplana was detected by pyrosequencing but not by Illumina, while Clostridiales and CaulobacteraceaeACCEPTED were detected by Illumina MANUSCRIPT but not by pyrosequencing. In the SF1 sample, Bacillales, Streptococccus and Peptostreptococcaceae were detected by pyrosequencing but not by Illumina, while Planococcaceae was detected by Illumina but not by pyrosequencing.

These taxa ranged from 0.0019% (Streptococcus in SF1-454) to 7.4% (Clostridiales in MF-

Illumina) of the total population. Despite these discrepancies, closely related taxa belonging to the same order or family as that of the missing taxa were detected. For example,

Peptostreptococcaceae belong to the order Clostridiales. Illumina sequencing found 7.4% of

Clostridiales in the MF sample, which was not detected by pyrosequencing, but

ACCEPTED MANUSCRIPT

pyrosequencing found 11.3% of Peptostreptococcaceae. Similarly, pyrosequencing found

4.9% of Peptostreptococcaceae in the SF1 sample, which was not detected by Illumina, but

Illumina found 4.8% of Clostridiales (Fig. 3). Therefore, valid comparisons can be made with results across the two sequencing platforms.

3.3. Initial bacterial composition of raw milk varies with source

The initial bacterial compositions of raw milk from the five sources are shown in Fig. 4. At the phylum level, it can already be seen that the DP1 sample is distinct from the other four samples, being overwhelmingly dominated by Proteobacteria (90–94%). In contrast, the initial microbiota from the other four sources are dominated by Proteobacteria and Firmicutes at roughly similar proportions (32–40% and 28–60% respectively), with substantial but more variable composition of Actinobacteria (4.6–33%) (Fig. 4). This difference could be related to PMA treatment: all samples were treated with PMA except those from DP1. The bacterial populations of the DP1 samples are also more homogeneous than the other four samples at the family and genus level. The dominant genus in DP1, Herbaspirillum (52–56%), was only found in SF2-CO2 among the other four sources at 1.2%. Although the initial bacterial compositions of the other four samples are quite similar at the phylum level, there are vast differences at theACCEPTED family and genus level (Fig. MANUSCRIPT 4). The only taxa common to the four samples are Propionibacterium (0.82–3.6%), Bifidobacterium (0.67–3.9%) and Alicyclobacillus (1.2–

8.5%). Samples from SF1 and SF2 have different predominant taxa, namely Staphylococcus

(29–31%) and Peptostreptococcaceae (39–44%), respectively. In contrast, the populations of

DP2 and MF are more diverse. As raw milk microbiota originate from cow skin microflora, farm environment and farming equipment (Quigley, O'Sullivan, et al., 2013), it is conceivable that variations in one or more of these aspects can lead to the widely different profiles seen here. The heterogeneous initial bacterial populations identified between our samples provide

ACCEPTED MANUSCRIPT

an ideal baseline to determine whether CO2 selects for certain groups of bacteria during storage.

Three independent studies carried out in Denmark, Ireland and Mexico have found lactic acid bacteria (LAB), in particular Lactococcus or Streptococcus, to be the predominant bacterial group in raw milk microbiota (Aldrete-Tapia et al., 2014; Masoud et al., 2012; Quigley,

McCarthy, et al., 2013). LAB were found in all day 0 samples except those from SF2, but they were only present in relatively minor proportions, with Lactococcus and Streptococcus being the only genera common to these four sources (0.081–8.2%) (Fig. 4). In two studies where raw milk samples were collected from multiple sources in the same country, unique microbiota profiles were found for each source. However, the profiles in one study are much more homogeneous across the sources, with Lactococcus forming 30-90% of the microbiota

(Quigley, McCarthy, et al., 2013), whereas the profiles in the other study vary extensively with source and season (Aldrete-Tapia et al., 2014). The heterogeneity in the latter study is similar to our findings. It is worth noting that the primers used in the studies cited above target different regions in the 16S rRNA gene compared to the primers we used (V1-V3, V3-

V4 or V4 as opposed to V5-V8), thus this cannot be ruled out as a factor in the differences seen in the bacterialACCEPTED communities. MANUSCRIPT

3.4. Control raw milk was spoiled by common milk spoilage bacteria

All non-carbonated controls were spoiled by Gammaproteobacteria (Fig. 5). Pseudomonas is the dominant spoilage genus in four out of the five non-carbonated samples. The spoilage

Pseudomonas OTUs could not be resolved to single species, but they all belong to the

Pseudomonas fluorescens group. SF1-control was spoiled by both Serratia and Pseudomonas, the former being slightly more dominant (50% vs 42%). The spoilage bacterial community of

ACCEPTED MANUSCRIPT

SF2 also contains a substantial proportion of Erwinia (24%) (Fig. 5). All three genera are minor members of the initial population: Pseudomonas was present at 1.79% or lower,

Serratia was present only in the DP1 and SF samples (3.1% or lower), and Erwinia was undetected in all samples. Dominant colony types on the spread plates of spoiled samples were identified by Sanger sequencing of the 16S rRNA gene. All the selected isolates were found to belong to the P. fluorescens group.

Pseudomonas, Serratia and Erwinia are psychrotrophs, and the dominance of the first two genera in spoiled raw milk has already been well characterised in culture-dependent studies

(Boor and Fromm, 2009; Heyndrickx et al., 2010; Machado et al., 2015; Sørhaug and

Stepaniak, 1991; Ternström et al., 1993). Erwinia is more often associated with spoilage of fruits and vegetables (Kahala et al., 2012; Tournas, 2005).

3.5 CO2 selects for different spoilage organisms in three raw milk samples

The carbonated samples from both SF1 and SF2 share similar spoilage bacteria as their corresponding controls (Fig. 5). Serratia has been reported be able to grow at high CO2 concentrations (Schuerger et al., 2013), thus the higher proportion of Serratia compared to

Pseudomonas in ACCEPTEDspoiled SF1-CO2 could possibly MANUSCRIPT be due to selection by CO2. A similar phenomenon was also found in carbonated raw Mozzarella whey that was spoiled during cold storage (Lo et al., 2016). In contrast, the other three carbonated samples were spoiled by different bacteria than their controls. The microbiota of spoiled carbonated samples from DP1,

DP2 and MF are dominated by Leuconostoc (62–95%) (Fig. 5). Spoiled DP2-CO2 also contains 17% Carnobacterium, which belongs to the same family as Leuconostoc

(Leuconostocaceae). Gluconacetobacter, comprising a substantial 8.4% of the spoiled SF2-

CO2 bacterial community, was found only in one spoiled control sample: SF2-control, at a

ACCEPTED MANUSCRIPT

miniscule proportion of 0.083%. BLAST analysis of the most dominant OTU sequences narrowed down the classification of Leuconostoc and Carnobacterium to Leuconostoc lactis/garlicum and Carnobacterium maltaromaticum respectively. Similar to the spoilage genera of the control samples, these genera are minor members of the day 0 populations.

Carnobacterium was present only in day 0 DP2-CO2 at 0.87%, while Leuconostoc and

Gluconacetobacter were undetected in all day 0 samples (Fig. 4). Although Herbaspirillum constitutes 23% of the bacterial population of the spoiled DP1-CO2 sample (Fig. 5), it is not considered as a major spoilage bacterium as it is a dominant member in the day 0 population

(52%) (Fig. 4) and has decreased in proportion when spoilage occurred.

Leuconostoc and Carnobacterium were found to be culturable psychrotrophs in raw milk

(Hantsis-Zacharov and Halpern, 2007). These genera, among other LAB, have been also been well established as major spoilage bacteria of refrigerated meat products stored in vacuum and modified atmosphere packaging which contains higher concentrations of CO2 (Borch et al., 1996; Ercolini et al., 2011; Stoops et al., 2015). Furthermore, it has been reported that

CO2 has little effect on a range of LAB, including Leuconostoc (Espie and Madden, 1997;

Ruas-Madiedo, Alonso, et al., 1998; van Hekken et al., 2000; Vinderola et al., 2000).

Therefore, it is notACCEPTED surprising that CO2 selected MANUSCRIPT for the growth of Leuconostoc and Carnobacterium in these three carbonated samples. To our knowledge, this is the first report of Leuconostoc as a major spoilage bacterium of raw milk treated with CO2.

The identities of dominant colony types determined by Sanger sequencing of 16S rRNA genes from the spoiled CO2 samples largely agree with the pyrosequencing results. Dominant spoilage isolates from SF1 and SF2 were found to be Hafnia, which is closely related to

Serratia, and a member of the P. fluorescens group respectively. However, recovery of

ACCEPTED MANUSCRIPT

dominant spoilage isolates from DP1, DP2 and MF varied with culture media. The isolates grown on nutrient agar obtained from DP1, DP2 and MF were identified as Le. lactis/garlicum, Lactococcus raffinolactis and Streptococcus parauberis respectively. In contrast, all dominant isolates grown on MRS agar were identified as Leuconostoc spp.: Le. lactis/garlicum and Leuconostoc citreum/holzapfelii. On nutrient agar under aerobic conditions, Leuconostoc grew very poorly (DP1 sample) or failed to grow (DP2 and MF samples), whereas Lactococcus and Streptococcus could grow, albeit not optimally, under these conditions. Therefore, Leuconostoc as a dominant spoilage genus would have been undetected if only a culture-dependent method using a general purpose culture medium such as nutrient agar had been adopted. These results highlight the importance of incorporating a culture-independent approach in community profiling studies.

The bacterial population of carbonated raw milk during cold storage has been studied using

PCR clone libraries and molecular fingerprinting of the 16S rRNA gene (Rasolofo et al.,

2011; Rasolofo et al., 2010). Pseudomonas was found to be the dominant spoilage genus in both studies. However, one study showed a higher proportion of Gram-positive bacteria in the carbonated sample than the untreated control after storage at 4°C for 7 days (39.2% vs

4.8%). LAB formedACCEPTED 13.1% of the day 7 CO 2MANUSCRIPT population (mainly Streptococcus, 8.2%), but they were undetected in the untreated control (Rasolofo et al., 2010). Our current results are consistent with those reported in this study.

It would be of interest to determine whether there were any distinctive features of the samples which led to systematic selection of LAB by CO2. Based on our results, initial bacterial composition and diversity, length of spoilage retardation, and geographical location

(Queensland vs Victoria) are factors that can likely be excluded. One feature that stood out

ACCEPTED MANUSCRIPT

was scale of operation: the carbonated samples not spoiled by LAB both came from small scale farms. However, the relationship between scale of operation and bacterial selection is unclear in this context, and a greater number of samples would need to be analysed to confirm this possible correlation.

Le. lactis is often isolated from fermented foods including traditional fermented milk products and brined products such as kimchi and stinky tofu (Dan et al., 2014; Liu et al.,

2011). It is also important for aroma and texture production in dairy products (Hemme and

Foucaud-Scheunemann, 2004; Marshall, 1987). There have been reports of Leuconostoc causing infections including bacteraemia, endocarditis and meningitis (Ogier et al., 2008).

However, with a few exceptions, these infections were limited to immunocompromised patients, and none of the cases were associated with consumption of foods containing

Leuconostoc. Furthermore, Le. lactis was rarely isolated in these cases, the most common species being Leuconostoc mesenteroides. Therefore, the use of CO2 in raw milk can be regarded as safe even if Leuconostoc will be enriched.

4. Conclusion This is the first studyACCEPTED in which pyrosequencing MANUSCRIPT and Illumina sequencing was used to analyse the bacterial populations of carbonated raw milk. Five raw milk samples obtained from geographically distinct sites with different initial bacterial populations were analysed. CO2 was effective in inhibiting bacterial growth in all samples studied, delaying spoilage for at least 7 days. The non-carbonated controls were spoiled by Pseudomonas and Serratia. CO2 selected for different spoilage organisms in three out of the five samples tested, namely, Le. lactis/garlicum. This is consistent with LAB being major spoilage bacteria of meat products in modified atmosphere packaging with higher concentrations of CO2. No pathogenic bacteria

ACCEPTED MANUSCRIPT

were enriched by CO2 treatment. This is the first study in which LAB were found to be

dominant spoilage organisms in carbonated milk. The inhibition of bacterial growth and the

lack of selection of harmful bacteria by CO2 support the introduction of carbonation as a

processing aid of raw milk. CO2 is already routinely used in processing cottage cheese in the

US to increase its shelf-life. Field trials in dairy processing plants will determine its

feasibility in raw milk in an industrial setting.

Acknowledgements

This project is funded by Dairy Innovation Australia (Grant number 2013000041). The

authors would like to thank Brooke Clarke, Mark Schleyer, Zivka Cholich and Chris Pillidge

for their assistance.

References

Afgan, E., Baker, D., van den Beek, M., Blankenberg, D., Bouvier, D., Čech, M., Chilton, J.,

Clements, D., Coraor, N., Eberhard, C., Grüning, B., Guerler, A., Hillman-Jackson, J.,

Von Kuster, G., Rasche, E., Soranzo, N., Turaga, N., Taylor, J., Nekrutenko, A.,

Goecks, J., 2016. The Galaxy platform for accessible, reproducible and collaborative biomedicalACCEPTED analyses: 2016 update. Nucleic MANUSCRIPT Acids Research DOI: 10.1093/nar/gkw343. Aldrete-Tapia, A., Escobar-Ramírez, M.C., Tamplin, M.L., Hernández-Iturriaga, M., 2014.

High-throughput sequencing of microbial communities in Poro cheese, an artisanal

Mexican cheese. Food Microbiology 44, 136-141.

Altschul, S.F., Gish, W., Miller, W., Myers, E.W., Lipman, D.J., 1990. Basic local alignment

search tool. Journal of Molecular Biology 215, 403-410.

Amigo, L., Olano, A., Calvo, M.M., 1995. Preservation of raw milk with CO2. Zeitschrift für

Lebensmittel-Untersuchung und Forschung 200, 293-296.

ACCEPTED MANUSCRIPT

Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: A flexible trimmer for Illumina

sequence data. Bioinformatics.

Boor, K., Fromm, H., 2009. Managing microbial spoilage in the dairy industry, in: Blackburn,

C.d.W. (Ed.), Food Spoilage Microorganisms. Woodhead Publishing, Cambridge.

Borch, E., Kant-Muermans, M.-L., Blixt, Y., 1996. Bacterial spoilage of meat and cured meat

products. International Journal of Food Microbiology 33, 103-120.

Caporaso, J.G., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, F.D., Costello, E.K.,

Fierer, N., Pena, A.G., Goodrich, J.K., Gordon, J.I., Huttley, G.A., Kelley, S.T.,

Knights, D., Koenig, J.E., Ley, R.E., Lozupone, C.A., McDonald, D., Muegge, B.D.,

Pirrung, M., Reeder, J., Sevinsky, J.R., Turnbaugh, P.J., Walters, W.A., Widmann, J.,

Yatsunenko, T., Zaneveld, J., Knight, R., 2010. QIIME allows analysis of high-

throughput community sequencing data. Nature Methods 7, 335-336.

Caporaso, J.G., Lauber, C.L., Walters, W.A., Berg-Lyons, D., Lozupone, C.A., Turnbaugh,

P.J., Fierer, N., Knight, R., 2011. Global patterns of 16S rRNA diversity at a depth of

millions of sequences per sample. Proceedings of the National Academy of Sciences

108, 4516-4522.

Chen, J.H., Hotchkiss, J.H., 1991. Effect of dissolved carbon dioxide on the growth of psychrotrophicACCEPTED organisms in cottage cheese.MANUSCRIPT Journal of Dairy Science 74, 2941-2945. Chen, J.H., Hotchkiss, J.H., 1993. Growth of Listeria monocytogenes and Clostridium

sporogenes in cottage cheese in modified atmosphere packaging. Journal of Dairy

Science 76, 972-977.

Dan, T., Liu, W., Sun, Z., Lv, Q., Xu, H., Song, Y., Zhang, H., 2014. A novel multi-locus

sequence typing (MLST) protocol for Leuconostoc lactis isolates from traditional

dairy products in China and Mongolia. BMC Microbiology 14, 150.

ACCEPTED MANUSCRIPT

de Filippis, F., la Storia, A., Stellato, G., Gatti, M., Ercolini, D., 2014. A selected core

microbiome drives the early stages of three popular Italian cheese manufactures.

PLoS ONE 9, e89680. de Pasquale, I., di Cagno, R., Buchin, S., de Angelis, M., Gobbetti, M., 2014. Microbial

ecology dynamics reveal a succession in the core microbiota involved in the ripening

of pasta filata Caciocavallo Pugliese cheese. Applied and Environmental

Microbiology 80, 6243-6255.

Edgar, R.C., 2010. Search and clustering orders of magnitude faster than BLAST.

Bioinformatics 26, 2460-2461.

Ercolini, D., De Filippis, F., La Storia, A., Iacono, M., 2012. ―Remake‖ by high-throughput

sequencing of the microbiota involved in the production of water buffalo Mozzarella

cheese. Applied and Environmental Microbiology 78, 8142-8145.

Ercolini, D., Ferrocino, I., Nasi, A., Ndagijimana, M., Vernocchi, P., La Storia, A., Laghi, L.,

Mauriello, G., Guerzoni, M.E., Villani, F., 2011. Monitoring of microbial metabolites

and bacterial diversity in beef stored under different packaging conditions. Applied

and Environmental Microbiology 77, 7372-7381.

Espie, W.E., Madden, R.H., 1997. The carbonation of chilled bulk milk. Milchwissenschaft 52, 249-253.ACCEPTED MANUSCRIPT Fairbairn, D.J., Law, B.A., 1986. Proteinases of psychrotrophic bacteria: their production,

properties, effects and control. Journal of Dairy Research 53, 139-177.

Glass, K.A., Kaufman, K.M., Smith, A.L., Johnson, E.A., Chen, J.H., Hotchkiss, J., 1999.

Toxin production by Clostridium botulinum in pasteurized milk treated with carbon

dioxide. Journal of Food Protection 62, 872-876.

ACCEPTED MANUSCRIPT

Guidone, A., Zotta, T., Matera, A., Ricciardi, A., De Filippis, F., Ercolini, D., Parente, E.,

2016. The microbiota of high-moisture mozzarella cheese produced with different

acidification methods. International Journal of Food Microbiology 216, 9-17.

Hantsis-Zacharov, E., Halpern, M., 2007. Culturable psychrotrophic bacterial communities in

raw milk and their proteolytic and lipolytic traits. Applied and Environmental

Microbiology 73, 7162-7168.

Hemme, D., Foucaud-Scheunemann, C., 2004. Leuconostoc, characteristics, use in dairy

technology and prospects in functional foods. International Dairy Journal 14, 467-494.

Hendricks, M.T., Hotchkiss, J.H., 1997. Effect of carbon dioxide on the growth of

Pseudomonas fluorescens and Listeria monocytogenes in aerobic atmospheres.

Journal of Food Protection 60, 1548-1552.

Heyndrickx, M., Marchand, S., Jonghe, V.D., Smet, K., Coudijzer, K., Block, J.D., 2010.

Understanding and preventing consumer milk microbial spoilage and chemical

deterioration, in: Griffiths, M.W. (Ed.), Improving the Safety and Quality of Milk,

Volume 2 - Improving Quality in Milk Products. Woodhead Publishing, Cambridge,

pp. 96-135.

Hotchkiss, J.H., Werner, B.G., Lee, E.Y.C., 2006. Addition of carbon dioxide to dairy products toACCEPTED improve quality: A comprehensive MANUSCRIPT review. Comprehensive Reviews in Food Science and Food Safety 5, 158-168.

Kahala, M., Blasco, L., Joutsjoki, V., 2012. Molecular characterization of spoilage bacteria as

a means to observe the microbiological quality of carrot. Journal of Food Protection

75, 523-532.

King, J.S., Mabbitt, L.A., 1982. Preservation of raw milk by the addition of carbon dioxide.

Journal of Dairy Research 49, 439-447.

ACCEPTED MANUSCRIPT

Kosikowski, F.V., Brown, D.P., 1973. Influence of carbon dioxide and nitrogen on microbial

populations and shelf life of cottage cheese and sour cream. Journal of Dairy Science

56, 12-18.

Liu, S.-n., Han, Y., Zhou, Z.-j., 2011. Lactic acid bacteria in traditional fermented Chinese

foods. Food Research International 44, 643-651.

Lo, R., Xue, T., Weeks, M., Turner, M.S., Bansal, N., 2016. Inhibition of bacterial growth in

sweet cheese whey by carbon dioxide as determined by culture-independent

community profiling. International Journal of Food Microbiology 217, 20-28.

Loss, C.R., Hotchkiss, J.H., 2003. The use of dissolved carbon dioxide to extend the shelf-life

of dairy products, in: Smit, G. (Ed.), Dairy Processing. Woodhead Publishing,

Cambridge, pp. 391-415.

Machado, S.G., da Silva, F.L., Bazzolli, D.M.S., Heyndrickx, M., Costa, P.M.d.A., Vanetti,

M.C.D., 2015. Pseudomonas spp. and Serratia liquefaciens as predominant spoilers in

cold raw milk. Journal of Food Science 80, M1842-M1849.

Maniar, A.B., Marcy, J.E., Bishop, J.R., Duncan, S.E., 1994. Modified atmosphere packaging

to maintain direct-set cottage cheese quality. Journal of Food Science 59, 1305-1308.

Margulies, M., Egholm, M., Altman, W.E., Attiya, S., Bader, J.S., Bemben, L.A., Berka, J., Braverman,ACCEPTED M.S., Chen, Y.-J., Chen, MANUSCRIPT Z., Dewell, S.B., Du, L., Fierro, J.M., Gomes, X.V., Godwin, B.C., He, W., Helgesen, S., Ho, C.H., Irzyk, G.P., Jando, S.C.,

Alenquer, M.L.I., Jarvie, T.P., Jirage, K.B., Kim, J.-B., Knight, J.R., Lanza, J.R.,

Leamon, J.H., Lefkowitz, S.M., Lei, M., Li, J., Lohman, K.L., Lu, H., Makhijani,

V.B., McDade, K.E., McKenna, M.P., Myers, E.W., Nickerson, E., Nobile, J.R., Plant,

R., Puc, B.P., Ronan, M.T., Roth, G.T., Sarkis, G.J., Simons, J.F., Simpson, J.W.,

Srinivasan, M., Tartaro, K.R., Tomasz, A., Vogt, K.A., Volkmer, G.A., Wang, S.H.,

ACCEPTED MANUSCRIPT

Wang, Y., Weiner, M.P., Yu, P., Begley, R.F., Rothberg, J.M., 2005. Genome

sequencing in microfabricated high-density picolitre reactors. Nature 437, 376-380.

Marshall, V.M., 1987. Lactic acid bacteria: starters for flavour. FEMS Microbiology Letters

46, 327-336.

Martin, J.D., Werner, B.G., Hotchkiss, J.H., 2003. Effects of carbon dioxide on bacterial

growth parameters in milk as measured by conductivity. Journal of Dairy Science 86,

1932-1940.

Masoud, W., Takamiya, M., Vogensen, F.K., Lillevang, S., Al-Soud, W.A., Sørensen, S.J.,

Jakobsen, M., 2011. Characterization of bacterial populations in Danish raw milk

cheeses made with different starter cultures by denaturating gradient gel

electrophoresis and pyrosequencing. International Dairy Journal 21, 142-148.

Masoud, W., Vogensen, F.K., Lillevang, S., Abu Al-Soud, W., Sørensen, S.J., Jakobsen, M.,

2012. The fate of indigenous microbiota, starter cultures, Escherichia coli, Listeria

innocua and Staphylococcus aureus in Danish raw milk and cheeses determined by

pyrosequencing and quantitative real time (qRT)-PCR. International Journal of Food

Microbiology 153, 192-202.

McDonald, D., Price, M.N., Goodrich, J., Nawrocki, E.P., DeSantis, T.Z., Probst, A., Andersen,ACCEPTED G.L., Knight, R., Hugenholtz, MANUSCRIPT P., 2012. An improved Greengenes taxonomy with explicit ranks for ecological and evolutionary analyses of bacteria and

archaea. The ISME Journal 6, 610-618.

Moir, C.J., Eyles, M.J., Davey, J.A., 1993. Inhibition of pseudomonads in cottage cheese by

packaging in atmospheres containing carbon dioxide. Food Microbiology 10, 345-351.

Nelson, M.C., Morrison, H.G., Benjamino, J., Grim, S.L., Graf, J., 2014. Analysis,

optimization and verification of Illumina-generated 16S rRNA gene amplicon surveys.

PLoS ONE 9, e94249.

ACCEPTED MANUSCRIPT

Nocker, A., Cheung, C.-Y., Camper, A.K., 2006. Comparison of propidium monoazide with

ethidium monoazide for differentiation of live vs. dead bacteria by selective removal

of DNA from dead cells. Journal of Microbiological Methods 67, 310-320.

Ogier, J.C., Casalta, E., Farrokh, C., Saïhi, A., 2008. Safety assessment of dairy

microorganisms: The Leuconostoc genus. International Journal of Food Microbiology

126, 286-290.

Prasad, P., Turner, M.S., 2011. What bacteria are living in my food?: An open-ended

practical series involving identification of unknown foodborne bacteria using

molecular techniques. Biochemistry and Molecular Biology Education 39, 384-390.

Quigley, L., McCarthy, R., O'Sullivan, O., Beresford, T.P., Fitzgerald, G.F., Ross, R.P.,

Stanton, C., Cotter, P.D., 2013. The microbial content of raw and pasteurized cow

milk as determined by molecular approaches. Journal of Dairy Science 96, 4928-4937.

Quigley, L., O'Sullivan, O., Beresford, T.P., Ross, R.P., Fitzgerald, G.F., Cotter, P.D., 2012.

High-throughput sequencing for detection of subpopulations of bacteria not

previously associated with artisanal cheeses. Applied and Environmental

Microbiology 78, 5717-5723.

Quigley, L., O'Sullivan, O., Stanton, C., Beresford, T.P., Ross, R.P., Fitzgerald, G.F., Cotter, P.D., 2013.ACCEPTED The complex microbiota MANUSCRIPTof raw milk. FEMS Microbiology Reviews 37, 664-698.

Rasolofo, E.A., LaPointe, G., Roy, D., 2011. Assessment of the bacterial diversity of treated

and untreated milk during cold storage by T-RFLP and PCR-DGGE methods. Dairy

Science and Technology 91, 573-597.

Rasolofo, E.A., St-Gelais, D., LaPointe, G., Roy, D., 2010. Molecular analysis of bacterial

population structure and dynamics during cold storage of untreated and treated milk.

International Journal of Food Microbiology 138, 108-118.

ACCEPTED MANUSCRIPT

Riquelme, C., Câmara, S., Enes Dapkevicius, M.d.L.N., Vinuesa, P., da Silva, C.C.G.,

Malcata, F.X., Rego, O.A., 2015. Characterization of the bacterial biodiversity in Pico

cheese (an artisanal Azorean food). International Journal of Food Microbiology 192,

86-94.

Roberts, R.F., Torrey, G.S., 1988. Inhibition of psychrotrophic bacterial growth in

refrigerated milk by addition of carbon dioxide. Journal of Dairy Science 71, 52-60.

Rothberg, J.M., Leamon, J.H., 2008. The development and impact of 454 sequencing. Nature

Biotechnology 26, 1117-1124.

Ruas-Madiedo, P., Alonso, L., González de Llano, D., de los Reyes-Gavilán, C.G., 1998.

Growth and metabolic activity of a cheese starter in CO2-acidified and non-acidified

refrigerated milk. Zeitschrift für Lebensmitteluntersuchung und -Forschung A 206,

179-183.

Ruas-Madiedo, P., Bada-Gancedo, J.C., Fernandez-Garcia, E., De Llano, D.G., de los Reyes-

Gavilan, C.G., 1996. Preservation of the microbiological and biochemical quality of

raw milk by carbon dioxide addition: A pilot-scale study. Journal of Food Protection

59, 502-508.

Ruas-Madiedo, P., Bascarán, V., Braña, A.F., Bada-Gancedo, J.C., de los Reyes-Gavilán, C.G., 1998.ACCEPTED Influence of carbon dioxide MANUSCRIPT addition to raw milk on microbial levels and some fat-soluble vitamin contents of raw and pasteurized milk. Journal of Agricultural

and Food Chemistry 46, 1552-1555.

Schuerger, A.C., Ulrich, R., Berry, B.J., Nicholson, W.L., 2013. Growth of Serratia

liquefaciens under 7 mbar, 0°C, and CO2-enriched anoxic atmospheres. Astrobiology

13, 115-131.

ACCEPTED MANUSCRIPT

Sierra, I., Prodanov, M., Calvo, M., Olano, A., Vidal-Valverde, C., 1996. Vitamin stability

and growth of psychrotrophic bacteria in refrigerated raw milk acidified with carbon

dioxide. Journal of Food Protection 59, 1305-1310.

Sørhaug, T., Stepaniak, L., 1991. Spoilage of milk and dairy products, in: Fox, P.F. (Ed.),

Food Enzymology. Elsevier Science Publishers, London, pp. 169-218.

Sørhaug, T., Stepaniak, L., 1997. Psychrotrophs and their enzymes in milk and dairy products:

Quality aspects. Trends in Food Science and Technology 8, 35-41.

Stead, D., 1986. Microbial lipases: their characteristics, role in food spoilage and industrial

uses. Journal of Dairy Research 53, 481-505.

Stoops, J., Ruyters, S., Busschaert, P., Spaepen, R., Verreth, C., Claes, J., Lievens, B., Van

Campenhout, L., 2015. Bacterial community dynamics during cold storage of minced

meat packaged under modified atmosphere and supplemented with different

preservatives. Food Microbiology 48, 192-199.

Ternström, A., Lindberg, A.M., Molin, G., 1993. Classification of the spoilage flora of raw

and pasteurized bovine milk, with special reference to Pseudomonas and Bacillus.

Journal of Applied Bacteriology 75, 25-34.

Tournas, V.H., 2005. Spoilage of vegetable crops by bacteria and fungi and related health hazards. CriticalACCEPTED Reviews in Microbiology MANUSCRIPT 31, 33-44. van Hekken, D.L., Rajkowski, K.T., Tomasula, P.M., Tunick, M.H., Holsinger, V.H., 2000.

Effect of carbon dioxide under high pressure on the survival of cheese starter cultures.

Journal of Food Protection 63, 758-762.

Vinderola, C.G., Gueimonde, M., Delgado, T., Reinheimer, J.A., Reyes-Gavilán, C.G.d.l.,

2000. Characteristics of carbonated fermented milk and survival of probiotic bacteria.

International Dairy Journal 10, 213-220.

ACCEPTED MANUSCRIPT

Werner, B.G., Hotchkiss, J.H., 2002. Effect of carbon dioxide on the growth of Bacillus

cereus spores in milk during storage. Journal of Dairy Science 85, 15-18.

ACCEPTED MANUSCRIPT

Figure captions

Fig. 1 Inhibition of bacterial growth by CO2 in raw milk stored at 4°C. Results from five raw milk samples collected from different sources (Table 1) are shown. Plate counts were performed until spoilage levels of bacteria were reached (106 CFU/mL).

Fig. 2 Rarefaction analysis of the sequencing reads obtained from dairy samples used in this study. The day 0 MF-CO2 and day 0 SF1-control samples were analysed by Illumina sequencing, while the rest of the samples were analysed by pyrosequencing. See section 3.2 for details.

Fig. 3 Comparison of 16S rRNA community profiling with 454 pyrosequencing and Illumina sequencing. Taxon names are only shown for OTUs with an occurrence of 1% or more in at least one sample. The rest of the OTUs are grouped into "others". The taxa are grouped by phylum, each denoted by a different bar pattern (from top to bottom, left to right of legend): v-pattern— Euryarchaeota; vertical stripes—Actinobacteria; horizontal stripes—

Bacteroidetes; solid—Firmicutes; brick pattern—Fusobacteria; polka dots –

Gemmatimonadetes; diagonal stripes—Proteobacteria (with subgroups of AlphaproteobacteriaACCEPTED, Betaproteobacteria and MANUSCRIPT Gammaproteobacteria); diamond grid—TM7. The rank of each taxon is shown: p__, phylum; c__, class; o__, order; f__, family; g__, genus.

Fig. 4 16S rRNA community profiling of day 0 raw milk from five different sources stored at

4°C. The sequencing platform was pyrosequencing, except for MF-CO2 and SF1-control

(Illumina sequencing). Taxon names are only shown for OTUs with an occurrence of 1% or more in at least one sample. The rest of the OTUs are grouped into "others". The taxa are grouped by phylum, each denoted by a different bar pattern (from top to bottom, left to right

ACCEPTED MANUSCRIPT

of legend): v-pattern— Euryarchaeota; diagonal brick pattern— Acidobacteria; vertical stripes—Actinobacteria; horizontal stripes—Bacteroidetes; checks— Chloroflexi; solid—

Firmicutes; brick pattern—Fusobacteria; polka dots – Gemmatimonadetes; grid—

Planctomycetes; diagonal stripes—Proteobacteria (with subgroups of Alphaproteobacteria,

Betaproteobacteria, Deltaproteobacteria and Gammaproteobacteria); diamond grid—TM7.

The rank of each taxon is shown: p__, phylum; c__, class; o__, order; f__, family; g__, genus.

Fig. 5 16S rRNA community profiling by pyrosequencing of spoiled raw milk from five different sources stored at 4°C. Taxon names are only shown for OTUs with an occurrence of

1% or more in at least one sample. The rest of the OTUs are grouped into "others". The taxa are grouped by phylum (from top to bottom of legend): Gammaproteobacteria,

Betaproteobacteria, Alphaproteobacteria and Firmicutes. Proteobacteria are denoted by a solid bar pattern and Firmicutes by solid. The rank of each taxon is shown: c__, class; o__, order; f__, family; g__, genus.

ACCEPTED MANUSCRIPT

Figure 1

ACCEPTED MANUSCRIPT

Figure 2

ACCEPTED MANUSCRIPT

Figure 3

ACCEPTED MANUSCRIPT

Figure 4

ACCEPTED MANUSCRIPT

Figure 5

ACCEPTED MANUSCRIPT

Table 1

Details of raw milk sample sources

Sample source Locationa Scale Month of sample collection (season) Dairy processing plant 1 (DP1) Queensland Large scale, one of major February 2013 (summer) national dairy producers Dairy processing plant 2 (DP2) Queensland Large scale, one of major March 2014 (autumn) national dairy producers Medium farm (MF) Queensland ~250 cows March 2014 (autumn) Small farm 1 (SF1) Queensland ~50 cows March 2014 (autumn) Small farm 2 (SF2) Victoria 4 cows May 2014 (autumn - winter) astate in Australia

Table 2

Characteristics of pyrosequencing reads

Sample source Number of samples Number of reads after quality filtering analysed Total Minimum Maximum Mean DP1 4 9174 539 3435 2293 DP2 4 20510 361 7984 5127 MF 4a 44916 3207 17779 11229 SF1 3b 18417 3363 11376 6139 SF2 4 27939 4571 10729 6984 a Anomalous results were obtained from day 0 MF-CO2, so this sample was resubmitted for Illumina sequencing (Table 3). bThe sequencing reaction of day 0 SF1-control failed, so this sample was resubmitted for Illumina sequencing (Table 3).

Table 3

Characteristics of llumina reads

Sample source (time point) Sample treatment Number of reads after quality filtering MF (day 0) no CO2 control 22950 +CO2 28934

SF1 (day 0) no CO2 control 25223 +CO2 18673

ACCEPTED MANUSCRIPT

Highlights

 Five carbonated raw milk samples were analysed by next-generation sequencing.  Three samples were spoiled by Leuconostoc, unlike the non-carbonated controls.  Two samples had the same spoilage bacteria as the controls (Pseudomonas, Serratia).  No harmful bacteria were selected by CO2 treatment.  CO2 increased storage life of raw milk stored at 4°C for at least 7 days.

ACCEPTED MANUSCRIPT