Inhibition of Listeria monocytogenes in Fresh Cheese Using a Bacteriocin-Producing Lactococcus lactis CAU2013 Strain

Abstract

In recent years, biocontrol of foodborne pathogens has become a concern in the food industry, owing to safety issues. Listeria monocytogenes is one of the foodborne pathogens that causes listeriosis. The major concern in the control of L. monocytogenes is its viability as it can survive in a wide range of environments. The purpose of this study was to isolate lactic acid bacteria with antimicrobial activity, evaluate their applicability as a cheese starter, and evaluate their inhibitory effects on L. monocytogenes. Lactococcus lactis strain with antibacterial activity was isolated from raw milk. The isolated strain was a low acidifier, making it a suitable candidate as an adjunct starter culture. The commercial starter culture TCC-3 was used as a primary starter in this study. Fresh cheese was produced using TCC-3 and L. lactis CAU2013 at a laboratory scale. Growth of L. monocytogenes (5 Log CFU/g) in the cheese inoculated with it was monitored during the storage at 4°C and 10°C for 5 days. The count of L. monocytogenes was 1 Log unit lower in the cheese produced using the lactic acid bacteria strain compared to that in the cheese produced using the commercial starter. The use of bacteriocin-producing lactic acid bacteria as a starter culture efficiently inhibited the growth of L. monocytogenes. Therefore, L. lactis can be used as a protective adjunct starter culture for cheese production and can improve the safety of the product leading to an increase in its shelf-life.

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TITLE PAGE
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- Korean Journal for Food Science of Animal Resources -
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ARTICLE INFORMATION
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Article Type
Research article
Article Title
Inhibition of Listeria monocytogenes in fresh chees using a bacteriocin-
producing Lactococcus lactis CAU2013 strain
Running Title (within 10 words)
Biocontrol of L. monocytogenes in fresh cheese
Author
Sung-Hee Yoon1, Geun-Bae Kim1
Affiliation
1 Chung-Ang University, Anseong, Korea
Special remarks – if authors have
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ORCID (All authors must have ORCID)
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Sung-Hee Yoon (https://orcid.org/0000-0002-6826-0840)
Geun-Bae Kim (https://orcid.org/0000-0001-8531-1104)
Conflicts of interest
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interest for all authors.
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The authors declare no potential conflict of interest.
Acknowledgements
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sources, equipment, and supplies). Include
name and number of grant if available.
(This field may be published.)
Author contributions
(This field may be published.)
Conceptualization: Kim GB
Data curation: Yoon SH, Kim GB
Investigation: Yoon SH, Kim GB
Writing - original draft: Yoon SH
Writing - review & editing: Yoon SH, Kim GB
Ethics approval (IRB/IACUC)
(This field may be published.)
This manuscript does not require IRB/IACUC approval because there are
no human and animal participants.
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Geun-Bae Kim
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kimgeun@cau.ac.kr
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8

2
Abstract
9
In recent years, biocontrol of foodborne pathogens has become a concern in the food industry,
10
owing to safety issues. Listeria monocytogenes is one of the foodborne pathogens that causes
11
listeriosis. The major concern in the control of L. monocytogenes is its viability as it can survive
12
in a wide range of environments. The purpose of this study was to isolate lactic acid bacteria
13
with antimicrobial activity, evaluate their applicability as a cheese starter, and evaluate their
14
inhibitory effects on L. monocytogenes.
15
Lactococcus lactis strain with antibacterial activity was isolated from raw milk. The isolated
16
strain was a low acidifier, making it a suitable candidate as an adjunct starter culture. The
17
commercial starter culture TCC-3 was used as a primary starter in this study. Fresh cheese was
18
produced using TCC-3 and La. lactis CAU2013 at a laboratory scale. Growth of L.
19
monocytogenes (5 log CFU/g) in the cheese inoculated with it was monitored during the storage
20
at 4℃ and 10℃ for 5 days. The count of L. monocytogenes was 1 log unit lower in the cheese
21
produced using the lactic acid bacteria strain compared to that in the cheese produced using the
22
commercial starter.
23
The use of bacteriocin-producing lactic acid bacteria as a starter culture efficiently inhibited
24
the growth of L. monocytogenes. Therefore, La. lactis can be used as a protective adjunct starter
25
culture for cheese production and can improve the safety of the product leading to an increase
26
in its shelf-life.
27
Keywords: Lactococcus lactis, bacteriocin, Listeria monocytogenes, cheese starter culture,
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foodborne pathogen
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Introduction
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Listeriosis is a foodborne disease caused by Listeria monocytogenes. It can lead to sepsis,
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meningitis, encephalitis, and even death (de Noordhout et al., 2014). Despite its low incidence
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compared with that of other foodborne illnesses, listeriosis is one of the major issues in the food
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industry because of its high fatality rate. L. monocytogenes is found in dairy products,
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particularly in ready-to-eat cheese products. As L. monocytogenes survives in various
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environments such as a those with a wide range of temperature (0–45℃) and pH (4.1–9.6), it
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can contaminate cheese at several stages of production; therefore, its growth is difficult to
37
control (Lungu et al., 2008; Melo et al., 2014).
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Several methods have been used to control the growth of L. monocytogenes in cheese,
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including using bacteriocin or bacteriocin-producing lactic acid bacteria (LAB). Bacteriocins
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are peptides or proteins, ribosomally synthesized by bacteria, which have antimicrobial ability
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against closely related species. The application of bacteriocin-producing bacteria is
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advantageous as they are stable, cost-effective, and safe. Anti-listerial activity of LAB in cheese
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have also been reported (Coelho et al., 2014; Dal Bello et al., 2012; Kondrotiene et al., 2018).
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In the present study, we aimed to determine the effects of bacteriocin-producing LAB isolated
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from raw milk on the growth of L. monocytogenes in milk broth and cheese.
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Materials and Methods
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Isolation of bacteriocin-producing lactic acid bacteria
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Potential bacteriocin-producing LAB were isolated from raw bovine milk, obtained from a
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Chung-Ang University-affiliated farm (Anseong, Republic of Korea). The sample was serially
51
diluted ten-fold and plated on MRS agar (BD Difco, USA). The plates were incubated at 37℃
52
for 24–48 h, and a total of 90 well-isolated colonies were collected. Each colony was inoculated
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into MRS broth for 24 h at 37℃.
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To screen for antimicrobial activity, the cell-free supernatant (CFS) was obtained after
55
neutralization with 1N NaOH, centrifugation at 13,000 rpm for 10 min at 4℃, and filtered
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through 0.45 µm filters to remove bacterial cells. Then, each supernatant was spotted on the
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tryptic soy agar (TSA, BD Difco, USA) plate inoculated with a lawn of Listeria monocytogenes
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ATCC 19115 as an indicator strain. The plates were incubated at 30℃ for 12 h, and antibacterial
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activity was confirmed with the presence of inhibition zone. The strains with antibacterial
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activity were routinely cultured in MRS broth at 37℃ overnight and were preserved in 10%
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skim milk supplemented with 25% (v/v) glycerol, stored at -80℃ for further use.
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Identification of bacteriocin-producing strain
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The bacteriocin-producing strains were identified by Gram staining, carbohydrate
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fermentation profile (analytical profile index (API) test), and 16S rRNA gene sequencing
66
analysis. Gram staining and API analysis was performed using a Gram-stain kit (BD Difco,
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USA) and API 50 CHL kit (Biomérieux, France), respectively, according to the manufacturer’s
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instructions.
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For 16S rRNA analysis, the genomic DNA was extracted using QIAamp PowerFecal DNA
70
Kit (Qiagen, Germany) and amplified using 2X H-star Taq PCR Master Mix (BioFACT,
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Republic of Korea). Polymerase chain reaction (PCR) was performed using the universal
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bacterial primers 27F (5′-AGAGTTTGATCMTGG CTCAG-3′), 1492R (5′-
73
TACGGYTACCTTGTTACGACTT-3′), 785F (5′-GGATTAGA TACCCTGGTA-3′), and 805R
74
(5′-GACTACCAGGGTATCTAATC-3′). The PCR products were purified using a PCR
75
purification kit (Qiagen, Germany) and sequenced by SolGent Co. Ltd. (Daejon, Republic of
76
Korea).
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The analyzed sequences were confirmed using the EzTaxon-e server (www.ezbiocloud.net/)
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(Kim et al., 2012) and NCBI GenBank database using the Basic Local Alignment Search Tool
79
(BLAST) algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al., 1990).
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Antibacterial activity of bacteriocin
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Bacteriocin activity was assessed using a spot-on-lawn method as described previously
83
(Phumisantiphong et al., 2017) with minor modifications. Briefly, each indicator strain was
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inoculated to 4 mL of molten TSA and overlaid on the base TSA plate. After solidification, 20
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µL of the neutralized CFS of LAB strains was spotted onto the indicator lawn. After incubation
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at 30℃ for 12 h, a clear inhibition zone was observed. The foodborne pathogens used as
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indicator strains were cultured in tryptic soy broth (TSB, BD Difco, USA) at 37℃ overnight
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before use. The experiment was conducted in triplicates.
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Evaluation of acid production
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The acid production of the La. lactis CAU2013 was evaluated and compared with that of the
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commercial starter TCC-3 (Chr. Hansen, Denmark), which consisted of Lactobacillus
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delbrueckii subsp. bulgaricus and Streptococcus thermophilus. The cultures were grown in
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MRS broth at 37℃ overnight. Then, individual cultures and mixture of La. lactis CAU2013
95
and TCC-3 (1:1 ratio) were inoculated in 10% skim milk broth (Harrington and Hill, 1991) and
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whole milk. The pH and titratable acidity (TA) were measured every three hours for 12 h while
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incubating at 30℃. To determine TA, 0.1% phenolphthalein was used as an indicator, and 0.1
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N sodium hydroxide (NaOH) for titration.
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Anti-listerial activity of strain CAU2013 as an adjunct starter in milk
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To determine the anti-listerial properties of strain CAU2013 when used as an adjunct starter
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in milk, 10% skim milk broth and whole milk media were inoculated with an overnight culture
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of CAU2013 and 1:1 ratio of CAU2013 and TCC-3 starter (final concentration of 7 log
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CFU/mL). Additionally, the overnight culture of L. monocytogenes ATCC 19115 was
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inoculated to each setup (final concentration of 5 log CFU/mL). The inoculated milk media
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were incubated at 30℃ for 12 h. The viable cell count of L. monocytogenes was determined
107
every three hours. Samples were diluted serially in ten-fold increments using 1×phosphate-
108
buffered saline (PBS, pH7.5) and plated on Oxford agar (BD Difco, USA).
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Manufacture of laboratory-scale cheese
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The lab-scale cheese was manufactured following the methods of Mills et al.(2011) with
111
some modifications. TCC-3 was used as the primary starter and La. lactis CAU2013 as an
112
adjunct culture. The starter cultures were initially grown in MRS broth at 37℃ for 24 h before
113
inoculation into 10% skim milk broth and incubated for 18 h at 37℃ before use. Additionally,
114
L. monocytogenes ATCC19115 was cultured in TSB for 18 h at 37℃ before use.
115
Milk (400 mL) (Seoul milk, Republic of Korea) was heated to 31℃ before the inoculation of
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starter culture. The starter cultures were inoculated as follows: TCC-3 and La. lactis CAU2013
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and TCC-3 (1:1 ratio), both at a final concentration of 7 log CFU/mL. Subsequently, 0.01% L.
118
monocytogenes at a level of 5 log CFU/mL was inoculated into both treatments. After 30 min,
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0.2 g/L of rennet was added, and the mixture was stirred for 2 min. Once coagulum formed
120
firmly, the curd was cut into cubes, and the mixture was stirred for 10 min. Then, the mixture
121
was heated to 36℃ for 10 min and stirred for 20 min. The whey was drained off, and curd was
122
distributed into the sterile dish. The samples were stored at 4℃ and 10℃ for 5 days. The
123
procedure of cheese production is illustrated in Figure 1.
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125
Microbial analysis of laboratory-scale fresh cheese
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The viable cell counts of LAB and L. monocytogenes in the lab-produced cheese were
127
determined in duplicate every day during storage at 4℃ and 10℃. For microbial analysis, 1 g
128
of cheese was homogenized in 9 mL of PBS buffer and were serially diluted ten-fold in the
129
same buffer and plated on the appropriate agar plate. The LABs were enumerated on MRS agar
130
after incubation at 37℃ for 3 days, and L. monocytogenes on Oxford agar after incubation at
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37℃ for 24 h. All of the experiments were conducted in triplicates.
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Results and discussion
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Isolation and identification of bacteriocin-producing strains
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Among the 90 colonies isolated from raw milk, one isolate exhibited antibacterial activity
135
against L. monocytogenes. The strain CAU2013 was characterized as a gram-positive, coccus-
136
shaped bacterium. The biochemical characteristics determined using the API 50 CHL kit are
137
described in Table 1. 16S rRNA gene sequence analysis revealed that strain CAU2013 is most
138
likely a strain of Lactococcus lactis (Table 2), which commonly produce nisin (Shin et al.,
139
2016). Neighbor-joining (NJ) phylogenetic tree of the strain CAU 2013 and related type strains
140
based on 16S rRNA gene sequences also clearly show that this strain belongs to Lactococcus
141
lactis (Supplementary Figure 1). La. lactis strains are historically used in the fermentation and
142
preservation of food and are generally recognized as safe (GRAS) (Cook et al., 2018).
143
Therefore, La. lactis CAU2013 was selected for downstream applications in the study.
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L. monocytogenes ATCC 19115 was used as an indicator strain for all experiments because it
145
belongs to the serotype 4b, which causes most cases of listeriosis.
146
147
Antibacterial activity of bacteriocin
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The bacteriocin produced by La. lactis CAU2013 had antibacterial activity against all Listeria
149
strains as well as Staphylococcus aureus, which are common foodborne pathogens (Yoon,
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2020). However, no antibacterial activity was observed against other gram-positive foodborne
151
pathogens, such as Salmonella enteritidis and Escherichia coli (Table 3). Generally, nisin is
152
highly effective against gram-positive bacteria by binding to lipid Ⅱ, which leads to the
153
inhibition of cell wall biosynthesis or pore formation in the membrane. However, nisin cannot
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bind to its target lipid II in gram-negative bacteria, because of the presence of the outer
155
membrane (Li et al., 2018).
156
157
Characterization of acid production
158
The changes in pH and TA values in 10% skim milk broth and in whole milk are presented
159
in Figure 2. La. lactis CAU2013 reduced the pH of skim milk broth from 6.41 to 5.77 and that
160
of whole milk from 6.65 to 6.20. Additionally, TA value increased to 0.25 in both broths. Ayad
161
et al. (2004) described fast, medium, or slow-acidifying strains as △pH(=pHat time-pHzero time)
162
of 0.4 U achieved after 3 h, 3–5 h, and > 5 h, respectively. Also, Raquib et al. (2003) classified
163
strains with titratable acidity as low, moderate, or fast when the TA values were < 0.5, between
164
0.5 and 0.6, and > 0.6, respectively. Therefore, La. lactis CAU2013 can be classified as a low
165
acidifier strain. This result is consistent with other studies that reported poor acid production
166
from La. lactis strains (Ayad et al., 2004; Coelho et al., 2014).
167
The pH values measured corresponded with the calculated TA and were generally similar for
168
skim milk broth and whole milk. The mixed starter, consisting of TCC-3 and La. lactis
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CAU2013, accelerated the acidification in milk. Nevertheless, bacteriocin-producing strains
170
delay acidification (Garde et al., 1997); however, the strain CAU2013 did not show similar
171
properties. The accelerated acidification might be because of the interaction between the strains;
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however, the underlying mechanisms need further research. Ávila et al. (2005) observed that
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enterocin-producing adjunct starter enterococci enhanced milk acidification, which may be
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stimulated by the low-molecular-weight nitrogen compounds produced by primary starter,
175
Lactobacillus helveticus LH92.
176
The rapid decline in pH during the initial stage of cheese production is crucial for curd
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formation and prevention of the growth of undesirable microorganisms. Therefore, the fast-
178
acidifying strains can be used as primary starters, while the slow-acidifying bacteria can be used
179
as adjunct starters. As the strain CAU2013 has antibacterial property but has low acid
180
production ability, it is better suited as an adjunct starter culture.
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Anti-listerial activity of strain CAU2013 as an adjunct starter in milk
183
The growth of L. monocytogenes was monitored in skim milk broth and whole milk during
184
incubation at 30℃. In skim milk broth with La. lactis CAU2013, the concentration of L.
185
monocytogenes count was reduced by 3 log units more compared with that of other samples
186
after 3 h and not detected following 6 h of fermentation (Figure 3a). In the whole milk with the
187
strain CAU2013, L. monocytogenes count was reduced by 0.5 log unit after 6 h, and 1 log unit
188
after 9 h compared with that of other samples (Figure 3b).
189
The results support the findings from several studies that reported that the addition of
190
bacteriocin affects the biocontrol of spoilage bacteria. Muñoz et al. (2007) investigated that E.
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faecalis-produced enterocin in milk and found that it could control the growth of
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Staphylococcus aureus. In addition, according to Arqués et al. (2011), the addition of nisin in
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milk decreased L. monocytogenes count by 3 log units after 4 h.
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The efficiency of the combined starter cultures in the inhibition of L. monocytogenes was
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lower in whole milk than in skim milk. The difference in the composition between the two milk
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media could be a factor responsible for the difference. In addition, Muñoz et al. (2007) stated
197
that low effectiveness in foods could be attributed to higher retention of the bacteriocin
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molecules by milk components, resulting in slower diffusion. However, in both cases, inhibition
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of L. monocytogenes growth was observed. The results suggest the potential application of La.
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lactis CAU2013 in various food systems to control L. monocytogenes growth.
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Inhibition of L. monocytogenes in laboratory-scale fresh cheese
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The cell count of the starter cultures was determined during the storage at 4℃ and 10℃
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(Figure 4). In both the cases, LAB reached a final concentration of 9 log CFU/g during cheese
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manufacture.
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During the storage at 4℃, the cheese treated with TCC-3 starter culture maintained L.
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monocytogenes count at 7.5 to 7.7 log CFU/g. In contrast, the cheese treated with TCC-3 and
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La. lactis CAU2013 had less L. monocytogenes count, approximately 0.5 log unit at 0 h and 1
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log unit after 5 days with a final concentration of 6.4 log CFU/g. Besides, during the storage at
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10℃, the cheese treated with TCC-3 starter culture maintained the bacterial count between 6.86
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and 7.31 log CFU/g (Figure 4a). within contrast, the cheese treated with TCC-3 and CAU2013
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had less L. monocytogenes count, approximately 1 log unit at 0 h and 1.5 log unit after 5 days,
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with a final concentration of 5.76 log CFU/g (Figure 4b). This result is consistent with a study
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that reported that 2 log unit reduction was observed in cheese with La. lactis strain (Coelho et
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al., 2014). Moreover, Kondrotiene et al. (2018) showed that nisin-producing La. lactis strains
216
decreased the growth of L. monocytogenes in fresh cheese during 7 days of storage at 4℃.
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Therefore, the results support that manufacturing cheese using a bacteriocin-producing starter
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reinforced the inhibition of growth of L. monocytogenes, and it would be effective in controlling
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contamination during cheese production. Additionally, after storage at temperatures of 4℃ and
220
10℃, L. monocytogenes count was reduced, which may confirm the potential of LAB in
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controlling the growth of L. monocytogenes during storage at refrigeration temperature.
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Conflicts of interest
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The authors declare no potential conflicts of interest.
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226
Author contributions
227
Conceptualization: Kim GB. Data curation: Yoon SH, Kim GB. Investigation: Yoon SH, Kim
228
GB. Writing - original draft: Yoon SH. Writing - review & editing: Yoon SH, Kim GB.
229
230
Ethics Approval
231
This article does not require IRB/IACUC approval because there are no human and animal
232
participants.
233
234
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producing lactic acid bacteria on a Lactobacillus helveticus adjunct cheese culture. J Food
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Ayad EHE, Nashat S, El-Sadek N, Metwaly H, El-Soda M. 2004. Selection of wild lactic acid
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Coelho MC, Silva CC, Ribeiro SC, Dapkevicius ML, Rosa HJ. 2014. Control of Listeria
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Dal Bello B, Cocolin L, Zeppa G, Field D, Cotter PD, Hill C. 2012. Technological
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A, Speybroeck N. 2014. The global burden of listeriosis: a systematic review and meta-
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Garde S, Gaya P, Medina M, Nuñez M. 1997. Acceleration of flavour formation in cheese by
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Malakauskasa M. 2018. Characterization and application of newly isolated nisin producing
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Lactococcus lactis strains for control of Listeria monocytogenes growth in fresh cheese.
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LWT Food Sci Technol 87:507–514.
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Li Q, Montalban-Lopez M, Kuipers OP. 2018. Increasing the antimicrobial activity of nisin-
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Listeria monocytogenes under low oxygen or anaerobic conditions: A review. Food
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Table 1. Carbohydrate fermentation patterns of the two isolated bacteriocin-producing lactic
294
acid bacteria. The test was performed with API 50 CHL kit and all data are from this study. +,
295
positive; -, negative
296
Carbohydrates
Carbohydrates
Glycerol
-
Salicin
+
Erythritol
-
D-cellobiose
+
D-arabinose.
-
D-maltose
+
L-arabinose
+
D-lactose (bovine origin)
+
D-ribose
+
D-melibiose
-
D-xylose
+
D-saccharose (sucrose)
+
L-xylose
-
D-trehalose
+
D-xylose
-
Inulin
-
Methyl-beta-D-xylopyranoside
-
D-melezitose
+
D-galactose
+
D-raffinose
-
D-glucose
+
Amidon (starch)
+
D-fructose
+
Glycogen
-
D-mannose
+
Xylitol
-
L-sorbose
-
Gentiobiose
+
L-rhamnose
-
D-turanose
-
Dulcitol
-
D-lyxose
-
Inositol
-
D-tagatose
+
D-mannitol
+
D-fucose
-
D-sorbitol
-
L-fucose
-
Methyl-alpha-D-
mannopyranoside
-
D-arabitol
-
Methyl-alpha-D-
glucopyranoside
-
L-arabitol
-
N-acetylglucosamine
+
Potassium gluconate
-
Amygdalin
+
Potassium 2-ketogluconate
-
Arbutin
+
Potassium 5-ketogluconate
-
Esculin ferric citrate
+
297

16
Table 2. Identification of bacteriocin-producing strains by BLAST and Ez-Taxon.
298
BLAST
EzTaxon
Strain
Taxon name
Similarity
(%)
Taxon name
Similarity
(%)
CAU2013
Lactococcus lactis subsp. lactis
100
Lactococcus lactis subsp. lactis
100
Lactococcus lactis subsp.
hordniae
99.86
Lactococcus lactis subsp.
hordniae
99.86
Lactococcus lactis subsp. tructae
99.39
Lactococcus lactis subsp. tructae
99.39
299
300
301
Table 3. Antimicrobial spectrum of bacteriocin from L. lactis CAU2013. +, < 10 mm; ++, >
302
10 mm; -, no inhibition zone.
303
Indicator strain
Inhibition activity
Gram positive
L. monocytogenes ATCC
15315
+
L. monocytogenes ATCC 7644
++
L. monocytogenes ATCC
19111
+
L. monocytogenes ATCC
19114
++
L. monocytogenes ATCC
19115
++
Staphylococcus aureus
RN6390
+
Gram negative
Salmonella enteritidis YHS
383
-
Escherichia coli ATCC 25922
-

17
Figure 1. The procedure of lab-scale fresh cheese production.
304
305

18
306
Figure 2. The values of pH and titratable acidity (TA) of strains grown in 10% skim milk and
307
whole milk at 30℃. (A) pH values and (B) TA values in 10% skim milk, (C) pH values and
308
(D) TA values in whole milk. TCC-3 (■), L. lactis CAU2013 (●), and the combination of
309
TCC-3 with CAU2013 (▲).
310
311
312
313
314
315
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
0 3 6 9 12 15
pH
Time (h)
TCC-3 CAU2013 TCC+CAU2013
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0 3 6 9 12 15
TA (%)
Time (h)
TCC-3 CAU2013 TCC+CAU2013
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0 3 6 9 12
TA (%)
Time (h)
TCC-3 CAU2013 TCC+CAU2013
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
0 3 6 9 12
pH
Time (h)
TCC-3 CAU2013 TCC+CAU2013
(A)
(B)
(D)
(C)

19
Figure 3. Biocontrol of L. monocytogenes in 10% skim milk broth and whole milk. L.
316
monocytogenes was inoculated in milk broth (A) and whole milk (B), and incubated at 30℃
317
without starter (●), or with 1 % of TCC-3 (▲), or the combination of TCC-3 and CAU2013
318
(■).
319
320
321
322
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
0 3 6 9 12
Log (CFU/mL)
Time (h)
control TCC TCC + CAU2013
4.00
5.00
6.00
7.00
8.00
0 3 6 9 12
Log (CFU/mL)
Time (h)
control TCC TCC + CAU2013
(B)
(A)

20
Figure 4. The viable cell counts of L. monocytogenes and LAB in fresh cheese manufactured
323
with TCC-3 starter and combination of L. lactis CAU2013 and TCC-3 and then stored for 5
324
days. During the storage at 4℃ (A) and 10℃ (B), LAB in cheese produced with TCC-3 (●);
325
TCC-3 and CAU2013 (■) were measured. Also, L. monocytogenes was measured in cheese
326
with TCC-3 (◆) and cheese with mixed starter (▲).
327
328
5.00
6.00
7.00
8.00
9.00
10.00
0 1 2 3 4 5
Log (CFU/g)
Time (Day)
TCC TCC+CAU2013 TCC(LAB) TCC+CAU2013(LAB)
5.00
6.00
7.00
8.00
9.00
10.00
012345
Log (CFU/g)
Time (Day)
TCC TCC+CAU2013 TCC(LAB) TCC+CAU2013
(B)
(A)

21
329