Ganesan et al. J. Appl. Micro., 2013
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Supplementary data
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MATERIALS AND METHODS
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Cheese manufacture. Cow’s milk (pH 6.6-6.7) was obtained from the Utah State
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University’s George B. Caine Dairy Research and Teaching Center (Wellsville, UT). The
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protein/fat ratio of the milk was standardized to 0.83, 1.9, and 5.0 for full, reduced, and low fat
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cheese respectively. Cheese was manufactured from standardized, pasteurized milk in open
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horizontal cheese vats at the Gary H. Richardson Dairy Products Laboratory (Utah State
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University, Logan, UT). Double strength chymosin rennet (Maxiren) and single strength annatto
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cheese color were obtained from DSM Food Specialties USA Inc. (Eagleville, PA). Calcium
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chloride was obtained from Danisco USA, Inc. (Madison, WI).
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Full-fat cheese. Cheese milk (160 kg) was warmed to 31°C, the starter and probiotic
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cultures (Table 1) were added, and allowed to ripen for 40 min. Annatto (12 ml) and calcium
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chloride (18 ml) were added followed by rennet (12 ml) to coagulate the milk within 30 min.
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The curd was cut manually using 1 cm knives, allowed to heal for 5 min, and then gently stirred
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manually for 25 min. The temperature of the curd and whey was raised to 39°C over 25 min.
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Stirring was continued for approximately 35 min, until the pH reached 6.3. The whey was
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drained and the curd allowed to mat. The matted cheese was cut into slabs approximately 15  30
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cm, and turned every 10 min. The temperature of the slabs was maintained at 35°C until the pH
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reached 5.4. The slabs were then milled, and salt (470 g) was added in three separate
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applications, 5 min apart. The set-to-salting time was ~ 4 h 35 min. Curd (13 kg) was packed into
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plastic cheesecloth-lined stainless steel hoops and pressed (55 kPa) overnight at ambient
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temperature, to yield one 10-kg block. Cheeses were vacuum-packaged and stored at 3C.
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Reduced-fat cheese. Milk (135 kg) was warmed to 31°C and the starter and probiotic
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cultures were added (see Table 1) and allowed to ripen for 40 min. Annatto (12 ml) was added
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and then 12 ml of rennet was added to coagulate the milk and produce a firm set in 30 min. The
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curd was cut manually using 1.5 cm knives, allowed to heal for 5 min, then gently stirred
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manually for 25 min. The temperature of the curd and whey was raised to 35.5°C over 15 min.
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Stirring was continued for approximately 60 min, until the pH reached 6.35. The whey was
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drained and sufficient water at 18°C was added to bring the temperature of the curd to 29°C. The
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curd was stirred manually for 10 min, the wash water drained, and the curd was stirred until the
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pH reached 5.9. Salt was added at the rate of 113 g per 4.5 kg of curd in three applications, 5 min
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apart. The set-to-salting time was ~ 2 h 55 min. Curd (13 kg) was packed into plastic
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cheesecloth-lined stainless steel hoops and pressed (55 kPa) overnight at ambient temperature to
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yield one 10-kg block. Cheeses were vacuum-packaged and stored at 3C.
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Low-fat cheese. Vinegar was slowly added to 135 kg of milk at 12°C until the pH
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reached 6.4. The milk was warmed to 32°C and the starter and probiotic cultures were added (see
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Table 1) and allowed to ripen for 40 min. Annatto (18 ml) was added and then 12 ml of rennet
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was added to coagulate the milk and produce a firm set in 30 min. The curd was cut vertically
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only, using 1.5 cm knives, allowed to heal for 5 min, then gently stirred manually for 15 min.
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The temperature of the curd and whey was raised to 38°C over 15 min. Stirring was continued
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for approximately 25 min, until the pH reached 6.2. The whey was drained and sufficient water
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at 18°C was added to bring the temperature of the curd to 26.5°C. The curd was stirred manually
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for 10 min, the wash water drained, and the curd was stirred until the pH reached 5.9. Salt was
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added at the rate of 118 g per 4.5 kg of curd in three applications, 5 min apart. The set-to-salting
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time was ~ 2 h 50 min. Curd (13 kg) was packed into plastic cheesecloth-lined stainless steel
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hoops and pressed (55 kPa) overnight at ambient temperature to yield one 10-kg block. Cheeses
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were vacuum-packaged and stored at 3C.
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Proximate Analysis. Proximate analysis was conducted on all cheeses at 5 d of age.
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Moisture content was determined in triplicate by weight loss using a microwave oven (CEM
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Corp., Indian trail, NC) at 70% power with an endpoint setting of <0.4 mg weight change over 2
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s. Fat content was determined in duplicate using a modified Babcock method (Richardson,
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1985). Salt was measured using by homogenizing grated cheese with distilled water for 4 min at
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260 rpm in a Stomacher 400 (Seward, England). The slurry was filtered through a Whatman #1
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filter paper, and the filtrate was analyzed for sodium chloride using a chloride analyzer (model
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926, Corning, Medfield, MA). The pH was measured using a glass electrode after stomaching 20
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g grated cheese with 10 g distilled water for 1 min at 260 rpm.
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RESULTS
Cheese manufacture. The average composition of cheeses was consistent within type,
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whereas the physico-chemical conditions across the cheese types were different at the start of
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aging (Table S1). For example, the reduced-fat Cheddar cheese had 18% more moisture and 25%
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more salt-in-moisture than the full fat cheese; whereas the low-fat cheese had 10% more
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moisture than the reduced fat cheese, but both cheeses had comparable salt-in-moisture levels.
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Hence, the differences observed in different cheeses’ bacterial populations are attributable to the
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overall physico-chemical differences derived from different fat, moisture, and salt contents.
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Optimization and validation of probiotic LAB qPCR. Primers used for detecting
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probiotic bacterial species in this study were selected from previous studies (Table 2), but were
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further tested and optimized to ensure specificity against the probiotic strains added to cheese.
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Under our qPCR conditions, some primers from other studies did not detect the species used
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optimally (data not shown) and were replaced by the primer sets shown in Table 2. All qPCR
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primers used were pre-optimized for different levels of probiotic species by adding the bacteria
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at known levels to cheese (detection range of 102-108 CFU/g; standard curves with R2≥0.9 for all
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species; see Fig. S1 in supplementary material). Specificity of primers was also verified against
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plate counting and proved to be superior, a good example of which is enumerating bifidobacteria
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where media plates of the control cheeses (no added probiotic) also exhibited colonies in
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purported bifidobacteria-specific media(Oberg et al. 2011) whereas the species were not
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detected by qPCR (data not shown). Some primer sets previously indicated to be species-specific
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were found to also detect multiple lactobacilli, thus providing a genus-level estimate of their
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population in cheese (Table 2). Hence, the availability of genus- and species-level primer sets for
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Lactobacillus allowed us to determine that in all cheeses, the levels and survival patterns of other
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lactobacilli were distinct from that of added probiotic lactobacilli (see Fig. S2 in supplementary
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material). By using the genus primer set for lactobacilli, we also assessed the impact of added
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probiotic species on NSLAB.
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Table S1. Proximate composition (averages) of Cheddar cheese made at different fat levels.
Cheese type
Moisture (%)
Fat
Salt
% Salt-in-moisture % Fat in dry matter
(%)
(%)
Full fat
38.8
31.5
1.2
3.0
51.5
Reduced fat
45.8
17.1
1.9
4.1
31.5
Low fat
50.5
7.5
2.0
3.9
15.2
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Table S2. Limit-of-detection for different probiotic bacteria using species primers in Cheddar
cheese.
Strain
CFU/gm cheese CV% at limit-of-detection
Bif6
12
3.0
BB12
17
3.0
L-26
120
1.9
CRL-431 110
1.6
L-10
340
1.1
F-19
410
1.0
LA-5
480
2.2
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Statistical analysis of probiotic survival
Generalized linear models (GLM), normal distribution
α=0.05
Table S3. Analysis of main effects across all probiotics added
Effects: Time, fat, Time * fat
Probiotic
Statistically significant (p≤0.05) effects and interactions
NSLAB levels
Probiotic levels
LA-5
-
-
L-10
Fat
-
F 19
Fat
Fat
L-26
Fat
-
CRL-431
-
Time
BB-12
-
Fat
Bif-6
-
-
Control – probiotic not added 8
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Not applicable
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Table S4. Differences in probiotic survival by genus
Effects: Time, fat, probiotic, Time * fat, fat * probiotic, time * probiotic, time * fat * probiotic
Probiotics
Statistically significant (p≤0.05) effects and interactions
All
Fat, probiotic, fat * probiotic
Lactobacillus probiotics Fat, probiotic, fat * probiotic
Bif probiotic
4
5
6
7
Fat, time * fat
Table S5. Impact of probiotic Lactobacillus strains on NSLAB levels in cheese
Effects: Time, fat, probiotic, Time * fat, fat * probiotic, time * probiotic, time * fat * probiotic
Probiotic
Statistically significant (p≤0.05) effects and interactions
Probiotic addition
Time, Time * fat
Probiotic
Statistically significant (p≤0.05) effects and interactions
LA-5
Probiotic
L-10
Fat, time*fat
F 19
Fat
L-26
Fat, Probiotic
CRL-431
Time*fat
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9
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Table S5. MIQE checklist for authors, reviewers and editors*
IMPORTA
NCE
CHECKLIST
Definition of experimental and control groups
E
Yes
Number within each group
E
Yes
Assay carried out by core lab or investigator's lab?
D
Yes
Acknowledgement of authors' contributions
D
Yes
E
Yes
Volume/mass of sample processed
D
Yes
Microdissection or macrodissection
E
NA
E
Yes
If frozen - how and how quickly?
E
NA
If fixed - with what, how quickly?
E
NA
E
Yes
E
Yes
Name of kit and details of any modifications
E
Yes
Source of additional reagents used
D
Yes
Details of DNase or RNAse treatment
E
Yes
Contamination assessment (DNA or RNA)
E
Yes
Nucleic acid quantification
E
Yes
Instrument and method
E
Yes
Purity (A260/A280)
D
Yes
Yield
D
Yes
ITEM TO CHECK
EXPERIMENTAL DESIGN
SAMPLE
Description
Processing procedure
Sample storage conditions and duration (especially
for FFPE samples)
NUCLEIC ACID EXTRACTION
Procedure and/or instrumentation
Ganesan et al. J. Appl. Micro., 2013
RNA integrity method/instrument
E
NA
RIN/RQI or Cq of 3' and 5' transcripts
E
NA
Electrophoresis traces
D
NA
E
Yes
E
NA
E
NA
Priming oligonucleotide (if using GSP) and
concentration
E
NA
Reverse transcriptase and concentration
E
NA
Temperature and time
E
NA
Manufacturer of reagents and catalogue numbers
D
NA
Cqs with and without RT
D*
NA
Storage conditions of cDNA
D
NA
If multiplex, efficiency and LOD of each assay.
E
Singleplex
Sequence accession number
E
No
Location of amplicon
D
No
Amplicon length
E
Yes
In silico specificity screen (BLAST, etc)
E
Yes
Pseudogenes, retropseudogenes or other
homologs?
D
NA
D
No
Secondary structure analysis of amplicon
D
NA
Location of each primer by exon or intron (if
applicable)
E
NA
E
NA
Inhibition testing (Cq dilutions, spike or other)
REVERSE TRANSCRIPTION
Complete reaction conditions
Amount of RNA and reaction volume
qPCR TARGET INFORMATION
Sequence alignment
What splice variants are targeted?
qPCR OLIGONUCLEOTIDES
Ganesan et al. J. Appl. Micro., 2013
Primer sequences
E
Yes
RTPrimerDB Identification Number
D
NA
D**
NA
Location and identity of any modifications
E
NA
Manufacturer of oligonucleotides
D
Yes
Purification method
D
Yes
E
Yes
Reaction volume and amount of cDNA/DNA
E
Yes
Primer, (probe), Mg++ and dNTP concentrations
E
Yes
Polymerase identity and concentration
E
Not supplied by
manufacturer
Buffer/kit identity and manufacturer
E
Not supplied by
manufacturer
Exact chemical constitution of the buffer
D
Not supplied by
manufacturer
Additives (SYBR Green I, DMSO, etc.)
E
SYBR Green I
Manufacturer of plates/tubes and catalog number
D
-
Complete thermocycling parameters
E
Yes
Reaction setup (manual/robotic)
D
Manual
Manufacturer of qPCR instrument
E
Yes
Evidence of optimisation (from gradients)
D
NA
Specificity (gel, sequence, melt, or digest)
E
Yes
For SYBR Green I, Cq of the NTC
E
Yes
Standard curves with slope and y-intercept
E
Yes
Probe sequences
qPCR PROTOCOL
Complete reaction conditions
qPCR VALIDATION
Ganesan et al. J. Appl. Micro., 2013
PCR efficiency calculated from slope
E
Yes
D
-
E
Yes
E
Yes
Cq variation at lower limit
E
Yes
Confidence intervals throughout range
D
-
Evidence for limit of detection
E
Yes
If multiplex, efficiency and LOD of each assay.
E
Singleplex
E
Yes
Cq method determination
E
Yes
Outlier identification and disposition
E
NA
Results of NTCs
E
Yes
Justification of number and choice of reference genes
E
NA
Description of normalisation method
E
NA
Number and concordance of biological replicates
D
Yes
Number and stage (RT or qPCR) of technical
replicates
E
Yes
Repeatability (intra-assay variation)
E
Yes
Reproducibility (inter-assay variation, %CV)
D
Yes
Power analysis
D
No
Statistical methods for result significance
E
Yes
Software (source, version)
E
Yes
Cq or raw data submission using RDML
D
No
Confidence interval for PCR efficiency or standard
error
r2 of standard curve
Linear dynamic range
DATA ANALYSIS
qPCR analysis program (source, version)
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* According to MIQE standards, all essential information (E) must be submitted with the
manuscript. Desirable information (D) should be submitted if available. If using primers
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obtained from RTPrimerDB, information on qPCR target, oligonucleotides, protocols and
validation is available from that source.
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Figure S1. Standard curves of bacterial counts for qCR-based estimation.
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Figure S2. Different methods for DNA extraction yield different results for lactococcal detection
by qPCR. Methods used for full fat Cheddar cheese over age were glass bead lysis (filled
squares) and lysozyme-mutanolysin lysis (filled circles and triangles).
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Figure S3. Levels of added probiotic Lactobacillus in Cheddar cheeses over 9 mo of aging,
estimated by qPCR. Error bars represent standard deviations of population estimates from two
biological replicates of cheese making.
Lactobacillus acidophilus La-5
Lactobacillus acidophilus L10
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Lactobacillus paracasei F-19
Lactobacillus casei L-26
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Lactobacillus casei CRL-431
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Figure S4. Levels of added probiotic Bifidobacterium lactis in Cheddar cheeses over 9 mo of
aging, estimated by qPCR. Error bars represent standard deviations of population estimates from
two biological replicates of cheese making.
Bifidobacterium lactis Bif-6
Bifidobacterium lactis BB12
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Figure S5. Levels of total lactobacilli in probiotic Lactobacillus-added Cheddar cheeses over 9
mo of aging, estimated by qPCR. Error bars represent standard deviations of population
estimates from two biological replicates of cheese making.
Lactobacillus acidophilus La-5
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Lactobacillus acidophilus L10
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Lactobacillus paracasei F-19
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Lactobacillus casei L-26
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Lactobacillus casei CRL-431
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REFERENCES
Oberg, C.J., Moyes, L.V., Domek, M.J., Brothersen, C. and McMahon, D.J. (2011) Survival of
probiotic adjunct cultures in cheese and challenges in their enumeration using selective media. J
Dairy Sci 94, 2220-2230.