probiotic bacteria survive in cheddar cheese and modify populations of other lactic acid bacteria
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ORIGINAL ARTICLE
Probiotic bacteria survive in Cheddar cheese and modifypopulations of other lactic acid bacteriaB. Ganesan1,2, B.C. Weimer3, J. Pinzon3, N. Dao Kong3, G. Rompato4, C. Brothersen1,2 andD.J. McMahon1,2
1 Dairy Technology and Innovation Laboratory, Western Dairy Center, Utah State University, Logan, UT, USA
2 Department of Nutrition, Dietetics, and Food Sciences, Utah State University, Logan, UT, USA
3 Department of Population Health and Reproduction, University of California, Davis, CA, USA
4 Center for Integrated BioSystems, Utah State University, Logan, UT, USA
Keywords
Cheddar cheese, low fat, nonculturability,
probiotic, survival.
Correspondence
Balasubramanian Ganesan, Dairy Technology
and Innovation Laboratory, Western Dairy
Center, Utah State University, 8700 Old Main
Hill, Logan, UT 84322, USA.
E-mail: g.balsu@usu.edu; and
Bart C. Weimer, University of California,
Davis School of Veterinary Medicine 1089
Veterinary Medicine Dr. VM3B, Room 4023
Davis, CA 95616, USA.
E-mail: bcweimer@ucdavis.edu
2013/2047: received 8 October 2013, revised
6 January 2014 and accepted 12 February
2014
doi:10.1111/jam.12482
Abstract
Aims: Starter lactic acid bacteria in Cheddar cheese face physico-chemical
stresses during manufacture and ageing that alter their abilities to survive and
to interact with other bacterial populations. Nonstarter bacteria are derived
from milk handling, cheese equipment and human contact during
manufacture. Probiotic bacteria are added to foods for human health benefits
that also encounter physiological stresses and microbial competition that may
mitigate their survival during ageing. We added probiotic Lactobacillus
acidophilus, Lactobacillus casei, Lactobacillus paracasei and Bifidobacterium
animalis subsp. lactis to full-fat, reduced-fat and low-fat Cheddar cheeses,
aiming to study their survival over 270 days of ageing and to determine the
role of the cheese matrix in their survival.
Methods and Results: Probiotic and other lactic acid bacterial populations
were enumerated by quantitative PCR using primers specifically targeting the
different bacterial genera or species of interest. Bifidobacteria were initially
added at 106 CFU g�1 cheese and survived variably in the different cheeses
over the 270-day ageing process. Probiotic lactobacilli that were added at
107 CFU g�1 cheese and incident nonstarter lactobacilli (initially at 108 CFU
g�1 cheese) increased by 10- to 100-fold over 270 days. Viable bacterial
populations were differentiated using propidium monoazide followed by
species-specific qPCR assays, which demonstrated that the starter and probiotic
microbes survived over ageing, independent of cheese type. Addition of
probiotic bacteria, at levels 100-fold below that of starter bacteria, modified
starter and nonstarter bacterial levels.
Conclusions: We demonstrated that starter lactococci, nonstarter lactobacilli
and probiotic bacteria are capable of surviving throughout the cheesemaking and
ageing process, indicating that delivery via hard cheeses is possible. Probiotic
addition at lower levels may also alter starter and nonstarter bacterial survival.
Significance and Impact of the Study: We applied qPCR to study multispecies
survival and viability and distinctly enumerated bacterial species in
commercial-scale Cheddar cheese manufacture.
Introduction
Probiotic bacteria are defined as ‘live micro-organisms
which when administered in adequate amounts confer a
health benefit on the host’ (FAO/WHO 2002; Morelli and
Capurso 2012). The consumption of probiotic bacteria is
reported to confer many health benefits such as prevent-
ing gut inflammation, immunomodulation, preventing
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology1642
Journal of Applied Microbiology ISSN 1364-5072
food allergy and also potentially provide anticarcinogenic
activity (Kailasapathy 2002; Caglar et al. 2005; Mengheri
2008). As early as infancy, probiotic bacterial species that
are components of the native microbiota of breast milk
enter the human colon. Probiotic bacteria are also sup-
plemented via infant formula (Moreno Villares 2008; de
Vrese and Schrezenmeir 2008) to aid milk oligosaccharide
digestion (Macfarlane et al. 2008), whereas adults typi-
cally source probiotics from fermented milks and nutri-
tional supplements. The consumption of probiotic-
supplemented cheese boosts innate immunity of geriatric
patients (Ibrahim et al. 2010). However, probiotic bacte-
ria must survive in foods to reach the human gastrointes-
tinal system and to further modify gut microbiota
(Kramer et al. 2009; Yu et al. 2009). Guidelines for pro-
biotic bacteria recommend that identity at the species
level must be established (FAO/WHO 2002).
Probiotic bacteria such as Bifidobacterium longum,
Bif. lactis, Lactobacillus acidophilus, Lact. casei and
Lact. paracasei are usually added to yoghurt and other
fermented milks (Heller 2001), and more recently to
cheese (Gardiner et al. 1998) as delivery vehicles for
human consumption. Lactococcus lactis is used as a starter
culture to produce lactic acid for pH reduction during
Cheddar cheese manufacture, whereas nonstarter lactic
acid bacteria (NSLAB) and other microbes are either
present in cheese milk or are added purposefully (Peter-
son and Marshall 1990; Trepanier et al. 1991; Habibi-
Najafi and Lee 1996; Swearingen et al. 2001). Probiotic
bacteria are usually added to cheese milk and thus
sequentially undergo physico-chemical stresses such as
heat, acid, salt and cold during initial manufacture, as
well as changes in redox potential over storage and distri-
bution (Rallu et al. 1996; van de Guchte et al. 2002), as
do other adventitious or added lactic acid bacteria (LAB).
When cheeses are manufactured from milk of different
fat contents (Bertoni et al. 2001), the sequence of abiotic
stresses varies due to the different processing conditions
used (Fox and Wallace 1997; Banks 2007; Coolbear et al.
2011), which may alter subsequent LAB survival during
ageing. As NSLAB survive in cheese and grow over age-
ing, of which lactobacilli are the dominant species, probi-
otic lactobacilli species may also remain viable in
Cheddar cheese during ageing until consumption to pro-
vide health benefits.
Estimates of bacterial viability in different foods and
environments vary based on the enumeration techniques
used. Growth media-based enumeration discounts possi-
ble alternate physiological states of bacteria, such as non-
culturability (Fenelon et al. 2000; Ganesan et al. 2007).
Such growth-based observations led to a previous
hypothesis that starter bacteria die and lyse to subse-
quently provide substrates that accelerate NSLAB growth
(Branen and Keenan 1969; Crow et al. 1995; Buist et al.
1997, 1998). However, lactococci, NSLAB and brevibacte-
ria become nonculturable in carbohydrate-depleted media
while remaining metabolically active (Ganesan et al.
2004, 2007) and maintain substrate transport for produc-
ing energy from alternate substrates (Ganesan et al.
2007). The declining lactococcal counts in cheese may
represent a subpopulation of replicating cells, while a
nonculturable population of cells that is unable to divide
and is hence not enumerated on growth media (Kilcawley
et al. 2011) coexists. Additionally, the enumeration of an
entire genus using selective growth media does not accu-
rately reflect the metabolic contributions of multiple spe-
cies of a diverse genus, such as Lactobacillus (Peterson
and Marshall 1990; Trepanier et al. 1991), that shares
metabolic capabilities with other Lactobacillales (Makar-
ova et al. 2006). Hence, alternate population assessment
methods are needed to better estimate LAB species diver-
sity in cheese and survival of probiotic bacterial species
in foods used for delivery.
Multiple studies at laboratory- or pilot-scale cheese
manufacture have demonstrated probiotic bacterial sur-
vival in hard cheeses such as Cheddar, a representative
set of which are listed in Table 1. According to these
studies, even the same strains or species survive variably,
with one group showing survival throughout ageing, but
another demonstrating loss of viability of the same in 6–8 weeks. Some studies were conducted in smaller scale
(10–20 l of milk), which do not represent issues in com-
mercial-scale manufacture such as adventitious NSLAB
and bacteriophages’ presence. Notably, none of these
studies enumerated survival of probiotic bacteria at the
species level. Neither did these studies assess the impact
of adding a microbe not usually found in cheese on the
starter or NSLAB of cheeses. In one study that used
qPCR for detecting bacteria (Achilleos and Berthier
2013), only soft cheeses were assessed over 24 h and did
not address the challenge of detecting bacteria in aged
semi-hard cheeses.
To meet regulatory requirements, bacteria are isolated
from foods (Giraffa and Rossetti 2004; Rossetti and Giraf-
fa 2005) prior to species identification based on 16S or
23S rRNA genes using organism-specific primers (Salama
et al. 1991; Klijn et al. 1995), single-gene sequencing, ran-
dom DNA amplification, use of restriction enzyme diges-
tion patterns (Byun et al. 2004; Rossetti and Giraffa
2005), gel electrophoresis by varying electric fields (pulse
field) (Klein et al. 1998; Henri-Dubernet et al. 2008), tem-
perature (Vasquez et al. 2001; Ogier et al. 2002) or chemi-
cal gradients (denaturing gradient) (Fasoli et al. 2003);
however, these methods do not enumerate bacteria nor
conclusively establish species identity. With advances in
PCR amplification technology and the use of fluorescent
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology 1643
B. Ganesan et al. LAB survival in probiotic-added cheese
Table
1Previousstudiesofprobioticad
ditionto
Ched
dar
cheese
Study
Probioticbacteriaused
Scaleof
cheese
man
ufacture
Targeted
levelof
probiotics
(CFU
g�1)
Detectionmethodin
cheese
Viability
assessmen
tin
cheese
Detectedlevels
ofprobiotics
(CFU
g�1)
Cheese
ageing
period
(months)
Detection
limitsof
assays
(CFU
g�1)
Dinakar
and
Mistry(1994)
Bifidobacterium
bifidum
(immobilized;ad
ded
duringsalting)
100kg
106
Microbialplatingonselectivemed
iaMicrobialplatingonselectivemed
ia106to
107
625
Gardiner
etal.(1998)
Lactobacillussalivarius,
Lact.paracasei
25,450l
(2reps9
2strains
only)
NS
RAPD
-PCR/gel
electrophoresison
DNAextractedfrom
growncolonies
Microbialplatingonselectivemed
ia108
825
Daigle
etal.(1999)
Bifidobacterium
infantis
250l
59
107
Microbialplatingonselectivemed
iaMicrobialplatingonselectivemed
ia107
325
Gardiner
etal.(1999a)
Enterococcusfaecium
450l
49
108
Antibioticresistan
cemutant
Microbialplatingonselectivemed
ia49
108
15
25
Gardiner
etal.(1999b)
Ent.faecium
450l
108
Antibioticresistan
cemutant
Microbialplatingonselectivemed
ia39
108
925
McBrearty
etal.(2001)
Bif.sp.
450l
108
RAPD
-PCR/gel
electrophoresison
DNAextractedfrom
growncolonies
Microbialplatingonselectivemed
ia105to
108
625
Auty
etal.(2001)
Lact.paracasei,
Bif.sp.BB-12
450l
108
Microbialplatingonselectivemed
ia,
stainingan
dconfocalmicroscopy
Microbialplatingonselectivemed
ia,
stainingan
dconfocalmicroscopy
NS
108(confocal)
Phillips
etal.(2006)
Lactobacillusacidophilus,
Bifidobacterium
sp.,Lact.casei,
Lact.paracasei
andLact.rham
nosus
10l
108
Microbialplatingonselectivemed
iaMicrobialplatingonselectivemed
ia103to
108
925
Sharp
etal.(2008)
Lact.casei
136kg
107
Antibioticresistan
cemutant
Microbialplatingonselectivemed
ia107
325
Ongan
dSh
ah
(2008)
Lact.acidophilusan
d
Lact.helveticus
20l
NS
Microbialplatingonselectivemed
iaMicrobialplatingonselectivemed
ia108
625
Achilleo
san
d
Berthier(2013)
Lact.paracasei
Not
disclosed
108
Microbialplatingonselective
med
iaan
dqPC
R
Microbialplatingonselective
med
iaan
dqPC
R
108
0200
NS,
notspecified
.
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology1644
LAB survival in probiotic-added cheese B. Ganesan et al.
labelling, reliable estimates of bacterial populations for a
single genus or species are easily obtained using quantita-
tive PCR (qPCR) (Matsuki et al. 2004; Kramer et al.
2009). Recent studies have established the applicability of
this technology to other fermented dairy products such as
yoghurt (Fasoli et al. 2003; Rademaker et al. 2006; Sieuw-
erts et al. 2008; Garcia-Cayuela et al. 2009; Sheu et al.
2009), and its applicability to cheese has been demon-
strated.
Nucleic acid extraction directly from a food matrix was
previously limited by high carbohydrate, protein and
lipid contents, but methods are now available to extract
high-quality DNA for direct use in identification strate-
gies. Challenges in direct nucleic acid extraction from
cheese were previously circumvented by prior microbial
cell separation from the cheese matrix for nucleic acid
extraction (Rudi et al. 2005; Fernandez et al. 2006; Mon-
net et al. 2006; Fukushima et al. 2007) or using large
sample quantities (≥10 g cheese) (Monnet et al. 2006,
2008; Sohier et al. 2012). Else, the methods only provided
higher detection limits (≥1000 CFU g�1) (Baruzzi et al.
2005; Rantsiou et al. 2008; Sheu et al. 2009) or are inca-
pable of distinguishing added probiotic lactobacilli from
NSLAB lactobacilli (Gardiner et al. 1998). Such
approaches cannot determine changes of species levels in
long-term aged cheeses. However, using genus- and spe-
cies-specific qPCR, population changes of distinct groups
of LAB specifically induced by probiotic addition can be
determined.
We hypothesized that the addition of specific probiotic
bacteria to cheese during manufacture modifies starter
and NSLAB lactobacilli survival in Cheddar cheese at dif-
ferent fat levels. In this study, three cheese types that had
different fat contents were made to determine the role of
the physico-chemical conditions in such cheeses in allow-
ing survival of probiotic lactobacilli and bifidobacteria by
applying a qPCR-based bacterial detection method. We
also assessed whether addition of probiotics at levels
below that of starter bacteria altered starter or NSLAB
levels. Further, the viability of the three groups of bacte-
ria was also determined using propidium iodide-based
qPCR assays. DNA extraction from small quantities of
cheese (0�2–1 g) was optimized by coupling physico-
chemical and enzymatic lysis of bacteria to release nucleic
acids, followed by phenol–chloroform-based applications
to remove protein and lipid. We found that starter,
NSLAB lactobacilli and probiotic bacteria survive
throughout Cheddar cheese ageing with limited reduction
in viability and that probiotic addition even at levels
lower than starter LAB, altered levels of starter lactococci
and NSLAB lactobacilli.
Materials and methods
Probiotic-added cheese manufacture
Starter culture, probiotic adjunct, suppliers and the
amount of culture used are indicated in Table 2. The tar-
geted range of probiotic bacteria in the finished cheese
was 3�6 9 106–107 CFU g�1 (based on the requirement
of ≥108 CFU per 28 g serving), and direct-vat-set cultures
were added proportionally based on manufacturer’s speci-
fications and expected cheese yields to achieve the target
levels. The control cheese was made with Lc. lactis
DVS850 starter culture only, while the probiotic cheeses
each contained DVS850 and one probiotic culture.
Table 2 Starter culture, probiotic adjunct, suppliers and the amount of freeze-dried culture used
Supplier Organism Name
Amount used in cheese (g)*
Full fat (160 kg
cheese milk)
Reduced fat (135 kg
cheese milk)
Low fat (135 kg
cheese milk)
Chr. Hansen, Milwaukee,
WI, USA
Lactococcus lactis DVS850 17�0 30�0 26�0
Cargill Inc., Waukeshaw,
WI, USA
Bifidobacterium lactis Bif-6 14�0 12�0 12�0
Chr. Hansen Bif. lactis BB-12 14�0 12�0 12�0Chr. Hansen Lactobacillus acidophilus LA-5 14�0 12�0 12�0DSM Food Specialties,
Logan, UT, USA
Lact. acidophilus L10 7�8 6�7 6�7
DSM Food Specialties Lactobacillus casei L26 6�4 5�5 5�5Chr. Hansen Lact. casei CRL-431 14�0 12�0 12�0Chr. Hansen Lactobacillus paracasei
subsp. paracasei
F19 35�0 30�0 30�0
*Inoculation levels were adjusted by milk quantity and manufacturer-provided information about cultures on numbers of viable bacteria in
freeze-dried cultures to achieve the desired target ranges of 107 to 108 CFU g�1 probiotic bacteria in cheese.
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology 1645
B. Ganesan et al. LAB survival in probiotic-added cheese
Manufacturing procedures for full-fat, reduced-fat and
low-fat Cheddar cheeses are described in the supplemen-
tary materials and methods section, along with proximate
analysis procedures for cheese.
Sampling
After ageing for 5 days, the cheese was cut into 10 blocks
of c. 1 kg each, vacuum packaged and stored at 3°C. Foreach replicate treatment, one block was randomly chosen
for analysis at 5 days, 1, 2, 3, 4, 6 and 9 month of age.
Extraction of genomic DNA from cheese
To extract DNA from all three types of cheese, finely
grated cheese (0�25 g) was suspended in TrizolLS (500 ll;Invitrogen, Carlsbad, CA, USA) along with glass beads
(0�3 g, size 0�1 mm; sterile, acid-washed; BioSpec Prod-
ucts Inc., Bartlesville, OK, USA). The cheese-TrizolLS sus-
pension was disrupted on a Mini-Beadbeater (BioSpec
Products) for 30 s to lyse bacteria and further shaken
with chloroform (200 ll) for 30 s to allow phase separa-
tion. The top aqueous phase was removed after centrifu-
gation (12 000 g for 15 min at room temperature), and
DNA was precipitated from the lower organic phase with
100% ethanol for 3 min. DNA was collected by centrifu-
gation (2000 g for 5 min at 4°C), washed twice with
0�1 mol l�1 sodium citrate in 10% ethanol (30 min at
room temperature), once with 75% ethanol (15 min at
room temperature), pelleted, air-dried and resuspended
in sterile double-deionized water (ddH2O) This DNA was
used to measure population changes of lactobacilli and
bifidobacteria.
Due to poor efficacy of glass bead lysis for lactococcal
detection (Fig. S2), lactococcal lysis was separately
accomplished by an enzymatic approach in which finely
grated cheese (0�25 g) was treated with bacterial lytic
enzymes (initially 50 mg ml�1 lysozyme + 10 U ml�1
mutanolysin at 37°C for 1 h, followed by 9 U ml�1
proteinase K at 50°C for 1 h). This was followed by phe-
nol–chloroform extraction of DNA and qPCR for enu-
merating lactococci using genus-specific primers. This
lysis approach was also applied to cheeses tested for via-
ble bacterial levels (see below).
DNA quality and yield
DNA quality and yield were checked on a Nanodrop
spectrophotometer (Agilent Technologies, Santa Clara,
CA, USA). All samples used in qPCR had a A260/A280
ratio ≥1�8.
Quantitative PCR
Bacterial levels in cheese were determined by qPCR in
triplicate reactions. Compliance to MIQE guidelines was
ensured throughout the process of qPCR analyses, data
extraction and data analysis (Table S5). All bacterial
primers were selected from previous studies that designed
genus- and species-specific primers for the starter and
probiotic organisms (Table 3) and were subsequently val-
idated by assays against the strains used in this study
prior to use (see Fig. S1). Briefly, the total DNA extracted
from cheese was used in a 25-ll reaction that included
qPCR master Mix (HotStart-IT� SYBR� Green; USB
Corp., Cleveland, OH, USA) and genus- and species-spe-
cific primers targeting the 16s rRNA gene (Table 3). Each
final reaction mixture contained: template DNA, 500 ng,
MgCl2, 2�5 mmol l�1, primers, 10 pmol and each dNTP,
0�4 mmol l�1. The qPCR was performed on a DNA
Engine OPTICON2 (Bio-Rad Labs Inc., Hercules, CA,
USA) with initial enzyme activation at 95°C for 5 min,
followed by 40 cycles of: denaturation at 95°C for 15 s,
annealing at 50°C for 30 s and extension at 72°C for
1 min. All DNA samples from probiotic-added cheeses
were always assayed simultaneously with DNA samples
from control cheeses (no probiotic added) using species-
specific primers to confirm lack of amplification. Following
Table 3 Sequences of primers used for genus- or species-specific 16s ribosomal gene qPCR for different cheese bacteria
Organism Specificity Primer sequence Cheeses applied to References
Lactococcus lactis Genus F: 50- GCGGCGTGGCTAATACATGC-30
R: 50- CTGCTGCGTCCCGTAGGAGT-30All Klijn et al. (1995)
Bifidobacterium lactis Species F: 50-GTGGAGACACGTTTCCC-30
R: 50-CACACACACAATCAATAC-30Bif-6, BB-12 Ventura et al. (2001)
Lactobacillus Genus F: 50-TGGAAACAGRTGCTAATACCG-30
R: 50-GTCCATTGTGGAAGATTCCC-30Control, LA-5, L10, L26, F19 Byun et al. (2004)
Lactobacillus acidophilus Species F: 50-GAGGCAGCAGTAGGGAATCTTC-30
R: 50-GGCCAGTTACTACCTCTATCCTTCTTC-30LA-5, L10 Delroisse et al. (2008)
Lactobacillus casei/paracasei Species F: 50-GCACCGAGATTCAACATGG-30
R: 50-GGTTCTTGGATYTATGCGGTATTAG-30L26, F19 Byun et al. (2004)
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology1646
LAB survival in probiotic-added cheese B. Ganesan et al.
MIQE-compliant procedures, results from all qPCR
assays were extracted using OPTICON monitor software
v2.2 (Bio-Rad).
Viable bacterial qPCR assay using propidium monoazide
The viability of bacteria present in cheese was verified by
a qPCR-based assay, using the DNA-chelating reagent
propidium monoazide (PMA; Biotium Inc., Hayward,
CA, USA). Briefly, the assay was conducted as follows: a
small portion of cheese (0�25 g) was thoroughly minced,
suspended in an equal amount (w/v) of sterile ddH2O,
and a stock of 20 mmol l�1 PMA was added to the
cheese suspension to obtain a final concentration of
50 lmol l�1, as described by Nocker et al. (2007). The
addition and mixing was carried out in light-transparent
tubes that were then kept in dark for 10 min to allow
PMA-nucleic acid binding, followed by exposure to a
650 W halogen lamp for 5 min, with samples kept on
ice, to quench the reaction. After light exposure, samples
were treated with lysozyme–mutanolysin and DNA was
extracted for qPCR-based enumeration. For live-dead
assays by qPCR using PMA, the difference in Cq times
with and without PMA addition, denoted as DCq, wasmonitored over cheese age.
Statistical analysis
All cheeses were made in two replicates that were manu-
factured from different batches of milk. Cheese samples
were collected for qPCR enumeration at 5 days, 1, 2, 3,
4, 6 and 9 months. Each sample was analysed by qPCR
in triplicate, and the results of bacterial counts from
qPCR assays were analysed as a nested factorial repeated-
measures design (Ganesan et al. 2007) to understand the
impact of cheese type on survival of different added pro-
biotics and the impact of cheese type and strain on sur-
vival of NSLAB. JMP v7 statistical software (The SAS
Institute, Cary, NC, USA) was used for repeated-mea-
sures statistical analyses. For the time series analyses, sig-
nificance was assigned at a = 0�05/n, where n is the
number of time points. Two-sample statistical compari-
sons within a particular condition when necessary were
performed by two-tailed Student’s t-tests with Microsoft
Excel software (Redmond, CA, USA), and significance
was assigned at a = 0�05.
Results
LAB population changes during ageing
Three types of Cheddar cheeses—full, reduced and low
fat—were manufactured (Table S1), and seven probiotic
bacterial species were added to the different cheeses.
Changes in specific LAB populations over ageing were
assessed by qPCR using a species primer set that corre-
sponded to the species added to the cheeses (validation
and variability results in supplementary material). We
developed a sensitive qPCR assay based on previously val-
idated primers that provided reliable detection even at
10–500 CFU g�1 from Cheddar cheese (Table S2). The
targeted levels of probiotics in cheese were at least
10 000-fold higher than our detection limits.
Primarily, probiotic bacteria were added targeting a
final level of 3�6 9 106–107 CFU g�1 cheese, which was
accomplished in all full- and low-fat cheeses (Fig. S3).
Interestingly, added probiotic lactobacilli populations
were consistently lower than the target level by 10-fold
initially in reduced-fat Cheddar cheeses, but rose to attain
levels of c. 107–108 CFU g�1 cheese within a month of
ageing (Fig. S2), indicating that they grew in the cheese
by 10- to 100-fold. However, at any time, probiotic lacto-
bacilli levels were only 1–10% of that of total lactobacilli
(Figs S2 and S4), confirming that added probiotic and
NSLAB lactobacilli can be distinctly enumerated using
qPCR.
Probiotic Lact. casei/paracasei survived through
270 days of ageing in all Cheddar cheeses either at com-
parable levels or grew by 10- to 70-fold (Table 4), with
differences in final levels attained within each fat level
(P ≤ 0�05). Added Lact. acidophilus exhibited a 16- to 37-
fold reduction in population (P ≤ 0�05) from the initial
level of addition during ageing in low-fat cheese
(Table 4). However, Lact. acidophilus strains did not dif-
fer in survival patterns over time across the different fat
Table 4 Fold change of different probiotic bacterial populations in
cheeses over 270 days of ageing
Probiotic organism
Fold change in populations (CFU g�1 ratio of
270 days/0 days)*
Full fat Reduced fat Low fat
Lactobacillus
acidophilus LA-5
�1�9 � 2�3 13 � 0�9 �16 � 0�4
Lact. acidophilus L10 2�3 � 1�0 230 � 0�9 �37 � 1�4Lactobacillus
casei CRL-431
4�1 � 2�0 9�3 � 0�9 71 � 0�6
Lact. casei L26 16 � 0�4 7�4 � 0�2 3�5 � 0�3Lactobacillus
paracasei F19
1�5 � 0�5 2�2 � 1�0 5�2 � 0�8
Bifidobacterium
lactis Bif-6
�12 � 10 2�8 � 1�1 �6900 � 10
Bif. lactis BB-12 �3200 � 100 1500 � 10 �2�7 � 1�0
Negative sign indicates reduction.
*Values are indicated followed by standard deviations after the ‘�’
sign.
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology 1647
B. Ganesan et al. LAB survival in probiotic-added cheese
levels (P > 0�05). In contrast, Bif. lactis strains exhibited
varying survival patterns (P ≤ 0�05) within each fat level,
with the Bif-6 strain populations declining by 6900-fold
in low-fat cheese within 270 days, whereas the BB-12
strain declined by 3200-fold in full-fat cheese (Table 4).
Bif-6 remained at similar levels to initial inoculum in
reduced-fat cheese (P > 0�05), whereas BB-12 increased
by 1500-fold (P ≤ 0�05) (Table 4). Notably, estimates of
bifidobacterial levels varied by 100- to 1000-fold within
replicates for 43% of cheeses across age and fat level,
which precluded any strong conclusions about their pop-
ulation variations. Such variation was much less prevalent
in probiotic lactobacilli (9% cheeses with 100- to 1000-
fold variation). However, bifidobacteria were exclusively
detected by qPCR throughout cheese ageing, which con-
firms their viability in cheese.
To understand the role of cheese fat content on micro-
bial survival, we directly compared the probiotic levels in
reduced-fat and low-fat cheeses. Interestingly, the lower
fat level appeared to reduce bacterial survival, as high-
lighted by significant decrease (P ≤ 0�05) in numbers of
Lact. acidophilus over ageing in low-fat cheese. However,
Lact. casei/paracasei grew in both reduced- and low-fat
cheeses. These results indicated that fat in cheese may aid
survival of selected species of lactobacilli during ageing.
As probiotic bacteria were added at levels of 107 CFU
g�1 and survived variably, they may alter the levels of
starter LAB and NSLAB in ageing Cheddar cheese. We
assessed the effect of probiotic addition by measuring
changes in overall lactobacilli populations using a primer
set targeting the Lactobacillus genus. Notably, in all
cheeses tested, levels of total lactobacilli were 10- to 100-
fold higher than levels of added probiotic, and the pat-
terns of survival of these two groups were different
(P ≤ 0�05) (Figs S2 and S4). However, survival and
growth of total lactobacilli in the probiotic lactobacilli-
added cheeses differed due to the different strains added
and fat level (P ≤ 0�05). While the overall populations of
total lactobacilli remained at 108 CFU g�1 or higher in
all cheeses, the addition of Lact. acidophilus La-5 and
Lact. casei CRL431 significantly (P ≤ 0�05) altered the
levels of total lactobacilli, dependent on cheese fat level.
For example, at 270 days of ageing, La-5 addition
reduced total lactobacilli by eightfold in the reduced-fat
cheese, but there was no change in the low-fat cheese
(P ≤ 0�05; Table 5). Addition of CRL431 decreased total
lactobacilli by sevenfold in reduced-fat Cheddar cheese
and increased total lactobacilli by sevenfold in low-fat
cheese (P ≤ 0�05; Table 5). Addition of other probiotic
organisms did not alter total lactobacilli levels (P > 0�05),but cheese fat level altered (P ≤ 0�05) the total lactobacilli
levels. One such example is the cheeses to which Lact. pa-
racasei F19 was added, in which total lactobacilli levels
had increased 34-fold in 270-day-old low-fat cheese
(P ≤ 0�05), but had decreased sixfold in reduced-fat
cheese (Table 5). These results indicate that fat reduction
in cheeses and probiotic addition alters survival of
NSLAB lactobacilli over cheese ageing.
As NSLAB levels were altered by addition of probiotic
lactobacilli and bifidobacteria, we also measured changes
in lactococcal populations using PCR targeting Lc. lactis
species in all three cheese types in a limited set of sam-
ples. Lactococcal populations in the control (no probiotic
added) cheeses were compared to levels in Lact. acidophi-
lus L-10-added and Bif. lactis Bif-6-added cheeses. Lacto-
coccal counts in control cheeses ranged between
7�5 9 107 and 8 9 108 CFU g�1 and did not change
over time. Addition of different probiotics significantly
(P ≤ 0�05) altered lactococcal populations and also the
effects varying within each fat level. For example, addi-
tion of Bif-6 did not change lactococcal levels within
270 days in low-fat cheeses, but decreased lactococcal
populations in reduced-fat cheeses by 10-fold compared
to without Bif-6 addition (P ≤ 0�05; Fig. 1), whereas L-10addition consistently allowed lactococci to survive at 10-
to 100-fold higher levels than without probiotic addition
in low-fat cheeses (P ≤ 0�05), but did not alter lactococ-
cal levels in reduced- and full-fat cheeses.
Viability of LAB in cheese
LAB that do not survive the harsh cheese conditions may
die, and their cell walls and membranes may rupture,
releasing DNA and intracellular contents into the cheese
matrix where it may remain stable and be detected using
PCR, which could artificially elevate the bacterial esti-
mates during qPCR assays. Such extracellular DNA can
be removed by treating samples with DNase enzyme prior
Table 5 Fold change of total lactobacilli populations in cheeses
added with different probiotic lactobacilli over 270 days of ageing
Probiotic organism
Fold change in populations (CFU g�1
ratio of 270 days/0 days)*
Full fat
(n = 2)
Reduced fat
(n = 2)
Low fat
(n = 2)
Lactobacillus
acidophilus LA-5
�1�6 � 2�7 �8�1 � 1�3 1�4 � 0�4
Lact. acidophilus L10 3�5 � 0�7 �8�3 � 1�0 �6�0 � 1�6Lactobacillus
casei CRL-431
1�6 � 0�8 �6�9 � 0�7 6�9 � 0�7
Lact. casei L26 5�8 � 0�5 1�7 � 0�2 1�5 � 0�4Lactobacillus paracasei F19 1�6 � 0�4 �5�6 � 0�7 34�2 � 1�3
Negative sign indicates reduction.
*Values are indicated followed by standard deviations after the ‘�’
sign.
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology1648
LAB survival in probiotic-added cheese B. Ganesan et al.
to cellular lysis to degrade prereleased DNA. However,
DNase-treated 3-month-old cheeses containing added
Lact. casei L26 and Bif. lactis Bif-6, when tested by qPCR
detection using species-specific primers, did not differ
(P > 0�05) in qPCR Cq times from nontreated cheeses
(Fig. 2), indicating that extracellular DNA was not avail-
able for PCR detection and did not contribute to the
total count using qPCR. This indicated that qPCR assays
are exclusively based on DNA from live bacteria (includ-
ing bacteria that are nonculturable) and that DNA from
dead bacteria is not being enumerated.
Secondarily, bacterial cells may exist in a physiologi-
cally dead state wherein they are partially autolysed or
their cell walls or membranes are partly compromised to
create a leaky membrane, but still contain intact DNA
that could be co-extracted along with live cells’ DNA and
cause overestimation. To assess this potential interference,
we conducted qPCR assays on all three cheese types at
5 days, 4, 6 and 9 months age, to which PMA, a DNA-
chelating dye, was added prior to DNA extraction. PMA
selectively permeates across and binds DNA in dead/com-
promised cells and thus limits PCR amplification from
the chelated DNA, which consequently will increase the
DCq over ageing if cell membrane is compromised. The
procedure of PMA treatment for cheese was validated by
adding 107–108 logarithmic growth phase cells to a cheese
sample obtained from another source prior to PMA treat-
ment, and DNA extraction was followed by qPCR enu-
meration. With and without PMA treatment, the DCqbetween samples was similar (P > 0�05), which proved
that the added cells were live (data not shown). We con-
ducted qPCR assays targeting the starter lactococci in
cheese without added probiotics and two cheeses with
5
6
7
8
9
10
CF
U g
m–1
CF
U g
m–1
CF
U g
m–1
5
6
7
8
9
10
0 90 270Days
5
6
7
8
9
10
120
0 90 270Days
120
0 90 270Days
120
Figure 1 Lactococcal starter bacteria population levels in Cheddar
cheeses estimated by qPCR. Vertical error bars represent standard
deviations of log10 (CFU g�1) assessed from two replicates of cheese.
Top panel, full-fat cheese, middle panel, reduced-fat cheese, and bot-
tom panel, low-fat cheese. (●) Control—probiotic not added; (■) pro-biotic Bifidobacterium lactis Bif-6 added; (♦) probiotic Lactobacillus
acidophilus L10 added.
0
5
10
15
20
25
Genus Species
Cq
Lactobacillus primer type
Figure 2 Quantitative PCR Cq times of Lactobacillus genus and spe-
cies determined from cheeses treated with DNase enzyme prior to
bacterial lysis to remove extracellular DNA. Cheese age was 3 months
and contained added probiotic Lactobacillus casei L26. White bar, no
DNase treatment, grey bar, DNase treated prior to lysis and DNA
extraction.
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology 1649
B. Ganesan et al. LAB survival in probiotic-added cheese
different probiotic species (Lact. acidophilus La-5 and
Bif. lactis Bif-6) in full-, reduced- and low-fat cheeses
over 9 months of cheese ageing. In almost all cases, the
DCq did not change significantly (P > 0�05) over cheese
age for lactococci (Fig. 3a), suggesting that over ageing
up to 9 months, starter lactococci survive the ageing pro-
cess without loss in viability no change in the membrane
permeability, despite declines in plate counts as demon-
strated by other studies (Crow et al. 1995; Buist et al.
1998; Oberg et al. 2011). The DCq for lactococci did,
however, increase slightly in the low-fat cheese with
added bifidobacteria between 0 and 120 days (Fig. 3a)
and remained at similar levels up to 270 days, suggesting
that the membrane permeation increase was insignificant
over long-term ageing of cheese. Lactococci survived even
after probiotic addition without loss in viability, suggest-
ing that the impact of added probiotics is likely minimal.
This approach also verified that LAB levels assessed by
qPCR identified viable and intact cells that were present
in the cheese matrix and not dead or compromised cells.
Assessing probiotic survival using the PMA-added
cheese samples showed that added lactobacilli also sur-
vived in Cheddar cheese over age, demonstrated by either
a significant reduction in DCq (P ≤ 0�05, full-fat cheese,
Fig. 3b) or modest increases in DCq (P ≤ 0�05, reduced-and low-fat cheeses, Fig. 3b). Similarly, small but signifi-
cant changes in DCq (P ≤ 0�05) were also observed with
bifidobacteria-added cheese (Fig. 3c), showing that bifi-
dobacteria also survived, with some loss of viability on
one strain, which matches assessment by standard qPCR
that showed some loss of bifidobacterial viability in 270-
day-old Cheddar cheeses (Table 4 and Fig. S3). Addi-
tional bifidobacterial species need to be assessed for their
viability in cheese. However, application of the PMA
assay confirmed that qPCR detected viable LAB and bifi-
dobacteria in the cheese matrix and that starter lactococ-
ci, NSLAB and probiotic lactobacilli, and bifidobacteria
remained viable throughout 270 days of Cheddar cheese
ageing.
Discussion
To consistently produce commercial cheeses, the manu-
facturing process of aged semi-hard and hard cheeses is
designed to physico-chemically control their microbial
diversity. For example, Cheddar cheese is initially inocu-
lated with high numbers of starter lactococci (105–106 CFU ml�1 milk) that replicate rapidly during cheese
(a)
(b) (c)
14
12
10
8
6
4
2
00
9876543210
0 180
50 100 150 200 250 300
Time (days)
Time (days)
ΔCq
(with
- w
ithou
t PM
A)
ΔCq
(with
- w
ithou
t PM
A) 18
1614121086420
0 180Time (days)
ΔCq
(with
- w
ithou
t PM
A)
Figure 3 Comparisons of live lactococcal (a),
probiotic Lactobacillus acidophilus (b), and
probiotic Bifidobacterium lactis (c) populations
by determining qPCR Cq times from cheese
samples’ DNA extracted with and without
propidium monoazide addition. Data are
presented as the difference in Cq times, DCq,
as explained in Materials and Methods.
Vertical error bars represent standard
deviations of DCq assessed from two
replicates. In panel a, □ full-fat control
(probiotic bacteria not added during
cheesemaking), ■, low-fat control (probiotic
bacteria not added during cheesemaking), 4,
Bif. lactis Bif-6 added to full-fat cheese, ♢
Bif. lactis Bif-6 added to low-fat cheese, ○,Lactobacillus casei L10 added to full-fat
cheese, ●, Lact. casei L10 added to low-fat
cheese. In panel b, white bars, full-fat cheese,
grey bars, reduced-fat cheese, and black bars,
low-fat cheese, all containing
Lact. acidophilus LA-5 added to cheese. In
panel c, white bars, full-fat cheese, grey bars,
reduced-fat cheese, and black bars, low-fat
cheese, all containing Bif. lactis Bif-6 added
to cheese. Statistical significance over time
was determined only within each cheese.
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology1650
LAB survival in probiotic-added cheese B. Ganesan et al.
manufacture and chiefly degrade lactose to produce lactic
acid and reduce cheese pH during early ageing. When
adjunct or probiotic bacteria are included, the balance of
LAB populations may be altered.
Chiefly, the added probiotics must survive in sufficient
numbers (target 3�6 9 106 to 3�6 9 107 CFU g�1 cheese,
to deliver 109 bacteria per serving) throughout long-term
ageing of cheese to provide desired health benefits upon
human consumption (Medici et al. 2004; Ouwehand
et al. 2012). Among the added lactobacilli, Lact. casei and
Lact. paracasei are commonly found in Cheddar cheese,
are even used as adjuncts and are expected to survive
throughout cheese ageing (Peterson and Marshall 1990).
To detect added probiotic bacteria, we applied primers
previously designed in other studies (Table 3) after select-
ing for specificity against the strains used in our study.
Better sensitivity was achieved compared to previous
studies by improving DNA extraction from Cheddar
cheese using phenol–chloroform-based methods (min.
100 CFU g�1 detected; Fig. S1). By applying genus- and
species-specific primers, we were able to distinctly enu-
merate probiotic lactobacilli and NSLAB lactobacilli. The
consistent presence of NSLAB lactobacilli at 10- to 100-
fold higher levels precluded any interference by added
probiotic lactobacilli in NSLAB enumeration. The use of
qPCR also provided a specific and reliable quantitative
assay throughout cheese ageing, as opposed to growth
media-based enumeration techniques (McSweeney et al.
1993; Gardiner et al. 1998; Crow et al. 2001; Oberg et al.
2011) where the distinction between starter and NSLAB
becomes difficult beyond 90 days of age. At certain cheese
ages, bacterial levels estimated by qPCR varied by as
much as 10- to 1000-fold, which appears to be common
in the cheese matrix based on other studies that used
media-based enumeration approaches (Table 1).
Total lactobacilli were present at much higher levels of
108 to 109 CFU g�1 initially than expected (<100 CFU
g�1) postmanufacture. This indicates insufficient sanita-
tion, which led to high levels of NSLAB in the 136-kg
milk cheese vats. In higher scale manufacture (>500 kg
milk per batch), equipment is frequently subject to clean-
ing-in-place, which provides excellent control over
unwanted NSLAB presence. However, even at this high
initial NSLAB level, we could still distinguish added pro-
biotic from NSLAB, which demonstrated the strength of
using qPCR to detect added bacterial species.
Added probiotic lactobacilli survived in cheese over
270 days of ageing, even growing 10 to 1000-fold
(Table 4, Fig. S2). This observation is not surprising,
considering that NSLAB lactobacilli are acquired from
different sources and only found at low levels (<100 CFU
g�1) in early stages of cheesemaking, but attain levels of
105–107 CFU g�1 over 3–6 months of ageing (Beresford
et al. 2001). Other studies that compared survival of dif-
ferent lactobacilli in cheese have confirmed the presence
of Lact. paracasei up to 300 days of cheese age (Gardiner
et al. 1998; Fitzsimons et al. 2001). Potentially, members
of the same genus likely share survival and metabolic
characteristics that allow them to adapt to the cheese
environment. Comparative genomics studies have verified
that members of the genus Lactobacillus share 383 orthol-
ogous genes across 20 distinct genomes (Makarova et al.
2006), which is likely a common core gene set that allows
survival of lactobacilli in a dairy matrix. However, sur-
vival of probiotic lactobacilli differed across cheese types.
The pattern of Lact. acidophilus strains’ survival was
not influenced by cheese fat levels, whereas Lact. casei
and Lact. paracasei strains survived differently due to fat,
although all three species increased in numbers over
cheese age. The rationale for variable survival of species is
not evident, because other studies have shown that
Lact. casei survives equally well in all these cheese types
(Fenelon et al. 2000; Midje et al. 2000). Both Lact. casei
and Lact. paracasei are common species of NSLAB in
Cheddar cheese and likely are better adapted to cheese
environments. Many previous studies have used Lact. ca-
sei and Lact. paracasei strains as adjuncts for reduced-fat
cheeses (Midje et al. 2000; Swearingen et al. 2001; Dasen
et al. 2003), but none have yet reported the associated
challenges for survivability in such conditions. One likely
explanation is lower salt-in-moisture (3%) in full-fat
cheese compared to reduced- or low-fat cheeses (4%)
that may allow starter bacteria to metabolize lactose fas-
ter, leading to sugar starvation and further, an earlier
shift into the nonculturable state (Ganesan et al. 2007).
The nondividing lactococci may hence be a lesser chal-
lenge to the added lactobacilli, whereas the later the lac-
tose reduction, starter nonculturability is delayed and so
is growth of lactobacilli. However, this explanation only
fits the increase in levels of strains CRL-431 and F19, and
not L26, which survived better in full-fat cheese. Addi-
tional genes in the genomes of lactobacilli outside the
common core set (Makarova et al. 2006) may be involved
in the ability of probiotic lactobacilli to survive differently
in cheese.
Bifidobacteria are common inhabitants of animal and
bird gastrointestinal systems (Gomez-Donate et al. 2012).
Their survival in cheese has been previously demonstrated
(Mc Brearty et al. 2001) but needs further comprehen-
sion, as cheese contains lactose as the abundant sugar
that is utilized by the starter bacteria and NSLAB (Crow
et al. 1993), thus limiting available sugar for bifidobacte-
ria. The species, Bif. lactis, used in this study is putatively
a dairy-adapted derivative of Bif. animalis (Garrigues
et al. 2010) and may survive effectively in cheese. How-
ever, the two Bif. lactis strains added to cheese showed
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology 1651
B. Ganesan et al. LAB survival in probiotic-added cheese
differing survival patterns in cheeses, with one surviving
poorly in full-fat cheese and the other exhibiting low sur-
vival in low-fat cheese. The variable survival of bifidobac-
terial species in even one cheese type has been reported
previously (Mc Brearty et al. 2001). Our observation and
that of Mc Brearty et al. (2001) suggests that alteration of
fat level effectively changes physico-chemical conditions
inside the cheese matrices and thus alters the survival of
members of the same genus.
We also verified that the addition of different probiotic
lactobacilli influenced the levels of total lactobacilli in
Cheddar cheese, as for example, inclusion of Lact. aci-
dophilus La-5 and Lact. casei CRl431. This is interesting,
considering that probiotics were added at 10- to 1000-
fold lower levels than total lactobacilli, and highlights that
even minor components of the cheese flora are likely to
alter the major component flora, though not universally.
This is likely due to the initial inoculum of an adjunct at
a higher level than its adventitious entry into the cheese
matrix that consequently favours its later competitive
existence. Total lactobacilli levels also changed in other
cheeses influenced by cheese type. This observation has
been partially confirmed by other groups using microbial
plating for enumeration (Gardiner et al. 1998; Swearingen
et al. 2001; Dasen et al. 2003) that were, however, limited
in distinguishing added adjuncts from other NSLAB.
In relation to LAB viability in cheese, the ‘lactococcal
lysis’ theory proposes that starter lactococcal species usu-
ally die during Cheddar cheese ageing as evidenced by
microbial selective enumeration (Fenelon et al. 2000).
Supposedly, the lysed cells release intracellular enzymes
into the cheese matrix that further degrade the caseins
into peptides and amino acids for NSLAB metabolism
(Crow et al. 1995; Buist et al. 1997, 1998). In a complex
ecology such as cheese, with multiple species present,
assessing the release of intracellular enzymes into the
cheese serum phase is nonspecific (O’Sullivan et al. 2000)
and thus not reliable. However, bacterial lysis may release
DNA into cheese that remains stable and contributes to
live bacterial estimates during qPCR. The absence of such
extracellular DNA was established by attempting qPCR
detection from DNase-treated cheeses using species-spe-
cific primers (Fig. 2). Hence, extracellular DNA did not
contribute towards qPCR amplification. Further, using
the selectively permeating PMA that binds intracellular
nucleic acids in dead or membrane-compromised cells,
we found that lactococci, other NSLAB and probiotic lac-
tobacilli all remained viable in Cheddar cheese over
270 days of ageing (Fig. 3). The reduced viability of bifi-
dobacteria was expected based on qPCR assays and was
further confirmed by PMA assays. These findings cumula-
tively demonstrated that qPCR, even without PMA addi-
tion, only estimates viable cell populations in cheese and
not that of dead cells. More importantly, these findings
highlight the ability of lactococci to survive in Cheddar
cheese throughout ageing, which was expected based on
prior studies that demonstrated lactococcal nonculturabil-
ity in culture (Ganesan et al. 2006, 2007). Potentially,
with casein-derived amino acids being abundant in
cheese, lactococci may survive in the nonculturable state
in cheese and acquire metabolic energy via previously
proposed mechanisms, such as Arg (Chou 2001; Chou
et al. 2001) and branched chain amino acid degradation
(Ganesan et al. 2006). While probiotic lactobacilli share
similar genetic repertoires to lactococci, their ability, or
that of the genetically distinct bifidobacteria, to use such
mechanisms for survival remains to be established.
In conclusion, different strains of probiotic lactobacilli
and bifidobacteria were added to full-, reduced- and low-
fat Cheddar cheeses to understand their survivability
under these diverse physico-chemical environments. We
demonstrated that both the starter LAB and probiotic
bacteria survived throughout ageing, indicating the suit-
ability of semi-hard, aged cheeses as suitable vehicles for
probiotic delivery. Probiotic lactobacilli survived differ-
ently at the species level. Some added probiotics also
altered the levels of total lactobacilli and starter bacteria,
even when present at levels 10- to 1000-fold lower. Fur-
ther, we also demonstrated that starter, NSLAB and pro-
biotic bacteria remained viable with an intact cell
membrane throughout the period of cheese ageing.
Acknowledgements
The authors acknowledge the assistance of and thank
Brian Pettee for cheesemaking and sampling and Reed
Gann for qPCR primer design.
Conflict of Interest
No conflict of interest declared.
Funding source
The study was accomplished by a grant to CB and DJM
that was funded by Dairy Management Inc. and adminis-
tered by the Dairy Research Institute (Rosemont, IL).
Authors’ contributions
CB, DJM and BW conceived the project; CB and DJM
made cheese with added probiotic bacteria; BW guided
JP, ND and GR, who performed DNA extraction and
qPCR; BG and BW conducted data analyses and authored
the manuscript.
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology1652
LAB survival in probiotic-added cheese B. Ganesan et al.
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B. Ganesan et al. LAB survival in probiotic-added cheese
Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Data S1 Materials and methods.
Table S1 Proximate composition (averages) of Ched-
dar cheese made at different fat levels.
Table S2 Limit of detection for different probiotic bac-
teria using species primers in Cheddar cheese.
Table S3 Analysis of main effects across all probiotics
added.
Table S4 Differences in probiotic survival by genus.
Table S5 Impact of probiotic Lactobacillus strains on
NSLAB levels in cheese.
Figure S1 Standard curves of bacterial counts for qCR-
based estimation.
Figure S2 Different methods for DNA extraction yield
different results for lactococcal detection by qPCR.
Figure S3 Levels of added probiotic Lactobacillus in
Cheddar cheeses over 9 months of ageing, estimated by
qPCR.
Figure S4 Levels of added probiotic Bifidobacterium
lactis in Cheddar cheeses over 9 months of ageing, esti-
mated by qPCR.
Figure S5 Levels of total lactobacilli in probiotic Lacto-
bacillus-added Cheddar cheeses over 9 months of ageing,
estimated by qPCR.
Journal of Applied Microbiology 116, 1642--1656 © 2014 The Society for Applied Microbiology1656
LAB survival in probiotic-added cheese B. Ganesan et al.
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